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Development of protocols for avocado tissue culture: somatic embryogenesis, protoplast culture, shoot proliferation and photoplast fusion

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Development of protocols for avocado tissue culture: somatic embryogenesis, protoplast culture, shoot proliferation and photoplast fusion
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Witjaksono, 1961-
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xv, 228 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Avocados ( jstor )
Embryogenesis ( jstor )
In vitro fertilization ( jstor )
Liquids ( jstor )
Nitrogen ( jstor )
Protoplast culture ( jstor )
Protoplasts ( jstor )
Somatic embryogenesis ( jstor )
Somatic embryos ( jstor )
Species ( jstor )
Avocado -- Cytology ( lcsh )
Avocado -- Genetics ( lcsh )
Avocado -- Physiology ( lcsh )
Dissertations, Academic -- Horticultural Science -- UF ( lcsh )
Horticultural Science thesis, Ph.D ( lcsh )
Plant protoplasts ( lcsh )
City of Gainesville ( local )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph.D.)--University of Florida, 1997.
Bibliography:
Includes bibliographical references (leaves 203-227).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Wtijaksono.

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DEVELOPMENT OF PROTOCOLS FOR AVOCADO TISSUE CULTURE: SOMATIC EMBRYOGENESIS, PROTOPLAST CULTURE, SHOOT PROLIFERATION AND PROTOPLAST FUSION











By


WITJAKSONO


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

























Copyright 1997

by

Witj aksono


























To my wife Nia,
for her tremendous sacrifice during these long years of study,
and to my sons, Lintang and Edgar,
for my not being there during their early days in this world.















ACKNOWLEDGMENTS


wish to express my


deepest


gratitude to


Dr. Richard


E. Litz,


my


academic


advisor, for all his encouragement, advice, guidance, help and patience during my entire


doctoral training and especially during the preparation of this dissertation.


His interest and


his stimulating discussions involving many topics will always be remembered.

I would like to extend my sincere gratefulness to the members of my dissertation


committee, Drs. Jude W.


Grosser, Dennis J. Gray, Michael E. Kane and Randy C. Ploetz,


for their help,


guidance and encouragement during the course of this study


and


their


constructive criticism in the writing of this dissertation.


A million thanks are extended to Mr.


Gray Martin, formerly of the University of


California,


Riverside,


for


his


help


in


supplying


avocado


fruitlets


for this


dissertation


research and also for his encouragement.


Sincere thanks are also extended to Dr. John


Menge and


Brandon McKee


(UC


Riverside)


for


providing Persea


seeds


and


to Dr.


Fernando


Pliego-Alfaro


of the


Universidad


de


Malaga,


Malaga,


Spain,


for


his


initial


involvement in the birth of this dissertation topic and for providing avocado shoot cultures for this work.

The invaluable assistance of the thoughtful Ms. Pamela Moon and her help with


statistical analyses,


graphics,


computers and


other matters is


gratefUlly acknowledged.


Help with statistical analysis and in photosynthesis measurements provided by Dr. Bruce


Schaffer and Mr. Angel Colt is highly valued.


I also would like to thank Dr. Ray Schnell,


I










Khalid bin Mohamad Zin, Levi Barros and Lad, and to Arlene, Fahad, Carmen, Hilmig and


Isabel for making the lab and


the neighborhood more colorful.


Invaluable


help from


Divina for picking up books and other literature from Gainesville is recognized.


The help


of other staff and faculty members at TREC is acknowledged.

The opportunity to pursue this doctoral degree was granted by the management of my office, the Research and Development Center for Biology, The Indonesian Institutes of Sciences (LIPI), and I am honored to acknowledge a scholarship awarded by my country, the Republic of Indonesia, administered by the Agency for the Assessment and Application


of Technology (BPP Teknologi).


Last but not least, a research assistantship and research


support from the California Avocado Society have been critical for the completion of this dissertation and therefore are deeply appreciated.
















TABLE OF CONTENTS

A CNW L DM ET S .. . .. . . .. . .. . . .. . .. . ... . .. . . ........... ................................. .. . . .. *. .. 1'v~



LIS jsF uA LE....................... . . .. ...*....................................................... ................

L6Ii S T 1 O F h( I F I U RS .. . .. . .. .... .......................... ....................... . . . . . . . . . . . .............x



CHAPTERS


I.

2.


Av.'c ad ....................


S *ma~tic~ Emb~r gernesi s.... ..........................................
Protoplast Isolation, Culture and Regeneration.
Somatic H11yridization.......... .......................... .


.* * .4


28 36 48


INITIATION AND MAINTENANCE OF AVOCADO


EMBRYOGENIC CULTURES....


I nt rodci ~ tion ......... Materials and Methods


.....*......................... ... 54j


.. . . . . .. . . . . . .......................... ..........................


Results ......


D iscu ssio n . . . . . ............................................................................................. ...............

SOMATIC EMBRYO DEVELOPMENT. MATURATION AND


GERMINATION ........


Materials and Methods


54 55
63 82


* .............................88


88 89


3.


4.


INTRODUCTION ..................

LITERATURE REVIEW............................ 4










PROTOPLAST ISOLATION, CULTURE AM) SOMATIC
EMBRYO REGENERATION OF AVOCADO ......................... 114


Introduction.........
Materials and Methods


114 117 123 145


AVOCADO SHOOT CULTURE AND PLANTLET DEVELOPMENT AN4D NET PHOTOSYNTHESIS IN A NONELEVATED AND ELEVATED CO2 ENVIRONMENT ................... 149


Materials and Methods

Discussion . .. . .. ......


..*............ ....................................


7.


PROTOPLAST FUSION BETWEEN AVOCADO AM) ITS RELATIVES, INCLUDING NECTANDRA CORJA CEA AND
PPER SEASPP ....... . .....*..*.......*................................... 1 74


Materials and Methods RIIesults ......... .....
Discussion . . .. .. ......


8.


174 175 181 188


SSUM M ARY AND CONCCLU SION ............................ .. .. ............................... ... .. ... 19.1


CAP P E N D IX ...........* .. .. .* .. .......................................................................*..............................194



L ISLISTE 1 .iI O F R E FE R E N C E S .....................................................................................................................................................................2 2 103


5.


6.


149 151 157
169


................................
.................................
.................................................. ..............
.................................................. ..............


.....................................
....................................
.........................................
..........................................

















LIST OF TABLES


Table


Nutrient composition of edible pulp of 'Fuerte' avocado per 100 g. .............


Comparison of the three horticultural races of Fersea americana.


Average avocado production 1992-1996. ........................................................................


.. .. .. .. .. .. .. ..7


I1


Avocado breeding objectives for special characters.............................................................. 12

List of Persea and related genera and their resistance to Phytophihora root-


rot.


......................... .. ................ 14


Summary


of in


vitro


studies


of


avocado, Persea americana


Mill.,


related species ini the famri1y of LIauraceae. ... ........................ ... ......................................... .. . .. 1 7

Enzymesiris usesedfoforrcprtoilatina.s....s.......................................................................................................... 38


2-7.


Avocado cultivars used for the experiments,


Length of 'Thomas'


their botanical varieties and


avocado fruitlet (measured without calyx) in relation


to zygotic embryo length, stage of development and morphology. ................ 56


Percentage


of


embryogenic


culture


induction


from


immature


zygotic


embryos from several avocado cultivars on different initiation media............... 64


Effect of major salt composition and gelling agent on necrosis and somatic embryogenesis of 'Thomas' nucellar culture. ...........................


Summary of the value of Pr>F from analysis of variances for the effect of


major


salt


composition,


and


gelling


agent


on


the


proliferation


* .~i il~r ~P * L~. C~. CT I.. -


2-1.


2-2.


2-3.

2-4.

2-5.


2-6.


3-1.


3-2.


and


3-3.


3-4.


3-5.


70


of


.....................................................................................56


PMe


their sources..................











4-1.


ANOVA of the effect of 'Isham'


embryogenic suspension culture-derived


somatic


embryo


size


and


stage


of


development


on


development


secondary somatic embryos on semisolid medium................................................................ 96


ANOVA of effect of Ge1-GrJA concentration on development of opaque


and hyperhydric somatic embryos and


size of opaque somatic embryos


from 'T362' proembryonic masses cultured on semisolid medium..


. . .. . ..98


ANOVA


of


the


effect


of


sucrose


concentration


proembryonic masses on production of opaque somatic embryos of various


101


The effect of total N and % N03


on color and morphology of avocado


'T362' suspension cultures.


ANOVA


for


the


effect


of


nitrogen


concentration


and


%/


N03~


proembryonic mass fresh weight gain and medium pH in liquid medium.......... 106


Protoplast yield per gram fresh weight of avocado embryogenic suspension
cu tu e s fro m' s eve al avocado cu~ltivTars ............ . .................. . ...................................... .. ..... .


ANOVA of the effect of medium osmolarity and protoplast density on the


plating


efficiency,


length


and


width


of


microcalli


derived


from


'T362'


avocado protoplasts cultured in agarose disc type method, after culture................................................


three weeks


125


ANOVA


of


the


effect


of


nitrogen


sources,


medium


osmolarity


protoplast density on number of microcalli and proembryonic masses from avocado 'T362'protoplasts, one month after culture.....................


ANOVA of the effect of medium osmolarity.


nitrogen source and plating


density on the percentages of microcalli and somatic embryo development from avocado 'T362' protoplasts, one month after culture..................


133


Percentage of protoplasts that divided on day 1,


5,


8 and 12 after culture. .....135


ANOVA of the effect of subculture age and dilution rate on culture fresh


weight and number of somatic embryos that developed derived 'T3 62' protoplasts, one month after culture. .....


from


nucellus-


4-2.


of


4-3.


4-4.


and


the


size


of


4-5.


5-1.


5-2.


105


on


5-3.


124


5-4.


and


5-5.


5-6.


132


139











The effect of NO3~:NH4*


avocado culture....


shoot


cultures


ratio at 20 mM on the growth of


maintained


in an incubator,


eight


'Guaram 13' weeks after


Effect of medium NO3~:NH42


ratio at 20 pm on the growth variables and


net photosynthesis of avocado shoot cultures after ten weeks in culture............ 162


Effect of atmospheric CO2 environment on the growth variables and net


photosynthesis


of avocado shoot cultures after ten weeks in culture.............. 163


The effect of atmospheric CO2 concentration on the growth variable and net photosynthetic rate of avocado shoot cultures after ten weeks in culture..... 165


Net photosynthesis (pmol CO2 g-2 s1) of intact avocado proliferating shoot


cultures,


their


subcultured


microcuttings


and


subtended


callus


in two


atmospheric CO2 concentrations, and intact plantlets and plantlet-derived shoots in an elevated CO2 concentration before and after cutting........................ 168


'Guaram


13'


avocado


plantlet


development


in two


atmospheric


Co2


environments, nine weeks after cultur... ..........


.......169


Leaf mesophyll protoplast yield from in vitro seedlings of Nectandra and


The growth and development of protoplasts after fusion and subculture of the microcalli/proembryonic masses following subculture in fresh .edi.......... 187


A-L.


Avocado tissue culture media.


A-2.


A-3.


Stock


solutions


of


8P


organoc


addenda


(7Kao


&


Michayluk, 1975)


modified by Grosser and Gmitter (1990) and their final concentration in


protoplast culture medium. .................


A-4.


Enzyme Solution for Protoplast Isolation.


.. . . . .. . . . .. . . . .. . . . .. . . . .. . . . . 2(2 1


A-5.


CPW salts Stock Solutions and Their Final Concentration in the Solution.. ....201


Amnnt


Af CPW


ite


Salti an nA


n a..


1inn


.l


c-A na:-.-


.S*uuI* n *n numn e N*m**5. * nDr m~I n.. *~I~


6-1.


6-2.


6-3.


6-4.


6-5.


6-6.


7-1.


7-2.


as


A-6


............ 160


......................................................................... 197


Avocado protoplast culture medium MS-8P. ................................................... 199


........ 200


G ti















LIST OF FIGURES


Figure


Development of secondary somatic embryos from zygotic embryos and of proembryonic masses from nucellar explants of 'Thomas' avocado. .............65


66


Morphological variations of avocado embryogenic cultures.


Effect of gelling agent, major salts and glutamine on the proliferation of


proembryonic avocado.....


masses


and


somatic


embryo


development


of


'Isham'


Growth response of 'Esther'


avocado


embryogenic


suspension


cultures


Effect


of


picloram


(P)


on


growth


response


of


'M425864'


avocado


embryogenic suspension cultures after 14 days.....


Effect


of


sucrose


(S)


on the


growth


response


of 'M25 864'


avocado


embryogenic suspension cultures after 14 days. ......


Effect of thiamine HCl (T) on the growth response of 'M25 864' embryogenic suspension culture after 14 days................


avocado ..............8 0


Effect


of


medium


sterilization


protocol


on


the


growth


of


avocado


embryogenic suspension cultures.


Avocado


somatic


embryo


development


from


proembryonic


masses


subsequent plant regeneration...........


Opaque


cotyledonary


stage


inoculum type derived from


somatic 'Isham'


embryo


production


as affected


by


avocado liquid embryogenic cultures


after one month on semisolid medium........


Effect of Ge1-GroJ


concentration on


develooment


of somatic embryos


3-1.


3-2.


3-3.


3-4.


3-5.


71


3-6.


77


3-7


78


3-8.


79


4-1.


4-2.


and


4-3.


94


96


page


.................................................81









Somatic embryo development as affected by high sucrose concentration. .......... 102


Effect


of


carbon


source


on fresh


weight


and


volume


of


embryogenic


nucellar 'Thomas' avocado cultures.


Effect of total nitrogen concentration and NO3I/NHIC weight gain (x 100%) and medium pH{........................


......................................................1 104'


ratio on culture fresh


..........107


Viability of protoplasts isolated from embryogenic cultures derived from
zygotic embryos of different avocado genotypes......................................................... 124


Effect


efficiency


of medium


of 'T362'


osmolarity


avocado


and


protoplast


protoplasts


plating


cultured


density


m agarose


on plating disc type


method after three weeks. ....


......126


Effect of medium osmolarity and protoplast plating density on length of


microcalli


that


developed


from


'T362'


avocado


protoplasts


cultured


agarose disc type method aftr treewes...............................


......126


The growth and differentiation of embryogenic culture-derived protoplasts


zygotic-derived


'T362'


avocado


cultured


in agarose


medium


with


medium osmolarity of 0.4 M as affected by plating density....................................... 128


A culture dish containing organized proembryonic masses and somatic embryos that differentiated from agarose-embedded protoplasts........... 130


Effect density


of


nitrogen


on the


source,


number


of


medium


microcalli


osmolarity


and


and


protoplast


proembryonic


masses


plating


that


developed from 'T362'


avocado protoplasts after one month in culture............. 134


Effect density


of


nitrogen


on the


source,


medium


percentage of microcalli


osmolarity


and


and


protoplast


proembryonic


masses


plating


that


developed from 'T362'


avocado protoplasts after one month in culture. .............. 134


Somatic


embryogenesis


from


avocado


protoplasts


cultured


in


liquid


...... ........ .. 1317


m edium ...... . *.................. *


Effect of subculture age and dilution rate in medium of low osmolarity


(0.15 M MS&8P) on the formation of large (>
em bryo s ... . . ..... . . . . .. ........ . . . .. . ...


2


mm diameter) somatic


......... 140


4-5.


4-6.


4-7


5-1.


5-2.


5-3.


5-4.


of


In


5-5.


5-6.


5-7


5-8.


5-9.









5 -11.


Somatic embryo development from protoplasts derived cultures as affected


by culture method.


. . .. ... . .. .. .. . ... . .. .. . ................. ** * *................. ............... . * *. .. . ... ...... . 4


In vitro shoot


growth of 'Guaram


13'


avocado in response to


varying


concentrations of KNO3 (N) as the sole inorganic nitrogen source in the


158


In vitro leaf growth of 'Guaram 13'


avocado shoots in response to varying


concentrations of KNO3 (N) as the sole inorganic nitrogen source in the
medum...................................................................................... ..


159


In vitro growth of 'Guaram


13'


avocado shoots in response to varying


concentrations of nitrogen (N) in the form of 3 NO3-


: 1 NH+. ..


Dry


matter


content


of


avocado


shoots in


vitro


in response


concentration of total nitrogen (N) in the form of 3 NO3~
w~~eekcs in cu~hlt.u re. .. .. .. .. .. .. . ... .. . .. .. ... .. . .. ......... * ............... ..


to


: 1NHt


varying after 10


Shoot


proliferation


of 'Guaram


13'


avocado


on media


of


different


content under elevated and ambient CO2 concentration, after ten weeks.


Plantlets of 'Guaram 13' concentration, after nine


avocado grown under elevated and ambient CO2
w e k . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . .... . . . . .


Protoplast Ne clandra


fusion between zygotic embryo-derived


conzacea


and


subsequent


somatic


'T362'


embryo


avocado


and


and


plantlet


regeneration and its shoot proliferation..................


183


RAPD banding patterns of leaf from a somatic embryo that developed from protoplasts fusion between embryogenic cultures of a zygotic-derived avocado line 'T362' and Nectandra coriacea, and its parental sources............185


RAPD banding patterns of proembryonic masses of avocados and their
Fui.isin wr vithI other avoc~cadoc and P. pachypodac).k . . ....* . .... ............................................................1 186


6-1.


6-2.


6-3.


6-4.


6-5.


161


6-6.


164


7-1.


N


166


167


7-2.


7-3.















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy


DEVELOPMENT OF PROTOCOLS FOR AVOCADO TISSUE CULTURE: SOMATIC


EMBRYOGENESIS


PROTOPLAST CULTURE, SHOOT PROLIFERATION AND


PROTOPLAST FUSION

By

Witj aksono


December, 1997 Chairman: Richard E. Litz Major Department: Horticultural Science


Avocado, Persea americana Mill.,


is an important fruit crop and is cultivated in


tropical


and


subtropical


regions.


Despite


its


importance,


commercial


production


mostly depended on only a few rootstock and scion cultivars.


Avocado improvement by


conventional breeding has been slow due to a long juvenile period, low fruit set, genetic heterogeneity, lack of genetic information regarding horticultural traits and inefficiency of


breeding


techniques.


Biotechnology,


including


somatic


cell


genetics


and


genetic


transformation,


has


great


potential


for


improving


perennial


fruit


speci


es,


including


avocado.


The use of biotechnology


for improving avocado


is dependent


on in vitfro


protocols, including efficient somatic embryogenesis, protoplast isolation and subsequent


has










Embryogenic cultures have been induced from several avocado genotypes and elite


cultivars.


Conditions for maintenance of embryogenic cultures have been determined, and


efficient somatic embryo development from


embryogenic cultures


has been


described.


Protoplasts


have


bn


isolated


from


embryogenic


cultures,


and


somatic


embryo


development from protoplast-derived cultures has been obtained.


Although plant recovery


from somatic embryos has been achieved, the efficiency of conversion, or germination has been low.


Interspecific protoplast


flhsion


between


embryogenic avocado


cultures


mesophyll protoplasts of Nectandra coriacea (Sw.) Griseb.


,P. borbonia (L) Spreng. and


P. pachypoda has been attempted in an effort to produce interspecific somatic hybrids


with


resistance


to phytophithora root


rot caused


by


P hyt op hihora


crnnamomi


Rands;


however, putative hybrid plants of the former and embryogenic cultures of the latter could


not be confirmed by RAPD analysis.


The somatic hybridization experiments were limited


availability


of


protoplasts


from


the


non-avocado


parents.


Whether


or not


protoplast


fusion


is


a viable


method


for


overcoming


sexual


incompatibility


between


avocado and its wild relatives remains unresolved.


The in vitro protocols


for avocado


that


have


been


developed


have


significant


implications for avocado improvement using biotechnology.


A research collaboration has


already demonstrated the feasibility of genetic transformation of embryogenic cultures of avocado.


and


leaf


by


the




















world.


CHAPThR 1
INTRODUCTION

Avocado, Persea americana Mill., is one of the most important fruit crops of the It has been consumed to some extent as a replacement for meat by native peoples


of tropical


America


since


antiquity


(Popenoe,


1927).


The


avocado


fruit


has a


high


nutritional value and energy content and is a source of antioxidants,


fruit protein and


soluble fiber (Bergh,


1992 b).


The high energy content of the avocado fruit is due to its


high


"good"


fat content,


which ranges from 3


to 30% fresh weight,


depending on the


cultivar.


Avocado


fat


is


82%


monounsaturated,


of which


95%


is oleic


acid;


polyunsaturated and


10% is


saturated


fat (Bergh,


I1992b).


Avocado oil


is used


as a


cosmetic (Purseglove, 1968; Bergh, 1992a, b).


World avocado production in the last


5


years has averaged Ca.


2


million MT (FAO,


1997).


Among other fruits,


world production of avocado ranks 10th after Musa (banana


and plantain), citrus, grape, apple, mango, pear, plum, peach and papaya (FAO, 1992).


the USA alone, avocado


's contribution to the economy during 1989-1992 was Ca. $211


million annually.


Among fruit crops, this


is


the sixth after citrus, grapes, apples, peaches


and pears (Anonymous, 1992).

Despite its nutritional and economic importance, avocado genetics are not well


understood.


This


is


in large part due to typical problems of breeding perennial species,


e g., low fruit set, high heterogeneity and a long juvenility period.


These difficulties have


impeded conventional breeding for addressing problems faced by the avocado industry


8%


is


In






2


chance seedlings (Bergh,


1976).


The major breeding


objectives


for


avocado


involve


development of better quality fruit and scion shoot, and rootstock (Bergh,


1975


,1976;


Bergh & Lahav,


1996).


Development of rootstocks that are tolerant of root rot disease


caused


by


Phytophthora


cinnamomi


(PR(R),


for


example,


is


urgently


needed.


Phytophthora root rot is the largest production problem around the world (Gustafson,


1976) PRR.


In the USA alone, the avocado industry has been losing ca. 200 ha annually due to Over 60% of avocado groves in the USA are affected by PRR, and losses exceed


$30 million each year (Coffey, 1987).

Biotechnology offers some novel approaches for generating variability for plant


improvement.


These have special relevance for perennial fruit species, because existing


superior cultivars could be altered for one or more specific traits


Using tissue culture


techniques,


somaclonal


vanants


with


resistance


to


bacterial


eaf


spot


caused


Xanthomonas


campestris


pv.


Pruni


(E..


Sm.)


Dows


have


been


produced


In


peach


(Hammerschlag,


1992).


Genetic variability that would


otherwise be unavailable using


conventional breeding approaches can also be exploited by somatic hybridization involving


distantly related and sexually incompatible species, e.g.,


citrus (Grosser & Grnitter,


1990;


Gmitter et al., 1992).


Tetraploid plants produced by somatic hybridization can be used as


parents for triploid scion breeding (Grosser & Gmitter,


1990).


Gene transfer by genetic


recombination


can also


generate


variability


by


bridging


the


sexual


barriers


between


species, phyla and kingdoms.


Genes from bacteria, e.g., the Bt gene for the endotoxin


from Bacillus thuringiensis (Williams et al., 1992) and the VHp gene for hemoglobin from


obligate aerobic, Gram negative Vitreocilla (Holmberg et al., 1997); from viruses, genes for the viral coat protein and viral nonstructural genes (Beachy et al., 19


eg.,


CP


90); and


by






3


Cloning horticulturally important genes, i.e.,


ripening genes and transformation of


plants with these genes in antisense (e.g., tomato) has marked the development of new


approaches for addressing post-harvest problems.


In fruit trees, stable integration and


expression of foreign genes, nos and npt


II genes,


and their inheritance in Mendelian


fashion have been demonstrated recently in apple (James et al.


1996).


This breakthrough


suggests that this technology might have direct application for single gene improvement of elite clones, e.g., for pest and disease resistance and for altered ripening for quality and shelf life improvement.

The potential for biotechnology can only be realized after development of efficient


protocols for plant regeneration from cell and tissue cultures.


only preliminary studies have been published,


Bergh (1992).


With respect to avocado,


which were reviewed by Pliego-Alfaro &


These studies were undertaken in order to develop protocols for avocado


somatic cell genetics and included:

1. initiation and maintenance of embryogenic cultures in semisolid and liquid medium;

2. somatic embryo maturation and gennination;

3. protoplast isolation, culture and regeneration from embryogenic cultures;

4. shoot proliferation from juvenile materials;

5. protoplast fusion between avocado and Nectandra coriacea and small-seeded Persea

species.















CHAPTER 2
LITERATURE REVIEW


Avocado


Use and Importance


Avocado is a fruit that is usually consumed fresh as a salad,


dessert or garish.


Unlike


other


fruits,


the


avocado


does


not


provide


refreshing


satisfaction


upon


consumption, but it gives the fUllness sensation of a staple food.


Avocado pulp is smooth


and nutty, neither sweet nor acid, and of a bland nature (Bergh & Lahav, 1996).


Superior


cultivars have a nutty or anise-like flavor.


Avocado


has


been


an important


food


in


Mexico


and


Central


America


since


antiquity 1927).


1,


where it takes the place of meat in the diet of Central Americans (Popenoe,


Avocados are appetizing, nourishing, cheap and available throughout most of the


year (Popenoe, 1927).


all fruit.


According to Purseglove (1968), avocado is the most nutritious of


Analysis of the nutrient composition of avocado (Table 2-1) indicates that the fat


content


is


very high and can be ca. 30%.


Avocado fat is 82% monounsaturated, of which


95% is oleic acid, 8% is polyunsaturated and 10% is saturated fat (Bergh,


therefore shares some of the benefits of olive oil.


1992b), and it


Coiquhoun (1990) demonstrated that


inclusion of 15-20% avocado in low fat diets lowered blood cholesterol and preserved


levels of high density liorotein (HDLV.


which is swod for the heart.


whereas a low fat









Table 2-1.


Nutrient composition of edible pulp of 'Fuerte' avocado per 100 g


Component Amount


74.O g


Water Energy component
Protein


17.O g


Lipid Carbohydrate


Fiber


Vitamins (mg or unless stated otherwise)


290 I.U. 4.00 mg 0.11 mg


A
C


Thiamine Riboflavin


Niacin


Mineral (mg)
Calcium


0 mg


L.60 mg


10.0 mg


Phosphorus


Iron


Sodium


Potassium


42.


0 mg


0.60 mg 4.0 mg 604.0 mg


Source:


Scora and Wolstenholme (1998) (in press).


Taxonomy


Avocado


,Persea americana (2n


=2x


= 2


4), is a member of the Lauraceae,


which


is mostly comprised of tree species,


except for the plant


parasite Cassytha filiformzs.


Avocado


is


the


only


economically


important


food


species


in


this


family.


Other


economically important species are used as spices,


e.g.,


Cinnamomum zeylanicum Blume


cassia


(Nees)


Nees


and


Eberm.


Ex Blume;


as medicinal


plants,


such


as C.


5


2


.2


g


6.0


.5


g
g


0.2


and


C.






6


Kopp (1966) divided the genus Persea into


2


subgenera, Persea and Eriodaphne,


based on the morphology of reproductive structures of herbarium materials.


Persea includes a small number of species characterized by large fruits,


The genus


while the subgenus


Eriodaphne consists of a large number of species, most of which have small fruits.


distinct


demarcation


of the


two


groups


has


been


further


confirmed


by


grafting


hybridization


studies.


The


members


within


each


subgenus


are graft


and


sexually


compatible with each other,


but are incompatible with members of the other subgenus


(Bergh & Ellstrand, 1986; Bergh & Lahav, 1996: are mostly resistant to Phytophihora root-rot,


The members of subgenus Eriodaphne


while members of subgenus Persea are


susceptible (Zentmeyer, 1980)


The subgenus Persea includes the commercial avocado, ' pulp, and other closely related species, which have thin pulp, e.g.,


which has a thick edible


P .


schiedeana Nees, P.


prnmatogena Williams and Molina, P. parv flora Williams and P. zentmyeri Scieber and


Bergh.


The commercial avocado has been long recognized to have three horticultural


races that are adapted to three different climatic conditions, i.e., the Mexican race,


which


is adapted


to high elevation


in the tropics,


the Guatemalan


race


which is adapted to


medium elevations in the tropics,


and the


West


Indian race which is adapted to low


tropical


elevations


(P openoe,


1941).


Popenoe


(1941)


dev eloped


systematic


keys


distinguish the three races and referred to the Mexican race as Per-sea drym Jolla,


Guatemalan race as P.


guatemalensis and the West Indian race as P.


americana.


races also differ in many horticultural traits (Table 2-2).


classification


of the


horticultural


races


of


commercial


avocado


and


other


Per-sea species is still in dispute.


Kopp (1966) recognized only 4 species, P. schiedeana,


Thi


s


and


to


The


the


The










Table


2-2.


Comparison of the three horticultural races of Persea americana.


Characteristic Race Mexican Guatenmalan West Indian


General


Color Surface Thickness Stone cells Pliability Peeling


Mexican highlands

subtropical most least intermediate less


Native Region

Climatic adaptation Cold tolerance Salinity tolerance Iron chlorosis tolerance Alternate baig
Internode Twig lenticels Bark roughness Stem pubescence
Size
Color
Flush color Anise
Underside waxiness
Season Bloom to maturity Perianth persistence

Length Thickness Shape
Size
Shape


Guatemalan highlands subtropical intermediate intermediate least more

long absent less less

large green reddest absent less
late 10-18 months less

long thick comical
small-large mostly round


longest pronounced less
more
smallest green greenest present (usually) more

early 5-7 months greater
short medium cylindrical
tiny-medium mostly elongated
usually purple waxy coating very thun absent membranous no


green rough


Tropical lowlands

tropical least most most less


shortest absent more less

largest pale green yellowish-green absent less
intermediate 6-8 months less
short thin nailhead
medium-very large variable


pale green-maroon shiny medium slight leathery yes


Size ratio Coats Tightness in cavity Surface


Pulp


Flavor


large thin often loose smooth
amise-like, spicy


often small usually thin tight
smooth
often rich


large thick often loose rough sweet, mild


7


black or variable thick present stiff variable


Form Leaf Flower Fruit Stem Fruit


Fruit Skin


Seed






8


with two


varieties,


the


native


species and its


progemtor.


The


Mexican


avocado, P.


americana var. dymrfolia, is the progenitor of the West Indian race, P. americana var.


americana),


while the Guatemalan avocado (P.


americana var.


guatemalensis)


is the


progemitor of P. nubigena var. nub igena.


The most recent


classification has been suggested by


Scora and Bergh (1990)


based


on new


data,


including


isozymes,


leaf terpenes,


morphology,


physiology,


field


observations and molecular markers as follows:

Persea americana Mill.
var. americana Mill.


var. drymffolia (Select.


&


Chain.) Blake


var. guatemalensis Williams
var. nubigena (Williams) Kopp
var. steyermarkii Allen
var. floccasa Mez
Persea zentmeyeri Scieber & Bergh "Aguacate de Montana" "Aguacate de mico"


Persea parv4/lora Williams,


"Aguacate cimarron"


Perseaprimatogena Williams & Molina


'Guaslipe"


"Aguacate de anis"
Persea schiedeana Nees


In this new classification, the horticultural races are given botanical varietal status since the difference between them is not far enough (based on isozymes) to give the races


species standing but is too far to be considered forms (Bergh & Elistrand,


1986).


three horticultural races were also as different from one to another as to other varieties (Scora & Bergh, 1990). Origin. Domestication and Distribution


The






9



avocado may have evolved in different climatic conditions in geographical isolation from


one another (Storey et al.


1986).


The Mexican race is thought to have originated in the


highlands of south-central Mexico, since primitive forms of Mexican avocado are found in


that area (Storey et at.,1986).


The Guatemalan race was believed to have originated in the


interior valley of the highlands of Guatemala, north of Guatemala City.


The West Indian


race did not originate in the West Indies, since there is no record of the avocado from


early explorations of the West Indies (Storey et al. in the Pacific lowlands of Central America (ca.


,1986). 82*-92*o


Instead, it may have developed west longitude [Storey et al.,


1986])


William (1976; 1977) argued that the West Indian race evolved from the Mexican


race and probably became adapted to a warmer climate in northern Central America.


This


argument was based on the morphological similarity of the two races and the archeological remains of avocado in Peru that date from Ca. 1500 BC. Domestication


Avocado


has


been


domesticated


in


Mexico


since


time


immemorial


(Popenoe,


1927).


The word avocado is


derived from the


S


panish word a/macate or aguacate, which


is a corruption of the Aztec ahuacati which is still used in parts of Mexico.


pa/wa, from the Aztec pauatl meaning fruit,


is


Guatemalan races of avocado in certain parts ol


The word


used with reference to the West Indian and f Mexico. The word palta (in Quechuan) is


used with reference to avocado in western South America.


Archeological remains of avocado seeds as old as


7000 BC have been recovered


from caves of the


Tehuacin area in Mexico.


Other avocado seeds


with younger carbon


dating ranging from 6600 BC,


4000 BC


32


00 BC, 500 BC, 300 BC to 300-1500


have also been recovered (Smith,


1966;


1969).


Interestingly, seed


size


appears to have


AD






10


Distribution


When the Spanish arrived in the Americas,


the avocado was being cultivated from


Mexico


to


northern


Peru


(Hodgson,


1950;


Storey


et


at.,


1986).


The


Spanish


conquistadors brought the avocado to Venezuela, the West Indies, and the Canary Islands


(Bergh & Lahav, 1996).


Eventually, avocado was cultivated in all tropical and subtropical


regions.


The avocado reached Spain in 1600,


Africa in Ghana in 175


0 (Smith et aL, 1992).


and was established on the east coast of [he avocado was introduced to Singapore


ca. 183


0 and to the Philippines Ca.


1890 (Burkill,


1935).


Avocado was brought from


Mexico to Florida in 1833 and to California in 1848 (Gustafson, 1976). Production


World avocado production in the last


5


years has averaged ca.


2


million MT (FAQ,


1997) and ranks 10th after Musa (banana and plantain), citrus, grape, apple, mango, pear,


plum, peach and papaya (FAG,


1992).


The major production areas are Mexico,


USA,


Brazil, Dominican Republic and Indonesia, respectively (Table 2-3).


Other countries that


are not leading


producers


but


which


export


significant


amounts


of


avocado


include


Australia, South Africa and Israel. Avocado Breeding and Advances


Avocado breeding objectives have been directed


toward


improvement


of scion


(fruit and tree) quality and rootstock quality as summarized in


Table


2


.4 (Bergh,


Bergh, 1976; Bergh & Lahav, 1996).


According to


Bergh &


Lahav (1996),


most of the morphological


varnability in


4 4.5 4 4 4 4 fl 4 * 4 44 4


19


75









Table


2-3.


Average avocado production 1992-1996.


Region Country Production (tons)


North and Central America


Mexico


U.


S .


A.


El Salvador Costa Rica Guatemala


767,904 179,073 40.600


1


Dominican Republic


Haiti Cuba


2
2
5
4


3.480


2.


895


6.000


5.


000


8.100


South America


Brazil


108.037


45.


Venezuela Colombia


Chile


Ecuador


Peru


940


73.963


5


3.400


13.376


5


4


5


5


9


Asia


Indonesia


Israel


Africa


Philippines South Africa Cameroon


96,926 54,494
24,100


40.9


43


2


5


0


3


0


Zaire


Congo


46.900


24


5


00


Madagascar


21.300


Europe


S


pain


Portugal Australia


Australia


44,008 14.940


2


970


WORLD


2,080,08


Source: FAO (1997) adapted from Bergh & Lahav (1996)


1


1


8










Table 2-4.


Avocado breeding objectives for special characters


Special Character


Special Character


Fruit Qualities


Medium size (200-300 g) Uniformity


- Skin


-Medium thickness


-Readily peelable
-Insect, disease tolerance
-Free from blemishes


-Attractive color Long tree storage Seed:.
-Small
-Tight in its cavity


Thick ovate shape Pulp:
-Proper softening
-Appetizing color
-Absence of fibers
-Pleasing flavor
-Long shelf life
-Slow oxidation
-Chilling tolerance
-High oil content
-High nutritional value


Shoot Qualities


Spreading habit Easy to propagate Strong grower


Tolerant to pests and diseases Tolerant of wind Tolerant of cold


Tolerant of heat Tolerant of salinity


Tolerant of chlorosis Tolerant of other stresses Short fruit maturation period Precocious Regular bearing Wide adaptability


Heavy bearer


Rootstock Qualities


Conducive to high quality fruit Conducive to healthy, productive trees Free from sunblotch viroid Dwarfing or semi-dwarfing Genetically uniform Hardy and vigorous Easily propagated


Easily grafted Tolerant to Phytophthora root-rot and other
disease
Tolerant of salinity Tolerant of chlorosis


Tolerant of drought


Tolerant of other adverse soil condition


Source: Bergh (1975)


12






13



inflorescence length, seed size, and softening time, had significant nonadditive variances


(Lavi et al., 1991).


This reflected the insignificantly low value of narrow-sense heritability


but the significant value of broad sense heritability (Lavi et al.,


993).


These results


indicated that hybridization should be aimed at increasing the genetic variance in progenies


by selecting parents that are not only of superior phenotype, but which also include


10-


30% of parents with inferior performance (Lavi


et al.,1993).


More recently,


genetic


associations between DNA fingerprint fragments and loci controlling important traits in


avocado,


e.g.,


fruit


color,


have been reported


(Mhameed


et al.,


1995).


Selection


progenies having these traits, e.g.,


fruit skin color, may be carried out early in the seedling


stage using markers associated with these traits. Breeding scion cultivars


Avocado


production


mostly


relies


on a few


cultivars.


Scion


cultivars


dominated by 'Hass'


and 'Fuerte'


which have been cultivated commercially for 40 years in


subtropical regions.


Both cultivars were chance seedlings of unknown parentage (Bergh,


1976; Bergh & Lahav,


1996).


Hass has several commercial weakness,


for example, it


produce


fruits


with


size


variability,


some


proportion


of


which


are too small


to be


marketable and this problem is aggravated with tree age (Bergh & Lahav,


1996).


Several


selections have been made to replace 'Hass'


(Bergh & Lahav,


1996), e.g.,


'Gwen'.


'Jim'


'Reed'


and


'Lamb'.


Cultivation of these new cultivars is still limited due to consumer


preference for 'Hass'


(Bergh & Lahav


1996).


Several local selections have been made,


including 'Ettinger'


and 'Iriet'


(Israel),


'Ardith' (USA) and 'Sarwill' (Australia) (Bergh &


Lahav, 1996). Breeding rootstock cultivars


of


are







1


the early


1950s.


Persea species and related genera collected from Mexico,


4


Guatemala,


Honduras and Nicaragua were tested for resistance to Phytophthora root-rot (PRR).


The


results indicated that most of the Persea species that are resistant to PRR belong to the subgenus Eriodaphne, while Persea species in subgenus Persea (including avocado) were


susceptible (Table


Table


2


-5.


2-5


The resistant species in subgenus Eriodasphne


List of Persea and related genera and their resistance to Phytophthora root-


rot.


Species Origin of collection Resistance


Persea subgenus Persea


Cultivars in California


Honduras, Mexico, Guatemala,


low low


P . fiocassa


Mez


P. gigantea P. schiedeana


P. nubigena L. 0. Wilim (= P. gigantea L. 0. Wilim.; = P americana var. nub igena Kopp)
Persea subgenus Eriodaphne
P. aiba Nees & Mart P. borbonia (L.) K. Spreng. P. caerulea (Ruiz & Pavon) Mez P. donnel-smithil Mez. P. haenkeana Mez. (= P. durifolia Mez. P. indica (L.) K. Spreng P. lingue (Ruiz & Pavon) Nees P. pachypoda P. Ion gipes
P. liebmani


P.


cmnerascens


P. skutchil


Nicaragua Mexico Honduras


Mexico, Guatemala, UCLA clone Guatemala, Honduras, Mexico, Nicaragua, El Salvador


Brazil


southern United States Venezuela, Costa Rica Guatemala, Honduras Peru


Canary Islands Chile


Mexico Mexico Mexico Mexico


Honduras, Guatemala


Various species of Nectandra,


Latin America


low low low


generally low with some exceptions.


high
usually high, some variabilitye high
moderately high, variable moderate


very low


low


high low


high high high

usually moderately high


Ocotea


and Phoebe


P. americana P. americana






15


were graft incompatible with avocado and other species of the subgenus Persea, but were


graft compatible with members of their own subgenus (Frolich et at.,


1958).


The same


relation hold true for sexual hybridization (Bringhurst, ,94 Bergh & Lahav,


1996).


The program at


UC, Riverside produced the PRR-tolerant


'Duke


7' ,


rootstock,


which was a seedling of 'Duke'


'Duke


7'


(Zentmeyer


has become the most important


&


Thorn,


rootstock


1956; Zentmeyer et a.,


in


California,


and


1963).


is propagated


clonally using the etiolation technique (Platt,


1976).


Other selections-that are promising


include


'Barr


Duke'


(a


seedling


of


'Duke 6'),


'D9'


that


induces


dwarfing


(from


irradiated


'Duke'


parent scions),


'Thomas'


(a survivor from a root-rot affected area),


'Martin Girande'


(a hybrid of avocado and Fersea schiedeana)-are all still in trial (Bergh &


Lahav,


1996).


The level of tolerance to


PRR of those selections are better than the


earlierly used rootstock 'Topa Topa'


but not as high as the PRR-resistant Persea species


in the subgenus Eriodaphne.


The absence of complete PRR resistance in the subgenus Persea,


together with


lack of information regarding the genetics of PRLR-tolerance, has made breeding for PRR tolerant/resistant rootstock difficult. Biotechnology and Its Potential for Avocado Improvement


Despite its importance, breeding avocado has been slow due to its


long juvenile


period,


high


genetic


heterozygosity,


low


fruit


set,


lack


of


genetic


information


inefficient breeding method (Lavi et al.


1991ib;


1993).


Systematic studies of the genetics


(Lavi et a.,


1991 b) and development of molecular markers for some horticultural traits,


including fruit skin color (Lavi et a.,


1991 a) may have a significant impact on breeding


an


and






16


pooL.


Biotechnology,


involving somatic cell genetics and


gene transfer,


may have an


important role in widening genetic variability.
Genetic transformation with antifungal genes such as glucanase has been proposed


as an alternative to combat disease (War develop a root-rot resistant rootstock.


1992) and might be a viable way to


Somatic hybridization via protoplast fusion has


been used to overcome sexual barriers in citrus (Grosser & Gmitter,


1990).


Protoplast


fusion technology could be an alternative way of combining the root-rot resistance traits of Persea species that are sexually and graft incompatible with avocado (Pliego-Alfaro &


Bergh, 1992; Bergh & Lahav, 1996).


Somatic embryogenesis and plant regeneration from


nucellar explants have been reported for woody tropical and subtropical fruit species, e.g.,


citrus (Rangan & Murashige,


1969) and mango (Litz et a.,


1982; Litz et a.,


199


5


).


This


approach may have direct application for


cheap clonal


propagation of a PRR tolerant


rootstock.

Avocado tissue culture has not been developed in comparison with other tropical


tree fruit species, i.e., citrus or mango.


Avocado tissue culture has been considered as


either being in its infancy (Pliego-Alfaro & Bergh,


1992) or recalcitrant (Gardner,


1993),


although tissue culture studies of avocado were initiated 50 years ago.


To realize the


potential of modern biotechnology for improvement of avocado, tissue culture protocols, including plant regeneration via somatic embryogenesis and from protoplasts, protoplast fusion and plant propagation through shoot proliferation, need to be developed.

Previous work on avocado tissue culture, including callus initiation, shoot culture,


somatic


embryogenesis


and


protoplast


isolation


are summarized


in


Table


2-6.


Early


reports


were


intended


to study


growth


responses


of


fruit


pericarp


tissue


in


vitro












Table


2-6.


Summary of in vitro studies of avocado, Persea americana Mill., and related species in the family of Laur


Reference Species, cultivars Purpose of Explant Physical Medium2 Respoti
studies environment

Callus culture
Schroeder, P. americana post harvest fruit pericarp not reported not reported callus(
1956 'Fuerte' physiology

Desjardins, cv. was not Sunbiotch stem 28 -30*C Liquid medium callus
1958 reported viroid irradiance was BM: inorganic salts similar to had cai
replication not reported Gautheret (1942) and White (1954), callus'a
study glycine, nicotinic acid, pyridoxine, brown
thianmin, calcium pantothenate, LCE,
2% sucrose
PGR: NAA

Schroeder, P. americana cv. callus anatomy pericarp not reported BM: Nitch, White cell pr<
1961 was not reported PGR: 10 mg 1-' IAA like an
cells

Schroeder, P. americana physical factor pericarp light and BM: Nitsch with FeEDTA in callus
1971 'H-ass' requirement temperature exchange for iron citrate dark, (
were tested PGR: 10 mg 1~1 IAA was ca
Blumenfeld & P. americana cytokinin cotyledon, 27*C BM: Miller, 1963 for cell
Gazit, 1971 'Fuerte' requirement mesocarp darkness PGR: as treatment mesoci
RH =80% exoger
cotylc











Table 2-6-continued


Reference Species, cultivars Purpose of Explant Physical Mediumt Respor
studies environment


Schroeder, 1975


P. americana
cv. was not


ex vivo floral behavior


floral parts


27 2*C


dark


reported


BM: Nitsch or MS with LCE PGR: Kin, LAA, NAA (concentration not specified)


extensicallus


Schroeder, 1977


P. americana
cv. was not


callus longevity


cotyledon, stem, peduncle, petiole


25 90


- 27*C
- 150 foot


BM: Nitsch POR: not specified


Callus occurei


reported


candle


Schroeder, 1978


P. americana
cv. was not


UV irradiation


fruit pieces


25 - 270C


Nitsch medium


90 foot candle


callus by UV


reported


Schroeder,


1979


P. americana


cv.


was not reported


callus longevity


2


cm stem


segment with or without bud (etiolated and non etiolated seedlings)


250C 100-400 foot candle


Nitsch as modified by Schroeder


(1977)


massiv CUt -enl shoot/!


24 h


Young, 1983


P. americana


'Lula',


propagation


'Waldin'


leaf, from seedling


270C 1800 lux,


16 h


Buickle et al.,


P. americana


1988


sunblotch viroid


seedling stems


not applicable


study


BM: Anderson (1970) salts and 'vitamine, 1 casein hydrolysate, 30 g/l sucrose
PGR: 1 mgF1 BA, 1 mgU1' 2,4 D BM: MS, 7 g/l agar PGR: 1 mg 1-' IAA and 0.3 mg 1-' BA


callus


callus










Table 2-6-continued


Reference Species, cultivars Purpose of Explant Physical Mediumz Respaii
studies environment


Aaouine, 1986 P. americana
cv. 'Topa Topa'
P. borbonia


callus formation seedling stems and


27*C, darkness


proliferation


P. indica P. nubigena


BM: % MS major salts (except MgSO4), MS minor salts, 15 mg/i Suc, 30 mg/I i-inositol, o.4 mg/i thiamine.HCl, 1 mg/i pyridoxine.HCl,
1 mg/i nicotinic acid, 2 g/l Geirite.


Callus: freshiw fold of


Kane et aL,


1989


P. palustris
(Raf.) Sarg.


organogenesis


2


cm3 cotyledon


pieces


4 weeks dark, 4


weeks light 25 _20 C


PGR: 0.3 mg/i 2,4-D, I BM: MS
PGR: 0.1 NAA, 10 211'


mg/i 2iP


profuse no orgt


Shoot cultures


Schroeder,


1980


P americana cv. was not reported


propagation


same as Schroeder


(1979)


25


-- 270C


100 - 200 foot


candle 24 h


EM: Nitsch and 4 tug F' caseine hydrolysate or MS PGR: 6 mg F'1 and 30 mg 1 isopentyl adenine for Nitsch and MS BM


masive stem s elongal


respectively


Pliego-Alfaro, 1981


Young, 1983


P. americana
'Topa-Topa'


P. americana


'Lula',


'Waldin'


shoot proliferation


organogemc study


2-3 cm stem tips from in vitro elongated shoot from decapitated plumule-radicle axes


bud


27*C, 40 gmol m 15 h.


-2 s1


27*C


1800 lax,


EM (MS salts, 0.4 mg F' ,thiamineHCl, 100 mg F'1 i-inositol, 30 g/l sucrose, 8 g/l TC agar), 170 mg 1F NaH2PO4. H20, 1 g/l neutralized, activated charcoal. PGR 50 mg 14' BA BM:
PGR: 0.2 mg i-1 BA, 0.2 mg F' IBA


Shoot< frequel abscisi


poor e


16 h











Table 2-6-continued


Reference Species, cultivars Purpose of Explant Physical Medium2 Respc
studies environment

Skene & P. americana Embryo rescue immature 27*/20*C, BM: MS liquid medium germi
Barlass, 1983 Open pollinated embryos from 50 snol m2 s-', PGR: 0.5 mg F' BA shoot
'Fuerte' abseised fruiflets 16 li. or embryo
(open and cont- darkness later
rolled pollination)
Skene & P. americana shoot plumule-radicle 270/200C, 10 ml liquid medium: multil
Barlass, 1983 Open pollinated proliferation axes of mature 50 pimol m- s-, BM: 2
'Fuerte' embryos 16 h PGR: 0.5 mg 1-' BA
Skene & P. americana shoot 2 cm shoots from 270/20*C, Shoots were pretreated with 20 s dip 30 - !
Barlass, 1983 Open pollinated proliferation proliferating 50 pmolm-2 s"1 , inl1N NaOH.
'Fuerte' from resqued shoots 16 h BM: White's medium, pH = 9.0, Agar
embryos PGR: 1 mg F' IBA
Gonzales- P americana var. shoot nodal and stem 27 1*C BM: MS 90%<
Rosas & americana R. proliferation tips of 2 -3 cm 500 lux PGR: 0.3 - 10 fIA and 0.01 - 3 Kin multi1
Salazar- antillana from seedlings 16 h cm w:
Garcia, 1984
rooting nodal and stem 27 1*C BM: MS 50%
tips of 2- 3 cm 500 lux PGR: 7.5 -10 LBA develt
from seedlings 16 h
Harty, 1985 P. americana shoot shoot apices with 270C IBM: MS inorganic salts modified by shoot
'Duke 7' proliferation 4 - 6 leaf irradiance - Dixon & Fuller (1976), 1000 myoprimordia from reported inositol, 30 g/l sucrose, 0.5 mg 1-'
green-house 16 h pyridoxine HCl, 5 mg F' thiamine
grown seedlings HC1, 5 mg F' nicotinic acid, 40 mg F'
L-glutamine, 40 mg F~' L-arginine
PGR: 10 mg F' Kin











Table 2-6-continued


Reference Species, cultivars Purpose of Explant Physical Mediumz Respot1
studies environment


Pliego-Atfaro et a]., 1987


P. americana
'GA-13'


shoot proliferation


'IV-8'


rooting assay


PliegoAlfaro &


P. americana
'Topa-Topa'


embryo gennination


nodal stem from heavily pruned trees ('IV-8') and grafted adult buds ('GA-13')


2-- 3 cm shoot tips from shoot cultures

plumule-radicle axes


Murashige, 1987


not reported


BM: Margara major salts (1984), MS minor, 0.4 mg F' thiamineHCl, 100 mg 1' i-inositol, 30 g/l sucrose, 8 g/l TC agar


PGR:


not reported


270C, 32gmol m2s' 16 h


mg F' BA


two-step rooting method (PliegoAlfaro, 1988)


BM (MS salts, 0.4 mg F' ,thiamineHCl, 100 mg 1 i-inositol, 30 g/l sucrose, 8 g/l TC agar), 1 g/l neutralized, activated charcoal.


shoot r declinii apical .
differci require cultivar 30% of 5% of'


shoot c pLu mul


PliegoAlfaro & Murashige,


shoot proliferation


1987


Pliego-Alfaro, 1988


P. americana
'Topa-Topa'


rooting experiment


1 --1.5 cm nodal stems developed from greenhousegrafted adult buds 2-3 cm stem tips from in vitro elongated shoot from decapitated plumule-radicle


270C, 32 pnolnf-2 s"' 16 h.


270C, 32gsmol m-2s"' 16 h.


BM, 170 mg 1-1 NaH2PO4. H120, neutralized, activated charcoal.


g/l


bud edc stem (V


PGR: 50 mg F' BA


Induction (3 days):


BM with 0.3x


PGR:


100%1


MS salts


25 mg F' IBA.


Development: IBM, no PGR, 1 g/l activated charcoal


axes











Table 2-6-continued


Reference Species, cultivars Purpose of Explant Physical Medium' Respc
studies environment

Pliego- P. americana rooting test of 2 - 3 cm stem tips 27*C, 32 pnmol 3 days in induction: juveni
Alfaro & 'Topa-Topa' different phases from juvenile, m2 s-1 16 h. B_:- 3 M at adult
Murashige, of avocado adult and 4x PGR: 25 rug F' IBA. rejuvc
1987 shoots grafted adult Development: BM, no PGR, 1 g/l 30%
materials activated charcoal
(rejuvenated)
Schall, 1987 P. americana shoot shoot tips and 27 *C B~M (according to Ned et at.,1982): %2 cultux
'Fuerte' proliferation nodal stem from light MS salts, Morel (1948) vitanun, 100 multij
2-4 yr. old tree 16 h myo-inositol, 30 g/l sucrose, 25 mg F' with I
grafted trees FeNa EDTA, 170 mg F' NaH2PO4.
H20
PGR: 1 -2BA
rooting shoot tips BM, 0.5 mg/i LEA, 0.5 mg/i AC 77%i<
days e
upon
Cooper, 1987 P. americana shoot shoot tips and BM: WPM multi]
'Fuerte' proliferation nodal stem from PGR: 0.1 mg/i BA shoot:
seedling
rooting EM, 3 g/l NAA 90-iC

Vega- P. americana propagation etiolated 25*C BM1: %A MS, 100 mg il myo-inositol, 3 mu
Solarzano, 'Colin V-33' and buds/shoots (the 3000 lux 0.4 mg F' thiamin HCL, 30 g/l explag
1989 a West Indian phase of the 16 h sucrose, 2 g/l Geirite
race selection shoots were not PGR: 2 mg F' BA, 2 mg F' GA (filter
reported) sterilized)










Table 2-6-continued


Reference Species, cultivars Purpose of Explant Physical Mediumz Respoi
studies environment


Biasi et al.,


1994.


P. americana
'Ouro-verde'


shoot proliferation


rooting


nodal stem from


seedlings shoot tips


not reported


not reported


BM: PGR:


'A MS Salts


3


axillary


mg V~' BA


according to Pliego-Alfaro (1988 )


Barringer et at., 1996


P. americana
West Indian


shoot proliferation


embryonic axis


races:


280C 4O molm-2 s'I 16 h.


BM: MS
PGR: 13.2 pM BA, 0.05 pM NAA


'Dade' 'Maxima', 'Tower 2' 'Choquette'


shoot


in vitro shoots


proliferation


rooting


in vitro shoots


BM: MS
PGR: 4.4 pM BA, 0.05 sM NAA


BM: MS PGR: 4.9 or 9.8 pM lEA


multip nodal subcul 30% r


GonzalesRosas et a.,


P. schiedeana


1985


propagation


1 cm nodal stem segment and stem


tips with


rooting


bud


27 *C 800 lux


BM: MS PGR: l - 3IBA andO.0l


- 1Kin


16 h


BM: MS PGR:l1I


shoot I existix1 callus
rooted segmc callus


BA, 3 Kin


Net et al,


P. indica


1982


propagation: shoot
proliferation


5


mm stem tips or


1 -2 node stem from I year-old


EM:


240C light 16 h


MS major, MS minor, 100


myo-inositol, 30 g/l sucrose, 25 mg 1
FeNa EDTA, 170 mg F~' NaH2PO4


Shoot shoot/


seedlings


H20, 80 mg F-' adenine sulfate,


25


mg


~ ascorbic acid, 2 glycine, 1 for each pyridoxine HCl, thiamine HCl,
nicotinic acid, panthothenic acid.


PGR:


2


mg ' BA


rooting


format











Table 2-6-continued


Reference Species, cultivars Purpose of Explant Physical Medium2 Responi
studies environment


Nel et al,


propagation,


1982


rooting


BM, 2 mg F' IBA (liquid medium with filter paper bridge)


65% ro


Kane et al.,


P. palustris


1989


P. pa/us tris .


Campos & Pais, 1996


P. indica


Propagation, shoot
proliferation propagation, vit~ro rooting
propagation, shoot
proliferation propagation,


rooting


shoot tips from in vitro seedlings


cx


15d2*0C 90 pmol m 16 h


3 cm cuffing


BM: MS medium


.2s', PGR: 0.25 mg 1' BA,0.5 mg F~' GA
Liquid medium (10 ml), 100 rpm


dip for


22


nodal stem from in vitro seedling


2*C


11.5 16 h


Wr m-2


terminal shoot cuffing 2 - 8 cm from shoot


5


min in 500 mg '1 IBA, plug


trays with Vegro KMay Mix A
BM: MS, 30 g/l sucrose, 8 g/l agar, pH =7.0
PGR: 1 mg F~' BA


induction: 1-2


s dip in 1-5 mg/nil


shoot ir increase


100%xr


shoot el


100%:


IBA


development: BM with


cultures


medium, no PGR


Wang & Ru, 1984


Sassafras randajense


Propagation: shoot
multiplication


1 cm shoot tips from 5 yr-old grafted trees


18


- 200C,


2


klx cool white fluorescent light, 12 h


BM (MS salts with 2x FeEDTA and LS organic, 5% sucrose), 30% LCE, 100 mg F' malt extract, 50 mg F' glutamine, 50 mg 1'1 arginine PGR: 5 mg I' Kin, mg F~', 0.05 NAA (60 mg F~' Kin required for stage II)


shoot ii


propagation: rooting


in vitro shoot


cm


EBM PGR:


rooting


5


- 10 IBA


liquid medium with filter paper support


% MS











Table 2-6-continued


Reference Species, cultivars Purpose of Explant Physical Mediumz Respc
studies environment


Nel et al,


propagation,


1982


BM,


rooting


Kane et al.,


P . palustris


1989


P. palustris.,


Campos &


Pais,


P. indica


1996


Propagation, shoot
proliferation propagation, vitro rooting

propagation, shoot
proliferation propagation,


shoot tips from in vitro seedlings


ex


3 cm cutting


nodal stem from in vitro seedling


2


mg F~' IBA (liquid medium


with filter paper bridge)


15 20CBM:MSmedium 90 pnnol m2 s 1, PGR: 0.25 mg i~1 BA,O.5 mg 1-' GA 16 h Liquid medium (10 ml), 100 rpm


dip for


22


2*C


11.5 16 h


'W m-2


terminal shoot


5


min in 500 mg F~' IBA, plug


trays with Vegro Klay Mix A
BM: MS, 30 g/l sucrose, 8 g/l agar, pH =7.0
PGR: 1 mg F-1 BA induction: 1-2 s dip in 1-5 mg/mn


65%ir


shoot increa 100%


shoot 1000*,


rooting


cutting


2


- 8cm


from shoot


development: EM with


% MS


cultures


medium, no PGR


Wang & Hu, 1984


Sassafras randajense


Propagation: shoot
multiplication


18


1 cm shoot tips from 5 yr-old grafted trees


- 200C,


2


kMx cool white fluorescent light, 12 h


BM (MS salts with 2x FeEDTA and LS organic, 5% sucrose), 30% LCE, 100 mg F'1 malt extract, 50 mg ''


glutamine, 50 mg F-'


shoot


arginine


propagation: rooting


in vitro shoot cm


PGR: 5 mg j-1 Kin, mg F'1, 0.05 NAA (60 mg F'l Kin required for stage II) BM
PGR: 5 -1OIBA liquid medium with filter paper


rootitl


support


IBA











Table 2-6-continued


Reference Species, cultivars Purpose of Explant Physical Mediumnz RespC:
studies environment

Protoplast Isolation
Buickle et at., P. americana cv. protoplast stem-derived Preplasmolyzation for 45 min in 2 - 3
1988 was not reported isolation callus: 500 mg solution: 10 ml of 0.7 M mannitol, callus
0.01 M MES, 0.4% PEG 6000, MS salts, pH 5.3
Digestion in preplasmolyzation solution and 1% Cellulase Onozuka RS and 0.1% Pectolyase Y23 for 3 h at 50 rpm at 29*C

Percival et al., P. americana ripening studies mesocarp from not applicable Predigestion solution (45 mini): 0.6 M protol 1991 'Hlass" mature fruits mannitol, O.5%IBSAO.1 mM per gi
dithiothreitol and mineral salts (Patnaik et al., 1981). Digestion solution (overnight, 18 hi): predigestion solution, 1% Rhozyme HP-i5O, 1% Cellulysin, 0.5% Macerase


2BM = basal medium, MS


= Murashige & Skoog (1962), WPM


= Woody Plant Medium (Lloyd & McCown, 1980), LCE


= liquid cocor


morpholino ethane sulfoxide, PEG = polyethylene glycol


PGR 2,4-D


=plant growth regulator in pM except stated otherwise, IAA


= 2,


4-dichiorophenoxyacetic acid, BA


=benzyladenine, 2iP


= indoleacetic acid, NAA


= naphthaleneacetic acid, IBA


= 2-isopentenyladenosine , Kin = kinetin, GA3


=1i


= Gibberellic






27


and P. indica (Aaouine,


1986) and cotyledons of P. palustris (Raf.) Sarg. (Kane et aL.,


1989).


Regardless of the objectives of these studies,


shoot


organogenesis was never


reported.

Schroeder (1980) attempted to initiate shoot growth and proliferation of shoot


cultures of avocado; however,


shoot


growth


was poor


on MS


medium.s


Suboptimal


growth responses, i.e., die back and leaf abscission (Pliego-Alfaro, 1981; Pliego-Alfaro et


al., 1987), scaly leaf formation (Kane et aL, 1989: Gonzales-Rosa et al., 1985),


extensive


callus formation at the base of explants (Schroeder,


1980; Gonzales-Rosas


et al.,


1985)


and survival rate <100% (Gonzales-Rosas & Salazar-Garcia,


1984; Schall,


1987) were


reported.


Better


proliferation


has


been


achieved


mostly


by


reducing


the


major


salts


content in MS medium (Schall,


1987:


Ned et al.,


1982:


Campos & Pais,


1996).


Variable


rooting frequencies


have been


reported


(Table


2-6)


from


100% (Pliego-Alfaro,


Kane et aL, 1986; Campos & Pais, 1996) to 30% (Skene & Barlass, 1983; Bemrnger et al.,


1996).


The difference in rooting frequency may be genotype-dependent or related to


degree of juvenility and rooting method.


Nevertheless,


tissue culture propagation and


plant establishment in soil have been reported for commercial avocado and several species


belonging to


Lauraceae


(Table


2-6),


although


the reported


procedures


have involved


juvenile phase explants.


Therefore, the practical application of this technique is limited to


propagation of germplasm for breeding programs or propagation of endangered species


(Campos &


Pais,


1996).


Attempts to


culture adult materials


have been


unsuccessful


(Pliego-Alfaro,


1981


)although


limited


shoot


proliferation


followed


by


low


rooting


frequencies can


be obtained


from


adult


phase


shoot


cultures


that


have been


partially


rejuvenated (Pliego-Alfaro & Murashige, 1987


;Schall, 1987).


19


88;






28


Avocado protoplasts have been isolated from callus (Blickle et aL, 1988), in order to study sunbiotch viroid replication, and from fruit mesocarp (Percival et aL, 1991), for


studying fruit ripening,


Consequently, morphogenic responses of protoplasts


have not


been reported


Somatic Embryogenesis


Since


the


first


description


of


somatic


embryogenesis


by


Reinert


(1958)


Steward (1958), this regeneration pathway has been widely used as a tool to study plant


development (Zimmerman,


1993), for clonal plant propagation and for development of


technology


for


plant


improvement


through


somatic


cell


genetics


and


genetic


transformation.


Somatic


embryogenesis


refers


to


the


developmental


pathway


from


somatic


cells/tissues that mimics zygotic embryogenesis (Zimmerman,


1993).


From a practical


standpoint, somatic embryogenesis


is a process whereby embryos develop from somatic


cells/tissues in tissue culture and can develop to maturity and subsequently


germinate,


forming normal plants (Wann,


1988).


Low frequency of root emergence and even lower


shoot development from somatic embryos is common in many species.


In the view of


many, the concept of plant conversion is therefore more operational than germination for


in vitro somatic embryogenesis (Redenbaugh,


1993).


Germination


n this


dissertation


defined as the development


of roots from somatic embryos,


while plant


conversion


defined as the development


of both


shoots and


roots


from


the same


somatic embryo


regardless of the plantlets survival during acclimatization and production two new leaves as required under Redenbaugh's criteria (1993).


and


is


s






29


3.

4.


somatic embryo development or maturation, and somatic embryo germination or conversion.


Initiation of Embryogenic Cultures

Somatic embryos can develop from single cells within an explant (direct somatic embryogenesis), or differentiation can be preceded by proliferation of callus cells (indirect


somatic embryogenesis).


In the indirect pathway, cells undergo a directive induction event


that involves a change of competence.


Competent cells undergo a permissive induction


results


in


the


embryogenic


pathway.


In


directive


induction,


embryogenically


predetermined


cells


simply


require


permissive


induction


to


express


the


embryogenic


pathway,


e.g.,


cells in explanted nucellar cultures of polyembryonic plants (Ammirato,


1987). Indirect Pathway


indirect


pathway,


differentiated


explants,


leaves,


flower


buds,


hypocotyls,


etc., are explanted


onto


inductive


plant


growth


medium


containing


auxin


(usually 2,4-D) in order to obtain an embryogenic culture.


Embryogenic cultures consist


of different cell types: parenchymatous, elongated and vacuolated cells, highly cytoplasmic


isodiametric cells and cell clusters that resemble proembryos (Halperin,


1966).


In the


presence


of


auxin,


cells


within


the


rapidly


dividing


cell


population


are stimulated


undergo unequal divisions.


This results in the formation of two sister cells: a large, highly


vacuolated cell and a small, cytoplasmically rich cell that is competent for embryogenesis


(Litz & Gray,


1995).


Unequal cell division or segmentation of elongated cells has been


documented in carrot (Backs-Huseman & Reinedt,


1970; Street &


Withers,


1974), Safix


that


In


the


to






30


proembryos.


The embryogenic masses that develop from proembryos in the presence of


2,4-D are proembryonic or proembryogenic. More than one somatic embryo can develop


from each proembryonic mass when it is transferred to


2.4-D-free medium


(Halperin,


1966).


In tropical woody species,


the indirect pathway of somatic embryogenesis has


been reported to occur from nucellar explants of monoembryonic mango species (Litz et


1982), arnd from leaf explants of coffee (Sondahi & Sharp,


1977) and longan (Litz,


1988). Direct Pathway


In the direct pathway, somatic embryos or proembryonic masses can develop from the explant (immature zygotic embryo, nucellus, hypocotyl, etc.) without a callus phase. Somatic embryos or proembryonic masses can be initiated from embryogenic cells that are


present in the explant.


In pearl millet,


Vasil and Vasil (1980) demonstrated that somatic


embryos can originate from subepidermal cells of the scutellum of immature embryos.


the presence of 2,4-D,


those


scutellar cells enlarge and


undergo internal


segmentation


resulting in the formation of proembryos.


In polyembryonic mango, the embryogenic cells


that are already present


In


nucellar tissue


are similar


morphologically


to proembryos


(DeWald,


1987).


Auxin in the medium can stimulate the cloning of such cells (Carman,


1990; Wann, 1990; Ammirato,


1985).


Direct development of disorganized proembryonic


masses


or somatic


embryos


from


carrot


explants


can be


controlled


by


manipulating


medium pH (Smith & Krikorian, 1990).


Induction


of


the


direct


embryogenic


pathway


in


woody


perennials


has


been


reported from the nucellus of polyembryonic mango (Litz,


1984; DeWald,


198


7:


Litz et


,1995) and monoembryonic mango (Jana et al.,


1994); cotyledons of wild pear (Prunus


al.,


In


al.






31


Maintenance of Embryogenic Cultures and Morphological Variability of Proembryonic
Masses


Embryogenic cultures are subcultured as proembryonic masses and not callus (Litz


& Gray,


1992).


Proliferation of proembryonic masses (and somatic embryos) has been


referred to as repetitive embryogenesis (Ammirato, 1987;


Litz & Gray,


1992).


Repetitive


embryogenesis


involves


continuous


cycles


of


secondary


somatic


embryogenesis


from


proembryonic masses that have lost their integrative ability to form single somatic embryos


(Williams & Maheswaran, 1986).


This has been described with walnut (Preece, 1995), big


leaf magnolia


(Merkie &


Watson-Pauly,


1993),


mango


(Litz


et al.


,1995)


and


citrus


(Button et aL, 1974; Cabasson et a.,


1995; Gavish et aL, 1991)


The loss


of integrative development at


the proembryo


stage may result in


continuous proliferation of proembryos to form disorganized proembryonic masses, i.e.,


citrus (Cabasson et al., 1995).


The loss of integrative development at the globular to heart


stage may result in the proliferation of organized proembryonic masses,


i.e., mango.


cells of the protoderm and subepidermal layer become embryogenic and may slough off from the primary proembryos (Button et at., 1974).

Repetitive somatic embryogenesis by production of singulated maturing somatic


embryos usually is slow (Gray,


1995;


Merkle,


1995)


and


not as amenable to


genetic


manipulation through genetic transformation and to mass propagation as proliferation of


embryogenic cultures through proembryonic masses (Merkie, 1995).


Therefore repetitive


embryogenesis


through


proembryonic


masses


proliferation


is desirable,


not only


maintenance of cultures but for somatic cell genetics.

Auxin is one of the most significant inductive factors for embryogenic cultures, and


the


The


for






32


& Komamine,


1995).


Habituation may also occur, e. g., in citrus proembryonic masses


(Button & Kochba, 1974; Grosser & Gmitter, 1990).

Proliferation of proembryonic masses has been reported to be affected by genotype


in mango (Litz et aL,


1995) and sweet gum (Merkie,


1995a).


Very often, a whole range


different


sizes and morphologies


of proembryonic masses


and


somatic


embryo


present


in cultures of


different species of


sweet


gum


(Liquidam bar


styracpfolia)


proembryonic masses can be preferentially selected for further subculture (Merkie, I 995a). Medium pH can affect the proliferation of wild carrot cultures, so that low pH favors the


proliferation


of


proembryonic


masses


while


high


pH


favors


the


proliferation


development of somatic embryos (Smith & Krikorian, 1990).

The total nitrogen concentration and the ratio of NH4C


:NO)~ have been reported


to affect proembryonic mass proliferation.


In citrus,


total nitrogen concentration of 60


mM nitrogen resulted in significantly higher proembryonic mass fresh weight compared to


30 mM,


while high fresh weight was maintained with a NH4A


:NO)~


ratio of 1


to 1:9


(Niedz, 1993). Somatic Embryo Development


Somatic


embryo


development


is comparable


to that


of


zygotic


embryos,


includes distinct morphological stages, e.g.,


globular, heart, torpedo and cotyledonary.


addition,


formation


of morphologically


abnormal


somatic


embryos


has been


reported.


Morphological anomalies


include


monocotyly,


polycotyly,


fused


cotyledons,


fasciation,


and multiple somatic embryos (Ammirato,


1987


Litz et al.


,1995; Gray,


1995).


Sieving


and fractionation followed by culturing small polyembryonic masses on somatic embryo


of


s


is


and


and


and


In






33



Somatic embryo development is initiated alter transfer of embryogenic cultures to medium without or with low auxin content. Fujiwara & Komamine (1975) observed that cytokinins do not affect development of somatic embryos, although development of shoot


meristems is enhanced.


Abseisic acid has been demonstrated to affect the differentiation


of apical meristems and subsequent plant conversion (Nickel & Yeung, 1993).


Sucrose


at


concentrations


of


1-6%


Is


generally


used


for


somatic


embryo


development. Replacing sucrose with another carbon source, e.g.,


glycerol (Ben-Hayyim


& Neuman, 1983; Gavish et at., 1991; Vu et at., 1993), galactose (Cabasson et al.


lactose and raffinose has been reported (Kochba et al.,


1995),


978; 1982).


The


effect


of


the


mineral


composition


of


the


maturation


medium


has


been


documented.


DeWald et at. (1989a) demonstrated that medium based upon a modification


of B5


formulation


resulted


In


better


mango


somatic


embryo


development


than


MS.


Similar results were reported by Muralidharan & Mascarenhas (1995) with Eucalyptus citriodora.

Incomplete maturation of somatic embryos is one of the most significant factors


that account for low rates of plant conversion (Bormman,


1992).


Precocious germination


of immature Hevea brasilhensis Mull. AMg. somatic embryos has resulted in poor plant


regeneration


(Michaux-Ferribre


et


at.,


1991).


Somatic


embryo


maturation


controlled by treatments with ABA, sucrose and desiccation.


Abscisic acid has been used


to prevent


precocious


germination


(Ammirato,


1985),


to inhibit


secondary


somatic


embryogenesis (Monsalud et al.,


199


5


type somatic embryos (Kim & Janick,


), and to confer desiccation tolerance in orthodox


1989), thereby promoting accumulation of storage


lipids, starch and proteins, synchronous maturation and a high frequency of conversion


can


be






34


of conifers (Attree & Fowke,


1993), this approach has been unsuccessfUl for angiosperm


tree species (Merkie, 1995b; Vieitez, 1995; Pliego-Alfaro & Murashige, 1988).

Pence (1991) demonstrated that during maturation of recalcitrant zygotic embryos


of Theobroma cacao.


ABA levels increased and peaked at the beginning of maturation and


then dropped subsequently.


Water content of the embryo also decreased ca.


40-70% and


synthesis of anthocyanins and lipids increased up to the end of maturation period.


Similar


trends were observed by Etienne et al.


(1993) with recalcitrant H.


brasiliensis zygotic


embryos,


while


somatic


embryos


had


low


ABA


content,


which


varied


little


during


maturation.


Inclusion of ABA in maturation medium, however, did not significantly affect


desiccation tolerance of either cacao zygotic embryo


s (Pence,


1992) or mango somatic


nucellar


embryos (Pliego-Alfaro et al.,


1995a;


although Etienne et


al.


(1993)


claimed that high osmolarity (100 g/l sucrose) together with high levels of ABA (1 mM) dramatically improved H. basiliensis somatic embryo desiccation tolerance and resulted in 77% germination and 34% conversion.

Slow desiccation has been demonstrated to improve maturation of H. brasiliensis


somatic embryos (Etienne et al., 1993).


In walnut, desiccated orthodox somatic embryos


convert at a high rate (45%) (Deng & Cornu, 1992).

Merkle (1995a; b) was able to prevent precocious germination of yellow poplar


somatic embryos by allowing their


development


on filter


paper


overlain


on semisolid


medium.


These somatic embryos matured and converted to plantlets at frequency up to


100% upon transfer to germination medium.


Filter paper may impose a matrix stress on


somatic embryos that encourages maturation (Merkie, 1995a, 1995b).

a 4. a., a., . net
4: a ir .- .- - a r2. .-.. n i1~ . a n 4- * a .. var


and


b),






35


cytokinin (Mathews & Wetztein, acid (Deng & Cornu, 1992) an


1993), cold treatment (Arilaga et al.,


Ld high


1994), gibberellic


CO2 together with high irradiance (Figueira &


Janick, 1993).


Cold


treatment increased


plant conversion


of (orthodox)


black locust


somatic


embryos (Arrilaga et a.,


1994), walnut (Deng & Cornu, 1992), chesnut (Vieitez F,


1995),


Camehia japonica and


C .


reticulata (Vieitez A,


1995),


although recalcitrant


avocado


somatic embryos did not respond (Pliego-Alfaro & Murashige,


1988).


Cold treatment


may be essential for species that require cold stratification for optimum germination, i.e., temperate hardwood species with orthodox embryos (Merkle, 1995b).


Mathews and Wetzstein (1993) applied


100 piM BA and


3


mg 1-1 anti ethylene


silver


nitrate


topically


to orthodox


pecan


somatic


embryos


(Carya


ilinoensis)


increased germination from


3


to 47% and plant conversion from


2


to 13%.


Gibberellic


acid as a component in germination medium has increased the conversion rate of orthodox walnut somatic embryos with or without a cold treatment; however, the conversion rate of


desiccated walnut somatic embryos was reduced (Deng & Comnu, 1992).


Gibberellic acid


in combination with LAA increased the conversion rate of orthodox Came/hia japonica


reticulata


in comparison


with


gibberellic


acid


alone


(Vieitez


A,


1995).


regulation of orthodox seed germination by gibberellic acid has been widely used in cereals (Berrie, 1984), but its effects are not understood (Deng & Cornu, 1992).


Maintaining recalcitrant somatic embryos


of cacao under high CO2 (20,000 ppm)


high


irradiance


(159-299


pimol


m


s2)


(Figuera


&


Janick,


1993)


has


been


demonstrated


to


increase


the


conversion


and


vigor


of


regenerated


plantlets.


importance


of high


Co2


and


high


irradiance


has


been


to stimulate


photoautotrophy


and


and


C .


and


The


The






36


are associated


with


this


developmental


pathway,


such


as poor


embryo


organization,


embryo maturation and somatic embryo germination or plant conversion.


Some of the


problems have been solved by providing conditions that mimic the in vivo condition of the


zygotic embryo.


For recalcitrant species, e. g., mango, cocoa and avocado, however, their


seed physiology is not widely understood.


Consequently, attempts to resolve problems


associated with embryo development and maturation unavoidably are based on orthodox


seed


s/somatic embryos and therefore are often ineffective.


Protoplast Isolation. Culture and Regeneration


Protoplast technology


has


been


important


in


somatic


cell


genetics


for


genetic


transformation and for somatic hybridization.


The application of somatic cell genetics to


woody fruit species is especially important for supplementing conventional plant breeding.


Discrete genetic variability


could


be directly


selected


without


prolonged


crossing and


backcrossing because woody


fruit


species are propagated


vegetatively


(Janick,


1992).


The production of somatic hybrid


s


involving citrus and its relatives by protoplast fusion


can involve sexually incompatible species belonging to different genera or tribes (Grosser


et al.


,1992).


The potential of somatic hybridization requires the availability of an efficient plant


regeneration system from protoplasts.


Among woody plants, an efficient protoplast-to-


plant regeneration system was first reported for citrus (Vardi et al.,


1972). Plants have


also


been


regenerated


from


protoplasts


of


sandalwood


(Santa/urn


album


L.)


(Rao


Ozias-Akins,


1985),


from leaf protoplasts of species in the family


Rosaceae,


including


Ma/us,


Prunus and Pyrus (Ochatt,


1990; 1993b,


C;


Ochatt


et al.,


1992;


Patat-Ochatt,


&






37


Protoplast Isolation

The discussion that follows is intended as a review of those factors that affect


isolation and culture of protoplasts together with plant regeneration,


respect to woody perennials


particularly with


and fruit trees.


Protoplasts can be isolated


either mechanically


or enzymatically.


For


in vitro


studies, the latter method has been preferred, because a large number of protoplasts can be


released


from


various


tissues


(Cocking,


1972).


The


use of


enzymes


for


protoplast


isolation was pioneered by Cocking in


1960 who isolated protoplasts from tomato roots


following


digestion


with


cellulase


purified


from


a fungus,


Myrothe c/urn


verrucaria


(Cocking, 1983). Enzymes


The enzyme mixture


that


is utilized


for protoplast


isolation


generally


contains


pectinase for loosening the tissue and


cellulase


for


degrading


the


cell


wall.


Various


enzymes are available commercially with different levels of purity (Table 2-7).


Impure


enzyme preparations contain nucleases (especially ribonucleases), peroxidases, proteolytic


enzymes


and


phenolic


compounds


(Vasil


&


Vasil,


1980).


Partial


purification


commercially available enzyme precipitation with ammonium sulfate and elution through


Sephadex G-25 or Biogel is sometimes useful (Vasil & Vasil,


1980).


Very highly purified


and crystalline enzymes are not usefUl since they are unable to digest complex plant cell walls; therefore complex enzyme mixtures, cleansed of toxic substance and impurities are


recommended (Vasil &


Vasil,


1980).


Purified enzymes such as Cellulase Onozuka RS,


Cellulase Onozuka RiO


,macerozyme RiO and Pectolyase


Y23 have been recommended


for protoplast isolation from tissues of woody plants (Grosser & Gmitter,


1990:


Ochatt et


of















Table


2-7


Enzymes used for protoplast isolation.


Enzyme Concentration Purity Organismal source Commercial source
in culture

Cellulase
Cellulase Onozuka R-10 1.0-3.0 Trichoderma viride Yakult Honsha Co, Tokyo
Karlan Chemical, Santa Rosa, CA
Cellulase Onozuka RS 1.0 high T. viride Yakult Honsha Co Tokyo, Japan
Karlan Chemical, Santa Rosa, CA
Meicelase P-i Meiji Seika Kaisha, Tokyo, Japan
Meicelase 1.0-4.0
Driselase cmude Basidiomycetes Sigma
Driselase 1.0-2.5 Irpex lacteus Kyowa Hakko Kogyo, Tokyo,
Cellulase crude T. viride Sigma Chemical Co. St. Louis, MO, USA
Cellulase YC T. viride Seishin Pharmaceutical Co., Tokyo
Cellulysin 1.0-3.0 T. viride Calbiochem, San Diego, CA
Cellulase CEL high T. viride Cooper Biomedical Inc., Malvern, PA

Hemicellulase
Helicase Helix pomatia Industrie Biologique Franqaise, Genevilliers,
France
Hemicellulase 0.2-0.5 crmde Aspergilus niger Sigma Chemical Co. St. Louis, MO
Rhozyme HP 150 1.0-2.0 Aspergilus niger Genencor Inc., South San Franscisco, CA.
Corning Glass, Corning, NY













Table


2-7--continued


Enzyme Concentration Purity Organismal source Commercial source
in culture

Pectinases
Macerozyme R- 10 0.5-1.0 high Rhizopus arrhizus Yakult Honsha Co, Tokyo
Karlan Chemical, Santa Rosa, CA
Pectolyase Y-23 0.1 high Aspergillus japonicus Seishin Pharmaceutical Co., Tokyo
Karlan Chemical, Santa Rosa, CA
Macerase (macerozyme) 0.5-1.0 Rhizopus arrhizus Calbiochem, San Diego, CA, USA
Pectinase 0.5-1.0 Aspergilus niger Sigma Chemical Co. St. Louis, MO, USA
PATE (Pectic - acid- acetyl- 0.1 Bacillus polymixa Farbwerke-Hoechst AG, Frankfurt, FRG
transferase)
Pectolyase crude A. japonicum Sigma Chemical Co. St. Louis, MO, USA


Sources: Ishil (1989); Evans and Bravo (1983)






40


10% of the concentration of cellulase.


A mixture of enzymes is normally used.


Since cell


wall


composition of


different tissues of


different


species is variable (Ishii,


1989),


enzyme


composition


of


the


digestion


solution


may


vary


and


must


be


determined


empirically for different species (Cocking,


1972;


Vasil &


Vasil,


1980; Evans & Bravo,


1983; Davey & Kumar, 1983).


Ishli (1989) demonstrated that the cell wall composition of


monocotyledonous


and


dicotyledonous


species


was


different,


and


demonstrated


that


xylanase is required for optimum protoplast release from oat leaves while pectin lyase was


required for


optimum protoplast release from petunia leaves.


With


woody


species,


single enzyme mixture may be effective for several species, although slight variations may


be required, depending on the tissue (Ochatt, 1990).


A mixture of 1% Cellulase Onozuka


Rio,


1% Hemicellulase and 0.1% Pectolyase


Y23 has been used for protoplast isolation


from numerous fruit and nut genotypes (Revilla et at., 1987


Ochatt, 1990; 1993b),


while


2% Meicelase,


2%


Rhozyme HP15OO and


0.03%


Macerozyme


RiO has been used


solate protoplasts from suspension cultures and callus tissues of cherry, pear and apple


(Ochatt,


1990;


1993a, b).


An enzyme mixture that was developed initially for


Trgfolium


rubens (Grosser & Collins, 1984) and improved for citrus contains 1% Cellulase Onozuka


1% Macerase or macerozyme RiO and 0.2% Pectolyase


Y23 (Grosser


& Gmitter,


1990) and is effective for various tissues and species, including embryogenic cultures and suspension cultures of citrus, non-embryogenic callus and suspension culture of citrus and


citrus relatives (Muorao-Filho & Grosser, 1992;


Grosser & Gmitter, 1992),


eaves from in


vitro and greenh-ouse grown citrus and citrus relatives (Grosser & Chandler, 1987), flower bud tetrads, and for other species, including embryogenic suspension cultures of mango and grape (Grosser, 1993, personal communication).


the


a


RS,


to






41


(Grosser & Chandler, 1987; Grosser & Gmitter, 1990; Kobayashi et at., 1985; Sim et at., 1988) and Actinidia chinensis (Tsai, 1993) even though lower concentrations have been used for protoplast isolation from grape embryogenic cultures (Reustle et al., 1995).

A minimal salt mixture has been included in the enzyme mixture to reduce shock


following transfer of protoplasts to culture medium.


These media include CPW


salts


(Frearson et a.,


1973) and 0.


5


x MT


(Murashige &


Tucker,


1965) (Kobayashi et al.,


1985.


Sim et al., 1988).


Other addenda such as morpholinoethanesulfoxide (NIBS) at concentrations of 3-7


mM


(Grosser


&


Gmitter,


1990


Ochatt,


1990


1993a,


b)


has been used


to buffer the


acidification that often occurs during digestion.


Polyvinylpyrollidone (PVP) has been used


to prevent


phenolic


oxidation,


especially


when


protoplast


isolation


involves


suspension cultures of woody perennials (Ochatt,


1993a,


b,; Patat-Ochatt,


1993; Lee &


Wetzstein,


1994).


Other


sub stances,


i.e.


CaCI2


and


NaH2PO4,


are added


as plasma


membrane stabilizers of protoplasts (Vasil & Vasil 1984;


Grosser & Gmitter, 1990).


Other physical factors influencing protoplast isolation

Enhancement of enzyme penetration into tissue is usually required when clumps of


tissues or leaves are used.


With herbaceaous plants, this can be achieved by removing the


epidermis and chopping the tissues into small pieces (Evans & Bravo,


1983).


With woody


species,


the epidermis


is difficult to remove;


therefore,


leaf tissue can


be chopped


feathered (Grosser & Gmitter, 1990).


plants, 1990).


When leaf tissue


is


derived from greenhouse-grown


vacuum infiltration may also facilitate enzyme penetration (Grosser


&


G


Grinding of leaves can provide a larger surface area for enzyme digestion,


mtter, while


entrapment of protoplasts during purification can be prevented by washing the cell debri


leaf


or


or






42


The ratio of tissue:enzyme solution (wlv) can be critical for protoplast isolation


from peach leaves where ratios of 10-20 mg mV'


resulted in high protoplast yields while


ratios of 50-100 mg ml' resulted in low protoplast yields (Mills & Hammerschlag, 1994). High tissue:enzyme ratios, however, have been used for other tissues, i.e., 100 mg mU" for


pear leaves (Ochatt & Power,


1988), and 150 mg mE' for apple leaves (Doughty & Power,


1988).


Grosser


& Gmnitter (1990) used a tissue:enzyme ratio of


Ca. 250 mg mE'


embryogenic citrus cultures, and 30-50 mg mE' for leaves.


The negative effect of higher


tissue:enzyme ratios may be associated with release of protease from digested tissues that


inhibits enzyme activity (Mills & Hammerschlag,


1994) and phenolic compounds.


tissue:enzyme ratio may be genotype and tissue dependent. Protoplast purification

Protoplasts are generally purified by passing the digestion mixture through a nylon


or stainless steel sieve (45-90 pm mesh) or another material,


e.g., Miracloth�,


This is


sometimes followed by gradient centriftugation to remove cell debris, undigested cells and broken protoplasts that can reduce culture pH, protoplast viability and fUsion frequency


(Grosser, 1994).


Grosser & Gmitter (1990) have sieved with a 45 pim stainless steel mesh


screen followed by gradient centrifugation at 100 g in 0.7 M (25% w/v) sucrose and 0.7


M (13% wlv) mannitol supplemented with modified CPW


salts (Frearson et aL.,


1973).


The purified protoplasts in the interphase can be washed again (Grosser & Gmitter, Protoplast Culture


990).


Factors


affecting


protoplast


culture


and


subsequent


growth


and


development


include


medium


composition,


osmoticum


and


osmolarity,


plating


density


environmental


conditions,


.e.,


irradiance and


temperature (Dons &


Colijn-Hooymans,


for


The


and






43


Medium composition


Protoplast


culture


media


are generally


composed


of


major


and


minor


salts,


vitamins and other organic addenda,


plant growth substances,


a carbon source and an


osmoticum.


Plant growth media that have been developed for a particular genotype can


be used (Davey & Kumar, 1983).


This approach seems logical and efficient even though


some modification of the medium composition is sometimes needed.


Major and minor salts of MS (Murashige and Skoog,


1962), B5 (Gamborg et a.,


) and KMS or KM8P (Kao & Michayluk, 1975) media are satisfactory.


The success


of the


KM8P medium may be due to the presence of multivitamins,


sugar


and sugar


alcohol


additives


that


provide essential


metabolic


intermediates


(Grosser,


1994).


efficient


protoplast


culture


medium


for


species


within


a genus


can be


developed


supplementing optimal basal media that have been effective for cell culture of the genus


with SP multivitamins,


organic acid and sugar/alcohol additives (Grosser,


994).


This


approach has been successful for Trifolium (Grosser & Collins,


1984),


citrus (Grosser &


Gmitter, 1990; Niedz, 1993) and apple (Kouider et a.,


1984; Deng et a!., 1995; Doughty


& Power,


1988; Patat-Ochatt et at.,1993).


Ochatt & Power (1988),


however, reported


that pear (Pyrus communis L.) mesophyll protoplasts only regenerate cell walls in K8P or


KM8P or MS media, but divide in MS medium without ammonium.


The negative effect


of K8P medium on protoplast division has also been reported for root callus protoplasts of


sour cherry (Prunus cerasus L.) (Ochatt,


1990).


Ochatt (1993a, b) suggested that scion


and rootstock genotypes may require different organic additives,


i.e


.rootstock genotypes


require more organic compounds than scion genotypes in the same genus, i.e., Pyrus and Ma/us (Ochatt et al., 1992b).


r r ~ if flfl


I.


I I -


'-I-' 1


nfl flfltfldflC. .fl fl.trflta nnn*n.tt at aS e~..' art.. nt a - . I.... a a * a4 a se a - . n * ~


196


5


An


by










Populus


spp.


(Russel


&


McCown,


1986;


1988)


and


for


somatic


embryogenesis


subsequent


plant


regeneration


from


protoplasts


of


citrus


(Grosser


&


Gmitter,


1990;


Niedtz, 1993).


Plant growth substances, i.e., cytokinin (BA, zeatin) and auxin (


2,4 D, NAA) are


required for microcallus development when protoplasts are isolated from leaves, callus or


suspension


culture


of


pear


(Ochatt,


1990;


1 993b),


Prunus


spp.(l99O;


I1993 c),


apple


(Patat-Ochatt et at., 1993), cell suspensions of VI/s labruscana and V


thunbergii (Mii et


1991) and in vitro leaves of VIts spp.


(Lee &


Wetztein,


1988),


callus of kiwifruit


(Oliveira & Pais,


1991), leaves from greenhouse-grown passionfruit (d'Utra


1993) and peach suspension cultures (Matsuta et al.,


1986).


Vaz et al.,


Although sometimes also


required for microcallus development from protoplasts


of V/tis sp. (Reustle et at.,


solated from embryogenic cultures


1995), plant growth substances are not generally required for


somatic embryogenesis of protoplasts isolated


from


embryogenic cultures,


e.g.,


citrus


(Grosser & Gmitter,


1990; Niedz,


1993; Kunitake et a.,


1991


;Kobayashi et al.,


1985;


Hidaka & Kajiura,


988;


S


im et al.


1988:


Vardi &


Galun,


198


8;,


1989).


Protoplasts


isolated from embryogenic cultures derived from seedling parts of citrus relatives such as


Citropsis schweinfurt h/i, A ta/ant/a biloculoarns,


etc. reportedly


required plant


growth


substances


such


as BA


or GA4 7


for


microcallus


formation


(Jumin


&


Nito,


1995);


however,


protoplasts from similar tissues of Microcitrus did not require plant


substances (Vardi et aL,


1986).


growth


The plant growth regulator requirement for protoplast


growth and development is probably determined by whether regeneration from the source is by organogenesis or somatic embryogenesis.


44


and


a.,






45


Grosser


& Gmitter,


1990), Prunus spp.


(Ochatt,


1993c),


Pyrus spp.


(Ochatt,


I1993b),


Malus


sp.


(Patat-Ochatt,


1993),


kiwifruit


(Oliveira


&


Pals,


1991),


Pass flora


(Dornelas & Vieira, 1993; d'Utra Vaz, 1993) and Vitis spp.


(Mii et al., 1991).


Protoplast plating density affects plating efficiency in apple (Koulder et al.


1984)


root


callus protoplasts of sour


cherry (Prunus cerasus


L.) (Ochatt,


1990).


highest plating efficiency was used to determine the optimum plating density.


Determining


plating density on the basis of the highest plating efficiency may not be appropriate when


regeneration is via somatic embryogenesis,


e.g.,


citrus (Kobayashi et at.,


1985).


Citrus


protoplasts derived from embryogenic cultures and plated at a high plating density (ca.


resulted


in


a


high


plating


efficiency


(40%);


however,


only


microcallus


formed


(Kobayashi et al.


1985).


At a low plating density (ca.


2


x 14,


ow plating efficiency


resulted (5%); however, somatic embryogenesis occurred.


citrus protoplasts


Somatic embryogenesis from


isolated from embryogenic cultures cultured at low plating density was


also reported by Hidaka and Kajiura (1988).


Inhibition of somatic embryogenesis when


fised citrus protoplasts were grown at a high plating efficiency was also reported (Grosser & Gmitter, 1990; Grosser, 1994). Medium osmolaritv


Medium osmolarity generally is 0.4 to 0.7M and supplied


active sugar (sucrose, glucose,


with a metabolically


or sorbitol) or a metabolically inert sugar such as mannitol.


Combining metabolically active and inert sugars has been recommended since the former is metabolized by the growing protoplasts and therefore the medium osmolarity is gradually


reduced.


This would subsequently reduce shock when the protoplast-derived cell colonies


are transferred to regeneration medium (Vasil & Vasil,


1983).


and


spp.


The


105)


2


x






46


dependent upon protoplast density


although high medium osmolanity ( 0.35 M mannitol


+ 0.15


M


sucrose)


results


in


a slightly


higher plating


efficiency than


lower


medium


osmolarity (< 0.3


5


M mannitol + 0.1


5


M sucrose).


Low medium osmolarity combined


with


low plating


density resulted


in


the direct


development


of


somatic


embryo


from


protoplasts,


while


with


high


medium


osmolarity


protoplasts


give


rise


to microcallus.


Development


of


somatic


embryos


directly


from


protoplasts


cultured


at low


medium


osmolarity (0.04


M


sucrose,


0.08


M


glucose and


0.23


M


mannitol)


and


low plating


efficiency was also reported for Citrus yuko (Hidaka & Kajiura, 1988).


of somatic embryos at low medium osmolarity, however,


efficiency.


The development


was inhibited by high plating


The inhibitory effect of high plating density on somatic embryo development


could be overcome by lowering the density (Kobayashi et aL,


1985, Grosser & Gmitter,


1990).


Regardless of the positive effect of low medium osmolarity for somatic embryo


development,


protoplast


culture


medium


has


generally


been


supplemented


with


high


medium osmolarity of Ca.


0.6-0.7 M


of several


different


sugars.


Grosser


&


Gmitter


(1990) employed 0.6-0.7 M osmoticum that consisted of 0.15-0.25 M sucrose and 0.45


M mannitol,


while Vardi and Galun (1989) used 0.3 M sucrose and 0.3 M mannitol.


combination of 0.3 M sucrose and 0


.3


A


M sorbitol was used for culture of protoplasts of C.


madurensis (Ling et al.,


1989) and C. unshiu (Ling et al.,


1990).


Other reports include


the use of a only of 0.6 M sorbitol for culture of protoplasts isolated from callus initiated


from seedling stems (Jumin & Nito,


1996a; b), 0.15 M sucrose and 0.4


5


M glucose for


embryogenic


culture-derived


protoplasts


of


C .


mitts


(Sim


et aL,


988)


mannitol, 0.2 M glucose and 0.05 M sucrose for kiwifruit protoplasts (Tsai et al., 1993).


and


0.4


M






47


Method of culture


Protoplasts of woody species have been cultured in: liquid culture as a thin layer, i.e., citrus (Grosser & Gmitter, 1990; Hlidaka & Kajiura, 1988; Kobayashi et aL, 1985) and


pear (Ochatt &


Power,


1988a); in liquid


culture as a shallow pool,


i.e.,


fised


citrus


protoplasts (Grosser & Gmitter,


1990) and apple (Ding et at.,


1995); immobilized with


low melting agarose in disc culture, i.e., sour cherry (Ochatt, 1993b) and semisolid culture


for Pass flora spp.


(Dornelas &


Vieira,


1993; D'Utra


Vaz et al


.1993), grape (Lee &


Wetzstein,


1988) and sour cherry (Ochatt,


1 993b); immobilized with agar or gellan gum,


i.e., citrus relatives (Jumin & Nito, 1990) and C. unshiu (Ling et a.,


991; Kunitake at al.,


1991); immobilized in Ca-alginate,


e. g., grape (Reustle et al.,


1995), apple (Huancaruna-


Perales & Schieder,


1993) and citrus (Niedz, 1993); in double phase liquid over semisolid


agar/gellan gum, i.e., C. mitts (Sim et al.,


1988) and Vitis labruscana and V


thunbergii


(Mil et al., 1991).


Liquid


culture


is easy


to handle


and


facilitates


observation


with


the


inverted


microscope.


When solid culture is required, low melting agarose results in higher plating


efficiency (Grosser & Gmitter, 1990), and facilitates protoplasts to grow under low plating


density, i.e.,


660-1300 protoplasts per ml as demonstrated in Hyocyamus muticus and


Nicotiana


tabacum


(Shillito


et al.


,1983).


Similarly,


agarose


has


been


effective


culturing protoplasts of


calamondin (C.


madurensis Lour.) (Ling


et a.,


1989).


Sour


cherry protoplasts,


however,


had


the same plating efficiency regardless of the culture


method


utilized


(Ochatt,


1990),


while


protoplasts


derived


from


stems


and


leaves


haploid apple divided in liquid medium,


but not in agarose discs


or in molten agarose


(Patat-Ochatt,


1993).


Protoplasts


of the


shrubby


ornamental


honeysuckle


(Lonicera


nitida cv Marn) crew better in liri tban in1 anorca


mnin (frott 1001n


for


of






48


liquid medium, but divided and formed microcallus in alginate-solidified medium, although


microcalli


eventually


died.


Ca-alginate


has


been


successfully


used


to culture


protoplasts of embryogenic grape (Reustle et al.,


1995).


Culture environment


Protoplast cultures have generally been maintained at


2


5 0


& Gmitter, 1990), since protoplasts can be sensitive to light (Grosser, growth inhibition by light has also been reported for Lonicera nitida


C in darkness (Grosser


1994). P 'Maigrun'


'rotoplast (Ochatt,


1991).


Some protoplasts, i.e.,


sour cherry,


can tolerate more light and can grow and


differentiate under diffuse light (Ochatt, 1990).

Protoplast culture and regeneration has been reported with several woody species. Regeneration in species other than citrus has mostly been confined to organogenesis since


the source of the protoplasts has not been embryogenic tissue.


Protoplast culture and


regeneration


from


embryogenic


tissues


is


more


efficient


and


simpler


than


from


nonembryogenic tissues.


Somatic Hybridization


Since the


first


interspecific somatic


hybrids between Nicotiana


glauca


and N.


langdorffii (Carison et al.,


1972) were reported, somatic hybrids have been produced in


various plants, including potato, tomato, legumes, cereals, eggplant, petunia and several trees such as citrus.


Somatic hybridization by


protoplast fusion


has


been


utilized


to bypass genetic


barriers


between


sexually


incompatible


wild


species


and


economic


species,


thereby


transferring important traits from wild species into commercial species.


Somatic hybrids


the






49



(Solanium melongena L.) with S. khzasianum, . torvum and S. nigrum were difficult-toroot, grew poorly and were highly sterile, whereas, somatic hybrids between eggplant with more closely related S. aethiopicum demonstrated vigorous growth and had high fruit


production (Sihachakr et al.,


1994).


Similar results were reported for somatic hybrids


between


potato (Solanium


tuberosum) and


related


species


(Butenko


&


Kucko,


Jadari et al.,


1992).


Since the somatic hybrids are tetraploid,


their use in breeding of


diploid species


is


dependent on the ability to regenerate diploid plants from microspores


and/or intensive backcrossing to the cultivated species (Sihachakr et al., 1994). Somatic Hybridization and Its Application to Woody Species


The application of somatic hybridization to woody species has been limited to a few genera, mostly Citrus spp. and relatives (Grosser & Gimitter, 1990), with which more


than


150


somatic


hybrids


have


been


produced


(Grosser


et al.


1996


Grosser,


1997


personal communication).


Other somatic hybridizations have involved sexually- and graft-


incompatible cherry rootstock


'Colt'


(Prurnus avium x


Pse udocerasus)


with


wild


pear


(Pyrus


commuis


var. pyraster)


(Ochatt


et al.,


1989),


between


sexually


compatible


Pass flora edulis f flavicarpa and P. incarnata (Otoni et al., 1995), between P. edulis f.


flavicarpa with P. alata, P.


amethyst/na, P.


cincinata and P. giberti (Dornelas et al.,


1995).


Two


approaches


for


somatic hybridization have been


used


in


citrus


rootstock


improvement.


Somatic hybrid rootstocks have been produced from existing rootstocks


that have desirable and complementary characteristics.


These somatic hybrids would have


characteristics of both parents since somatic hybridization is an additive process with no


1994;






50



al., 1992), although the characterization of the inheritance of the parental characters in the somatic hybrids has not yet been reported.


Another


approach


involves


production


of


somatic


hybrids


between


existing


rootstocks


and


sexually


incompatible,


related


species


for


germplasm


enhancement.


Several somatic hybrids have been produced, including combinations of C. reticulata with


Citropsis


gilletiana,


and


C.


sinensis


' Hamlin'


sweet


orange


with


Severinia disticha,


Citropsis gillatiana, S.


lansium.


Somatic hybrids of the last two combinations did not


grow vigorously


eventually died.


This failure may be due to somatic incompatibility since C. lansium and


limonia cannot


be grafted


easily with


citmus (Louzada &


Grosser,


1994).


Recent


attempts involving


a vigorous


selection


of A.


ceylanica


with


'Succari


sweet


orange


resulted in prolific and more vigorous somatic hybrids (Mourao-Filho,


1995); however,


latent somatic incompatibility in these somatic hybrids has not been determined.

Mourao-Filho (1995) reported that somatic hybrid plants between four varieties of sweet orange (C. sinensis) + . disticha died following attack by an unidentified fungus,


although


previously reported


somatic


hybrids


of the


same


parental


combination


were


reportedly unaffected (Louzada & Grosser,


1994).


Analysis of the somatic hybrid plants


of 'Cleopatra'


mandarin (C.


reticulata) + Citropsis gillatiana showed a high level


susceptibility to an undetennined leat/stem fungal spotting disease that drastically reduced


tree vigor (Mourao-Filho,


1995).


It is not clear if this undesirable phenotype was due to


somaclonal variation or a negative genomic interaction (Mourao-Filho, 1995).


Grosser


et al.


(1992)


also


reported


that


somatic


hybrids


between


C .


sinensis


'Hamlin'


+ Severenia bux folla had chromosome numbers of 27 and it is not clear whether


bux Jolla, Atalantia ceylanica, Feronia limonia and


F.


Clausena


and


of






51



than the allotetraploid (2n = 4x = 32), have been produced from carrot and barley (Kisaka


et al


., 1997).


Somatic


hybridization


for


citrus


scion


improvement


has


been


intended


production of seedless triploid scion cultivars either by production of tetraploid somatic hybrids for crosses with diploids or by direct protoplast fusion between diploid and pollenderived haploid protoplasts (Grosser & Gmitter, 1990).

Somatic hybridization has been reported for producing somatic hybrids between


graft- and cross-incompatible cherry rootstock


'Colt'


(Prunus avium x Pseudocerasws)


and wild pear (Pyrus communis var. pyraster) (Ochatt et al.,


1989).


Further analysis of


the somatic hybrid plants demonstrated that one clone was graft-compatible with both parents (Ochatt & Patat-Ochatt, 1994).

Somatic hybridization has also been used to produce fertile allotetraploid somatic hybrids between Pass flora edulis f. fiavicarpa and P. incarnata that have cold tolerance


characteristics (Otoni et al.,


1995).


These somatic hybrid plants are fertile and showed


intermediate characteristics


of both


parents


for most


characters


observed;


the


natural


hybrids of these species are sterile and only their tetraploid (after colchicine treatment)


derivatives are fertile.


Other somatic hybrid


s


involving Pass flora species combinations


have been intended to introgress desirable characteristic of wild species into Pass flora


edulis f.


flavicarpa (Dornelas et al., 1995).


Somatic hybridization has also been use to produce cytoplasmic hybrids (cybrids).


Cybrid


production in


citrus involves the donor-recipient method


(Vardi


et al.,


1987).


Following


fusion


between


protoplasts


isolated


from


embryogenic


cultures


and


from


mesophyll protoplasts, diploid plants with morphological characteristics of the leaf parents


for






52



prerequisite for somatic embryogenesis from non morphogenic leaf mesophyll protoplasts


(Grosser


et al.,


1996).


To date there is no application for


citrus cybrid


production;


however,


cybridization


may


increase


the


availability


of


citrus


germplasm


for


further


somatic fUsions (Grosser et al.,


1996).


Fusion and Selection Methods and Confirmation of Somatic Hybrids


Protoplast fusion in woody plants is mostly achieved using either the polyethylene


glycol


(PEG)


method


or electrofiision.


Dextran


as a fusion


agent


was


reported


somatic hybridization of Populus species (Park & Soon,


1994).


PEG has been used to


produce


more


than


150 citrus


somatic hybrids (Grosser


et al.


1996:


Grosser,


1997


personal communication).


Electroflision has been used to produce at least


0 somatic


hybrids and cybrids (Saito et al.,


1993; Ling & Iwamasa,


1994; Moriguchi et al.,


1996;


Motomura et al.


,1995; Hidaka & Omura,


1992;


Yamamoto & Kobayashi,


1995).


The


widespread


use of PEG


for


somatic


hybridization


has


demonstrated


its


efficacy


efficiency.


Furthermore, it


is


inexpensive, simple and does not cause protoplast mortality


(Grosser & Gmitter, 1990).

Selection of somatic hybrids in citrus involves the use of embryogenic cultures of


one parent and non-morphogenic cell suspensions,


callus or leaves of the other parent.


The embryogenic cells confer regeneration potential to the fused protoplasts while unfrised protoplasts of the non-morphogenic parent cannot develop in plant growth regulator-free


medium.


Habituated nucellar derived embryogenic cultures can be used for one parent in


citrus somatic hybridization (Grosser


& Gmitter,


1990).


Although habituated cultures


have


ost embryogenic competence, somatic hybrids could be recovered following fusion.


for


and










obtain somatic hybrids between Prunus and Pyrus (Ochatt et al.,


1989) and Pass flora


(Otoni et al., 1995).


Somatic


hybrid


identity


is generally


confirmed


by


several


methods,


including


morphology


characters,


chromosome


counts


and


molecular


markers,


i.e.


nuclear


ribosomal DNA analysis,


mitochondrial DNA analysis (Grosser


& Gmitter,


1990) and


Polymerase Chain Reaction (PCR)-based Random Amplified Polymorphic DNA (RAPD)


analysis.


Confirmation of somatic hybridization using RAPD markers in citrus has been


shown in some cases to require more than one primer (Mourao-Filho, 1995).


General Conclusion of the Literature Review


Avocado is an important fruit crop; however, breeding new cultivars has been slow


and improvement


of


existing cultivars has not


occurred.


Biotechnology,


i.e.,


somatic


hybridization


and


genetic


transformation,


can


create


genetic


variability


otherwise


unavailable


through


conventional


breeding.


Application


of


these


technologies


dependent on the availability of efficie cultures, protoplasts or other tissues. T plants has been somatic embryogenesis.


nt plant regeneration protocols from suspension he most efficient regeneration pathway for woody Although preliminary studies regarding somatic


embryogenesi s


of


avocado


have


been


reported,


the


published


information


has


been


insufficient to utilize for improving avocado by somatic cell genetics.


It is clear that work


with other woody species (e.g., citrus, mango, etc.) might be applicable to similar in vitro studies with avocado.


53


is















CHAPThR 3
INITIATION ANT) MAINTENANCE
OF AVOCADO EMBRYOGENIC CULTURES


Introduction


The avocado (Persea americana Mill.) is one of the major fruit crops of the world. The species includes three horticultural races of different climatic adaptation; the tropical


americana


var.


americana


(West


Indian),


the


less


tropical


P .


americana


guatemalensis


(Guatemalan)


and


the


semitropical


P .


americana


var.


dymifolia


(Mexican).


Despite its great importance, there have been relatively few reports related to


cell and tissue culture of this species.


The initiation of callus from different plant parts


was described by Schroeder (1956; 1961


1971;


975; 1978), Blumenfeld & Gazit (1971)


and Desjardins


(1958);


however,


these cultures


were nonmorphogenic.


Embryogenic


cultures were initiated from zygotic embryos excised from abscised avocado fruits,


but no


further


somatic


embryo


development


was


reported


(B arlass


&


Skene,


1983).


Embryogenic


cultures


were


also


initiated


from


early


stage


'H-ass'


(Pliego-Alfaro


Murashige,


1988),


'Fuerte'


and 'Duke


7'


(Mooney & van Staden,


1987) zygotic embryo


explants.


Mature somatic embryos were recovered from embryogenic cultures, and low


frequency


plant


recovery


was


described.


The


effects


of


genotype


on


somatic


embryogenesis were not addressed in these reports, and the establishment of embryogenic suspension cultures was not attempted.


P.


var.


&






55


Materials and Method


Embryogenic Cultures on Semisolid Medium Induction of embryogenic cultures from zygotic embryos


Avocado


fruitlets,


0.3-2.0


cm length


without the calyx,


representing


different


cultivars of different races and hybrids involving two or more races were collected from the USDA-ARS Subtropical Horticultural Research Station (Miami, FL) national avocado germplasm repository, the germplasm collection of the University of California, Riverside,


CA and the University of Florida,


Tropical Research and Education Center (Homestead,


(Table


3-1).


After removal


of


sepals


and


peduncles,


the


fruitlets


were


surface-


disinfested in a 10--20% solution of commercial bleach containing 10-20 drops of Tween


20� per liter for 10-20 mn.


Fruitlets were rinsed with two changes of sterile, deionized


water.


The firuitlets were bisected under axenic conditions.


The zygotic embryo was


removed from each fruit (Pliego-Alfaro & Murashige,


growth medium, i.e., induction medium.


1988) and transferred onto plant


A single zygotic embryo was placed into culture


in each 60


x


15 mm Petri dish and sealed with Parafim@.


Cultures were maintained in


darkness


at


25


OC.


The


stage


of


development


of


the


zygotic


embryos


corresponding fruit size for


'Thomas' are indicated in Table 3


The induction media consisted of


) MS (Murashige & Skoog,


1962) major salts


(MSP) and three modifications of MS,


which included 2) omission of NIztNO3 (MT~), 3)


substitution of NaNO3 (MSNa) for NH4NO3 4) KNO3 (MSK) as the sole nitrogen source


(with the concentration of nitrogen being equivalent to that in MS) and


(Gamborg et al, 1968).


5


) B35 major salts


All of the major salts formulations were supplemented with MS


FL)


-2.


and


the






56


Table 3-1.


Avocado cultivars used for the experiments, their botanical varieties and their


sources.


Cultivar Botanical Variet/ Source

Booth 7 G xM TREC, UF
Booth 8 G xM TREC,UF
Duke 7 M UC Riverside
Esther Gx([(G xM)x G] UC Riverside
Hass Gi UC Riverside
Isham G USDA-ARS, Miami
Irwing 56 Complex hybrids USDA-ARS, Miami
Jose Antonio W USDA-ARS, Miami
Lamb Gi UC Riverside
M25 864 M USDA-ARS, Miami
T362 Gi UC Riverside
Thomas M UC Riverside
Waldn W SDA-RS, iam
Waln GW USDA-ARS, Miami


z


From Smith et at. (1992)


Table


3-2.


Length of


'Thomas'


avocado fruitlet (measured without calyx) in relation to


zygotic embryo length, stage of development and morphology.


Fruit Length Zygotic Embryo Developmental Morphology

(cm) Length (mm) Stage

<0.5 0.1-0.4 globular 0

0.5-O.8 0.5-0.8 early heart stage C )V

0.9-1.1 1-2 late heart stage

1.2-1.5 3-7 early torpedo

>1.5 >7 late torpedo, early






57



The pH of all media was adjusted to 5.7-5.8 with either KOH or HICI prior to


addition of agar and autoclaved at 121*C at


1.1


kg cm4 for


15 m'in.


The plant growth


media were dispensed in 10 ml aliquots into sterile disposable Petri dishes (60 x 15 mm). Binomial confidence intervals of 95% were computed for each treatment mean using SAS program (SAS Institute, 1992), and for 0% occurrence, the upper limit of 95% confidence


intervals were calculated using "the Rule of Three"


(Jovanovic & Levy, 1997)


Induction of embryogenic cultures from nucellar explants


Fruitlets


of


0.3- 5


cm length


(without


calices)


were


surface-disinfested


described above.


Each fruitlet was bisected longitudinally and the endo sperm and the


zygotic embryo was removed from each seed.


The nucellus together with the integument


were removed and plated on the surface of plant growth medium with the nucellus in contact with medium.


This experiment involved 4 genotypes.


For 'Thomas'


,there were two explants


from a single ovule in 60


x


15 mm sterile plastic Petri dishes containing induction medium.


There were five different induction media whose compositions were described above (see


Induction


of


embryogenic


cultures


from


zygotic


embryos);


however,


for


the


other


genotypes,


'Hass'


'Lamb'


and 'T3 62', six explants from 3 ovules were explanted in 60


15 mm sterile plastic Petri dishes containing B5 induction medium.


There were total of 36


explants (18 fruitlets) per treatment for 'Thomas',


100 explants (


5


0 fruitlets) for 'Lamb'


412 explant (206 fruitlets) and 36 explant (18 fruitlets) for 'T362'


Percent responses


were calculated based on the number of fruitlets, not on the number of explants used for the experiments

Maintenance of embryogenic cultures on semisolid medium


as


x






58


They were thereafter subcultured at 2-4 week intervals on MSP medium (25 ml per 100 x


20 mm Petri dish).


The proembryonic masses of 0.1-1 mm diameter were used to form


inocula


with 0.2-0


.5


cm diameter.


Up


to 7


inocula were plated


on each Petri dish.


Subculture intervals were 2-4 weeks.


The Petri dishes were sealed with Parafim@.


Effect of medium formulation and gelling agent on the growth and development of


'Thomas'


and


' Isham'


embryogenic cultures


Factorial


experiments


consisted


of three media


formulations and


two


types of


gelling agent.


The medium formulations included MS major salts, B5 major salts without


(N7H42S04


(B5~) and B5~


supplemented with 400 mg V'


glutamine;


the


gelling agents


tested were 8 g V


TC agar and


2


g F' Gel-Gro@ gellan gum (ICN Biochemicals).


major salts formulations were supplemented with MS minor salts, 0.4 mg V1 thiamine HCl,


100 mg V1 myo-inositol, 0.4 [M picloram and 30 g V


sucrose.


The pH of the media was


adjusted to


5.


7-5.8


with either 0.1-1.0 N KOH or HCl prior to addition of gelling agent.


The media were sterilized by autoclaving at 121 0C at 1.1 kg cm


for 15 mn.


Plant growth


media were dispensed in


25


ml aliquots into sterile disposable plastic Petri dishes (100


20 mm).


'Thomas'


the


inoculum


consisted


of


14-day-old


embryogenic


suspension


cultures growing in


80 m1


MSP


medium in 250


ml Erlenmeyer flasks.


Embryogenic


cultures were decanted into a sterile funnel layered with


2


layers of sterile Kimwipes tissue


paper.


Approximately


200-300 mg proembryonic masses were subdivided


to form


inocula of 0.3-0.5 cm diameter flattened on the surface of the growth medium.


'Isham'


the


inoculum


consisted


of


14-day-old


embryogenic


suspension


cultures maintained in 80 ml MSP medium in


2


50 ml Erlenmeyer flasks.


The cultures


4 4 4 - -


The


For


x


For


7






59


surface of the media.


There were seven inocula per Petri dish.


The Petri dishes were


sealed with Paraftim@ and maintained in darkness at 250C.


For 'Thomas'


the percent necrotic tissue and the number of somatic embryos that


developed


per inoculum


four weeks after transfer were


recorded


and


analyzed


using


ANOVA (SAS Institute, 1992).


For


'Isham'


the numbers of 1) proembryonic masses, 2)


globular


somatic


embryos,


3)


hyperhydric


and


opaque


heart


stage


somatic


embryos


(diameter (diameter


<5
5


mm) and 4) hyperhydric and opaque early torpedo stage somatic embryos mm) per inoculum were recorded after four weeks of culture.


Embryogenic Suspension Cultures


General procedures


following procedures


were applicable


for


all


experiments


unless


specified


otherwise.


cm


Medium sterilization was carried out either by autoclaving


at 121*0C at


for 15 min or by millipore filter-sterilization with a 0.2 pm sterile filter.


.1


kg


For filter-


sterilized media, the flasks were sterilized prior to use by autoclaving at 1210 C at 1.1 kg


cm2


for 20 mn.


The volume of liquid medium was either 40 ml in


1


25


ml Erlenmeyer


flask


(referred


to hereafter as 40 ml


medium)


or 8O


ml


in


2


50 ml Erlenmeyer flasks


(referred to hereafter as 80 ml medium).


The pH of all media was adjusted to


5.


7-5.8


with either 0.1-1 N KOH or HCI.


The cultures were sealed with heavy duty aluminum-


foil and secured with Parafilm@, and maintained in semi darkness at


2


5


C on a rotary


shaker at 125 rpm. Initiation


Embryogenic suspension cultures were estab


a.


shed by inoculatinQ 100-300 me of


The






60


Maintenance


Embryogenic suspension cultures were subcultured biweekly into filter-sterilized


80 nml MSP medium.


For cultures that consisted entirely of proembryonic masses without


later


stages


of


development


(PEM


type),


e.g.,


'Esther'


and


'M25864'


0.8-1.0


proembryonic masses was used as the inoculum.


For


cultures up


to 12 months old,


proembryonic masses were sieved


through nylon filtration


fabric (1.8


mm


or 0.8


mesh) and the smallest fractions were used as inocula.


For older cultures (>


12 months),


sieving was unnecessary since the size of proembryonic masses was more homogenous


and smaller than in newly established cultures.


'Thomas'


cultures were also maintained as


described for the PEM type.


newly


established


cultures


that


consisted


of


proembryonic


masses


differentiated as somatic embryos in maintenance medium (SE-type), only proembryonic masses and globular embryos that passed through 0.8 mm mesh nylon filtration fabric were


used as inocula.


With older cultures that contained dedifferentiated proembryonic masses,


0.8-1


g proembryonic masses that


passed


through


either


1.8


or 0.8


mm mesh nylon


filtration fabric were used as the inoculum.


The


morphology


of


embryogenic


cultures


of


different


cultivars


was


observed


during a 1-2 year period.


Growth of 'Esther'


embryogenic suspension cultures


Embryogenic suspension cultures of 'Esther'


were used to determine the growth


pattern


of


embryogenic cultures,


because


this


genotype


proliferated


in


liquid


growth


medium


without concurrent


production


of


early


stage cotyledonary


somatic embryos.


'Esther'


embryogenic suspension cultures (12-14-day-old) maintained in liquid MSP plant


-.1 I * I . I. * . -. *4


g


mm


For


that






61


proembryos were used as the inoculum in 40 ml autoclaved MSP medium. experimental units that consisted of 3 replicates of 8 medium flasks. Each


There were 24


medium flask in


a replicate was inoculated with 420 20 mg proembryonic masses that were randomly


harvested at day 0, 3, 6, 9, 1


2,


15, 20 and


Growth parameters that were recorded included the volume of the precipitated


culture,


fresh


weight and


dry weight.


Precipitated


culture


volume was measured


decanting cultures into sterile 50 ml


graduated centrifuge tubes and allowing them to


settle.


Fresh


weight


was


determined


by


pipetting


out the


liquid


medium


from


centrifuge tubes and transferring the proembryonic masses to preweighed weighing dishes.


Dry weights were obtained by drying the cell masses in an oven at weighing them 30 min after they were removed from the oven. Reg


55*0


C overnight, and


rression analyses were


fitted to the data using Sigma Plot (version


2.


0, JandeV'T' Scientific, San Rafael,


CA).


Effect of picloram on growth of 'M25864' suspension cultures


The effect


of picloram


on the


growth of


'M25864'


in suspension


culture was


examined at six concentrations: 0.


0.3


, 0.41, 1.


25


, 4.14 and 12.48 pM.


The plant growth


medium used for the experiment consisted of MS salts with 0.1 mg U' thiamine HCI,


mg 04


myo-inositol,


3O gU1


sucrose and


picloram as treatment.


Each treatment


replicated four times.


inoculum


for


the


experiment


was


14-day-old


'M25864'


embryogenic


suspension


cultures


that had


been


maintained in


suspension for


ca. one year.


Media


preparation,


preparation of the inoculum and


inoculation,


cultural


conditions and


data


collection


were


as described


for the


growth


response experiment


above.


Regression


analyses were fitted to the data using Sigma Plot (version 2.0, JandeI"M Scientific,


25.


by


the


The


100


was


San






62


Effect of sucrose on the growth of 'M25 864' suspension cultures

Sucrose concentration in liquid medium was examined at six different levels: 20,


40, 50, 60 and 70 g 0i


The plant growth medium consisted of MS salts,


0.4 ptM


picloram,


0.1


mg 1i1


thiamine Hfl,


100 mg 0i


myo-rnositol and sucrose as treatment.


There


were


4


replications


per treatment.


Medium


preparation,


source


of inoculum,


inoculum


preparation,


inoculation,


cultural


conditions,


data


collection


and


statistical


analysis were as described for the picloram experiment. Effect of thiamine HCI on growth of 'M25 864' suspension cultures


The effect of thiamine HCI on the growth of


'M25864'


embryogenic suspensions


was evaluated at six concentrations: 0


,0.4,


1, 4,


10 and 40 mg V1


The plant growth


medium consisted of MS salts, 0.4 p.M picloram,


45 g 0


sucrose,


00 mg F'


myo-inositol


and thiamine HCl.


Each treatment had four replicates.


Media preparation,


source of


inoculum,


inoculum


preparation,


inoculation,


cultural


conditions,


data


collection


statistical analysis were as described for the picloram experiment. Effect of ascorbic acid and medium sterilization on growth of suspension cultures

Ascorbic acid (100 mg F') in the plant growth medium and medium sterilization


approach, i.e., autoclaving or filter sterilization,


were evaluated in a block factorial design


with three replications for two embryogenic lines,


'M25864'


and


'Esther'


The plant


growth medium used for the experiment was MSP formulation (Table A-I).

The source of inoculum, inoculum preparation, inoculation, cultural conditions and


data collection were as described for the picloram experiment.


the data were computed using Proc.


Analysis of variances of


GLM, and t-test was also performed (SAS Institute,


1992).


30,


and






63


Results


Induction of Embryogenic Cultures on Semi-solid Medium


Embryogenic cultures were induced


from avocado


zygotic embryos


18-40


days after


explaning and were associated with zygotic embryos of different developmental stages


from globular (0.10 mm) to early torpedo stage (2.7


mm).


The frequency of somatic


embryogenesis of different genotypes on different medium formulations was generally low,


from 0 to 25% (Table 3


-3


No induction medium effect could be statistically inferred;


however,


there


was


an indication


that


induction


medium


containing


B5


major


salts


stimulated the greatest embryogenic response from the most genotypes.


Induction


of


embryogenic


cultures


generally


occurred


from


the


basal


part


zygotic embryos,


with


the single exception


of


explanted


globular zygotic embryos of


'Esther'


,which were completely embryogenic.


It was not clear whether or not the basal


part of the zygotic embryos included the hypocotyl region since this basal area was very


small. There were two distinct types of embryogenic cultures that were initiated from the explants. 1) Induction of proembryonic masses without heart and later developmental


stages of somatic embryos, i.e., PEM type.


and 'M25864'.


The PEM type response included 'Esther'


2) Induction and development of somatic embryos, including formation of


globular to heart stages from the surface of the zygotic embryo explants (Figure 3-1), i.e.,


SE type.


The SE type response included 'Booth 7'


'Booth 8'.


'T362'.


'Yon'


nucellar-


derived


'Thomas'


,zygotic-derived 'Thomas'


and 'Isham'.


Induction of embryogenuc cultures in nucellar explants occurred primarily from the


mycropylar end of the explants (Figure 3-1


C).


However,


embryogenic cultures were


of











Table


3-3. Percentage of embryogenic culture induction from immature zygotic embryos from several avocE on different induction media.


Cultivar


Zygotic Embryo Length


Rep.


Induction Media'


(mm)


MSP


MSNa


MS~


MSK


Booth 8


Booth Sa Esther


Isham M25864
T362 Thomas Yon


>.1-0
.6-1
.6-3
.8-3
.1-0
.6-0
.4-2
.0-5
.1-0
.1-2
.4-7


4


1
8
0


o (0-27)7 o (0--27) o (0-27) o (0-25) 6 (8-20) o (0-9) 25 (7-80' o (0-30) o (0-25) 22 (7-48' 0 (0-27)


0-27) 0-27) 0-27) 0-25)
0-9) 0-9) 0-60) 0-3 0) 3-41) 0-16) (3-61


)


)


0
0 2
9 0 0 2
1 0
6 ) 2


(0-27) (0-27)
0 (3-61 (3-41) (0-9)
(0-9)
5 (7-80
3 (4-53 (0-25)
(2-27) 0 (3-61


)


(0-27) (0-27) (0-27)
(0.3-4 (0-9)
(0-9) (0-60) (0-30) (0-25)
(2-27) (0-27)


)
)


)


1)


see Materials & Methods for details Number in the parentheses indicated binomial confidence interval at 95 %.


B


0 1 0 1 0 0 0 1 1 4
1







65


Fmire


3-]. Ievclnm-nt nE esnnnru cnmntik amhrvnc Trn


m


nwontir pnimnc ind






















Figure 3-2.


Morphological variations of avocado embryogenic cultures.


- - - -


SC - I


-4


Two distinct


p


type of embryogenesis: PEtM-type that is dominated by eaparneo disorganized proembryonic masses (A, D, G3 and J) SE-type that is dominated by highly organized proembryonic masses and somatic embryos (C, F, I and L), and


cultures that are undergoing morphological changes (B, E, H and K). embryogenic cultures on semisolid medium. Note the friable texture


(A) 'Esther' and granular


nature of the culture.


(D)


The suspension


culture of 'Esther'


(A).


Note


homogenous nature of the inoculum.


(G) Newly established suspension culture of


'Esther'


consisting


of


mostly


proembryonic


masses


and


few


organized


embryogenic masses (arrowhead).


'Esther'


embryogenic suspension without


organized proembryonic masses.
(C) 'Isham' embryogenic culture


This culture has been in culture longer than (G3).


eon semisolid medium.


Note the development of


globular


to heart


stage


somatic


embryos


covering


the


entire


surface


proembryonic masses.


(F) The development of various stages of somatic embryo


development


when


'Isham'


proembryonic


masses


were


transferred


to liquid


medium.


(I)


Another


example


of SE-type


of


culture


('Booth


8')


which


dominated by development of highly organized proembryonic masses or globular


somatic embryo.


(B)


nodular proembryonic proembryonic masses.


Embryogenic culture of 'T362'


masses (lower part)


and


consisting


fine granular


and


of


organized


disorganized


(E) Suspension culture of 'T362'consisted of organized


proembryonic proembryonic disorganization


masses/somatic


masses. at the


(H)
surface


embryos,


A


of


higher


the


fine


granular


magnification


proembryonic


and


of


masses.


disorgamzed


(E).


(K)


Note


the


'Booth


8',


suspension


culture consisting


of


disorganized


cell


clumps


incapable of


somatic


embryogenesis.


the


of


the


is


(H)













67







S
'V






68


of nucellar embryogenic cultures for 'Lamb'


'Hass'


and 'T362'


was also low, i.e. 2%,


0.5% and 6% respectively.

Maintenance of embryogenic cultures on semisolid medium
Transfer of embryogenic cultures (PEM-type) that developed on MSP onto fresh medium of the same composition resulted in continued proliferation of the cultures with occasional


development of heart and later stages of somatic embryos.


Morphologically


the cultures


were


friable


(Figure


3-2


A)


and


either


yellow-honey


('Esther')


or


white-brown


('M25864').


After several subcultures, the proembryonic masses were smaller and paler


and some of the proembryonic masses had become disorganized.


Two to three weeks


after routine transfer


onto


MSP


medium,


the upper part


of the cultures


was


brown;


however, tissue in contact with the medium was healthy and proliferated.


Cultures were


almost completely necrotic after 5-6 weeks.


Transfer


of


somatic


embryos


that


developed


directly


from


explanted


zygotic


embryos (SE-type) to medium of the same composition, e.g., B5, MSK, MSNa, resulted in maturation of the somatic embryos and formation of a few secondary somatic embryos


from the basal part of the somatic embryos.


MSP medium.


They were alternatively subcultured onto


After several transfers onto MSP medium, proliferation of proembryos,


proembryonic


masses


and


globular


somatic


embryos


was


enhanced


(Figure


3-2 B).


Proembryos were characterized by granular morphology and small size (ca. 0.1-0


.2


mm).


The globular somatic embryos were characterized by granular morphology with smooth surfaces and ca. 0.3-1 mm diameter and the formation of secondary somatic embryos at one pole instead of scattered around the entire surface. The proembryonic masses were characterized by their nodular morphology with diameter of Ca. 1-2 mm and the formation






69



that developed in this medium were mostly hyperhydric and their shapes were distorted. Early and late heart stage somatic embryos produced proembryonic masses and secondary


globular somatic embryos from their bases.


Therefore the gross morphology of this type


culture (SE-type)


was dominated


by


the


appearance of proembryonic


masses


hyperhydric somatic embryos at various stages of development.


SE-type


cultures


were


maintained


by


subcultuning


proembryos,


proembryonic


masses and globular somatic embryos.


After ca.


5-


10


subcultures,


depending on


genotype,


the


embryogenic


cultures


also


consisted


of


smaller


cell


masses


with


organization,


and


which


appeared


to be


disorganized


proembryonic


masses.


genotype,


'Isham'


,disorganized proembryonic masses did not develop.


Instead, globular,


heart


and


cotyledonary


stage


somatic


embryos


developed


from


the


entire


surface


proembryonic masses (Figure 3


brown


-2


C).


Distal to the culture medium, the cultures became


2-3 weeks after transfer.


Effect of medium composition and gelling agent on growth and development of nucellus-


derived 'Thomas'


and 'Isham'


embryogenic cultures.


Medium


composition


significantly


affected


the


percentage


of 'Thomas'


cultures


became necrotic after four weeks.


MS formulation resulted in the lowest frequency of


necrosis (Table


3-4).


Type of


gelling


agent


had


no significant


effect


on necrosis of


cultures


nor was


there an


interaction


of


gelling


agent


with


medium


with


respect


necrosis.


Somatic embryo development was not


significantly affected by the different


treatments.


Table


3-5


demonstrated that the proliferation of 'Isham'


proembryonic masses was


significantly affected by media formulation and the interaction of media formulation with


1 1 . - - ~. - - 4.


'T'L... Lt. J ~Mt lAn


of


and


the


less


In


one


of


that


to









Table 3-4.


Effect of major salt composition, and gelling agent on necrosis and somatic


embryogenesis of 'Thomas'


nucellar culture.


Major sal&s Gelling agent Necrotic tissue No. somatic
(%/) embryosb

(Mean+ SE) (Mean+ SE)
MS 8 gl1'TC Agar 17+8 0.28+0.15
2 g F' Gel-Gro 27+16 0.46 +0.28
B5 8 gF'*TC Agar 100 +0 0.74 + 0.21
2 g p' Gel-Gro 100+0 0.51+0.16
B5-G 8 g1-'TC Agar 100 +0 0.57+0.23
2 g F-' Gel-Gro 100+ 0 0.00 + 0.00
Anova Summary
Major salt (M) * NSY
Gelling agent (G) NS NS
M*G NS NS


a Major salt of MS (Murashige & Skoog,


962), E5 (Gamborg et at.


, 1968),


B5-G is B5 major


salts without (NH)2 SO4, with 400 mg


-1


glutanine.


b Number of early stage somatic embryos per inocuturn.


*Significant at a


= 1%; d Not Significant at a


= 1%


Table 3


-5. Summary


of the value of Pr>F


from


ANOVA for the effect of major


composition, and gelling agent on the proliferation of proembryonic masses and


development of somatic embryos from 'Isham


Dependent Variable


Proembryonic mas sCsI Globular SE'


Cotyledonary SE3<5mm


Opaque2 Hyperhydrous2


Total


Cotyledonary SE 5 mm


Opaque2 Hyperhydrous2 Total1


cultured on semisolid medium.


Component


Major Salts
(M)


. . . . . . .
0.0060 0.2620
0.000 1 0.004 1 0.0002
0 .0544
0.000 1 o 00029


Gelling


M*G


Agent (G)


0.1188 0.7634
0.1176 0.0840 0.0001
0.7294 0.000 1 0 0001


0.0037 0.0 10 1
0.0574
0.005 1 0.0400
0.1126 0.000 1 0 0400


70


salt


. . . . . Pr>F


.












Gel Gro-MS


Gel Gro-B5


Gel Gro-B5-G


A


K


+


Tl


F'


*
/
A


t


A


[ ]PEM


Glob SE Small Op SE


Small Hyp SE


Tot Small SE


1111Th Large Op SE E Large Hyp SE W Tot Large SE


Tot SE


TO Agar-MS


TO Agar-B5


TO Agar-B5-G


ng agent-Major salts/glutamine


Figure 3-3.


Effect of gelling agent, major salts and glutamine on the proliferation of proembryonic masse


somatic embryo development of 'Isham' avocado.


6


T.


I


*
.



E
z


4.

2.


6.


Ge


%
2


I






72


Somatic embryo growth and development, as indicated by different sizes, stages


hyperhydricity,


was


prolific with


'Isham'


The development


of


globular


somatic


embryos was not significantly affected by medium and gelling agent but was significantly


affected by their interaction.


Medium with B5~G solidified with Gel-GroTh resulted in the


highest number of globular somatic embryos.


Opaque cotyledonary somatic embryo <


mm diameter development was only affected by medium, with the highest value occumrng


on medium B5~G.


The presence of hyperhydric cotyledonary somatic embryos <


was significantly affected by medium and the interaction of medium with gelling agent, and


the highest number occurred with the treatment of B5 and Gel-Gro'M


The total number


cotyledonary


somatic


embryos


5


mm


was


significantly


affected


by


medium


formulation, gelling agent and their interaction, and the highest number was obtained with


medium consisting of B5~G and Gel-GroTMh


There was no significant effect of treatment


on development of opaque cotyledonary somatic embryos


> 5


mm diameter.


Hyperhydric


cotyledonary


somatic embryos


> 5


mm diameter were significantly


affected


by media


formulation, gelling agent and their interactions,


with the highest number obtained on MS


with Gel-Gro'M


Total number of cotyledonary somatic embryos


affected by media formulation (P>F


> 5


= 0.0002) and gelling agent (P


mm was significantly > F = 0.001) but not


by their interaction (P>F


= 0.04).


Duncan Multiple Range Test (DMRT) indicated that B35


medium


was optimum


for total


somatic


embryos


followed


insignificantly


significantly by B5~G.


T-test indicated that Gel-GroTM resulted in more somatic embryos


than TC agar (P>


T


- 0.0075).


The effect of treatment on number of somatic embryos (of


all stages) was the same as the effect on the total number of large somatic embryos.


Tnitintinnnofltmhrmnnanir Cxncncinn 'uiltiirnc


and


S


of


5


mm


by


MS


and






73



were established successfully on semisolid medium could also be established in liquid MSP medium. Maintenance


The two distinct types of cultures, PEM-type and SE-type, described on semisolid


medium,


retained


these


characteristics


in


newly-established


embryogenic


avocado


suspensions.


In the PEM-types, i.e.,


'Esther'


and 'M25 864'


the suspensions consisted of


less organized proembryonic masses of different sizes with a low frequency of globular


and heart stage somatic embryos (Figure 3-2 D,


G).


In the SE type,


the suspensions


consisted primarily of globular to cotyledonary somatic embryos (Figure


3-2


PEM-type could be maintained by subculturing any fraction of the cultures;


F, L).


The


however,


maintenance of the SE-type required the smallest fraction of cultures (<0.8 mm mesh size)


as inoculum.


Newly established cultures of both types of suspensions were characterized


by production of abundant cell debris.


With


time and


regular


subculture,


the morphology


of


embryogenic


suspension


cultures changed.


Globular, heart and early cotyledonary stage somatic embryos that


occurred at low frequency in the PEM-type, e.g.,


'Esther'


,started to disappear after


months


and


the


cultures


consisted


of increasingly


disorganized


proembryonic


masses


(Figure 3-2 J).


Similar morphological


changes also occurred in


SE-type cultures,


with several


variations.


For example, proembryonic masses or globular somatic embryos derived from


zygotic embryos of 'T362' and 'Lamb' lost their ability to organize as heart stage somatic


embryos,


but


retained


their


morphology


6-18


months


before


becoming


completely


12










developed


from


proembryonic


masses,


but


partially


dedifferentiated,


forming


organized proembryonic masses (nucellar-derived 'Thomas'


, 'ass') or formed partially


disorganized proembryonic masses and large, free vacuolate cells (zygotic embryo-derived


'Thomas'.


'Hass')


before


completely


disorganizing.


Completely


disorganized


proembryonic masses consisted of small isodiametric cells in clusters that were either slow


growing ('Lamb') or fast growing ('T362'


,zygotic embryo-derived


'Thomas') (Figure 3


K).


'Isham'


could not be maintained in suspension.


The proembryonic masses that


were inoculated developed into globular to cotyledonary stage somatic embryos in the


presence of picloram.


Therefore


'Isham'


was maintained by


alternating subculture of


semisolid with liquid media; proembryonic masses from semisolid medium were inoculated


liquid


medium and


globular somatic embryos and


proembryonic


masses in liquid


medium were subcultured onto semisolid medium before they could develop as later stage


somatic embryos.


Unorganized proembryonic masses were never recovered from cultures


of 'Isham'; however, the size of somatic embryos that developed on semisolid or liquid medium decreased with time.

Time required for organized proembryonic masses or globular somatic embryos to become completely disorganized was genotype-dependent (Table 3-6).


Growth responses of 'Esther'


Embryogenic Suspension Cultures


'Esther'


embryogenic suspension culture growth pattern could be


fitted with


curvelinear trend with respect to tissue volume, fresh weight and dry weight.


regression lines had high coefficient of correlation values (r2


The fitted


= 0.96-0.98) (Figure 3-4).


74


less


-2


into


a






75


Table 3-6. Characteristics of avocado embryogenic suspension cultures.


Cultivar of Mother Explant Embryogenesis Time Required for PEM
Tree Type Disorganization

Booth 7 zygotic SE 8-12 month
Booth 8 zygotic SE 8-12 months
Esther zygotic PEM 12-24 months
Hass nucellus SE 6-12 months
Isham zygotic SE never
Lamb nucellus SE 3-6 months
M25864 zygotic PEM 8-12 months
T362 zygotic SE 12-18 months
T362 nucellus SE 6-12 months
Thomas zygotic SE 12-18 months
Thomas nucellus SE 12-18 months
Yon zygotic SE 3-6 months


days of culture.


A trend similar to volume variable was observed for fresh weight, except


that the lag phase was ca 1 day longer and the exponential phase was 1 day shorter.


Dry


weight, however, increased without an apparent lag phase, but with a sharp exponential phase for ca 4 days, followed by a linear phase for 8 days, and progressively decelerating


phase for 6 days and declining thereafter. regression line to occur at different days. weight (20 days) and volume (21 days).


The peak for each variable was predicted by its Dry weight peaked at 18 days, followed by fresh Increases in volume, fresh weight and dry weight


under the growth conditions described were ca 14-fold, 6.4-fold and 7.9-fold, respectively.

During the declining phase, loss of dry weight from the peak to the end of culture









Effect of picloram. sucrose and thiamine HCI on the Rrowth of 'M25864'


embryoge.ic


suspension cultures.


Regression


analysis


of


volume


and


dry


weight


with


respect


to


picloram


concentration resulted in very low r2 values (< 0.05), indicating that picloram had no effect


on those growth variables.


The fresh weight of the cultures decreased curvelinearly (r2-


0.37) with picloram concentration (Figure 3-


5


).


Therefore, the concentration of 0.41


retained


as the


standard


concentration


of


picloram


for


suspension


cultures.


Sucrose affected


the


volume,


fresh


and


dry weight of 'M25 864'


embryogenic


suspension cultures curvelinearly; however, its coefficient of correlation was low (r2


0.34-0.35).


The volume and fresh weight of the embryogenic suspension culture peaked


at a sucrose concentration of Ca.


25


mg F


1, while the dry weight peaked at ca. 50 g F'


The increase in dry weight that resulted from increased sucrose concentration from 20 to


50 gE'i


may indicate increased starch content since it


coincided


with the decrease of


volume and fresh weight of cultures at a sucrose concentration of


Sucrose concentration of 30 g F


2


5


g l


and thereafter.


has been used as standard sucrose concentration in the


medium.


Regression analysis of the effect


of thiamine


HCl


concentration in the medium


demonstrated that r2 for volume, fresh weight and dry weight of the culture were very


low, i. e. 0.16, 0.11 and 0.004, respectively.


This low value indicated that thiamine HCl


was not very


critical for growth.


Nevertheless,


based


on the increasing trend


of the


volume and fresh weight with increasing thiamine HCI concentration from 0 to 10 mg F1


a thiamine HCl


concentration of


4


mg F'


has been adopted as standard for avocado


maintenance medium (MSP).


76


(0.


I -


mg


)


was


piM




























6


E


E


1


0)
4

0)
*
C

C 'A a,
I
Li-


0.25


0


20


0.15 0.10


0.05


0 0 C
0


C 0


S


.1.. ______________________


0 o0
0


r


I

w t Y


a
0 o 0

0


0


S

S
0


0


15 18


212427


Days


in culture


- -~ - ~, A P ~.t1.. - I 1 9 9 1.


77


0)

-c
0)
9
4)



0


0


3


6


9


1


2





















8


3


6


3.4-


3


0


2.8 0.38


0.36

0.34 -


0


0

0


.32

.30

28

0


0.1


3


II


V
V

I
V
w
V
V
V
V

V


II I


0
0


o *


.111


\ .25

0.41


4.14


12.48


Picloram concentration (pM)


78


0)

-c
I
U-


0)


I)
6
0








































































30


40


50


60


Sucrose concentration (g H-)


r4 ~ ~r 1%


r~ r


r, t-~


I-


I


4


4


a


, .


- - - - - -


4


I A... .. 4.-b- . 4 -. - - - - I t - A. L.. ~. - - - ...L L. 'ft Lit A'


79


E
U)
E
0








0)
4-i
-C
0)
U)

-C
U)
U)


0.4


* a

A
A


S
A


0
0


0


*


0)
4
-c
0) is


6
0


0.3


0.2


20


70

























8


E


6


0)
E

0


0)5


4-a
-c
a) ii

-C
(0 a,
IL


4



3


0.52 0.48 0.44


* A

A

A
A a

A.
a




III I



F



V
V yY V
V
V




II I

0
0
0
0
o 0 4


0
0 0
0 00 o 0 0
0



...IIt I


Thiamine HOI


(mg


P .
I (~1 1 VO


i--i


PW~~+ i-a' .k;0~,;.-~ tic


Ian e-L~ r,...ne..w4-L .nnnanna at ~& ,~ - . - .... -


8


0


0)
4-i
-c
0)




0


0


4


10


0.4


1


40


)



















atodae


itar


atodae


0.3 Q.2 0.12 0.04


atodae


filt&r


atodae


filter


IMredteiliin


MvbdumsterIiz2in


Figure 3-8. Effect of medium sterilization protocol on the growth of avocado emhryogemic
suspension cultures. Data from cultures in medium with and without ascorbic acid


were pooled to assess effects of medium sterilization protocol.


Medium was either


81


fits


2


1


i


(B)


(A)


I


(C) (D


__I


.6


Q.4 .2


i
LL


i
S


i
a


(D)









Effect of ascorbic acid and medium sterilization method on growth of suspension cultures


Ascorbic acid in


the medium and


its interaction


with the medium sterilization


method did not significantly affect (P


> 0.05) the growth of either


'Esther'


or 'M25 864'


embryogenic suspension cultures.


Medium sterilization method significantly affected fresh


weight (P


< 0.01) and dry weight (P


< 0.01) of 'Esther'


, and fresh weight (P


< 0.05) and


dry weight (P


< 0.05) of 'M25864'embryogenic suspension cultures.


Data from cultures


in plant growth media with or without ascorbic acid were pooled to determine affects of


medium


sterihzation


protocol.


The


t -test


showed


that


filter


sterilization


resulted


significantly higher fresh weight (P


< 0.01 for 'Esther'


P


< 0.05 for 'M25864)'


and dry


weight (P


< 0.01


for 'Esther'


P


< 0.05 for 'M25864') than autoclaving (Figure 3-8).


Therefore, filter sterilization has been used as the standard protocol for preparing liquid maintenance medium.


Discussion


Somatic embryogenesis


in avocado had been reported previously using immature


zygotic embryos as explants (Skene &


Barlass,


1983


Mooney


&


Van


Staden,


1987:


Pliego-Alfaro & Murashige,


988).


Using a similar method, embryogenic cultures have


been established both on semisolid and in liquid medium from immature zygotic embryos


and from nucellar explants obtained from 12


avocado cultivars/genotypes.


The establishment of avocado embryogenic cultures was characterized by direct formation of proembryonic masses or somatic embryos from the explant without apparent


development of callus.


This embryogenic culture establishment has been referred to as the


proembryogenic


determined


cell


(PEDC)


pathway


(Sharp


et al.,1980)


or permissive


4


in






83


was quite low.


Low frequency of embryogenic culture initiation from immature zygotic


embryos or nucellar explants has been reported for some other woody species, e.g.,


<1%


for big leaf magnolia


(Merkie &


Warson-Pauley,


1993),


<5%


for Cornus


fiorib unda


(Trigiano et


a.,


1989) and


pecan (Corte--Olivares et a.,


1990).


A low


embryogenic


response


was


also


observed


from


the


nucellus


of


monoembryonic


Citrus


spp.,


eg.,


microcitrus (Vardi et at.,


1986) and seedless


'Cohen'


citrange (Grosser


et aL.,


1993).


However, a high embryogenic response (60-80%) was reported from immature zygotic


embryo explants of Quercus robus (Chalupa, 1995).


Moore (198


5


) found that ovules of


monoembryonic


Citrus


spp.


did


not


proiduce


embryogenic


cultures


while


polyembryonic species produced somatic embryos.


Chaturvedi and Mitra (1972) indicated


a correlation


appeared


to exist


between


degree


of


polyembryony


and


somatic


embryogenesis.


The


size of zygotic embryo


explants


was


reported


to be critical


for


initiating


avocado embryogenic cultures (Pliego


Alfaro & Bergh,


1992;


Mooney


&


van Staden,


1987)


however this variable may be


genotype-dependent,


since


embryogenic


cultures


could be induced from zygotic embryos ca.


2 mm length in the current study.


Nucellar


tissues from very young fruit could also be used for initiating embryogenic cultures at low


frequency.


Induction of embryogenic avocado cultures from nucellar explants could have


significant


horticultural


implications.


Somatic


embryogenesis


from


avocado


nucellus


could


replace


the


expensive


etiolation


technique


that


is currently


used


for


clonally


propagating


avocado


rootstock


cultivars


(Frolich


&


Platt,


1971).


Single


character


Improvement


using


elite


scion


genotypes


could


also


be


accomplished


via


genetic


transformation.


that


the






84


a low


concentration


of reduced


inorganic


nitrogen


appears


to favor


somatic


embryo


development and has been used for mango somatic embryo development (Litz et at., 1991;


1995; Mathews & Litz,


1992).


Muralidharan & Mascarenhas (1995) also demonstrated


that Eucalyptus citriodora embryogenic masses proliferated in MS-based medium,


while


somatic embryo development (maturation) increased on B 5-based medium.


Another


characteristic


of


embryogenic


avocado


cultures


was


the


distinction


between the PEM-type (2 embryogenic culture lines) and the SE-type (12


embryogenic


culture lines) that was evident after culture initiation and which was consistent


maintenance on semisolid medium and in liquid medium.


during


This distinction could be an


important marker for predicting somatic embryogenesis frequency since the PEM-type is


associated with lower frequency of development


of somatic embryos (see Chapter


Proliferation of proembryonic masses requires an exogenous auxin and its


4).


withdrawal


results in somatic embryo development (Litz & Gray,


1995).


Therefore,


the different


types


of


avocado


embryogenic


cultures


reflect


differential


sensitivity


to auxin.


The


different types of somatic embryogenesis in avocado may also indicate differences in the integrated development of the somatic proembryos (William & Maheswaran, 1986).


Embryogenic cultures resembling avocado of the PEM-type,


characterized by


. .,


discreet globular or clusters of globular, organized structures, yellow or white, with gross morphology consisting of friable, granular/nodular structures, have also been reported for


Salix vim inalis (Grtanrros,


1995), Rosa hybrida (Robert et al.,


199


5


)cacao (Figuera &


Janick,


1995), apple (Wallin et a.,


1995) and cotton (Finer,


1988).


The histology of an


avocado embryogenic culture has been reported by Mooney


&


Van


Staden (1987) to


consist of somatic proembryos from 0.1-1.0 mm.


The nodular structures were actually






85


when proembryos are maintained in induction medium, they may not organize as somatic embryos but will continue to enlarge and form proembryonic masses that form secondary proembryos on their surface.


Avocado


embryogenic


cultures


can be


maintained


both


in


liquid


suspension


cultures and on solid medium.


The ease of proliferation and maintenance of avocado


embryogenic cultures would provide regenerable materials for further culture manipulation


involving somatic cell genetics or transformation.


Using zygotic-derived


embryogenic


culture of 'Thomas'


Cruz-Hernandez et al.


(1998)


reported Agrobacterium-mediated


transformation of avocado


with GUS


and NPT II


genes and recovery


of transformed


somatic embryos.


Changes


involving gross


morphology


of


embryogenic


cultures


of


the


SE-type


occurred after a relatively short time and can be implicated in the progressive loss of


embryogenic potential of these cultures.


William and Maheswaran (1986) suggested that


the loss of integrated organization of globular embryos is associated with appearance of


proembryonic masses in embryogenic cultures.


The morphology of disorganized avocado


proembryonic


masses


is


similar


to


embryogenic


cultures


that


have


become


"undifferentiated''


and which have been selected from


the most friable nucellar Citrus


deliciosa cultures (Cabasson et al.,


1995) and from habituated nucellar cultures of Citrus


aurantium L. (Gavish et a!., 1991) even though these cultures could be induced to form


somatic embryos.


Chaturvedi and Mitra (1975) found that after prolonged subculture the


organized globular embryogenic culture initiated from stems of Citrus grandis became highly friable, but embryogenic.


Embryogenic


avocado


suspension


cultures


of


the


PEM-type


grow


as typical




Full Text

PAGE 1

DEVELOPMENT OF PROTOCOLS FOR AVOCADO TISSUE CULTURE: SOMATIC EMBRYOGENESIS, PROTOPLAST CULTURE, SHOOT PROLIFERATION AND PROTOPLAST FUSION By WITJAKSONO . . A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1997

PAGE 2

Copyright 1997 Witjaksono

PAGE 3

0 To my wife Nia, for her tremendous sacrifice during these long years of study, and to my sons, Lintang and Edgar, for my not being there during their early days in this world. I

PAGE 4

ACKNOWLEDGMENTS I wish to express my deepest gratitude to Dr. Richard E. Litz, my academic advisor, for all his encouragement, advice, guidance, help and patience during my entire doctoral training and especially during the preparation of this dissertation. His interest and his stimulating discussions involving many topics will always be remembered. I would like to extend my sincere gratefulness to the members of my dissertation committee, Drs. Jude W. Grosser, Dennis J. Gray, Michael E. Kane and Randy C. Ploetz, for their help, guidance and encouragement during the course of this study and their constructive criticism in the writing of this dissertation. A million thanks are extended to Mr. Gray Martin, formerly of the University of California, Riverside, for his help in supplying avocado fruitlets for this dissertation research and also for his encouragement. Sincere thanks are also extended to Dr. John Menge and Brandon McKee (UC Riverside) for providing Persea seeds and to Dr. Fernando Phego-Alfaro of the Universidad de Malaga, Malaga, Spain, for his initial involvement in the birth of this dissertation topic and for providing avocado shoot cultures for this work. The invaluable assistance of the thoughtful Ms. Pamela Moon and her help with statistical analyses, graphics, computers and other matters is gratefully acknowledged. Help with statistical analysis and in photosynthesis measurements provided by Dr. Bruce SchafiFer and Mr. Angel Coll is highly valued. I also would like to thank Dr. Ray Schnell, Cecile Olano, Wilber Quitanilla and Wilhelmina Wasik from the USDA-ARS, Miami, for providing facilities to work in their lab and their help with RAPD analysis. Sincere appreciation also goes to other graduate students in the lab — Jay, MaryJoy and Andres — for sharing their knowledge and insights, and to visiting scientists Drs. iv

PAGE 5

Khalid bin Mohamad Zin, Levi Barros and Lad, and to Arlene, Fahad, Carmen, Hilmig and Isabel for making the lab and the neighborhood more colorful. Invaluable help from Divina for picking up books and other literature from Gainesville is recognized. The help of other stafiF and faculty members at TREC is acknowledged. The opportunity to pursue this doctoral degree was granted by the management of my oflBce, the Research and Development Center for Biology, The Indonesian Institutes of Sciences (LIPI), and I am honored to acknowledge a scholarship awarded by my country, the Republic of Indonesia, administered by the Agency for the Assessment and Application of Technology (BPP Teknologi). Last but not least, a research assistantship and research support from the California Avocado Society have been critical for the completion of this dissertation and therefore are deeply appreciated.

PAGE 6

TABLE OF CONTENTS ACKNOWLEDGMENTS iv LIST OF TABLES viu LIST OF FIGURES x ABSTRACT xii CHAPTERS 1. INTRODUCTION 1 2. LITERATURE REVIEW 4 Avocado 4 Somatic Embryogenesis 28 Protoplast Isolation, Culture and Regeneration 36 Somatic Hybridization 48 3 . INTTIATION AND MAINTENANCE OF AVOCADO EMBRYOGENIC CULTURES 54 Introduction 54 Materials and Methods 55 Results 63 Discussion 82 4. SOMATIC EMBRYO DEVELOPMENT, MATURATION AND GERMINATION 88 Introduction 88 Materials and Methods 89 Results 93 Discussion 108 vi

PAGE 7

5. PROTOPLAST ISOLATION, CULTURE AND SOMATIC EMBRYO REGENERATION OF AVOCADO 114 Introduction 1 14 Materials and Methods 117 Results 123 Discussion 145 6. AVOCADO SHOOT CULTURE AND PLANTLET DEVELOPMENT AND NET PHOTOSYNTHESIS JN A NONELEVATED AND ELEVATED CO2 ENVIRONMENT 149 Introduction 149 Materials and Methods 151 Results 157 Discussion 169 7. PROTOPLAST FUSION BETWEEN AVOCADO AND ITS • RELATIVES, INCLUDING NECTANDRA CORIACEA AND PERSEASP? 174 Introduction 174 Materials and Methods 175 Results 181 Discussion 188 8. SUMMARY AND CONCLUSION 191 Summary 191 Conclusion 194 APPENDIX 196 LIST OF REFERENCES 203 BIOGRAPHICAL SKETCH 228 vii

PAGE 8

LIST OF TABLES Table Eage 2-1 . Nutrient composition of edible pulp of 'Fuerte' avocado per 1 00 g 5 2-2. Comparison of the three horticultural races of Persea americana. 7 2-3. Average avocado production 1992-1996 11 2-4. Avocado breeding objectives for special characters 12 2-5 . List of Persea and related genera and their resistance to Phytophthora rootrot 14 2-6. Summary of in vitro studies of avocado, Persea americana Mill., and related species in the family of Lauraceae 17 27. Enzymes used for protoplast isolation 38 31. Avocado cultivars used for the experiments, their botanical varieties and their sources 56 3-2. Length of 'Thomas' avocado fruitlet (measured without calyx) in relation to zygotic embryo length, stage of development and morphology 56 3-3. Percentage of embryogenic culture induction from immature zygotic embryos from several avocado cultivars on different initiation media 64 3-4. Effect of major salt composition and gelling agent on necrosis and somatic • embryogenesis of 'Thomas' nucellar culture 70 3-5. Summary of the value of Pr>F from analysis of variances for the effect of major salt composition, and gelling agent on the proliferation of proembryonic masses and development of somatic embryos from 'Isham' cultured on semisolid medium 70 3-6. Characteristics of avocado embryogenic cultures in liquid medium 75 viii.

PAGE 9

4-1. ANOVA of the effect of Isham' embryogenic suspension culture-derived somatic embryo size and stage of development on development of secondary somatic embryos on semisolid medium 96 4-2. ANOVA of effect of Gel-Gro™ concentration on development of opaque and hyperhydric somatic embryos and size of opaque somatic embryos from 'T362' proembryonic masses cultured on semisolid medium 98 4-3. ANOVA of the effect of sucrose concentration and the size of proembryonic masses on production of opaque somatic embryos of various stage of development 101 4-4. The effect of total N and % NO3" on color and morphology of avocado 'T362' suspension cultures 105 45. ANOVA for the effect of nitrogen concentration and % NO3" on proembryonic mass fresh weight gain and medium pH in liquid medium 106 51. Protoplast yield per gram fresh weight of avocado embryogenic suspension cultures from several avocado cultivars 124 5-2. ANOVA of the effect of medium osmolarity and protoplast density on the plating eflBciency, length and width of microcalli derived from 'T362' avocado protoplasts cultured in agarose disc type method, three weeks after culture 125 5-3. ANOVA of the effect of nitrogen sources, medium osmolarity and protoplast density on number of microcalli and proembryonic masses from avocado 'T3 62 'protoplasts, one month after culture 132 5-4. ANOVA of the effect of medium osmolarity, nitrogen source and plating density on the percentages of microcalli and somatic embryo development from avocado 'T362' protoplasts, one month after culture 133 5-5. Percentage of protoplasts that divided on day 1, 5, 8 and 12 after culture 135 5-6. ANOVA of the effect of subculture age and dilution rate on culture fresh weight and number of somatic embryos that developed from nucellusderived 'T362' protoplasts, one month after culture 139 5-7. Relationship among genotypes, their embryogenic characteristics, protoplast yield and response after one month in liquid medium 0.4 M MS" 8P, at a plating density of 0.8-1.2 x lO' protoplast ml ' 142 ix

PAGE 10

6-1. The efifect of N03":NH4^ ratio at 20 mM on the growth of 'Guaram 13' avocado shoot cultures maintained in an incubator, eight weeks after culture 160 6-2. Effect of medium NOs iNHi^ ratio at 20 ^im on the growth variables and net photosynthesis of avocado shoot cultures after ten weeks in culture 162 6-3. Effect of atmospheric CO2 environment on the growth variables and net photosynthesis of avocado shoot cultures after ten weeks in culture 163 6-4. The effect of atmospheric CO2 concentration on the growth variable and net photosynthetic rate of avocado shoot cultures after ten weeks in culture 165 6-5. Net photosynthesis (|amol CO2 g"^ s"^) of intact avocado proliferating shoot cultures, their subcultured microcuttings and subtended callus in two atmospheric CO2 concentrations, and intact plantlets and plantlet-derived shoots in an elevated CO2 concentration before and after cutting 168 66. 'Guaram 13' avocado plantlet development in two atmospheric CO2 environments, nine weeks after culture 169 71. Leaf mesophyll protoplast yield from in vitro seedlings of Nectcmdra and Per sea sp 182 7-2. The growth and development of protoplasts after fiision and subculture of the microcalli/proembryonic masses following subculture in fresh medium 187 A-1 . Avocado tissue culture media 197 A-2. Avocado protoplast culture medium MS 8P 199 A-3. Stock solutions of 8P organic addenda (Kao & Michayluk,1975) as modified by Grosser and Gmitter (1990) and their final concentration in protoplast culture medium 200 A-4. Enzyme Solution for Protoplast Isolation 201 A-5. CPW salts Stock Solutions and Their Final Concentration in the Solution 201 A-6. Amount of CPW Stock Solution and Osmoticum per 100 ml Gradient Centrifiigation Solution 202 A-7. Protoplast Fusion Solution 202 X

PAGE 11

LIST OF FIGURES Figure page 3-1. Development of secondary somatic embryos from zygotic embryos and of proembryonic masses from nucellar explants of 'Thomas' avocado 65 3-2. Morphological variations of avocado embryogenic cultures 66 3-3. Effect of gelling agent, major salts and glutamine on the proliferation of proembryonic masses and somatic embryo development of 'Isham' avocado 71 3-4. Growth response of 'Esther' avocado embryogenic suspension cultures over time (T) 77 3-5. Effect of picloram (P) on growth response of 'M25864' avocado embryogenic suspension cultures after 14 days 78 3-6. Effect of sucrose (S) on the growth response of 'M25864' avocado embryogenic suspension cultures after 14 days 79 3-7. Effect of thiamine HCl (T) on the growth response of 'M25864' avocado embryogenic suspension culture after 14 days 80 38. Effect of medium sterilization protocol on the growth of avocado embryogenic suspension cultures 81 41. Avocado somatic embryo development from proembryonic masses and subsequent plant regeneration 94 4-2. Opaque cotyledonary stage somatic embryo production as affected by inoculum type derived from 'Isham' avocado liquid embryogenic cultures after one month on semisolid medium 96 4-3. Effect of Gel-Gro™ concentration on development of somatic embryos from proembryonic masses on semisolid medium after one month of culture 99 4-4. Effect of sucrose concentration on the production of opaque somatic embryos on semisolid medium 101 xi

PAGE 12

4-5. Somatic embryo development as afifected by high sucrose concentration 102 4-6. Effect of carbon source on fresh weight and volume of embryogenic nucellar 'Thomas' avocado cultures 104 47. Effect of total nitrogen concentration and 'NOi/NH/ ratio on culture fresh weight gain (x 100%) and medium pH 107 51. Viability of protoplasts isolated from embryogenic cultures derived from zygotic embryos of different avocado genotypes 124 5-2. Effect of medium osmolarity and protoplast plating density on plating efficiency of 'T362' avocado protoplasts cultured in agarose disc type method after three weeks 126 5-3. Effect of medium osmolarity and protoplast plating density on length of microcalli that developed from 'T362' avocado protoplasts cultured in agarose disc type method after three weeks 126 5-4. The growth and differentiation of embryogenic culture-derived protoplasts of zygotic-derived 'T362' avocado cultured in agarose medium with medium osmolarity of 0.4 M as affected by plating density 128 5-5. A culture dish containing organized proembryonic masses and somatic embryos that differentiated from agarose-embedded protoplasts 130 5-6. Effect of nitrogen source, medium osmolarity and protoplast plating density on the number of microcalli and proembryonic masses that developed from 'T362' avocado protoplasts after one month in culture 134 5-7. Effect of nitrogen source, medium osmolarity and protoplast plating density on the percentage of microcalli and proembryonic masses that developed from 'T362' avocado protoplasts after one month in culture 134 5-8. Somatic embryogenesis from avocado protoplasts cultured in liquid medium 137 5-9. Effect of subculture age and dilution rate in medium of low osmolarity (0.15 M MS'8P) on the formation of large (> 2 mm diameter) somatic embryos 140 5-10. Effect of subculture age and dilution rate in medium of low osmolarity (0.15 M MS'8P) on fresh weight accumulation of protoplast-derived proembryos after one month of culture 140

PAGE 13

,1^ 511. Somatic embryo development from protoplasts derived cultures as aflfected by culture method 143 61. In vitro shoot growth of 'Guaram 13' avocado in response to varying concentrations of KNO3 (N) as the sole inorganic nitrogen source in the medium 158 6-2. In vitro leaf growth of 'Guaram 13' avocado shoots in response to varying concentrations of KNO3 (N) as the sole inorganic nitrogen source in the medium 159 6-3. In vitro growth of 'Guaram 13' avocado shoots in response to varying concentrations of nitrogen (N) in the form of 3 NOs' : 1 NHt^ 161 6-4. Dry matter content of avocado shoots in vitro in response to varying concentration of total nitrogen (N) in the form of 3 NO3" : 1 NH4'^, after 10 weeks in culture 164 6-5. Shoot proliferation of 'Guaram 13' avocado on media of different N content under elevated and ambient CO2 concentration, after ten weeks 166 66. Plantlets of 'Guaram 13' avocado grown under elevated and ambient CO2 concentration, after nine weeks 167 71. Protoplast fiision between zygotic embryo-derived 'T362' avocado and Nectandra coriacea and subsequent somatic embryo and plantlet regeneration and its shoot proliferation 183 7-2. RAPD banding patterns of leaf from a somatic embryo that developed from protoplasts fiision between embryogenic cultures of a zygotic-derived avocado line 'T362' and Nectandra coriacea, and its parental sources 185 7-3. RAPD banding patterns of proembryonic masses of avocados and their fiision with other avocado and P. pachypoda 186 xiii

PAGE 14

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfilbnent of the Requirements for the Degree of Doctor of Philosophy DEVELOPMENT OF PROTOCOLS FOR AVOCADO TISSUE CULTURE: SOMATIC EMBRYOGENESIS, PROTOPLAST CULTURE, SHOOT PROLIFERATION AND PROTOPLAST FUSION By Witjaksono December, 1997 Chairman: Richard E. Litz > Major Department: Horticultural Science . -if : • • Avocado, Persea americana Mill., is an important fruit crop and is cultivated in tropical and subtropical regions. Despite its importance, commercial production has mostly depended on only a few rootstock and scion cultivars. Avocado improvement by conventional breeding has been slow due to a long juvenile period, low fruit set, genetic heterogeneity, lack of genetic information regarding horticultural traits and inefficiency of breeding techniques. Biotechnology, including somatic cell genetics and genetic transformation, has great potential for improving perennial fruit species, including avocado. The use of biotechnology for improving avocado is dependent on in vitro protocols, including efficient somatic embryogenesis, protoplast isolation and subsequent regeneration of plants from protoplasts, genetic transformation and shoot proliferation for propagating unique regenerants. This study was undertaken to develop the cell culture system for improving avocado. xiv

PAGE 15

Embryogenic cultures have been induced from several avocado genotypes and elite cultivars. Conditions for maintenance of embryogenic cultures have been determined, and efficient somatic embryo development from embryogenic cultures has been described. Protoplasts have been isolated from embryogenic cultures, and somatic embryo development from protoplast-derived cultures has been obtained. Although plant recovery from somatic embryos has been achieved, the efficiency of conversion, or germination has been low. Interspecific protoplast fusion between embryogenic avocado cultures and leaf mesophyll protoplasts of Nectandra coriacea (Sw.) Griseb. , P. borbonia (L.) Spreng. and P. pachypoda has been attempted in an effort to produce interspecific somatic hybrids v^th resistance to phytophthora root rot caused by Phytophthora cinnamomi Rands; however, putative hybrid plants of the former and embryogenic cultures of the latter could not be confirmed by RAPD analysis. The somatic hybridization experiments were limited by the availability of protoplasts from the non-avocado parents. Whether or not protoplast fiision is a viable method for overcoming sexual incompatibility between avocado and its wild relatives remains unresolved. The in vitro protocols for avocado that have been developed have significant implications for avocado improvement using biotechnology. A research collaboration has already demonstrated the feasibility of genetic transformation of embryogenic cultures of avocado. XV

PAGE 16

CHAPTER 1 INTRODUCTION Avocado, Persea cmericana Mill., is one of the most important fruit crops of the world. It has been consumed to some extent as a replacement for meat by native peoples of tropical America since antiquity (Popenoe, 1927). The avocado fruit has a high nutritional value and energy content and is a source of antioxidants, fruit protein and soluble fiber (Bergh, 1992 b). The high energy content of the avocado fruit is due to its high "good" fat content, which ranges from 3 to 30% fresh weight, depending on the cultivar. Avocado fat is 82% monounsaturated, of which 95% is oleic acid; 8% is polyunsaturated and 10% is saturated fat (Bergh, 1992b). Avocado oil is used as a cosmetic (Purseglove, 1968; Bergh, 1992a, b). World avocado production in the last 5 years has averaged ca. 2 million MT (FAO, 1997). Among other fiiiits, world production of avocado ranks 10th after Mw^a (banana and plantain), citrus, grape, apple, mango, pear, plum, peach and papaya (FAO, 1992). In the USA alone, avocado's contribution to the economy during 1989-1992 was ca. $211 million annually. Among fruit crops, this is the sixth after citrus, grapes, apples, peaches and pears (Anonymous, 1992). Despite its nutritional and economic importance, avocado genetics are not well understood. This is in large part due to typical problems of breeding perennial species, e.g., low fiiiit set, high heterogeneity and a long juvenility period. These difficulties have impeded conventional breeding for addressing problems faced by the avocado industry (Pliego-Alfaro & Bergh, 1992). Consequently, the avocado industry worldwide has mostly relied on a few scion cultivars such as 'Fuerte' and 'Hass' and a few rootstock cultivars such as 'Duke 7', 'Thomas', and 'Barr Duke', all of which originated from

PAGE 17

2 chance seedlings (Bergh, 1976). The major breeding objectives for avocado involve development of better quality fruit and scion shoot, and rootstock (Bergh, 1975, 1976; Bergh & Lahav, 1996). Development of rootstocks that are tolerant of root rot disease caused by Phytophthora cinnamomi (PRR), for example, is urgently needed. Phytophthora root rot is the largest production problem around the world (Gustafson, 1976). In the USA alone, the avocado industry has been losing ca. 200 ha annually due to PRR. Over 60% of avocado groves in the USA are affected by PRR, and losses exceed $30 miUion each year (Coffey, 1987). Biotechnology offers some novel approaches for generating variability for plant improvement. These have special relevance for perennial fruit species, because existing superior cultivars could be altered for one or more specific traits. Using tissue culture techniques, somaclonal variants with resistance to bacterial leaf spot caused by Xanthomonas campestris pv. Pruni (E.F. Sm.) Dows have been produced in peach (Hammerschlag, 1992). Genetic variability that would otherwise be unavailable using conventional breeding approaches can also be exploited by somatic hybridization involving distantly related and sexually incompatible species, e.g., citrus (Grosser & Gmitter, 1990; Gmitter et al., 1992). Tetraploid plants produced by somatic hybridization can be used as parents for triploid scion breeding (Grosser & Gmitter, 1990). Gene transfer by genetic recombination can also generate variability by bridging the sexual barriers between species, phyla and kingdoms. Genes from bacteria, e.g., the Bt gene for the endotoxin from Bacillus thuringiensis (Williams et al., 1992) and the VHp gene for hemoglobin from obligate aerobic, Gram negative Vitreocilla (Holmberg et al.,1997); from viruses, e.g., CP genes for the viral coat protein and viral nonstructural genes (Beachy et al., 1990); and from humans, e.g., 2-5 Aase (2-5 oligoadenylate synthetase) and RNase L (ribonuclease L) cDNA induced by interferon in human (Ogawa et al., 1996) can be transferred into and expressed in plants.

PAGE 18

Cloning horticulturally important genes, i.e., ripening genes and transformation of plants with these genes in antisense (e.g., tomato) has marked the development of new approaches for addressing post-harvest problems. In fruit trees, stable integration and expression of foreign genes, nos and npt n genes, and their inheritance in Mendelian fashion have been demonstrated recently in apple (James et al., 1996). This breakthrough suggests that this technology might have direct application for single gene improvement of elite clones, e.g., for pest and disease resistance and for altered ripening for quality and shelf life improvement. The potential for biotechnology can only be realized after development of eflBcient protocols for plant regeneration from cell and tissue cultures. With respect to avocado, only preliminary studies have been published, which were reviewed by PHego-Alfaro & Bergh (1992). These studies were undertaken in order to develop protocols for avocado somatic cell genetics and included: 1 . initiation and maintenance of embryogenic cultures in semisolid and liquid medium; 2. somatic embryo maturation and germination; 3. protoplast isolation, culture and regeneration from embryogenic cultures; 4. shoot proliferation from juvenile materials; 5. protoplast flision between avocado and Nectandra coriacea and small-seeded Persea species.

PAGE 19

CHAPTER2 LITERATURE REVIEW Avocado Use and Importance Avocado is a fruit that is usually consumed fresh as a salad, dessert or garnish. Unlike other fixiits, the avocado does not provide refreshing satisfaction upon consumption, but it gives the fullness sensation of a staple food. Avocado pulp is smooth and nutty, neither sweet nor acid, and of a bland nature (Bergh & Lahav, 1996). Superior cultivars have a nutty or anise-like flavor. Avocado has been an important food in Mexico and Central America since antiquity, where it takes the place of meat in the diet of Central Americans (Popenoe, 1927). Avocados are appetizing, nourishing, cheap and available throughout most of the year (Popenoe, 1927). According to Purseglove (1968), avocado is the most nutritious of all fiixit. Analysis of the nutrient composition of avocado (Table 2-1) indicates that the fat content is very high and can be ca. 30%. Avocado fat is 82% monounsaturated, of which 95% is oleic acid, 8% is polyunsaturated and 10% is saturated fat (Bergh, 1992b), and it therefore shares some of the benefits of olive oil. Colquhoun (1990) demonstrated that inclusion of 15-20% avocado in low fat diets lowered blood cholesterol and preserved levels of high density lipoprotein (HDL), which is good for the heart, whereas a low fat diet without avocado lowered both cholesterol and HDL levels. Avocado also has a very high density nutritional value, high protein level, vitamins and potassium. The health benefits of avocado consumption have been discussed by Bergh (1992a; b). 4

PAGE 20

5 Table 2-1. Nutrient composition of edible pulp of 'Fuerte' avocado per 100 g Component Amount Vitamins (mg or unless stated otherwise) A Water 74.0 g Energy component Protein 2.2 g Lipid 170 g Carbohydrate 6.0 g Fiber 15g 290 1.U. C , 14.00 mg Thiamine . 0.11 mg Riboflavin 0.20 mg Niacin 1.60 mg Mineral (mg) , Calcium 10.0 mg Phosphorus 42.0 mg Iron 0.60 mg Sodium 4.0 mg Potassium 604.0 mg Source: Scora and Wolstenholme (1998) (in press). Taxonomy Avocado, Persea americana (2n = 2x = 24), is a member of the Lauraceae, which is mostly comprised of tree species, except for the plant parasite Cassytha flliformis. Avocado is the only economically important food species in this family. Other economically important species are used as spices, e.g., Cinnamomum zeylanicum Blume and C. cassia (Nees) Nees and Eberm. Ex Blume; as medicinal plants, such as C. camphora (L.) J. Presl.; as timber, e. g., Nectandra Roland ex Rottb, Ocotea Aubl and Phoebe; and as ornamentals, i.e., Persea indica Spreng (Schroeder, 1995).

PAGE 21

Kopp (1966) divided the genus Persea into 2 subgenera, Persea and Eriodaphne, based on the morphology of reproductive structures of herbarium materials. The genus Persea includes a small number of species characterized by large fruits, while the subgenus Eriodaphne consists of a large number of species, most of which have small fruits. This distinct demarcation of the two groups has been fiirther confrrmed by grafting and hybridization studies. The members within each subgenus are graft and sexually compatible with each other, but are incompatible v^th members of the other subgenus (Bergh & EUstrand, 1986; Bergh & Lahav, 1996). The members of subgenus Eriodaphne are mostly resistant to Phytophthora root-rot, while members of subgenus Persea are susceptible (Zentmeyer, 1980) The subgenus Persea includes the commercial avocado, which has a thick edible pulp, and other closely related species, which have thm pulp, e.g., P. schiedeana Nees, P. primatogena Williams and Molina, P. parviflora Williams and P. zentmyeri Scieber and Bergh. The commercial avocado has been long recognized to have three horticultural races that are adapted to three different climatic conditions, i.e., the Mexican race, which is adapted to high elevation in the tropics, the Guatemalan race which is adapted to medium elevations in the tropics, and the West Indian race which is adapted to low tropical elevations (Popenoe, 1941). Popenoe (1941) developed systematic keys to distinguish the three races and referred to the Mexican race as Persea drymifolia, the Guatemalan race as P. guatemalensis and the West Indian race as P. americana. The races also differ in many horticultural traits (Table 2-2). The classification of the horticultural races of commercial avocado and other Persea species is still in dispute. Kopp (1966) recognized only 4 species, P. schiedeana, P. steyemarkii, P. floccasa and P. americana, the latter of which consisted of var. drymifolia (Mexican race), var. americana (West Indian race) and var nubigena, while the Guatemalan race was not discussed. William (1966; 1967), using morphological data and the fossil record, proposed that the commercial avocado consisted of two species, each

PAGE 22

Table 2-2. Comparison of the three horticultural races of Persea americana. Characteristic Race Mexican Guatemalan West Indian General Native Region Mexican highlands Guatemalan Tropical lowlands highlands Climatic adaptation subtropical subtropical tropical Cold tolerance most intermediate least Salinity tolerance least intermediate most Iron chlorosis tolerance intermediate least most ' ' A If Afnof A rv^OfincT /VlLCriUtlC UCoIUlg more less Form Intemode longest long J V ^ shortest Twig lenticels pronounced absent Rark roiiphnes^ less less more less less Leaf Size smallest large largest Color preen green pale green Flush color greenest reddest yellowish-green Anise present (usually) absent absent Underside waxiness more less less Qaqcoti early late intermediate Bloom to maturity 5—7 months 10-18 months 6-8 months Perianth persistence greater less less Fruit Stem Length Short long short Thickness medium thick tliin Shape cylindrical conical nailhead Fruit Size tiny-medium small-large medium-very large Shape mostly elongated mostly round variable Fruit Skin Color usually purple black or green pale green-maroon Surface waxy coating variable rough shiny Thickness very thin thick medimn OlUllC cells aUaClll Pliability membranous stiff leathery Peeling no variable yes Seed Size ratio large often small large Coats thin usually thin thick Tightness in cavity often loose tight often loose Surface smooth smooth , rough Pulp Flavor anise-like, spicy often rich sweet, mild Oil content highest high low Distinct fibers common less common intermediate Cold Tolerance more more less storage Source: Bergh &. Lahav (1996), Bergh (1975)

PAGE 23

with two varieties, the native species and its progenitor. The Mexican avocado, P. americam var. drymifolia, is the progenitor of the West Indian race, P. americana var. canericana), while the Guatemalan avocado (P. americana var. guatemalensis) is the progenitor of P. nubigena var. nuhigena. The most recent classification has been suggested by Scora and Bergh (1990) based on new data, including isozymes, leaf terpenes, morphology, physiology, field observations and molecular markers as follows: Persea americanayfiW. vzx. americana MilVl. ' ' ^ * , var. d>>7n//o//a (Select. & Cham.) Blake ^. var. ^ate/wa/e/i«5 Williams ^ v > var. nubigena (Williams) Kopp var. steyermarkii Allen war. Jloccasa Mez ' Persea zentmeyeri Scieber & Bergh "Aguacate de Montana" "Aguacate de mico" Persea parviflora Williams, "Aguacate cimarron" Persea pn/wa/ogena Williams & Molina 'Guaslipe" f "Aguacate de anis" Persea schiedeana'NQes In this new classification, the horticultural races are given botanical varietal status since the difference between them is not far enough (based on isozymes) to give the races species standing but is too far to be considered forms (Bergh & Ellstrand, 1986). The three horticultural races were also as different fi"om one to another as to other varieties ' (Scora & Bergh, 1990). Origin. Domestication and Distribution ' , Origin : ' " Avocado may have originated in the Chiapas-Guatemala-Honduras region where wild avocado is still found (Kopp, 1966); however, the three horticultural races of

PAGE 24

9 avocado may have evolved in different climatic conditions in geographical isolation from one another (Storey et aL, 1986). The Mexican race is thought to have originated in the highlands of south-central Mexico, since primitive forms of Mexican avocado are found in that area (Storey et al., 1986). The Guatemalan race was believed to have originated in the interior valley of the highlands of Guatemala, north of Guatemala City. The West Indian race did not originate in the West Indies, since there is no record of the avocado from early explorations of the West Indies (Storey et al., 1986). Instead, it may have developed in the Pacific lowlands of Central America (ca. 82°-92° west longitude [Storey et aL, 1986]). William (1976; 1977) argued that the West Indian race evolved from the Mexican race and probably became adapted to a warmer climate in northern Central America. This argument was based on the morphological similarity of the two races and the archeological remains of avocado in Peru that date from ca. 1500 BC. Domestication Avocado has been domesticated in Mexico since time immemorial (Popenoe, 1927). The word avocado is derived from the Spanish word ahuacate or aguacate, which is a corruption of the Aztec ahuacatl which is still used in parts of Mexico. The word pahua, from the Aztec pauatl meaning fiuit, is used with reference to the West Indian and Guatemalan races of avocado in certain parts of Mexico. The word palta (in Quechuan) is used with reference to avocado in western South America. Archeological remains of avocado seeds as old as 7000 BC have been recovered from caves of the Tehuacan area in Mexico. Other avocado seeds with younger carbon dating ranging from 6600 BC, 4000 BC, 3200 BC, 500 BC, 300 BC to 300-1500 AD have also been recovered (Smith, 1966; 1969). Interestingly, seed size appears to have increased over time, indicating selection for larger fruit (Smith, 1966). Avocado seeds excavated from 2 sites in the Moche Valley (Peru) have been carbon dated at 2000-1500 BC and 1500-1200 BC (Williams, 1976).

PAGE 25

Distribution When the Spanish arrived in the Americas, the avocado was being cuhivated from Mexico to northern Peru (Hodgson, 1950; Storey et al., 1986). The Spanish conquistadors brought the avocado to Venezuela, the West Indies, and the Canary Islands (Bergh & Lahav, 1996). Eventually, avocado was cultivated in all tropical and subtropical regions. The avocado reached Spain in 1600, and was established on the east coast of Africa in Ghana in 1750 (Smith et al., 1992). The avocado was introduced to Singapore ca. 1830 and to the Philippines ca. 1890 (Burkill, 1935). Avocado was brought from Mexico to Florida in 1833 and to California in 1848 (Gustafson, 1976). Production Worid avocado production in the last 5 years has averaged ca. 2 million MT (FAO, 1997) and ranks 10th after Mw^a (banana and plantain), citrus, grape, apple, mango, pear, plum, peach and papaya (FAO, 1992). The major production areas are Mexico, USA, Brazil, Dominican Republic and Indonesia, respectively (Table 2-3). Other countries that are not leading producers but which export significant amounts of avocado include Australia, South Africa and Israel. Avocado Breeding and Advances Avocado breeding objectives have been directed toward improvement of scion (fiaiit and tree) quality and rootstock quality as summarized in Table 2.4 (Bergh, 1975; Bergh, 1976; Bergh & Lahav, 1996). According to Bergh & Lahav (1996), most of the morphological variability in avocado is muhigenic and only one dwarfing character from P. schiedeana is controlled by a single gene. To combine desirable characters of different cultivars or to recover intermediate traits of two extreme phenotypes can be achieved efficiently by crossing the

PAGE 26

Table 2-3. Average avocado production 1992-1996. Region Country Production (tons) North and Central America Mexico 767,904 U.S. A. 179,073 Hi ooiVaUUl 40,600 23,480 rriiatPTtialjl vJLiaLdiicua > 22,895 156 000 Haiti 45,000 Cuba ' 8^100 1^1 €L£A1 \ 108 037 \lt^n AT1 1^19 V CliCZiLlCla 45 940 Colombia 73,963 Chile 53,400 Ecuador 13,376 Peru . J'* 54,559 A 915) 96,926 Israel 54^494 Philippines : ; 24,100 Africa South Africa 40,953 Cameroon 43,200 Zaire 4o,yuu Congo 24,500 Madagascar 21,300 Europe Spain 44,008 Portugal 14,940 Australia Australia % 12,970 WORLD 2,080,088 Source; FAO (1997) adapted from Bergh & Lahav (1996)

PAGE 27

12 Table 2-4. Avocado breeding objectives for special characters Special Character Special Character Fruit Qualities Medium size (200-300 g) Uniformity -Skin -Medium thickness -Readily peelable -Insect, disease tolerance -Free from blemishes -Attractive color Long tree storage Seed: -SmaU -Tight in its cavity Spreading habit Easy to propagate Strong grower Tolerant to pests and diseases Tolerant of wind Tolerant of cold Tolerant of heat Tolerant of salinity Thick ovate shape Pulp: -Proper softening -Appetizing color -Absence of fibers -Pleasing flavor -Long shelf life -Slow oxidation -Chilling tolerance -High oil content -High nutritional value Shoot Qualities Tolerant of chlorosis Tolerant of other stresses Short fruit maturation period Precocious Regular bearing Wide adaptability Heavy bearer Rootstock Qualities Conducive to high quality fiaiit Conducive to healthy, productive trees Free from sunblotch viroid Dwarfing or semi-dwarfing Genetically uniform Hardy and vigorous Easily propagated Easily grafted Tolerant to Phytophthora root-rot and other disease Tolerant of salinity Tolerant of chlorosis Tolerant of drought Tolerant of other adverse soil condition Source: Bergh(1975) cultivars (Bergh & Lahav, 1996). However, this conventional method has been challenged recently (Lavi et al. , 1 99 1 ; Lavi et al. , 1 993). Genetic studies from populations of selfand cross-pollinated avocados with respect to anise scent, fruit density, flowering intensity, fruit weight, harvest duration.

PAGE 28

13 inflorescence length, seed size, and softening time, had significant nonadditive variances (Lavi et aL, 1991). This reflected the insignificantly low value of narrow-sense heritability but the significant value of broad sense heritability (Lavi et al., 1993). These results indicated that hybridization should be aimed at increasing the genetic variance in progenies by selecting parents that are not only of superior phenotype, but which also include 1030% of parents with inferior performance (Lavi et al.,1993). More recently, genetic associations between DNA fingerprint fi-agments and loci controlling important traits in avocado, e.g., fioiit color, have been reported (Mhameed et aL, 1995). Selection of progenies having these traits, e.g., fiiiit skin color, may be carried out early in the seedling stage using markers associated with these traits. Breeding scion cultivars Avocado production mostly relies on a few cultivars. Scion cultivars are dominated by 'Hass' and 'Fuerte' which have been cultivated commercially for 40 years in subtropical regions. Both cultivars were chance seedlings of unknown parentage (Bergh, 1976; Bergh & Lahav, 1996). Hass has several commercial weakness, for example, it produce fiuits with size variability, some proportion of which are too small to be marketable and this problem is aggravated with tree age (Bergh & Lahav, 1996). Several selections have been made to replace 'Hass' (Bergh & Lahav, 1996), e.g., 'Gwen', 'Jim', 'Reed' and 'Lamb'. Cultivation of these new cultivars is still limited due to consumer preference for 'Hass' (Bergh & Lahav, 1996). Several local selections have been made, including 'Ettinger' and 'Iriet' (Israel), 'Ardith' (USA) and 'Sarwill' (Australia) (Bergh & Lahav, 1996). Breeding rootstock cultivars The threat of root-rot disease, caused by Phytophthora cinnamomi Rands, to the avocado industry in California was recognized as eariy as the 1920s (Coffey, 1986). To address this problem, a breeding program was established at the University of California in

PAGE 29

14 the early 1950s. Persea species and related genera collected from Mexico, Guatemala, Honduras and Nicaragua were tested for resistance to Phytophthora root-rot (PRR). The results indicated that most of the Persea species that are resistant to PRR belong to the subgenus Eriodaphne, while Persea species in subgenus Persea (including avocado) were susceptible (Table 2-5). The resistant species in subgenus Eriodaphne Table 2-5. List of Persea and related genera and their resistance to Phytophthora rootrot. Species Origin of collection Resistance r^iiltii/Qrc in f^QlifVimia \,_/lilUV
PAGE 30

> 15 were graft incompatible with avocado and other species of the subgenus Persea, but were graft compatible with members of their own subgenus (Frolich et al., 1958). The same relation hold true for sexual hybridization (Bringhurst, 1954; Bergh & Lahav, 1996). The program at UC, Riverside produced the PRR-tolerant 'Duke 7' rootstock, which was a seedling of 'Duke' (Zentmeyer & Thorn, 1956; Zentmeyer et aL, 1963). 'Duke 7' has become the most important rootstock in California, and is propagated clonally using the etiolation technique (Piatt, 1976). Other selections-that are promising include 'Barr Duke' (a seedling of 'Duke 6'), 'D9' that induces dwarfing (fi-om an irradiated 'Duke' parent scions), 'Thomas' (a survivor from a root-rot affected area), 'Martin Grande' (a hybrid of avocado and Persea schiedecma)-zxt all still in trial (Bergh & Lahav, 1996). The level of tolerance to PRR of those selections are better than the earlierly used rootstock 'Topa Topa' but not as high as the PRR-resistant Persea species in the subgenus Eriodaphne. The absence of complete PRR resistance in the subgenus Persea, together with lack of information regarding the genetics of PRR-tolerance, has made breeding for PRR tolerant/resistant rootstock difficult. Biotechnology and Its Potential for Avocado Improvement Despite its importance, breeding avocado has been slow due to its long juvenile period, high genetic heterozygosity, low fruit set, lack of genetic information and inefficient breeding method (Lavi et al., 1991b; 1993). Systematic studies of the genetics (Lavi et al., 1991b) and development of molecular markers for some horticultural traits, including fixiit skin color (Lavi et al., 1991a) may have a significant impact on breeding efficiency. Nevertheless, breeding root-rot resistant rootstocks would be very difficult if not impossible to achieve using conventional methods, due to the lack of a resistance gene

PAGE 31

pool. Biotechnology, involving somatic cell genetics and gene transfer, may have an important role in widening genetic variability. Genetic transformation with antifungal genes such as glucanase has been proposed as an alternative to combat disease (Lamb et al., 1992) and might be a viable way to develop a root-rot resistant rootstock. Somatic hybridization via protoplast fusion has been used to overcome sexual barriers in citrus (Grosser & Gmitter, 1990). Protoplast fusion technology could be an alternative way of combining the root-rot resistance traits of Persea species that are sexually and graft incompatible with avocado (Pliego-Alfaro & Bergh, 1992; Bergh & Lahav, 1996). Somatic embryogenesis and plant regeneration from nucellar explants have been reported for woody tropical and subtropical fruit species, e.g., citrus (Rangan & Murashige, 1969) and mango (Litz et al., 1982; Litz et al., 1995). This approach may have direct application for cheap clonal propagation of a PRR tolerant rootstock. Avocado tissue culture has not been developed in comparison with other tropical tree fruit species, i.e., citrus or mango. Avocado tissue culture has been considered as either being in its infancy (Pliego-Alfaro & Bergh, 1992) or recalcitrant (Gardner, 1993), although tissue culture studies of avocado were initiated 50 years ago. To realize the potential of modem biotechnology for improvement of avocado, tissue culture protocols, including plant regeneration via somatic embryogenesis and from protoplasts, protoplast fusion and plant propagation through shoot proliferation, need to be developed. Previous work on avocado tissue culture, including callus initiation, shoot culture, somatic embryogenesis and protoplast isolation are summarized in Table 2-6. Early reports were intended to study growth responses of fioiit pericarp tissue in vitro (Schroeder, 1956; 1961; 1971). Subsequently, callus initiation was reported from various tissues, including flower parts, cotyledons, seedling stems and leaves (Table 2-6). Callus also was initiated from stems of other Persea species, including P. nubigena, P. borbonia

PAGE 32

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PAGE 33

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PAGE 37

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PAGE 38

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PAGE 39

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PAGE 40

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PAGE 41

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PAGE 42

27 and P. indica (Aaouine, 1986) and cotyledons of P. palustris (Raf.) Sarg. (Kane et aL, 1989). Regardless of the objectives of these studies, shoot organogenesis was never reported. Schroeder (1980) attempted to initiate shoot growth and proliferation of shoot cultures of avocado; however, shoot growth was poor on MS medium. Suboptimal growth responses, i.e., die back and leaf abscission (Pliego-Alfaro, 1981; Pliego-Alfaro et al., 1987), scaly leaf formation (Kane et al., 1989: Gonzales-Rosa et al., 1985), extensive callus formation at the base of explants (Schroeder, 1980; Gonzales-Rosas et al., 1985) and survival rate <100% (Gonzales-Rosas & Salazar-Garcia, 1984; Schall, 1987) were reported. Better proliferation has been achieved mostly by reducing the major salts content in MS medium (Schall, 1987; Nel et al., 1982; Campos «& Pais, 1996). Variable rooting frequencies have been reported (Table 2-6) from 100% (Pliego-Alfaro, 1988; Kane et al., 1986; Campos & Pais, 1996) to 30% (Skene «fe Barlass, 1983; Berringer et al., 1996). The difference in rooting frequency may be genotype-dependent or related to degree of juvenility and rooting method. Nevertheless, tissue culture propagation and plant establishment in soil have been reported for commercial avocado and several species belonging to Lauraceae (Table 2-6), although the reported procedures have involved juvenile phase explants. Therefore, the practical application of this technique is limited to propagation of germplasm for breeding programs or propagation of endangered species (Campos & Pais, 1996). Attempts to culture adult materials have been unsuccessful (Pliego-Alfaro, 1981), although limited shoot proliferation followed by low rooting frequencies can be obtained from adult phase shoot cultures that have been partially rejuvenated (Pliego-Alfaro &. Murashige, 1987; Schall, 1987). Somatic embryogenesis derived from zygotic embryos has been reported, although plant recovery has been only 2.5-5% (Mooney & Van Staden, 1987; Pliego-Alfaro & Murashige, 1988). Only three genotypes have been regenerated, and suspension cultures were not obtained. These reports are therefore largely preliminary.

PAGE 43

28 Avocado protoplasts have been isolated from callus (Blickle et al., 1988), in order to study sunblotch viroid replication, and from fruit mesocarp (Percival et al., 1991), for studying fruit ripening. Consequently, morphogenic responses of protoplasts have not been reported. Somatic Embryogenesis ; : Since the first description of somatic embryogenesis by Reinert (1958) and Steward (1958), this regeneration pathway has been widely used as a tool to study plant development (Zimmerman, 1993), for clonal plant propagation and for development of technology for plant improvement through somatic cell genetics and genetic transformation. Somatic embryogenesis refers to the developmental pathway from somatic cells/tissues that mimics zygotic embryogenesis (Zimmerman, 1993). From a practical standpoint, somatic embryogenesis is a process whereby embryos develop from somatic cells/tissues in tissue culture and can develop to maturity and subsequently germinate, forming normal plants (Wann, 1988). Low frequency of root emergence and even lower shoot development from somatic embryos is common in many species. In the view of many, the concept of plant conversion is therefore more operational than germination for in vitro somatic embryogenesis (Redenbaugh, 1993). Germination in this dissertation is defined as the development of roots from somatic embryos, while plant conversion is defined as the development of both shoots and roots from the same somatic embryo regardless of the plantlets survival during acclimatization and production two new leaves as required under Redenbaugh's criteria (1993). Somatic embryogenesis can be divided into several stages; 1 . initiation of embryogenic culture, 2. maintenance of embryogenic culture.

PAGE 44

29 3. somatic embryo development or maturation, and 4. somatic embryo germination or conversion. Initiation of Embryogenic Cultures Somatic embryos can develop from single cells within an explant (direct somatic embryogenesis), or differentiation can be preceded by proliferation of callus cells (indirect somatic embryogenesis). In the indirect pathway, cells undergo a directive induction event that involves a change of competence. Competent cells undergo a permissive induction that results in the embryogenic pathway. In directive induction, embryogenically predetermined cells simply require permissive induction to express the embryogenic pathway, e.g., cells in explanted nucellar cultures of polyembryonic plants (Ammirato, 1987). Indirect Pathway In the indirect pathway, differentiated explants, i.e., leaves, flower buds, hypocotyls, etc., are explanted onto inductive plant growth medium containing auxin (usually 2,4-D) in order to obtain an embryogenic culture. Embryogenic cultures consist of different cell types: parenchymatous, elongated and vacuolated cells, highly cytoplasmic isodiametric cells and cell clusters that resemble proembryos (Halperin, 1966). In the presence of auxin, cells within the rapidly dividing cell population are stimulated to undergo unequal divisions. This results in the formation of two sister cells: a large, highly vacuolated cell and a small, cytoplasmically rich cell that is competent for embryogenesis (Litz & Gray, 1995). Unequal cell division or segmentation of elongated cells has been documented in carrot (Backs-Huseman & Reinert, 1970; Street & Withers, 1974), Salix (Gronroos, 1995) and Cqffea canephora (P. ex Fr.) (Berthouly & Michaux-Ferriere, 1996). The competent cells form proembryonic cell clusters. Subculture of embryogenic cultures on or in induction medium results in the loss of integrative organization of the

PAGE 45

30 y' proembryos. The embryogenic masses that develop from proembryos in the presence of 2,4-D are proembryonic or proembryogenic. More than one somatic embryo can develop from each proembryonic mass when it is transferred to 2,4-D-free medium (Halperin, 1966). In tropical woody species, the indirect pathway of somatic embryogenesis has been reported to occur from nucellar explants of monoembryonic mango species (Litz et al., 1982), and from leaf explants of coffee (Sondahl & Sharp, 1977) and longan (Litz, 1988). Direct Pathway In the direct pathway, somatic embryos or proembryonic masses can develop from the explant (immature zygotic embryo, nucellus, hypocotyl, etc.) without a callus phase. Somatic embryos or proembryonic masses can be initiated from embryogenic cells that are present in the explant. In pearl millet, Vasil and Vasil (1980) demonstrated that somatic embryos can originate from subepidermal cells of the scutellum of immature embryos. In the presence of 2,4-D, those scutellar cells enlarge and undergo internal segmentation resulting in the formation of proembryos. In polyembryonic mango, the embryogenic cells that are already present in nucellar tissue are similar morphologically to proembryos (DeWald, 1987). Auxin in the medium can stimulate the cloning of such cells (Carman, 1990; Wann, 1990; Ammirato, 1985). Direct development of disorganized proembryonic masses or somatic embryos from carrot explants can be controlled by manipulating medium pH (Smith & Krikorian, 1990). Induction of the direct embryogenic pathway in woody perennials has been reported from the nucellus of polyembryonic mango (Litz, 1984; DeWald, 1987; Litz et al., 1995) and monoembryonic mango (Jana et al., 1994); cotyledons of wild pear (Prunus avium) (De March et al.,1993); and nucellus of monoembryonic citrus such as Citrus grandis Osbeck, C. limon Burmf and C. reticulata Blanco x C. sinensis Osbeck (Rangan etal.,1969).

PAGE 46

> 31 Maintenance of Embrvogenic Cultures and Morpholo gical Variability of Proembryonic Masses Embryogenic cultures are subcultured as proembryonic masses and not callus (Litz & Gray, 1992). Proliferation of proembryonic masses (and somatic embryos) has been referred to as repetitive embryogenesis (Ammirato, 1987; Litz & Gray, 1992). Repetitive embryogenesis involves continuous cycles of secondary somatic embryogenesis from proembryonic masses that have lost their integrative ability to form single somatic embryos (Williams & Maheswaran, 1986). This has been described vnth walnut (Preece, 1995), big leaf magnolia (Merkle & Watson-Pauly, 1993), mango (Litz et al., 1995) and citrus (Button et al, 1974; Cabasson et al., 1995; Gavish et al., 1991) The loss of integrative development at the proembryo stage may result in the continuous proliferation of proembryos to form disorganized proembryonic masses, i.e., citrus (Cabasson et al., 1995). The loss of integrative development at the globular to heart stage may result in the proliferation of organized proembryonic masses, i.e., mango. The cells of the protoderm and subepidermal layer become embryogenic and may slough off from the primary proembryos (Button et al., 1974). Repetitive somatic embryogenesis by production of singulated maturing somatic embryos usually is slow (Gray, 1995; Merkle, 1995) and not as amenable to genetic manipulation through genetic transformation and to mass propagation as proliferation of embryogenic cultures through proembryonic masses (Merkle, 1995). Therefore repetitive embryogenesis through proembryonic masses proliferation is desirable, not only for maintenance of cultures but for somatic cell genetics. Auxin is one of the most significant inductive factors for embryogenic cultures, and because of its ability to inhibit embryo development, it is often critical for maintenance of embryogenic cultures (Litz & Gray, 1992). Auxin can disrupt the polarized cell division that is required for integrated development of proembryos to somatic embryos (Kawahara

PAGE 47

32 & Komamine, 1995). Habituation may also occur, e. g., in citms proembryonic masses (Button &Kochba, 1974; Grosser &Gmitter, 1990). , . Proliferation of proembryonic masses has been reported to be affected by genotype in mango (Litz et aL, 1995) and sweet gum (Merkle, 1995a). Very often, a whole range of different sizes and morphologies of proembryonic masses and somatic embryos is present in cultures of different species of sweet gum (Liquidambar styracifolia) and proembryonic masses can be preferentially selected for further subculture (Merkle, 1995a). Medium pH can affect the proliferation of wild carrot cultures, so that low pH favors the proliferation of proembryonic masses while high pH favors the proliferation and development of somatic embryos (Smith & Krikorian, 1990). The total nitrogen concentration and the ratio of NH4'^ : NO3' have been reported to affect proembryonic mass proliferation. In citrus, total nitrogen concentration of 60 mM nitrogen resulted in significantly higher proembryonic mass fresh weight compared to 30 mM, while high fi-esh weight was maintained with a NH4^ ; NO3' ratio of 1:1 to 1:9 (Niedz, 1993). Somatic Embryo Development Somatic embryo development is comparable to that of zygotic embryos, and includes distinct morphological stages, e.g., globular, heart, torpedo and cotyledonary. In addition, formation of morphologically abnormal somatic embryos has been reported. Morphological anomalies include monocotyly, polycotyly, fused cotyledons, fasciation, and multiple somatic embryos (Ammirato, 1987; Litz et al., 1995; Gray, 1995). Sieving and fractionation followed by culturing small polyembryonic masses on somatic embryo development medium has been used to reduce the frequency of multiple somatic embryos and to increase the frequency of singulated somatic embryo (Ammirato, 1987; Litz et al., 1995; Merkle, 1995b).

PAGE 48

33 Somatic embryo development is initiated after transfer of embryogenic cultures to medium without or with low auxin content. Fujiwara & Komamine (1975) observed that cytokinins do not affect development of somatic embryos, although development of shoot meristems is enhanced. Abscisic acid has been demonstrated to affect the differentiation of apical meristems and subsequent plant conversion (Nickel & Yeung, 1 993). Sucrose at concentrations of 1-6% is generally used for somatic embryo development. Replacing sucrose with another carbon source, e.g., glycerol (Ben-Hayyim & Neuman, 1983; Gavish et al., 1991; Vu et al., 1993), galactose (Cabasson et al., 1995), lactose and raffinose has been reported (Kochba et al., 1978; 1982). The effect of the mineral composition of the maturation medium has been documented. DeWald et al. (1989a) demonstrated that medium based upon a modification of B5 formulation resulted in better mango somatic embryo development than MS. Similar results were reported by Muralidharan & Mascarenhas (1995) with Eucalyptus citriodora. Incomplete maturation of somatic embryos is one of the most significant factors that account for low rates of plant conversion (Bomman, 1992). Precocious germination of immature Hevea brasiliemis Miill. Arg. somatic embryos has resulted in poor plant regeneration (Michaux-Ferriere et al., 1991). Somatic embryo maturation can be controlled by treatments with ABA, sucrose and desiccation. Abscisic acid has been used to prevent precocious germination (Ammirato, 1985), to inhibit secondary somatic embryogenesis (Monsalud et al., 1995), and to confer desiccation tolerance in orthodox type somatic embryos (Kim & Janick, 1989), thereby promoting accumulation of storage lipids, starch and proteins, sjmchronous maturation and a high fi-equency of conversion (Bomman, 1992). Nickel & Yeung (1993) suggested that ABA may induce shoot meristem differentiation or prevent precocious germination that includes precocious vacuolation of the cells in the apical notch that prevents meristem differentiation. Although application of ABA together with an osmoticum can increase plant conversion

PAGE 49

34 of conifers (Attree & Fowke, 1993), this approach has been unsuccessful for angiosperm tree species (Merkle, 1995b; Vieitez, 1995; Pliego-Alfaro & Murashige, 1988). Pence (1991) demonstrated that during maturation of recalcitrant zygotic embryos of Theobroma cacao, ABA levels increased and peaked at the beginning of maturation and then dropped subsequently. Water content of the embryo also decreased ca. 40-70% and synthesis of anthocyanins and lipids increased up to the end of maturation period. Similar trends were observed by Etienne et al. (1993) with recalcitrant H. brasiliensis zygotic embryos, while somatic embryos had low ABA content, which varied little during maturation. Inclusion of ABA in maturation medium, however, did not significantly affect desiccation tolerance of either cacao zygotic embryos (Pence, 1992) or mango somatic and nucellar embryos (Pliego-Alfaro et al., 1995a; b), ahhough Etienne et al. (1993) claimed that high osmolarity (100 g/1 sucrose) together with high levels of ABA (1 mM) dramatically improved H. basiliensis somatic embryo desiccation tolerance and resulted in 77% germination and 34% conversion. Slow desiccation has been demonstrated to improve maturation of H. brasiliensis somatic embryos (Etienne et al., 1993). In walnut, desiccated orthodox somatic embryos convert at a high rate (45%) (Deng & Comu, 1992). Merkle (1995a; b) was able to prevent precocious germination of yellow poplar somatic embryos by allowing their development on filter paper overlain on semisolid medium. These somatic embryos matured and converted to plantlets at fi-equency up to 100% upon transfer to germination medium. Filter paper may impose a matrix stress on somatic embryos that encourages maturation (Merkle, 1995a, 1995b). Somatic Embryo Germination and Plant Conversion Various treatments have been demonstrated to increase shoot development/plant conversion fi^om somatic embryos of woody plants, including localized application of

PAGE 50

35 cytokinin (Mathews & Wetztein, 1993), cold treatment (Arilaga et al., 1994), gibbereUic acid (Deng & Comu, 1992) and high CO2 together with high irradiance (Figueira & Janick, 1993). Cold treatment increased plant conversion of (orthodox) black locust somatic embryos (Arrilaga et al., 1994), walnut (Deng & Comu, 1992), chesnut (Vieitez F, 1995), Cornelia japonica and C. reticulata (Vieitez A, 1995), although recalcitrant avocado somatic embryos did not respond (Pliego-Alfaro & Murashige, 1988). Cold treatment may be essential for species that require cold stratification for optimum germination, i.e., temperate hardwood species with orthodox embryos (Merkle, 1995b). Mathews and Wetzstein (1993) applied 100 \iM BA and 3 mg 1-1 anti ethylene silver nitrate topically to orthodox pecan somatic embryos (Carya illinoensis) and increased germination fi-om 3 to 47% and plant conversion from 2 to 13%. GibbereUic acid as a component in germination medium has increased the conversion rate of orthodox walnut somatic embryos with or without a cold treatment; however, the conversion rate of desiccated walnut somatic embryos was reduced (Deng & Comu, 1992). GibbereUic acid in combination with lAA increased the conversion rate of orthodox Camellia japonica and C. reticulata in comparison with gibbereUic acid alone (Vieitez A, 1995). The regulation of orthodox seed germination by gibbereUic acid has been widely used in cereals (Berrie, 1984), but its effects are not understood (Deng & Comu, 1992). Maintaining recalcitrant somatic embryos of cacao under high CO2 (20,000 ppm) and high irradiance (159-299 |amol m"^ s"^) (Figuera & Janick, 1993) has been demonstrated to increase the conversion and vigor of regenerated plantlets. The importance of high CO2 and high irradiance has been to stimulate photoautotrophy through the promotion of the growth of chlorophyUous explants/plantlets (Kozai, 1991). In conclusion, somatic embryogenesis has been widely reported and studied for different plant species, and has great potential for clonal propagation and crop improvement. It is necessary to solve problems related to developmental anomalies that

PAGE 51

36 are associated with this developmental pathway, such as poor embryo organization, embryo maturation and somatic embryo gennination or plant conversion. Some of the problems have been solved by providing conditions that mimic the in vivo condition of the zygotic embryo. For recalcitrant species, e. g., mango, cocoa and avocado, however, their seed physiology is not widely understood. Consequently, attempts to resolve problems associated with embryo development and maturation unavoidably are based on orthodox seeds/somatic embryos and therefore are often ineffective. Protoplast Isolation. Culture and Regeneration Protoplast technology has been important in somatic cell genetics for genetic transformation and for somatic hybridization. The application of somatic cell genetics to woody fruit species is especially important for supplementing conventional plant breeding. Discrete genetic variability could be directly selected without prolonged crossing and backcrossing because woody fruit species are propagated vegetatively (Janick, 1992). The production of somatic hybrids involving citrus and its relatives by protoplast fusion can involve sexually incompatible species belonging to different genera or tribes (Grosser etal., 1992). The potential of somatic hybridization requires the availability of an efficient plant regeneration system from protoplasts. Among woody plants, an efficient protoplast-toplant regeneration system was first reported for citrus (Vardi et al., 1972). Plants have also been regenerated from protoplasts of sandalwood {Santalum album L.) (Rao & Ozias-Akins, 1985), from leaf protoplasts of species in the family Rosaceae, including Mains, Prunus and Pyrus (Ochatt, 1990;1993b, c; Ochatt et al., 1992; Patat-Ochatt, 1993), and of species such as grapevine (Reustle et al., 1995), Actinidia chinemis (Tsai et al., 1993) and Passiflora spp. (Domelas et al., 1993; De'Utra Vaz et al., 1993).

PAGE 52

37 Protoplast Isolation v The discussion that follows is intended as a review of those factors that affect isolation and culture of protoplasts together with plant regeneration, particularly with respect to woody perennials and fruit trees. Protoplasts can be isolated either mechanically or enzymatically. For in vitro studies, the latter method has been preferred, because a large number of protoplasts can be released from various tissues (Cocking, 1972). The use of enzymes for protoplast isolation was pioneered by Cocking in 1960 who isolated protoplasts from tomato roots following digestion with cellulase purified from a fungus, Myrothecium verrucaria (Cocking, 1983). Enzymes The enzyme mixture that is utilized for protoplast isolation generally contains pectinase for loosening the tissue and cellulase for degrading the cell wall. Various enzymes are available commercially with different levels of purity (Table 2-7). Impure enzyme preparations contain nucleases (especially ribonucleases), peroxidases, proteolytic enzymes and phenolic compounds (Vasil & Vasil, 1980). Partial purification of commercially available enzyme precipitation with ammonium sulfate and elution through Sephadex G-25 or Biogel is sometimes usefiil (Vasil & Vasil, 1980). Very highly purified and crystalline enzymes are not usefiil since they are unable to digest complex plant cell walls; therefore complex enzyme mixtures, cleansed of toxic substance and impurities are recommended (Vasil & Vasil, 1980). Purified enzymes such as Cellulase Onozuka RS, Cellulase Onozuka RIO, macerozyme RIO and Pectolyase Y23 have been recommended for protoplast isolation from tissues of woody plants (Grosser & Gmitter, 1990; Ochatt et al., 1992b). For the most part, enzymes for woody plant protoplast isolation have been used at concentrations of 0.3-2%, except for Pectolyase Y23 which is generally used at

PAGE 53

38 i2 6 o & •a iS o S o 3 O cT 9 2 i lb ^ ^ H o ;5 O U km. niger niger s cetci a hoderr, iride idiomy iride iride iride iride 'xpomi Aspergilus Aspergilus Trie > 1 ^> > ;^ > i; Hell 2 5 u •o 5 u JC3 o 3 o t o 2 o i in p Q Q (J U O U

PAGE 54

I 39 1 in •a S (3 D d 1 •§ I 1 E? o I I s: I. SI ^ ftj ^ 03 2 00 ON I I I v\ yr\ O l-H CO 00 ON 3 CO o 2 1/1 o 3 o 00

PAGE 55

40 10% of the concentration of cellulase. A mixture of enzymes is normally used. Since cell wall composition of dififerent tissues of different species is variable (Ishii, 1989), the enzyme composition of the digestion solution may vary and must be determined empirically for different species (Cocking, 1972; Vasil & Vasil, 1980; Evans & Bravo, 1983; Davey & Kumar, 1983). Ishii (1989) demonstrated that the cell wall composition of monocotyledonous and dicotyledonous species was different, and demonstrated that xylanase is required for optimum protoplast release from oat leaves while pectin lyase was required for optimum protoplast release from petunia leaves. With woody species, a single enzyme mixture may be effective for several species, although slight variations may be required, depending on the tissue (Ochatt, 1990). A mixture of 1% Cellulase Onozuka RIO, 1% Hemicellulase and 0.1% Pectolyase Y23 has been used for protoplast isolation from numerous fruit and nut genotypes (Revilla et al., 1987; Ochatt, 1990; 1993b), while 2% Meicelase, 2% Rhozyme HP1500 and 0.03% Macerozyme RIO has been used to isolate protoplasts from suspension cultures and callus tissues of cherry, pear and apple (Ochatt, 1990; 1993a, b). An enzyme mixture that was developed initially for Trifolium rubens (Grosser & Collins, 1984) and improved for citrus contains 1% Cellulase Onozuka RS, 1% Macerase or macerozyme RIO and 0.2% Pectolyase Y23 (Grosser & Gmitter, 1990) and is effective for various tissues and species, including embryogenic cultures and suspension cultures of citrus, non-embryogenic callus and suspension culture of citrus and citrus relatives (Muorao-Filho & Grosser, 1992; Grosser & Gmitter, 1992), leaves from in vitro and greenhouse grown citrus and citrus relatives (Grosser & Chandler, 1987), flower bud tetrads, and for other species, including embryogenic suspension cultures of mango and grape (Grosser, 1993, personal communication). Other components of enzvme mixtures To prevent protoplasts from bursting after release, increased osmolarity of the medium is essential. Mannitol (0.7 M) has generally been used satisfactorily for citrus

PAGE 56

41 (Grosser & Chandler, 1987; Grosser & Gmitter, 1990; Kobayashi et aL, 1985; Sim et a!., 1988) and Actinidia chinensis (Tsai, 1993) even though lower concentrations have been used for protoplast isolation from grape embryogenic cultures (Reustle et al., 1995). A minimal salt mixture has been included in the enzyme mixture to reduce shock following transfer of protoplasts to culture medium. These media include CPW salts (Frearson et al., 1973) and 0.5 x MT (Murashige & Tucker, 1965) (Kobayashi et aL, 1985; Simetal., 1988). Other addenda such as morpholinoethanesulfoxide (MES) at concentrations of 3-7 mM (Grosser & Gmitter, 1990; Ochatt, 1990; 1993a, b) has been used to buffer the acidification that often occurs during digestion. Polyvinylpyrollidone (PVP) has been used to prevent phenolic oxidation, especially when protoplast isolation involves leaf or suspension cultures of woody perennials (Ochatt, 1993a, b; Patat-Ochatt, 1993; Lee & Wetzstein, 1994). Other substances, i.e., CaCb and NaH2P04, are added as plasma membrane stabilizers of protoplasts (Vasil & Vasil 1984; Grosser & Gmitter, 1990). Other physical factors influencing protoplast isolation ^ Enhancement of enzyme penetration into tissue is usually required when clumps of tissues or leaves are used. With herbaceaous plants, this can be achieved by removing the epidermis and chopping the tissues into small pieces (Evans & Bravo, 1983). With woody species, the epidermis is difficult to remove; therefore, leaf tissue can be chopped or feathered (Grosser & Gmitter, 1990). When leaf tissue is derived from greenhouse-grown plants, vacuum infiltration may also facilitate enzyme penetration (Grosser & Gmitter, 1990). Grinding of leaves can provide a larger surface area for enzyme digestion, while entrapment of protoplasts during purification can be prevented by washing the cell debri (Russel & McCown, 1986), agitation of the digestion mixture and running the digested mixture up and down a Pasteur pipet (Mills & Hammerschlag, 1994).

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'42 The ratio of tissue:enzyme solution (w/v) can be critical for protoplast isolation from peach leaves where ratios of 10-20 mg ml'^ resulted in high protoplast yields while ratios of 50-100 mg ml * resulted in low protoplast yields (Mills & Hammerschlag, 1994). High tissue:enzyme ratios, however, have been used for other tissues, i.e., 100 mg ml"* for pear leaves (Ochatt & Power, 1988), and 150 mg ml ' for apple leaves (Doughty & Power, 1988) . Grosser & Gmitter (1990) used a tissue:enzyme ratio of ca. 250 mg ml"' for embryogenic citrus cultures, and 30-50 mg ml"' for leaves. The negative effect of higher tissue:enzyme ratios may be associated with release of protease from digested tissues that inhibits enzyme activity (Mills & Hammerschlag, 1994) and phenolic compounds. The tissue:enzyme ratio may be genotype and tissue dependent. Protoplast purification , Protoplasts are generally purified by passing the digestion mixture through a nylon or stainless steel sieve (45-90 \im mesh) or another material, e.g., Miracloth*. This is sometimes followed by gradient centrifiigation to remove cell debris, undigested cells and broken protoplasts that can reduce culture pH, protoplast viability and fiision frequency (Grosser, 1994). Grosser & Gmitter (1990) have sieved with a 45 nm stainless steel mesh screen followed by gradient centrifiigation at 100 g in 0.7 M (25% w/v) sucrose and 0.7 M (13% w/v) mannitol supplemented with modified CPW salts (Frearson et al., 1973). The purified protoplasts in the interphase can be washed again (Grosser & Gmitter, 1990). Protoplast Culture Factors affecting protoplast culture and subsequent growth and development include medium composition, osmoticum and osmolarity, plating density and environmental conditions, i.e., irradiance and temperature (Dons & Colijn-Hooymans, 1989) . These factors can be optimized on the basis of plating efficiency, i.e., the proportion of protoplasts that respond to culture conditions by undergoing division after a determined time period.

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Medium composition Protoplast culture media are generally composed of major and minor salts, vitamins and other organic addenda, plant growth substances, a carbon source and an osmoticum. Plant growth media that have been developed for a particular genotype can be used (Davey & Kumar, 1983). This approach seems logical and eflBcient even though some modification of the medium composition is sometimes needed. Major and minor salts of MS (Murashige and Skoog, 1962), B5 (Gamborg et al., 1965) and KM8 or KM8P (Kao & Michayluk, 1975) media are satisfactory. The success of the KM8P medium may be due to the presence of multivitamins, sugar and sugar alcohol additives that provide essential metabolic intermediates (Grosser, 1994). An eflBcient protoplast culture medium for species within a genus can be developed by supplementing optimal basal media that have been effective for cell culture of the genus with 8P multivitamins, organic acid and sugar/alcohol additives (Grosser, 1994). This approach has been successful for Trifolium (Grosser & Collins, 1984), citrus (Grosser & Gmitter, 1990; Niedz, 1993) and apple (Kouider et al., 1984; Deng et al., 1995; Doughty & Power, 1988; Patat-Ochatt et al.,1993). Ochatt & Power (1988), however, reported that pear (Pyrus communis L.) mesophyll protoplasts only regenerate cell walls in K8P or KM8P or MS media, but divide in MS medium without ammonium. The negative effect of K8P medium on protoplast division has also been reported for root callus protoplasts of sour cherry (Prunus cerasus L.) (Ochatt, 1990). Ochatt (1993a, b) suggested that scion and rootstock genotypes may require different organic additives, i.e., rootstock genotypes require more organic compounds than scion genotypes in the same genus, i.e., Pyrus and A/a/M5 (Ochatt et al., 1992b). The ammonium nitrate content of KM8P medium may be deleterious to protoplasts (Grosser, 1994). Elimination of NH4NO3 from culture medium has been important for microcallus formation of pear {Pyrus sp.) (Ochatt 1990; 1992; 1993; Ochatt & Power, 1988a, b; Ochatt & Caso, 1986), for plant regeneration from protoplasts of

PAGE 59

44 Populus spp. (Russel & McCown, 1986; 1988) and for somatic embiyogenesis and subsequent plant regeneration from protoplasts of citrus (Grosser & Gmitter, 1990; Niedtz, 1993). Plant growth substances, i.e., cytokinin (BA, zeatin) and auxin (2,4 D, NAA) are required for microcallus development when protoplasts are isolated from leaves, callus or suspension culture of pear (Ochatt, 1990; 1993b), Prunus spp.(1990; 1993c), apple (Patat-Ochatt et al., 1993), cell suspensions of Vitis labruscana and V. thunbergii (Mii et aL, 1991) and in vitro leaves of Vitis spp. (Lee & Wetztein, 1988), callus of kiwifruit (Oliveira & Pais, 1991), leaves from greenhouse-grown passionfiuit (d'Utra Vaz et al., 1993) and peach suspension cultures (Matsuta et al., 1986). Although sometimes also required for microcallus development from protoplasts isolated from embryogenic cultures of Vitis sp. (Reustle et al., 1995), plant growth substances are not generally required for somatic embryogenesis of protoplasts isolated from embryogenic cultures, e.g., citrus (Grosser & Gmitter, 1990; Niedz, 1993; Kunitake et al., 1991; Kobayashi et al., 1985; Hidaka & Kajiura, 1988; Sim et al., 1988; Vardi & Galun, 1988; 1989). Protoplasts isolated from embryogenic cultures derived from seedling parts of citrus relatives such as Citropsis schweinfurthii, Atalantia biloculoaris, etc. reportedly required plant growth substances such as BA or GA4+7 for microcallus formation (Jumin & Nito, 1995); however, protoplasts from similar tissues of Microcitrus did not require plant growth substances (Vardi et al., 1986). The plant growth regulator requirement for protoplast growth and development is probably determined by whether regeneration from the source is by organogenesis or somatic embryogenesis. Plating Density Protoplasts of woody species are generally cultured at plating densities of 0.5-1 x 10* protoplasts ml ' , which is considered optimum for citrus (Vardi & Galun, 1988; 1989;

PAGE 60

45 Grosser & Gmitter, 1990), Prurms spp. (Ochatt, 1993c), Pyrus spp. (Ochatt, 1993b), Malus sp. (Patat-Ochatt, 1993), kiwifiiiit (Oliveira & Pais, 1991), Passiflora spp. (Domelas & Vieira, 1993; d'Utra Vaz, 1993) and Vitis spp. (Mii et al., 1991). Protoplast plating density affects plating efficiency in apple (Kouider et al., 1984) and root callus protoplasts of sour cherry (Prunus cerasus L.) (Ochatt, 1990). The highest plating efficiency was used to determine the optimum plating density. Determining plating density on the basis of the highest plating efficiency may not be appropriate when regeneration is via somatic embryogenesis, e.g., citrus (Kobayashi et al., 1985). Citrus protoplasts derived from embryogenic cultures and plated at a high plating density (ca. 2 x lO') resulted in a high plating efficiency (40%); however, only microcallus formed (Kobayashi et al., 1985). At a low plating density (ca. 2 x 10*), low plating efficiency resulted (5%); however, somatic embryogenesis occurred. Somatic embryogenesis from citrus protoplasts isolated from embryogenic cultures cultured at low plating density was also reported by Hidaka and Kajiura (1988). Inhibition of somatic embryogenesis when fused citrus protoplasts were grown at a high plating efficiency was also reported (Grosser & Gmitter, 1990; Grosser, 1994). Medium osmolarity Medium osmolarity generally is 0.4 to 0.7M and supplied with a metabolically active sugar (sucrose, glucose, or sorbitol) or a metabolically inert sugar such as mannitol. Combining metabolically active and inert sugars has been recommended since the former is metabolized by the growing protoplasts and therefore the medium osmolarity is gradually reduced. This would subsequently reduce shock when the protoplast-derived cell colonies are transferred to regeneration medium (Vasil & Vasil, 1983). Systematic studies have evaluated the effect of medium osmolarity in factorial combinations with plating density of citrus {Citrus sinensis) protoplasts isolated from embryogenic cultures (Kobayashi et al., 1985). The optimum medium osmolarity is

PAGE 61

46 dependent upon protoplast density, although high medium osmolarity (> 0.35 M mannitol + 0.15 M sucrose) results in a slightly higher plating eflBciency than lower medium osmolarity (< 0.35 M mannitol + 0.15 M sucrose). Low medium osmolarity combined with low plating density resulted in the direct development of somatic embryo from protoplasts, while with high medium osmolarity protoplasts give rise to microcallus. Development of somatic embryos directly from protoplasts cultured at low medium osmolarity (0.04 M sucrose, 0.08 M glucose and 0.23 M mannitol) and low plating efficiency was also reported for Citrus yuko (Hidaka & Kajiura, 1988). The development of somatic embryos at low medium osmolarity, however, was inhibited by high plating efficiency. The inhibitory effect of high plating density on somatic embryo development could be overcome by lowering the density (Kobayashi et al., 1985; Grosser & Gmitter, 1990). Regardless of the positive effect of low medium osmolarity for somatic embryo development, protoplast culture medium has generally been supplemented with high medium osmolarity of ca. 0.6-0.7 M of several different sugars. Grrosser & Gmitter (1990) employed 0.6-0.7 M osmoticum that consisted of 0.15-0.25 M sucrose and 0.45 M mannitol, while Vardi and Galun (1989) used 0.3 M sucrose and 0.3 M mannitol. A combination of 0.3 M sucrose and 0.3 M sorbitol was used for culture of protoplasts of C. madurensis (Ling et al., 1989) and C. unshiu (Ling et al., 1990). Other reports include the use of a only of 0.6 M sorbitol for culture of protoplasts isolated from callus initiated from seedling stems (Jumin & Nito, 1996a; b), 0.15 M sucrose and 0.45 M glucose for embryogenic culture-derived protoplasts of C. mitis (Sim et al., 1988) and 0.4 M mannitol, 0.2 M glucose and 0.05 M sucrose for kiwifruit protoplasts (Tsai et al., 1993). Culture of protoplasts at low medium osmolarity is simpler than culture at high medium osmolarity since the latter requires reduction of medium osmolarity during the culture period (Grosser & Gmitter, 1990; Ochatt, 1993a). The tolerance of protoplasts to low medium osmolarity must be determined.

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47 Method of culture ^ v Protoplasts of woody species have been cultured in: liquid culture as a thin layer, i.e., citrus (Grosser & Gmitter, 1990; Hidaka & Kajiura, 1988; Kobayashi et aL, 1985) and pear (Ochatt & Power, 1988a); in liquid culture as a shallow pool, i.e., fused citrus protoplasts (Grosser & Gmitter, 1990) and apple (Ding et al., 1995); immobilized with low melting agarose in disc culture, i.e., sour cherry (Ochatt, 1993b) and semisolid culture for Passiflora spp. (Domelas & Vieira, 1993; D'Utra Vaz et al., 1993), grape (Lee & Wetzstein, 1988) and sour cherry (Ochatt, 1993b); immobilized with agar or gellan gum, i.e., citrus relatives (Jumin & Nito, 1990) and C. unshiu (Ling et al., 1991; Kunitake at al., 1991); immobilized in Ca-alginate, e. g., grape (Reustle et al., 1995), apple (HuancarunaPerales & Schieder, 1993) and citrus (Niedz, 1993); in double phase liquid over semisolid agar/gellan gum, i.e., C. mitis (Sim et al., 1988) and Vitis labruscana and V. thunbergii (Mii et al., 1991). Liquid culture is easy to handle and facilitates observation with the inverted microscope. When solid culture is required, low melting agarose resuUs in higher plating eflHciency (Grosser & Gmitter, 1990), and facilitates protoplasts to grow under low plating density, i.e., 660-1300 protoplasts per ml as demonstrated in Hyocyamus muticus and Nicotiana tabacum (Shillito et al., 1983). Similarly, agarose has been effective for culturing protoplasts of calamondin (C. madurensis Lour.) (Ling et al., 1989). Sour cherry protoplasts, however, had the same plating efficiency regardless of the culture method utilized (Ochatt, 1990), while protoplasts derived from stems and leaves of haploid apple divided in liquid medium, but not in agarose discs or in mohen agarose (Patat-Ochatt, 1993). Protoplasts of the shrubby ornamental honeysuckle (Lonicera nitida cv. Maigrun) grew better in liquid than in agarose medium (Ochatt, 1991) Niedz (1993) demonstrated higher plating eflBciencies for protoplasts embedded in Ca-alginate beads than in liquid medium when plating densities were high. Oliviera & Pais (1991) observed that callus-derived protoplasts of kiwifhiit 'Hayward' did not survive in

PAGE 63

48 liquid medium, but divided and formed microcallus in alginate-solidified medium, although the microcalli eventually died. Ca-alginate has been successfully used to culture protoplasts of embryogenic grape (Reustle et aL, 1995). Culture environment Protoplast cultures have generally been maintained at 25° C in darkness (Grosser & Gmitter, 1990), since protoplasts can be sensitive to light (Grosser, 1994). Protoplast growth inhibition by light has also been reported for Lonicera nitida 'Maigrun' (Ochatt, 1991). Some protoplasts, i.e., sour cherry, can tolerate more light and can grow and differentiate under diffiise light (Ochatt, 1990). Protoplast culture and regeneration has been reported with several woody species. Regeneration in species other than citrus has mostly been confined to organogenesis since the source of the protoplasts has not been embryogenic tissue. Protoplast culture and regeneration fi-om embryogenic tissues is more eflBcient and simpler than fi^om nonembryogenic tissues. Somatic Hybridization Since the first interspecific somatic hybrids between Nicotiana glauca and N. langdorffii (Carlson et al., 1972) were reported, somatic hybrids have been produced in various plants, including potato, tomato, legumes, cereals, eggplant, petunia and several trees such as citrus. Somatic hybridization by protoplast fiision has been utilized to bypass genetic barriers between sexually incompatible wild species and economic species, thereby transferring important traits fi-om wild species into commercial species. Somatic hybrids have been obtained within the genera Nicotiana, Solarium and Brassica (Bajaj, 1994); however, the usefulness of somatic hybrids in breeding has been impeded by sterility and undesirable characters from the wild species. For example, somatic hybrids of eggplant

PAGE 64

49 {Solanum melongena L.) with S. khasianum, S. torvum and S. nigrum were difficult-toroot, grew poorly and were highly sterile, whereas, somatic hybrids between eggplant with more closely related S. aethiopicum demonstrated vigorous growth and had high fruit production (Sihachakr et al., 1994). Similar results were reported for somatic hybrids between potato {Solanum tuberosum) and related species (Butenko & Kucko, 1994; Jadari et al., 1992). Since the somatic hybrids are tetraploid, their use in breeding of diploid species is dependent on the ability to regenerate diploid plants from microspores and/or intensive backcrossing to the cultivated species (Sihachakr et al., 1994). Somatic Hybridization and Its Application to Woody Species The application of somatic hybridization to woody species has been limited to a few genera, mostly Citrus spp. and relatives (Grosser & Gmitter, 1990), with which more than 150 somatic hybrids have been produced (Grosser et al., 1996; Grosser, 1997, personal communication). Other somatic hybridizations have involved sexuallyand graftincompatible cherry rootstock 'Colt' (Prunus avium x Pseudocerasus) with wild pear (Pyrus communis var. pyraster) (Ochatt et al., 1989), between sexually compatible Passiflora edulis f flavicarpa and P. incamata (Otoni et al., 1995), between P. edulis f flavicarpa with P. alata, P. amethystina, P. cincinata and P. giberti (Domelas et al, 1995). Two approaches for somatic hybridization have been used in citrus rootstock improvement. Somatic hybrid rootstocks have been produced from existing rootstocks that have desirable and complementary characteristics. These somatic hybrids would have characteristics of both parents since somatic hybridization is an additive process with no segregation. Therefore, traits that are dominant or codominant should be express in the somatic hybrids. Several somatic hybrids for this purpose have been produced (Gmitter et

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50 al., 1992), although the characterization of the inheritance of the parental characters in the somatic hybrids has not yet been reported. Another approach involves production of somatic hybrids between existing rootstocks and sexually incompatible, related species for germplasm enhancement. Several somatic hybrids have been produced, including combinations of C. reticulata with Citropsis gilletiana, and C. sinensis 'Hamlin' sweet orange with Severinia disticha, Citropsis gillatiana, S. buxifolia, Atalantia ceylanica, Feronia limonia and Clausena lansium. Somatic hybrids of the last two combinations did not grow vigorously and eventually died. This failure may be due to somatic incompatibility since C. lansium and F. limonia cannot be grafted easily with citrus (Louzada & Grosser, 1994). Recent attempts involving a vigorous selection of A. ceylanica with 'Succari' sweet orange resulted in prolific and more vigorous somatic hybrids (Mourao-Filho, 1995); however, latent somatic incompatibility in these somatic hybrids has not been determined. Mourao-Filho (1995) reported that somatic hybrid plants between four varieties of sweet orange (C. sinensis) + S. disticha died following attack by an unidentified flingus, although previously reported somatic hybrids of the same parental combination were reportedly unaffected (Louzada & Grosser, 1994). Analysis of the somatic hybrid plants of 'Cleopatra' mandarin (C. reticulata) + Citropsis gillatiana showed a high level of susceptibility to an undetermined leaf stem fiingal spotting disease that drastically reduced tree vigor (Mourao-Filho, 1995). It is not clear if this undesirable phenotype was due to somaclonal variation or a negative genomic interaction (Mourao-Filho, 1995). Grosser et al. (1992) also reported that somatic hybrids between C. sinensis 'Hamlin' + Severenia buxifolia had chromosome numbers of 27 and it is not clear whether these plants were triploid or aneuploid. Somatic hybridization involving distantly related species can result in asymmetric somatic hybrids due to chromosome elimination (Kao, 1977; Kumar & Cocking, 1987). Somatic hybrids with chromosome number of 24, less

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51 than the allotetraploid (2n = 4x = 32), have been produced from carrot and barley (Kisaka et al., 1997). Somatic hybridization for citrus scion improvement has been intended for production of seedless triploid scion cultivars either by production of tetraploid somatic hybrids for crosses with diploids or by direct protoplast flision between diploid and pollenderived haploid protoplasts (Grosser & Gmitter, 1990). Somatic hybridization has been reported for producing somatic hybrids between graftand cross-incompatible cherry rootstock 'Cok' (Prunus avium x Pseudocerasus) and wild pear (Pyrus communis var. pyraster) (Ochatt et al., 1989). Further analysis of the somatic hybrid plants demonstrated that one clone was graft-compatible with both parents (Ochatt & Patat-Ochatt, 1994). Somatic hybridization has also been used to produce fertile allotetraploid somatic hybrids between Passiflora edulis f flavicarpa and P. incamata that have cold tolerance characteristics (Otoni et al., 1995). These somatic hybrid plants are fertile and showed intermediate characteristics of both parents for most characters observed; the natural hybrids of these species are sterile and only their tetraploid (after colchicine treatment) derivatives are fertile. Other somatic hybrids involving Passiflora species combinations have been intended to introgress desirable characteristic of wild species into Passiflora eJw/w f y7avzca/7?a (Domelas et al., 1995). Somatic hybridization has also been use to produce cytoplasmic hybrids (cybrids). Cybrid production in citrus involves the donor-recipient method (Vardi et al., 1987). Following fiision between protoplasts isolated from embryogenic cultures and from mesophyll protoplasts, diploid plants with morphological characteristics of the leaf parents have been recovered in addition to somatic hybrids (Saito et al., 1993; Tusa et al., 1990; Moriguchi et al., 1996). These plants were cybrids since their mitochondrial DNA was similar to the embryogenic parents and their nuclear DNA was similar to the leaf parent. The acquisition of mitochodrial DNA from the embryogenic parent seems to be a

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I 52 prerequisite for somatic embryogenesis from non morphogenic leaf mesophyll protoplasts (Grosser et al., 1996). To date there is no application for citrus cybrid production; however, cybridization may increase the availability of citrus germplasm for further somatic fusions (Grosser et al., 1996). Fusion and Selection Methods and Confirmation of Somatic Hybrids Protoplast fusion in woody plants is mostly achieved using either the polyethylene glycol (PEG) method or electrofusion. Dextran as a fusion agent was reported for somatic hybridization of Populus species (Park & Soon, 1994). PEG has been used to produce more than 150 citrus somatic hybrids (Grosser et al., 1996; Grosser, 1997, personal communication). Electroflision has been used to produce at least 10 somatic hybrids and cybrids (Saito et al., 1993; Ling & Iwamasa, 1994; Moriguchi et al., 1996; Motomura et al., 1995; Hidaka & Omura, 1992; Yamamoto & Kobayashi, 1995). The widespread use of PEG for somatic hybridization has demonstrated its eflScacy and efficiency. Furthermore, it is inexpensive, simple and does not cause protoplast mortality (Grosser & Gmitter, 1990). Selection of somatic hybrids in citrus involves the use of embryogenic cultures of one parent and non-morphogenic cell suspensions, callus or leaves of the other parent. The embryogenic cells confer regeneration potential to the fused protoplasts while unfused protoplasts of the non-morphogenic parent cannot develop in plant growth regulator-free medium. Habituated nucellar derived embryogenic cultures can be used for one parent in citrus somatic hybridization (Grrosser & Gmitter, 1990). Although habituated cultures have lost embryogenic competence, somatic hybrids could be recovered following fusion. It has been possible to regenerate the somatic hybrid alone by using medium with 0.6 M sucrose osmoticum (Ohgawara et al., 1994). This type of selection relies on heterosisor hybrid vigor-like expression in somatic hybrid cells or embryos, and has also been used to

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"v > 53 obtain somatic hybrids between Prunus and Pyrus (Ochatt et al., 1989) and Passiflora (Otonietal., 1995). Somatic hybrid identity is generally confinned by several methods, including morphology characters, chromosome counts and molecular markers, i.e., nuclear ribosomal DNA analysis, mitochondrial DNA analysis (Grosser & Gmitter, 1990) and Polymerase Chain Reaction (PCR)-based Random Amplified Polymorphic DNA (RAPD) analysis. Confirmation of somatic hybridization using RAPD markers in citrus has been shown in some cases to require more than one primer (Mourao-Filho, 1995). General Conclusion of the Literature Review Avocado is an important fiiiit crop; however, breeding new cuhivars has been slow and improvement of existing cultivars has not occurred. Biotechnology, i.e., somatic hybridization and genetic transformation, can create genetic variability otherwise unavailable through conventional breeding. Application of these technologies is dependent on the availability of eflBcient plant regeneration protocols fi^om suspension cultures, protoplasts or other tissues. The most efficient regeneration pathway for woody plants has been somatic embryogenesis. Although preliminary studies regarding somatic embryogenesis of avocado have been reported, the published information has been insufficient to utilize for improving avocado by somatic cell genetics. It is clear that work with other woody species (e.g., citrus, mango, etc.) might be applicable to similar in vitro studies with avocado.

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CHAPTERS INITIATION AND MAINTENANCE OF AVOCADO EMBRYOGENIC CULTURES Introduction The avocado (Persea americana Mill.) is one of the major fruit crops of the world. The species includes three horticultural races of different climatic adaptation; the tropical P. americana var. americana (West Indian), the less tropical P. americana var. guatemalensis (Guatemalan) and the semitropical P. americana var. drymifolia (Mexican). Despite its great importance, there have been relatively few reports related to cell and tissue culture of this species. The initiation of callus from different plant parts was described by Schroeder (1956; 1961; 1971; 1975; 1978), Blumenfeld & Gazit (1971) and Desjardins (1958); however, these cultures were nonmorphogenic. Embryogenic cultures were initiated from zygotic embryos excised from abscised avocado fruits, but no further somatic embryo development was reported (Barlass & Skene, 1983). Embryogenic cultures were also initiated from early stage 'Hass' (Pliego-Alfaro & Murashige, 1988), 'Fuerte' and 'Duke 7' (Mooney & van Staden, 1987) zygotic embryo explants. Mature somatic embryos were recovered from embryogenic cultures, and low frequency plant recovery was described. The effects of genotype on somatic embryogenesis were not addressed in these reports, and the establishment of embryogenic suspension cultures was not attempted. Conditions for induction of embryogenic avocado cultures of different genotypes and their growth on semisolid medium and in suspension have been described. 54

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1 55 Materials and Methods Embryogenic Cultures on Semisolid Medium Induction of embryogenic cultures from zygotic embryos Avocado fruitlets, 0.3-2.0 cm length without the calyx, representing different cultivars of different races and hybrids involving two or more races were collected from the USDA-ARS Subtropical Horticultural Research Station (Miami, FL) national avocado germplasm repository, the germplasm collection of the University of California, Riverside, CA and the University of Florida, Tropical Research and Education Center (Homestead, FL) (Table 3-1). After removal of sepals and peduncles, the fruitlets were surfacedisinfested in a 10-20% solution of commercial bleach containing 10-20 drops of Tween 20® per liter for 10-20 min. Fruitlets were rinsed with two changes of sterile, deionized water. The fruitlets were bisected under axenic conditions. The zygotic embryo was removed from each fruit (Pliego-Alfaro & Murashige, 1988) and transferred onto plant growth medium, i.e., induction medium. A single zygotic embryo was placed into culture in each 60 x 15 mm Petri dish and sealed with Parafihn®. Cultures were maintained in darkness at 25°C. The stage of development of the zygotic embryos and the corresponding fruit size for 'Thomas' are indicated in Table 3-2. The induction media consisted of 1) MS (Murashige & Skoog, 1962) major salts (MSP) and three modifications of MS, which included 2) omission of NH4NO3 (MS ), 3) substitution of NaNOs (MSNa) for NH4NO3 4) KNO3 (MSK) as the sole nitrogen source (vAth the concentration of nitrogen being equivalent to that in MS) and 5) B5 major salts (Gamborg et al., 1968). All of the major salts formulations were supplemented with MS minor salts, 0.41 ^M (0.1 mg 1"') picloram and (in mg l ') thiamine HCl (0.4), myo-inositol (100) and sucrose (30,000). The various plant growth media were solidified with 8 g 1"' TC agar (Carolina Biological Supply Company).

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56 Table 3-1. Avocado cultivars used for the experiments, their botanical varieties and their sources. uuiiivar OOlalUCai VancLy Booth 7 GxM TREC, UF Booth 8 GxM TREC,UF Duke 7 M UC Riverside Esther G X [(G X M) X G] UC Riverside Hass G UC Riverside Isham G USDA-ARS, Miami Irwing 56 Complex hybrids USDA-ARS, Miami Jose Antonio W USDA-ARS, Miami Lamb G UC Riverside M25864 M USDA-ARS, Miami T362 G UC Riverside Thomas M UC Riverside Waldin W USDA-ARS, Miami Yon GxW USDA-ARS, Miami From Smith et al. (1992) Table 3-2. Length of 'Thomas' avocado fruitlet (measured without calyx) in relation to zygotic embryo length, stage of development and morphology. Fruit Length Zygotic Embryo Developmental Morphology (cm) Length (mm) Stage <0.5 0.1-0.4 globular 0.5—0.8 0.5-0.8 early heart stage 0.9—1.1 1-2 late heart stage 1.2—1.5 3-7 early torpedo >1.5 >7 late torpedo, early cotyledonary stage

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57 The pH of aU media was adjusted to 5.7-5.8 with either KOH or HCl prior to addition of agar and autoclaved at 12 IT at 1.1 leg cm"^ for 15 min. The plant growth media were dispensed in 10 ml aliquots into sterile disposable Petri dishes (60 x 15 mm). Binomial confidence intervals of 95% were computed for each treatment mean using S AS program (SAS Institute, 1992), and for 0% occurrence, the upper limit of 95% confidence intervals were calculated using "the Rule of Three" (Jovanovic & Levy, 1 997) Induction of embryogenic cultures fi'om nucellar explants Fruitlets of 0.3-0.5 cm length (without calices) were surface-disinfested as described above. Each fixiitlet was bisected longitudinally and the endosperm and the zygotic embryo was removed fi^om each seed. The nucellus together with the integument were removed and plated on the surface of plant growth medium with the nucellus in contact with medium. ^ This experiment involved 4 genotypes. For 'Thomas', there were two explants fi-om a single ovule in 60 x 15 mm sterile plastic Petri dishes containing induction medium. There were five different induction media whose compositions were described above (see Induction of embryogenic cultures fi^om zygotic embryos); however, for the other genotypes, 'Hass', 'Lamb' and 'T362', six explants fi-om 3 ovules were explanted in 60 x 15 mm sterile plastic Petri dishes containing B5 induction medium. There were total of 36 explants (18 fiuitlets) per treatment for 'Thomas', 100 explants (50 fiiiitlets) for 'Lamb', 412 explant (206 fi\iitlets) and 36 explant (18 fixiitlets) for 'T362'. Percent responses were calculated based on the number of fioiitlets, not on the number of explants used for the experiments Maintenance of embryogenic cultures on semisolid medium Embryogenic cultures, consisting of proembryonic masses and early cotyledonary somatic embryos that developed on the various induction media, were transferred onto fi-esh semisolid medium of the same formulation after 2-4 weeks for 1-2 subcultures.

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58 They were thereafter subcultured at 2-4 week intervals on MSP medium (25 ml per 100 x 20 mm Petri dish). The proembryonic masses of 0. 1-1 mm diameter were used to form inocula with 0.2-0.5 cm diameter. Up to 7 inocula were plated on each Petri dish. Subculture intervals were 2-4 weeks. The Petri dishes were sealed with Parafilm®. Effect of medium formulation and gelling agent on the growth and development of 'Thomas' and ' Isham' embryogenic cultures Factorial experiments consisted of three media formulations and two types of gelling agent. The medium formulations included MS major salts, B5 major salts without (NH4)2S04 (35 ) and B5' supplemented with 400 mg 1"^ glutamine; the gelling agents tested were 8 g 1"' TC agar and 2 g l ' Gel-Gro® gellan gum (ICN Biochemicals). The major salts formulations were supplemented with MS minor salts, 0.4 mg 1"' thiamine HCl, 100 mg myo-inositol, 0.4 jxM picloram and 30 g 1"^ sucrose. The pH of the media was adjusted to 5.7-5.8 with either 0.1-1.0 N KOH or HCl prior to addition of gelling agent. The media were sterilized by autoclaving at 12 TC at 1.1 kg cm'^ for 15 min. Plant growth media were dispensed in 25 ml aliquots into sterile disposable plastic Petri dishes (100 x 20 mm). For 'Thomas', the inoculum consisted of 14-day-old embryogenic suspension cultures growing in 80 ml MSP medium in 250 ml Erlenmeyer flasks. Embryogenic cultures were decanted into a sterile funnel layered with 2 layers of sterile Kimwipes tissue paper. Approximately 200-300 mg proembryonic masses were subdivided to form 7 inocula of 0.3-0.5 cm diameter flattened on the surface of the growth medium. For 'Isham', the inoculum consisted of 14-day-old embryogenic suspension cultures maintained in 80 ml MSP medium in 250 ml Erlenmeyer flasks. The cultures were double sieved through sterile 1.8 mm and 0.8 mm mesh nylon filtration fabric. The smallest proembryonic mass fi-action was decanted into a sterile funnel layered with double layers of Kimwipes and was subdivided to form 0.2-0.3 cm diameter inocula on the

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59 surface of the media. There were seven inocula per Petri dish. The Petri dishes were sealed with Parafihn® and maintained in darkness at 25°C. For 'Thomas', the percent necrotic tissue and the number of somatic embryos that developed per inoculum four weeks after transfer were recorded and analyzed using ANOVA (SAS Institute, 1992). For 'Isham', the numbers of 1) proembryonic masses, 2) globular somatic embryos, 3) hyperhydric and opaque heart stage somatic embryos (diameter <5 mm) and 4) hyperhydric and opaque early torpedo stage somatic embryos (diameter ^5 mm) per inoculum were recorded after four weeks of culture. Embryogenic Suspension Cultures General procedures The following procedures were applicable for all experiments unless specified otherwise. Medium sterilization was carried out either by autoclaving at 121° C at 1.1 kg cm"^ for 15 min or by millipore filter-sterilization with a 0.2 ^m sterile filter. For filtersterilized media, the flasks were sterilized prior to use by autoclaving at 121° C at 1.1 kg cm"^ for 20 min. The volume of liquid medium was either 40 ml in 125 ml Erlenmeyer flask (referred to hereafter as 40 ml medium) or 80 ml in 250 ml Erlenmeyer flasks (referred to hereafter as 80 ml medium). The pH of all media was adjusted to 5.7-5.8 with either 0. 1-1 N KOH or HCl. The cultures were sealed with heavy duty aluminumfoil and secured with Parafilm®, and maintained in semi darkness at 25°C on a rotary shaker at 125 rpm. Initiation Embryogenic suspension cultures were established by inoculating 100-300 mg of 8-10-day-old embryogenic cultures from semisolid MSP medium into either autoclaved or filter-sterilized 40 ml liquid MSP medium.

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60 Maintenance Embryogenic suspension cultures were subcultured biweekly into filter-sterilized 80 ml MSP medium. For cultures that consisted entirely of proembryonic masses without later stages of development (PEM type), e.g., 'Esther' and 'M25864', 0.8-1.0 g proembryonic masses was used as the inoculum. For cultures up to 12 months old, proembryonic masses were sieved through nylon filtration fabric (1.8 mm or 0.8 mm mesh) and the smallest fi-actions were used as inocula. For older cultures (>12 months), sieving was unnecessary since the size of proembryonic masses was more homogenous and smaller than in newly established cultures. 'Thomas' cultures were also maintained as described for the PEM type. For newly established cultures that consisted of proembryonic masses that diflFerentiated as somatic embryos in maintenance medium (SE-type), only proembryonic masses and globular embryos that passed through 0.8 mm mesh nylon filtration fabric were used as inocula. With older cultures that contained dedifferentiated proembryonic masses, 0.8-1 g proembryonic masses that passed through either 1.8 or 0.8 mm mesh nylon filtration fabric were used as the inoculum. The morphology of embryogenic cultures of different cultivars was observed during a 1-2 year period. Growth of 'Esther' embryogenic suspension cultures Embryogenic suspension cultures of 'Esther' were used to determine the growth pattern of embryogenic cultures, because this genotype proliferated in liquid growth medium without concurrent production of early stage cotyledonary somatic embryos. 'Esther' embryogenic suspension cultures (12-14-day-old) maintained in liquid MSP plant growth medium were used as inoculum. Suspension cultures were sieved through sterile nylon 1.8 mm mesh fihration fabric and were retained on 0.8 mm mesh nylon filtration fabric. The sieved cultures that were retained on 0.8 mm mesh filtration fabric were decanted onto a double layer of sterile tissue paper (Kimwipes) and 420 ± 20 mg

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61 proembryos were used as the inoculum in 40 ml autoclaved MSP medium. There were 24 experimental units that consisted of 3 replicates of 8 medium flasks. Each medium flask in a replicate was inoculated with 420 ± 20 mg proembryonic masses that were randomly harvested at day 0, 3, 6, 9, 12, 15, 20 and 25. Growth parameters that were recorded included the volume of the precipitated culture, fi-esh weight and dry weight. Precipitated culture volume was measured by decanting cultures into sterile 50 ml graduated centrifuge tubes and allowing them to settle. Fresh weight was determined by pipetting out the Uquid medium fi-om the centrifuge tubes and transferring the proembryonic masses to preweighed weighing dishes. Dry weights were obtained by drying the cell masses in an oven at 55° C overnight, and weighing them 30 min after they were removed from the oven. Regression analyses were fitted to the data using Sigma Plot (version 2.0, Jandel™ Scientific, San Rafael, CA). Effect of picloram on growth of 'M25864' suspension cultures . . The effect of picloram on the growth of 'M25864' in suspension culture was examined at sbc concentrations: 0, 0.13, 0.41, 1.25, 4.14 and 12.48 ^iM. The plant growth medium used for the experiment consisted of MS salts with 0.1 mg f' thiamine HCl, 100 mg myo-inositol, 30 g 1'' sucrose and picloram as treatment. Each treatment was replicated four times. The inoculum for the experiment was 14-day-old 'M25864' embryogenic suspension cultures that had been maintained in suspension for ca. one year. Media preparation, preparation of the inoculum and inoculation, cultural conditions and data collection were as described for the growth response experiment above. Regression analyses were fitted to the data using Sigma Plot (version 2.0, Jandel™ Scientific, San Rafael, CA).

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62 EflFect of sucrose on the growth of 'M25864' suspension cultures Sucrose concentration in liquid medium was examined at six diflFerent levels: 20, 30, 40, 50, 60 and 70 g 1"' . The plant growth medium consisted of MS salts, 0.4 |iM picloram, 0.1 mg 1'^ thiamine HCl, 100 mg 1"^ myo-inositol and sucrose as treatment. There were 4 replications per treatment. Medium preparation, source of inoculum, inoculum preparation, inoculation, cultural conditions, data collection and statistical analysis were as described for the picloram experiment. Effect of thiamine HCl on growth of 'M25864' suspension cultures The effect of thiamine HCl on the growth of 'M25864' embryogenic suspensions was evaluated at six concentrations: 0, 0.4, 1, 4, 10 and 40 mg r\ The plant growth medium consisted of MS salts, 0.4 ^M picloram, 45 g 1"' sucrose, 100 mg 1"' myo-inositol and thiamine HCl. Each treatment had four replicates. Media preparation, source of inoculum, inoculum preparation, inoculation, cultural conditions, data collection and statistical analysis were as described for the picloram experiment. Effect of ascorbic acid and medium sterilization on growth of suspension cultures Ascorbic acid (100 mg 1"') in the plant growth medium and medium sterilization approach, i.e., autoclaving or filter sterilization, were evaluated in a block factorial design with three replications for two embryogenic lines, 'M25864' and 'Esther'. The plant growth medium used for the experiment was MSP formulation (Table A-1). The source of inoculum, inoculum preparation, inoculation, cultural conditions and data collection were as described for the picloram experiment. Analysis of variances of the data were computed using Proc. GLM, and t-test was also performed (SAS Institute, 1992).

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> 63 Results Induction of Embryogenic Cultures on Semi-solid Medium Embryogenic cultures were induced from avocado zygotic embryos 18-40 days after explanting and were associated with zygotic embryos of different developmental stages from globular (0.10 mm) to early torpedo stage (2.7 mm). The frequency of somatic embryogenesis of different genotypes on different medium formulations was generally low, from 0 to 25% (Table 3-3). No induction medium effect could be statistically inferred; however, there was an indication that induction medium containing B5 major salts stimulated the greatest embryogenic response from the most genotypes. Induction of embryogenic cultures generally occurred from the basal part of zygotic embryos, with the single exception of explanted globular zygotic embryos of 'Esther', which were completely embryogenic. It was not clear whether or not the basal part of the zygotic embryos included the hypocotyl region since this basal area was very small. There were two distinct types of embryogenic cultures that were initiated from the explants. 1) Induction of proembryonic masses without heart and later developmental stages of somatic embryos, i.e., PEM type. The PEM type response included 'Esther', and 'M25864'. 2) Induction and development of somatic embryos, including formation of globular to heart stages from the surface of the zygotic embryo explants (Figure 3-1), i.e., SE type. The SE type response included 'Booth 7', 'Booth 8', 'T362', 'Yon', nucellarderived 'Thomas', zygotic-derived 'Thomas' and 'Isham'. : Induction of embryogenic cultures in nucellar explants occurred primarily from the mycropylar end of the explants (Figure 3-1 C). However, embryogenic cultures were rarely induced from the inner surface of the nucellus. Only two explants of 'Thomas' that were cultured on MSNa and B5 responded out of 18 ovules per treatment. The induction

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64 o O O O O ^ I-—* CO \^ \^ oooonooooovoo c o 3 (/3 f^t»,v£)^/-^^oo«n>nr~vo OO(S0\OOfSr-i 4) N 3 ooo>— imr^ooo .—Cr-H,— l^frj,— (Tj-OO— 1 — >— I in o 0-r ^. 1 00 o O o o 00 00 5 XI «-> ii o o o o PQ m \0 CO ^ a J3 o l-H E"' Hi T3 u c ,o c .S 1 3 2

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65 Figure 3-1. Development of secondary somatic embryos from zygotic embryos and proembryonic masses from nucellar explants of Thomas' avocado. (A) Secondary somatic embryos developed on the basal region of a cotyledonary zygotic embryo explant of 2 mm length. (B) Similar to (A) except that the zygotic embryo was at heart stage of 0.5 mm length. (C) Proembryonic masses (white) developed from a nucellar explant.

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Figure 3-2. Morphological variations of avocado embryogenic cultures. Two distinct type of embryogenesis: PEM-type that is dominated by the appearance of disorganized proembryonic masses (A, D, G and J), SE-type that is dominated by highly organized proembryonic masses and somatic embryos (C, F, I and L), and cultures that are undergoing morphological changes (B, E, H and K). (A) 'Esther' embryogenic cultures on semisolid medium. Note the friable texture and granular nature of the culture. (D) The suspension culture of 'Esther' (A). Note the homogenous nature of the inoculum. (G) Newly established suspension culture of 'Esther' consisting of mostly proembryonic masses and few organized embryogenic masses (arrowhead). (H) 'Esther' embryogenic suspension without organized proembryonic masses. This culture has been in culture longer than (G). (C) 'Isham' embryogenic culture on semisolid medium. Note the development of globular to heart stage somatic embryos covering the entire surface of the proembryonic masses. (F) The development of various stages of somatic embryo development when 'Isham' proembryonic masses were transferred to liquid medium. (I) Another example of SE-type of culture ('Booth 8') which is dominated by development of highly organized proembryonic masses or globular somatic embryo. (B) Embryogenic culture of 'T362' consisting of organized nodular proembryonic masses (lower part) and fine granular and disorganized proembryonic masses. (E) Suspension culture of 'T362'consisted of organized proembryonic masses/somatic embryos, fine granular and disorganized proembryonic masses. (H) A higher magnification of (E). Note the disorganization at the surface of the proembryonic masses. (K) 'Booth 8' suspension culture consisting of disorganized cell clumps incapable of somatic embryogenesis.

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68 of nucellar embryogenic cultures for 'Lamb', 'Hass' and 'T362' was also low, i.e. 2%, 0.5% and 6% respectively. Maintenance of embryogenic cultures on semisolid medium Transfer of embryogenic cultures (PEM-type) that developed on MSP onto fresh medium of the same composition resulted in continued proliferation of the cultures with occasional development of heart and later stages of somatic embryos. Morphologically, the cultures were friable (Figure 3-2 A) and either yellow-honey ('Esther') or white-brown ('M25864'). After several subcultures, the proembryonic masses were smaller and paler and some of the proembryonic masses had become disorganized. Two to three weeks after routine transfer onto MSP medium, the upper part of the cultures was brown; however, tissue in contact with the medium was healthy and proliferated. Cultures were almost completely necrotic after 5-6 weeks. Transfer of somatic embryos that developed directly from explanted zygotic embryos (SE-type) to medium of the same composition, e.g., B5, MSK, MSNa, resulted in maturation of the somatic embryos and formation of a few secondary somatic embryos from the basal part of the somatic embryos. They were alternatively subcultured onto MSP medium. After several transfers onto MSP medium, proliferation of proembryos, proembryonic masses and globular somatic embryos was enhanced (Figure 3-2 B). Proembryos were characterized by granular morphology and small size (ca. 0. 1-0.2 mm). The globular somatic embryos were characterized by granular morphology with smooth surfaces and ca. 0.3-1 mm diameter and the formation of secondary somatic embryos at one pole instead of scattered around the entire surface. The proembryonic masses were characterized by their nodular morphology with diameter of ca. 1-2 mm and the formation of secondary somatic embryos and proembryonic masses on their surfaces. These structures gave rise to a mixture of globular, heart and cotyledonary stage somatic embryos and proembryonic masses ca. 2-3 weeks after transfer. The somatic embryos

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69 that developed in this medium were mostly hyperhydric and their shapes were distorted. Early and late heart stage somatic embryos produced proembryonic masses and secondary globular somatic embryos from their bases. Therefore the gross morphology of this type of culture (SE-type) was dominated by the appearance of proembryonic masses and hyperhydric somatic embryos at various stages of development. SE-type cultures were maintained by subculturing proembryos, proembryonic masses and globular somatic embryos. After ca. 5-10 subcultures, depending on the genotype, the embryogenic cultures also consisted of smaller cell masses with less organization, and which appeared to be disorganized proembryonic masses. In one genotype, 'Isham', disorganized proembryonic masses did not develop. Instead, globular, heart and cotyledonary stage somatic embryos developed from the entire surface of proembryonic masses (Figure 3-2 C). Distal to the culture medium, the cultures became brown 2-3 weeks after transfer. Effect of medium composition and gelling agent on growth and development of nucellusderived 'Thomas' and 'Isham' embryogenic cultures. Medium composition significantly affected the percentage of 'Thomas' cultures that became necrotic after four weeks. MS formulation resulted in the lowest frequency of necrosis (Table 3-4). Type of gelling agent had no significant effect on necrosis of cultures nor was there an interaction of gelling agent with medium with respect to necrosis. Somatic embryo development was not significantly affected by the different treatments. Table 3-5 demonstrated that the proliferation of 'Isham' proembryonic masses was significantly affected by media formulation and the interaction of media formulation with gelling agent. The highest value of proembryonic mass proliferation occurred with MS major salt formulation and solidified with TC agar (Figure 3-3).

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70 Table 3-4. Effect of major salt composition, and gelling agent on necrosis and somatic embryogenesis of 'Thomas' nucellar culture. Major salts* Gelling agent Necrotic tissue {/o) fNO. somauc cmoryos (MeaniSE) (Mean ± SE) MS 8 e r' TC Agar 17 ±8 0.28 ±0.15 ^ ^ 2gl"'Gel-Gro 27± 16 0.46 ± 0.28 BS 8 g 1' TC Agar 100 ±0 0.74 ±0.21 2 g r' Gel-Gro 100 ±0 0.51 ±0.16 B5G Sgl'TCAgar 100 ±0 0.57 ±0.23 2 gl"' Gel-Gro 100 ±0 0.00 ± 0.00 Anova Summarv Major salt (M) **" NS" Gelling agent (G) NS NS M*G NS NS * Major salt of MS (Murashige & Skoog, 1962), B5 (Gamborg et aL, 1968), B5 G is 35 major salts without (NH4)2 SO4, with 400 mg l ' glutamine. Number of early stage somatic embryos per inoculum. ' Significant at a = 1%; ^ Not Significant at a = 1% Table 3-5. Summary of the value of Pr>F from ANOVA for the effect of major salt composition, and gelling agent on the proliferation of proembryonic masses and development of somatic embryos from 'Isham' cultured on semisolid medium. Dependent Variable Component Major Salts (M) Gelling Agent (G) M*G . . . Pr>F Proembryonic masses' 0.0060 0.1188 0.0037 Globular SE' 0.2620 0.7634 0.0101 Cotyledonary SE<5nim Opaque^ 0.0001 0.1176 0.0574 Hyperhydrous'^ 0.0041 0.0840 0.0051 Total' 0.0002 0.0001 0.0400 Cotyledonary SE^5 mm Opaque^ 0.0544 0.7294 0.1126 Hyperhydrous^ 0.0001 0.0001 0.0001 Total' 0.0002 0.0001 0.0400 Total Hyperhydrous SE^ 0.0001 0.0001 0.0157 Total Opaque SE' 0.0008 0.2811 0.0493 Total Cotyledonary SE' 0.0017 0.0004 0.0561 'data were normally distributed, therefore no transformation, ^data were not normally distributed and were transformed with arc sin transformation.

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C O I m iL (Q Oi < O E (0 (0 ' (0 (0 I— o to' m m iL CO < a> O) (D D) c O) = ? CD CO o < • O J L C>4 CO CM gsjp J8d jequjnN CA e o •a o -I o o. Cm O G O I o V »-> c o e<3 O T3 -5 o ^ E o c 2 OJ g MIS .S > rri B ^ O m ««

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. > 72 Somatic embryo growth and development, as indicated by different sizes, stages and hyperhydricity, was prolific with 'Isham'. The development of globular somatic embryos was not significantly affected by medium and gelling agent but was significantly affected by their interaction. Medium v^th B5'G solidified with Gel-Gro™ resulted in the highest number of globular somatic embryos. Opaque cotyledonary somatic embryo < 5 mm diameter development was only affected by medium, v^th the highest value occurring on medium B5"G. The presence of hyperhydric cotyledonary somatic embryos < 5 mm was significantly affected by medium and the interaction of medium with gelling agent, and the highest number occurred with the treatment of B5 and Gel-Gro™. The total number of cotyledonary somatic embryos < 5 mm was significantly affected by medium formulation, gelling agent and their interaction, and the highest number was obtained with medium consisting of B5"G and Gel-Gro™. There was no significant effect of treatment on development of opaque cotyledonary somatic embryos > 5 mm diameter. Hyperhydric cotyledonary somatic embryos > 5 mm diameter were significantly affected by media formulation, gelling agent and their interactions, with the highest number obtained on MS with Gel-Gro™. Total number of cotyledonary somatic embryos > 5 mm was significantly affected by media formulation (P>F = 0.0002) and gelling agent (P > F = 0.001) but not by their interaction (P>F = 0.04). Duncan Multiple Range Test (DMRT) indicated that B5 medium was optimum for total somatic embryos followed insignificantly by MS and significantly by B5"G. T-test indicated that Gel-Gro™ resulted in more somatic embryos than TC agar (P> T = 0.0075). The effect of treatment on number of somatic embryos (of all stages) was the same as the effect on the total number of large somatic embryos. Initiation of Embryogenic Suspension Cultures. Rapidly growing, 8-10 day-old proembryonic masses with a uniform white or yellow color were used as inoculum. All genotypes, with the exception of 'Isham', that

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were established successfully on semisolid medium could also be established in liquid MSP medium. Maintenance The two distinct types of cultures, PEM-type and SE-type, described on semisolid medium, retained these characteristics in newly-estabUshed embryogenic avocado suspensions. In the PEM-types, i.e., 'Esther' and 'M25864', the suspensions consisted of less organized proembryonic masses of different sizes with a low frequency of globular and heart stage somatic embryos (Figure 3-2 D, G). In the SE type, the suspensions consisted primarily of globular to cotyledonary somatic embryos (Figure 3-2 F, L). The PEM-type could be maintamed by subculturing any fraction of the cultures; however, maintenance of the SE-type required the smallest fraction of cultures (<0.8 mm mesh size) as inoculum. Newly established cultures of both types of suspensions were characterized by production of abundant cell debris. With time and regular subculture, the morphology of embryogenic suspension cultures changed. Globular, heart and early cotyledonary stage somatic embryos that occurred at low frequency in the PEM-type, e.g., 'Esther', started to disappear after 12 months and the cultures consisted of increasingly disorganized proembryonic masses (Figure 3-2 J). Similar morphological changes also occurred in SE-type cultures, with several variations. For example, proembryonic masses or globular somatic embryos derived from zygotic embryos of 'T362' and 'Lamb' lost their ability to organize as heart stage somatic embryos, but retained their morphology 6-18 months before becoming completely disorganized. The periphery of proembryonic masses and globular somatic embryos underwent limited disorganization (Figure 3-2 H), which eventually resulted in loss of organization (nucellar-derived 'T362', 'Booth 8', 'Yon'). Globular somatic embryos

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developed from proembryonic masses, but partially dedifferentiated, forming less organized proembryonic masses (nucellar-derived 'Thomas', 'Hass') or formed partially disorganized proembryonic masses and large, free vacuolate cells (zygotic embryo-derived 'Thomas', 'Hass') before completely disorganizing. Completely disorganized proembryonic masses consisted of small isodiametric cells in clusters that were either slow growing ('Lamb') or fast growing ('T362', zygotic embryo-derived 'Thomas') (Figure 3-2 K). 'Isham' could not be maintained in suspension. The proembryonic masses that were inoculated developed into globular to cotyledonary stage somatic embryos in the presence of picloram. Therefore 'Isham' was maintained by alternating subculture of semisolid with liquid media; proembryonic masses from semisolid medium were inoculated into liquid medium and globular somatic embryos and proembryonic masses in liquid medium were subcultured onto semisolid medium before they could develop as later stage somatic embryos. Unorganized proembryonic masses were never recovered from cultures of 'Isham'; however, the size of somatic embryos that developed on semisolid or liquid medium decreased with time. Time required for organized proembryonic masses or globular somatic embryos to become completely disorganized was genotype-dependent (Table 3-6). Growth responses of 'Esther' Embryogenic Suspension Cultures 'Esther' embryogenic suspension culture growth pattern could be fitted with a curvelinear trend with respect to tissue volume, fresh weight and dry weight. The fitted regression lines had high coefiBcient of correlation values (r^ = 0.96-0.98) (Figure 3-4). There was a short lag phase (ca. 1 day) with respect to volume of 'Esther' embryogenic suspension cultures, followed by 5 days of exponential growth, 9 days of linear phase, 5 days of progressive decelerating growth and stationary or declining growth through 25

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Table 3-6. Characteristics of avocado embryogenic suspension cultures. Cultivar of Mother Explant Embryogenesis Time Required for PEM Tree Type Disorganization Booth 7 zygotic SE 8-12 month Booth 8 zygotic SE 8-12 months Esther zygotic PEM 12-24 months Hass nucellus SE 6-12 months Isham zygotic SE never Lamb nucellus SE 3-6 months M25864 zygotic PEM 8-12 months T362 zygotic SE 12-18 months T362 nucellus SE 6-12 months Thomas zygotic SB 12-18 months Thomas nucellus SE 12-18 months Yon zygotic SE 3-6 months days of culture. A trend similar to volume variable was observed for fresh weight, except that the lag phase was ca 1 day longer and the exponential phase was 1 day shorter. Dry weight, however, increased without an apparent lag phase, but with a sharp exponential phase for ca 4 days, followed by a linear phase for 8 days, and progressively decelerating phase for 6 days and declining thereafter. The peak for each variable was predicted by its regression line to occur at different days. Dry weight peaked at 18 days, followed by fresh weight (20 days) and volume (21 days). Increases in volume, fresh weight and dry weight under the growth conditions described were ca 14-fold, 6.4-fold and 7.9-fold, respectively. During the declining phase, loss of dry weight from the peak to the end of culture period accounted for ca. 40%. This dry weight loss was much higher than the loss in volume and fresh weight, each of which accounted for 17%.

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76 Effect of picloram. sucrose and thiamine HCl on the prowth of 'M25864' embrvogenic suspension cultures. Regression analysis of volume and dry weight with respect to picloram concentration resuhed in very low r^ values (< 0.05), indicating that picloram had no effect on those growth variables. The fresh weight of the cultures decreased curvelinearly (r^ = 0.37) with picloram concentration (Figure 3-5). Therefore, the concentration of 0.41 (0.1 mg r^) was retained as the standard concentration of picloram for suspension cultures. Sucrose affected the volume, fresh and dry weight of 'M25864' embryogenic suspension cultures curvelinearly; however, its coefficient of correlation was low (r^ = 0.34-0.35). The volume and fresh weight of the embryogenic suspension culture peaked at a sucrose concentration of ca. 25 mg 1"\ while the dry weight peaked at ca. 50 g 1"'. The increase in dry weight that resulted from increased sucrose concentration from 20 to 50 g 1"^ may indicate increased starch content since it coincided with the decrease of volume and fresh weight of cultures at a sucrose concentration of 25 g 1"* and thereafter. Sucrose concentration of 30 g 1"' has been used as standard sucrose concentration in the medium. Regression analysis of the effect of thiamine HCl concentration in the medium demonstrated that r^ for volume, fresh weight and dry weight of the culture were very low, i. e. 0.16, 0.11 and 0.004, respectively. This low value indicated that thiamine HCl was not very critical for growth. Nevertheless, based on the increasing trend of the volume and fresh weight with increasing thiamine HCl concentration from 0 to 10 mg 1'* , a thiamine HCl concentration of 4 mg 1"' has been adopted as standard for avocado maintenance medium (MSP).

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77 Figure 3-4. Growth response of 'Esther' avocado embryogenic suspension cultures over time (T). The regression lines are represented by A) volume = 0.44 +0.013 T + 0.040 T2 0.0013 T3, r2 = 0.98, B) fresh weight = 0.46 0.03 T + 0.021 T2 0.00067 T3, r2 = 0.96 and C) dry weight = 0.03 +0.0081 T + 0.0015 T2 0.000063 T3, r2 0.96.

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78 8 • ' 0.28 0-^H25 4.14 12.48 0.13 041 Picloram concentration (jjM) Figure 3-5. Effect of picloram (P) on growth response of 'M25864' avocado embryogenic suspension cultures after 14 days. The growth responses and their regression lines are represented by: (a) volume, Y = 6.9 0.03 P 0.005 P^, r^ = 0.05; (b) Y = 3.4 0.1 P -0.004 P^ ^ = 0.37; (c) Y = 0.35 0.02 P 0.0002 P^ ^ = 0.03.

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79 Figure 3-6. EflFect of sucrose (S) on the growth response of 'M25864' avocado embryogenic suspension cultures after 14 days. The growth responses and their regression lines are represented by: (a) volume, Y = 4.9 0.09 S 0.0014 r^ = 0.35; (b) Y = 1.25 0.037 S -0.0005 r^ = 0.34; (c) Y = 0.1 0.01 S 0.0001 S^ r^ = 0.35.

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lilt I I L ,^/fl\4 10 40 0-4 1 Thiamine HCI (mg Figure 3-7. Eflfect of thiamine HCI (T) on the growth response of 'M25864' avocado embryogenic suspension culture after 14 days. The growth responses and their regression lines re represented by: (a) volume, Y = 7.1 0.07 T 0.002 T^, r^ = 0. 15; (b) Y = 4.0 0.04 T 0.008 T^ = 0. 1 1; (c) Y = 0.47 0.02 T 0.0005 T^ r^ = 0.004.

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autodave filter aiodave filter @2 1 0.6 0.4 0.2 (Q autodave filter Medimstetilizalion (B) (D) "T autodave filter Medumstedlizadon 0.3 0.2 0.0 0.12 0.06 0.04 Figure 3-8. Effect of medium sterilization protocol on the growth of avocado embryogenic suspension cultures. Data from cultures in medium with and without ascorbic acid were pooled to assess effects of medium sterilization protocol. Medium was either autoclaved at 1.1 kg cm"^, 121° C for 15 min or filtered with 0.2 ^m filter unit. (A) Fresh weight of 'Esther' avocado, (B) dry weight of 'Esther' avocado, (C) fi-esh weight of 'M25864' avocado, (D) dry weight of 'M25864' avocado. Bars in the histograms represent standard errors of the means.

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C2 Effect of ascorbic acid and medium sterilization method on growth of suspension cultures Ascorbic acid in the medium and its interaction with the medium sterilization method did not significantly affect (P > 0.05) the growth of either 'Esther' or 'M25864' embryogenic suspension cultures. Medium sterilization method significantly affected fi-esh weight (P < 0.01) and dry weight (P < 0.01) of 'Esther', and fi-esh weight (P < 0.05) and dry weight (P < 0.05) of 'M25864'embryogenic suspension cultures. Data fi^om cultures in plant growth media with or without ascorbic acid were pooled to determine affects of medium sterilization protocol. The t-test showed that filter sterilization resulted in significantly higher fi-esh weight (P < 0.01 for 'Esther'; P < 0.05 for 'M25864)' and dry weight (P < 0.01 for 'Esther'; P < 0.05 for 'M25864') than autoclaving (Figure 3-8). Therefore, filter sterilization has been used as the standard protocol for preparing liquid maintenance medium. Discussion Somatic embryogenesis in avocado had been reported previously using immature zygotic embryos as explants (Skene & Barlass, 1983; Mooney & Van Staden, 1987; Pliego-Alfaro & Murashige, 1988). Using a similar method, embryogenic cultures have been established both on semisolid and in liquid medium fi-om immature zygotic embryos and fi-om nucellar explants obtained fi-om 12 avocado cultivars/genotypes. The establishment of avocado embryogenic cultures was characterized by direct formation of proembryonic masses or somatic embryos fi-om the explant without apparent development of callus. This embryogenic culture establishment has been referred to as the proembryogenic determined cell (PEDC) pathway (Sharp et al.,1980) or permissive induction pathway (Ammirato, 1987), in contrast to the induced embryogenic determined cell (lEDC) pathway (Sharp et al., 1980) or directive induction pathway (Ammirato, 1987), which involves the formation of callus prior to the development of somatic embryos. The embryogenic response of avocado nucellar and zygotic embryo explants

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83 was quite low. Low frequency of embryogenic culture initiation from immature zygotic embryos or nucellar explants has been reported for some other woody species, e.g., <1% for big leaf magnolia (Merkle & Warson-Pauley, 1993), <5% for Comus floribunda (Trigiano et al., 1989) and pecan (Corte-Olivares et al., 1990). A low embryogenic response was also observed from the nucellus of monoembryonic Citrus spp., e.g., microcitrus (Vardi et al., 1986) and seedless 'Cohen' citrange (Grosser et al., 1993). However, a high embryogenic response (60-80%) was reported from immature zygotic embryo explants of Quercus robus (Chalupa, 1995). Moore (1985) found that ovules of monoembryonic Citrus spp. did not proiduce embryogenic cultures while the polyembryonic species produced somatic embryos. Chaturvedi and Mitra (1972) indicated that a correlation appeared to exist between degree of polyembryony and somatic embryogenesis. The size of zygotic embryo explants was reported to be critical for initiating avocado embryogenic cultures (Pliego Alfaro & Bergh, 1992; Mooney &. van Staden, 1987); however this variable may be genotype-dependent, since embryogenic cultures could be induced from zygotic embryos ca. 2 mm length in the current study. Nucellar tissues from very young fruit could also be used for initiating embryogenic cultures at low frequency. Induction of embryogenic avocado cultures from nucellar explants could have significant horticultural implications. Somatic embryogenesis from avocado nucellus could replace the expensive etiolation technique that is currently used for clonally propagating avocado rootstock cultivars (Frolich & Piatt, 1971). Single character improvement using elite scion genotypes could also be accomplished via genetic transformation. Establishing and maintaining embryogenic avocado cultures was based upon initiation of repetitive or secondary somatic embryogenesis from cultures on/in plant growth medium containing B5 major salts and then using the secondary somatic embryos as explants on medium containing MS major salts. B5 major salts-containing medium with

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84 a low concentration of reduced inorganic nitrogen appears to favor somatic embryo development and has been used for mango somatic embryo development (Litz et al., 1991; 1995; Mathews & Litz, 1992). Muralidharan & Mascarenhas (1995) also demonstrated that Eucalyptus citriodora embryogenic masses proliferated in MS-based medium, while somatic embryo development (maturation) increased on B5-based medium. Another characteristic of embryogenic avocado cultures was the distinction between the PEM-type (2 embryogenic culture lines) and the SE-type (12 embryogenic culture lines) that was evident after culture initiation and which was consistent during maintenance on semisolid medium and in liquid medium. This distinction could be an important marker for predicting somatic embryogenesis frequency since the PEM-type is associated with lower frequency of development of somatic embryos (see Chapter 4). Proliferation of proembryonic masses requires an exogenous auxin and its withdrawal results in somatic embryo development (Litz & Gray, 1995). Therefore, the different types of avocado embryogenic cultures reflect differential sensitivity to auxin. The different types of somatic embryogenesis in avocado may also indicate differences in the integrated development of the somatic proembryos (William & Maheswaran, 1986). Embryogenic cultures resembling avocado of the PEM-type, i.e., characterized by discreet globular or clusters of globular, organized structures, yellow or white, with gross morphology consisting of fnable, granular/nodular structures, have also been reported for Salix vimitmlis (Gronrros, 1995), Rosa hybrida (Robert et al., 1995), cacao (Figuera & Janick, 1995), apple (Wallin et al., 1995) and cotton (Finer, 1988). The histology of an avocado embryogenic culture has been reported by Mooney & Van Staden (1987) to consist of somatic proembryos from 0.1-1.0 mm. The nodular structures were actually proembryonic masses, since they lacked a discernible protoderm and proembryos continually formed on the surface of the proembryonic masses. These structures resemble proembryonic masses that constitute embryogenic cultures of citrus (Button et al., 1974) and mango (DeWald, 1987; Litz et al., 1995; 1992, etc.). Ammirato (1984) indicated that

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85 when proembryos are maintained in induction medium, they may not organize as somatic embryos but will continue to enlarge and form proembryonic masses that form secondary proembryos on their surface. Avocado embryogenic cultures can be maintained both in liquid suspension cultures and on solid medium. The ease of prohferation and maintenance of avocado embryogenic cultures would provide regenerable materials for fiirther culture manipulation involving somatic cell genetics or transformation. Using zygotic-derived embryogenic culture of 'Thomas', Cruz-Hernandez et al. (1998) reported Agrobacterium-mediated transformation of avocado with GUS and NPT n genes and recovery of transformed somatic embryos. Changes involving gross morphology of embryogenic cultures of the SE-type occurred after a relatively short time and can be implicated in the progressive loss of embryogenic potential of these cultures. William and Maheswaran (1986) suggested that the loss of integrated organization of globular embryos is associated with appearance of proembryonic masses in embryogenic cultures. The morphology of disorganized avocado proembryonic masses is similar to embryogenic cultures that have become "undifferentiated" and which have been selected from the most friable nucellar Citrus deliciosa cultures (Cabasson et al., 1995) and from habituated nucellar cultures of Citrus aurantium L. (Gavish et al., 1991) even though these cultures could be induced to form somatic embryos. Chaturvedi and Mitra (1975) found that after prolonged subculture the organized globular embryogenic culture initiated from stems of Citrus grandis became highly fiiable, but embryogenic. Embryogenic avocado suspension cultures of the PEM-type grow as typical suspension cultures, with a 2 week subculture period with a 5.5-fold increase in mass, etc. Most of the attempts to improve growth of PEM-type embryogenic cultures in liquid medium by altering the organic addenda of the plant growth medium were not successfiil. Increase in dry weight of the suspension cultures with 50 g 1"' sucrose was demonstrated

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t 86 but 30 g sucrose has been used routinely. Increased dry weight with sucrose concentrations above 30 g 1"^ only resulted in accumulation of starch in the cells. High starch content in embryogenic cells must be avoided if cultures are to be used as a source for protoplasts (Grosser, 1994). Filter sterilizing liquid medium significantly improved the growth of avocado embryogenic cultures. Medium autoclaving has been reported to cause the formation of toxic substance as the result of breakdown of sucrose and other carbohydrates (Shiao & Bomman, 1991). The loss of embryogenic potential of avocado embryogenic cultures in a relatively short time could be disadvantageous since 1) studies of somatic embryo development, maturation and germination require several months (see Chapter 4) so that the same cultures cannot be used indefinitely; 2) gene transformation or somaclonal variation experiments that require long periods of subculturing (selection) may result in loss of embryogenic potential and failure to recover somatic embryos. Therefore conservation methods are required to solve this problem. Preliminary experiments have indicated that avocado embryogenic cultures can be initiated fi"om embryogenic cultures on semisolid medium that have been stored for 6 months at 6-10° C. However, cryopreservation might be explored for this species, using proembryonic masses, microcalli and eariy globular stage somatic embryos. Cryopreservation protocols and subsequent plant regeneration have been developed for different tropical plant trees, (Withers, 1992; Engelman, 1991), including nucellar-derived embryogenic cultures (Kobayashi et al., 1990; Sakai et al., 1990), proembryonic masses and early stage somatic embryos ( Marin et al., 1993) of navel orange (Citrus sinensis (L.) Osb.), embryogenic cells of rice (Oryza sativa L) (Jain et al., 1996), embryogenic cell suspensions and callus cultures of cotton (Gossypium hirsutum L.) (Rajasekaran, 1996), and sweet potato (Ipomoea batatas (L.) Lam) (Blakesley etal., 1996). In conclusion, embryogenic cultures of avocado have been established on semisolid and maintained on semisolid or in liquid medium fi-om several genotypes (including four

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87 elite selections) with diverse genotypic background from either zygotic embryos or nucellar explants. Establishment of suspension cultures has been especially critical for further studies involving somatic cell genetics, protoplast isolation, culture and somatic hybridization and plant transformation. The loss of embryogenic potential of cultures after a few months may pose problems related to lack of homogeneity of experimental cultures over time. Conservation techniques, i.e., cryopreservation, may circumvent this problem.

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CHAPTER 4 SOMATIC EMBRYO DEVELOPMENT, MATURATION AND GERMINATION Introduction Pliego-Alfaro & Murashige (1988) reported that avocado somatic embryos were hard, white, glistening and egg shaped. The development of these somatic embryos from embryogenic cultures occurred in low frequency and was not affected by sucrose concentration, ABA, casein hydrolysate and activated charcoal (Pliego-Alfaro & Murashige, 1988). In Chapter 3, the establishment of avocado embryogenic cultures and their maintenance as suspension cultures were reported. The embryogenic cultures were characterized by the presence of proembryonic masses and various stages of hyperhydric somatic embryos on both semisolid proliferation medium and in suspension culture. Efficient incipient somatic embryo development of several embryogenic avocado genotypes in suspension culture has been demonstrated (Chapter 3); however, these somatic embryos failed to develop further. In addition, the ability of cultures to form somatic embryos in liquid medium diminishes with age of the culture. In this chapter, efficient production of good quality somatic embryos by manipulating some of the physical conditions of culture is described. 88

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89 Materials and Methods General Procedures Plant materials used in these experiments consisted of proembryonic masses derived from suspension cultures that were grown in 80 ml filter sterilized MSP medium (Table A-1) in 250 ml Erlenmeyer flasks maintained at 120 rpm in semidarkness for 14 days. Embryogenic cultures on semisolid medium were maintained in darkness. All cultures were maintained at 25°C. Effect of Embryogenic Suspension Culture-derived Somatic Embryo Stage and Size on the Development of Somatic Embryos on Semisolid Medium 'Isham' embryogenic suspension cultures were used as the source of inoculum. 'Isham' embryogenic cultures consisted of proembryonic masses and somatic embryos of different sizes at early stages of development. These cultures had been subcultured 2-3 times after inoculation with proembryonic masses that had been maintained on semisolid MSP (Table A-1) medium for over six months (2-4 week subculture period in liquid medium). The treatments consisted of three different sizes and developmental stages of somatic embryos that were used as the inoculum: 1) proembryonic masses with diameter <0.8 mm with or without globular to early heart stage somatic embryos attached; 2) heart stage somatic embryos with a cotyledon of 0.8-1.5 mm width and 0.3-0.5 cm length; 3) cotyledonary stage somatic embryos with cotyledons >2 mm width and >0.5 cm length. The inocula were cultured on SED medium (Table A-1). There were nine inocula per Petri dish (1 10 x 20 mm) and there were five Petri dishes per treatment. Data were subjected to analysis of variances to determine the treatment effect (SAS Institute, 1992). Treatment means were separated using standard errors and they were presented graphically.

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90 Effect of Gelling Agent Concentration on the Development of Somatic Embryos The effect of different gelling agent concentrations in the medium was evaluated with Gel-Gro gellan gum at 2, 3, 4, 5, 6, 7, 8, 9, 10 g 1"' and TC agar at 8 g 1"' as the control. The basal medium was SED medium (Table A-1) with the gelling agent as treatment. The source of inoculum was 14-day-old 'T362' embryogenic suspension cultures that had been maintained for >8 months with a 2-week subculture interval. 'T362' embryogenic suspension cultures consisted of different sizes of proembryonic masses and proembryonic mass clumps, but with no somatic embryo development. The inoculum for the experiment consisted of 5-10 mg proembryonic masses that passed through sterile nylon filtration fabric of 1.8 mm mesh and were retained on sterile nylon filtration fabric of 0.8 mm mesh. There were 9 inocula per Petri dish (110 x 20 mm) and there were 4 replications Petri dishes per treatment. The number of hyperhydric and opaque somatic embryos and their sizes were recorded after 1 month of culture. Analysis of variance was computed to determine the treatment effect. Treatment means were separated using standard errors and they were presented graphically. Effect of Sucrose Concentration and the Size of Proembryonic Masses on the Development of Opaque Somatic Embryos on Semisolid Medium A factorial experiment was carried out to determine the effects of sucrose concentration in the plant growth medium and size of proembryonic mass inoculum on the development of opaque somatic embryos grown on semisolid medium. The sucrose concentrations tested were 10, 30, 50, 70, 90, 110 and 130 g l'\ and the size of proembryonic masses ranged fi-om 0.8 to 1.8 mm diameter (passed through 1.8 mm mesh nylon filtration fabric and retained on 0.8 mm mesh nylon filtration fabric) and > 1.8 mm diameter (retained on 1.8 mm mesh nylon filtration fabric). The plant growth medium was SED formulation (Table A-1) with sucrose concentration as treatments.

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91 The source of inoculum consisted of 'T362' embryogenic suspension cultures as described previously. There were 9 inocula (0.05-0.12 g each) per replication and there were 4 replicates per treatment. t Data were subjected to analysis of variances to determine the treatment effect (SAS Institute, 1992). Treatment means were separated using standard errors and they were presented graphically. Effect of Carbon Source on the Growth and Development of Cultures that Have Lost Embryogenic Competence Galactose and glycerol at concentration of 5.4% (w/v) and 2.8% (v/v), respectively, in addition to a control (3% sucrose) were tested for their effect on somatic embryo development in liquid medium. The inoculum was derived from nucellar 'Thomas' cultures that had been maintained for over one year. The culture consisted of dedifferentiating proembryonic masses (350-1500 nm diameter). The inoculum, consisting of 200 mg proembryonic masses, was inoculated in 40 ml liquid medium in 125 ml Erlenmeyer flasks. The medium composition was SED (Table A-1) with carbon source as the treatment. Effect of Total Nitrogen Concentration and Ratio of NO^'iNIL"^ on the Growth and Development of 'T362' Avocado Embryogenic Cultures The effects of total inorganic nitrogen concentration (30 mM and 60 mM) and the ratio of N03'/NH4^ (1:0, 3:1, 1:1, 1:3 and 0:1) on the growth and development of embryogenic cultures that consisted of disorganized proembryonic masses were evaluated in suspension in a factorial experiment. The N03":NH4^ ratio was formulated using KNO3, NH4NO3 and (NH4)2S04 according to Niedz (1994). The composition of the medium was that of SED medium (Table A1) with inorganic nitrogen according to treatments. The

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92 inoculum consisted of 0.4 g dediflferentiating proembryonic masses of 'T362' in 40 ml liquid medium. There were 5 replicates per treatment. Fresh weight and pH of the cultures were observed after 2 weeks in culture. Analysis of variances were performed to determine interaction (SAS Institute, 1992). Regression analyses were fit to the data using Sigma Plot™ (Jandel Scientific, San Raphael, CA). For regression analyses, the NOs'iNHt^ ratios were expressed as NO3" percentage fi"om total inorganic nitrogen and set as the x axis. Somatic Embryo Maturation and Germination Opaque cotyledonary somatic embryos (> 0.8 cm diameter) that developed on SED medium were transferred individually onto semisolid Somatic Embryo Maturation and Germination (SEMG) medium (Table A-1). Aliquots of 25 ml medium were dispensed into 150 x 25 mm glass test tubes, closed with polypropylene Kaputs, autoclaved for 15 min at 12rc and 1.1 kg cm"^ and cooled as slants. After subculture of each somatic embryo, the tubes were closed with Suncaps™ and secured with rubber bands. The cultures were kept upright in darkness at 25°C. The somatic embryos were subcultured onto fi-esh medium of the same composition at 2-3 month intervals. When root or shoot growth was apparent, the cultures were transferred to 16 h light provided by cool white fluorescent tubes (80-100 ^mol s'^ m"^). Shoot Proliferation and Plantlet Regeneration fi-om Shoot-Derived Somatic Embryos The shoot that emerged from each somatic embryo that developed from a 'T362'derived embryogenic culture was decapitated 1-1.5 cm from the tip, cultured on Shoot Multiplication (SM) medium (Table A-1) according to Witjaksono (1991) and subcultured at 8 week intervals for several passages. They were thereafter subcultured on

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93 Avocado Shoot Proliferation (ASP) medium (Table A-1). Rooting of shoots has been described in Chapter 6 and follows the protocol of Pliego-Alfaro (1988). Results Subculturing proembryonic masses or globular somatic embryos on medium similar to proliferation medium without plant growth regulators resulted in low frequency occurrence of opaque, white somatic embryos that resembled zygotic embryos. On medium without plant growth regulators, the somatic embryos were generally hyperhydrous and distorted. Under optimal conditions, healthy opaque, white mature somatic embryos developed through globular, early and late heart, cotyledonary stages as indicated in Figure 4-1 A-G. These somatic embryos occasionally developed shoots without roots. The shoots could be propagated and rooted (Figure 4-1 H). The optimal conditions for this development were determined as follows. Effect of Embryogenic Suspension Culture-derived Somatic Embryo Stage and Size on Development of Somatic Embryos on Semisolid Medium This experiment was carried out in order to determine if developmental stage of somatic embryos that developed in liquid medium affected their further development on semisolid medium. Late heart stage somatic embryos that developed in liquid medium failed to develop further on semisolid medium. Their size remained the same and their color became dull and opaque; however, smooth, opaque secondary somatic embryos developed from the plated somatic embryo inocula. Development of proembryonic masses inoculated from liquid onto semisolid medium proceeded differently. Some of the proembryonic masses died; however, secondary somatic embryogenesis occurred from some of the cultures. The secondary somatic embryos were different in size, and they were a mixture of hyperhydric and opaque embryos. Since only opaque and large somatic

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Figure 4-1. Avocado somatic embryo development from proembryonic masses and subsequent plant regeneration. (A-E) Developmental stages of 'Booth 7' avocado somatic embryos from globular to cotyledonary. Note that in C, the two cotyledons were apart while in the zygotic counterpart, those cotyledons remain close to each other (see Table 3-2). (F) 'Booth 7' somatic embryos, mostly at cotyledonary stage, enlarged to 0.8-1.2 cm diameter after two months on SED medium. (G) Multiple shoot development from a 'Booth 7' somatic embryo ca. one year after its initiation. (H) A plantlet and a proliferating shoot that developed from a somatic embryo. The somatic embryo developed from zygotic embryoderived 'T362' avocado.

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> 96 Table 4.1. ANOVA of the effect of 'Isham' embryogenic suspension culture-derived somatic embryo size and stage of development on development of secondary somatic embryos on semisolid medium. Source DF Sum of Squares Mean Square F Value Pr > F Number of secondary opaque somatic embryos >0.8 cm diameter Treatment 2 525.733 262.867 66.83 0.0001 Error 12 47.200 3.933 Total 14 572.933 Number of somatic embryos that developed root Treatment 2 1.733 0.8667 2.60 0.1153 Error 12 4.000 0.333 Total 14 5.733 large SE medium SE Inoculum type PEM Figure 4-2. Opaque cotyledonary stage somatic embryo production as affected by inoculum type derived from 'Isham' avocado liquid embryogenic cultures after one month on semisolid medium. Large SE = cotyledonary somatic embryos with cotyledon size of > 2 mm width and > 5 mm length; Medium SE = cotyledonary somatic embryos with cotyledon size of > 0.8-1.5 mm width and > 3-5 mm length; PEM = proembryonic masses with diameter < 0.8 mm with or without globular to heart stage somatic embryos attached.

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97 embryos (>0.8 cm) matured normally, only the appearance of this type of somatic embryo was recorded. Table 4.1 indicated that inoculum size and developmental stages of somatic embryos used as the inoculum significantly affected the number of opaque somatic embryos produced per dish, but had no effect on root emergence of the somatic embryos. The largest number of opaque cotyledonary somatic embryos per dish was obtained when the proembryonic masses were used as the inoculum; and the lowest number was obtained when the largest somatic embryos were used as the inoculum (Figure 4-2). Somatic embryo development in this avocado embryogenic genotype was very efficient. Effect of Gelling Agent Concentration on Development of Somatic Embryos After 1 month of culture, the growth and development of the cultures were visually distinguishable among treatments. On treatments consisting of 8 g 1"^ TC Agar and 2 g 1"' Grel-Gro™ there was no somatic embryo development, although loose nodular and granular proembryonic masses and distorted heart to torpedo stage hyperhydric somatic embryos proliferated. With increasing Gel-Gro™ concentration, somatic embryos with well formed cotyledons were observed. While there was no necrotic tissue in the treatment 2 g 1"^ Gel-Gro™ and 8 g 1"^ TC Agar, the percentage of necrotic tissue that developed from the distal part of the culture increased with Gel-Gro™ concentration. Analysis of variance (Table 4-2) indicated that Gel-Gro™ concentration significantly affected the number of opaque, hyperhydric somatic embryos, the total number of somatic embryos and the size of somatic embryo (width and length) that developed on semisolid medium. Figure 4-3 demonstrated that the length of somatic embryos increased slightly on medium with 3 to 5 g l ' Gel-Gro™ and then decreased with a Gel-Gro™ concentration of

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98 10 g rV A significant decrease in somatic embryo length occurred with Gel-Gro™ at 7 g T V The number of hyperhydric somatic embryos increased slightly, while the number of Table 4-2. ANOVA of eflfect of Gel-Gro concentration on development of opaque and hyperhydric somatic embryos and size of opaque somatic embryo fi"om 'T362' proembryonic masses cultured on semisolid medium. Source . , ; DF , Sum of Mean Square F Value Pr>F ^ ?" ' Squares Number of opaque somatic embryos Treatment 8 69.60 8.70 12.21 0.0001 Error 27 19.23 0.71 Total 35 88.83 Number of hyperhydrous somatic embryo Treatment 8 121.32 15.16 13.02 0.0001 Error 27 31.45 1.16 Total 35 152.77 Total number of somatic embryo Treatment 8 277.85 34.73 23.44 0.0001 Error 27 40.00 1.48 Total 35 Width of somatic embryo Treatment 8 19.61 2.45 19.54 0.0001 Error 27 3.39 0.13 Total 35 22.97 Length of somatic embryo Treatment 8 39.84 4.98 22.06 0.0001 Error 27 6.09 0.23 Total 35 45.94

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99 Gel QoGDnDerttalkn(gr^) 2345678910 I I I I I 1 r I 2 \ J fl i 1 1 Qsl QDaonoertrsiicn (rrgf ^ r 8 9 10 Figure 4-3. Effect of Gel-Gro™ concentration on development of somatic embryos from proembryonic masses on semisolid medium after one month of culture.

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100 opaque somatic embryos increased sharply with increasing Gel-Gro concentration. Good quality opaque somatic embryos were obtained with 6 g 1"' Gel-Gro™. At 6 g 1"' Gel-Gro™, 0.12 opaque somatic embryos developed from each inoculum. Since there were 9 inocula per Petri dish, 1-2 opaque cotyledonary somatic embryos could be subcultured for somatic embryo germination. At 7 g 1'* Gel-Gro™, 5-7 opaque somatic embryos could be recovered. Since no significant difiference in somatic embryo size was detectable between 6 and 7 g l ' Gel-Gro™, these Gel-Gro™ concentrations were used as standards. With Gel-Gro™ concentrations of 6-7 g r\ good quality cotyledonary somatic embryo development has been obtained for cultures that were initiated from zygotic embryos of 'Booth 7' (Figure 4-1 F), 'Booth 8', 'Yon' and 'Thomas'. Somatic embryo development was also obtained from embryogenic suspensions of 'M25864' that could not form somatic embryos in liquid medium. Somatic embryo development on this GelGro™-modified medium was also obtained with 'Hass', 'Thomas' and 'Lamb' cultures that were initiated from nucellar explants. Somatic embryo development was not observed on this medium from cultures that were derived from 'Esther' zygotic embryos. Effect of Sucrose Concentration and the Size of Proembrvonic Masses on the Development of Opaque Somatic Embryos on Semisolid Medium The development of opaque somatic embryos of various stages of development (from heart to cotyledonary) was affected neither by size of proembryonic mass inocula nor its interaction with sucrose concentration, but was significantly affected by sucrose concentration (Table 4-3). No opaque somatic embryos developed at sucrose concentrations of 10 g fV Opaque somatic embryos developed only at sucrose concentrations of at least 30 g 1"'. The number of opaque somatic embryos increased with a sucrose concentration of 90 g 1'^ and then decreased with sucrose concentrations up to 130 g after 1 month in culture (Figure 4-4).

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101 Table 4-3. ANOVA of the effect of sucrose concentration and the size of proembiyonic masses on production of opaque somatic embryos of various stages of development/ Source DF Sum of Squares Mean Squares F Value Pr>F Sucrose 6 4296.66 716.11 25.33 0.0001 Size 1 59.62 59.62 2.11 0.1539 Sucrose* Size 6 383.58 63.93 2.26 0.0557 Error 42 1187.58 28.27 Total 55 5927.43 ^from suspension culture-derived proembryonic masses of 'T362' on semisolid medium. 1 \ \ i — — — I — — — I — 10 30 50 70 90 110 130 Sijcrose (xrioentnation (g H) Figure 4-4. EflFect of sucrose concentration on the production of opaque somatic embryos on semisolid medium.

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102 i Figure 4-5. Somatic embryo development as affected by high sucrose concentration. (A) Globular to early heart stage somatic embryos developed on the entire surface of zygotic-derived 'T3623' cultured on SED with 13% sucrose 3 months after culture (B) Development of late heart to cotyledonary stage somatic embryos one month after transfer from 13% sucrose to 3% sucrose containing SED medium.

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With increasing sucrose concentration, smaller and earlier stages of somatic embryos developed; at a concentration of 90 g 1'^ there were only globular and heart stage somatic embryos. Therefore, the sucrose concentration of 30 g f' was retained as the standard. The proembryonic masses on media with high sucrose concentrations (90-130 g 1"^) become compact and deep yellow in color, and there was a low frequency of early stage somatic embryo (globular to heart) development on their surface. After 3 months, globular somatic embryos covered the entire inoculum (Figure 4-5 A). Transfer of these cultures onto SED medium with 30 g 1'^ sucrose and 6 g 1"^ Gel-Gro™ resulted in development of clusters of somatic embryos from heart to early cotyledonary stages (Figure 4-5 B). Effect of Carbon Source on the Growth and Development of Thomas' Cultures that Had Lost their Embryogenic Potential Microscopic observation revealed that cultures maintained in liquid medium with sucrose consisted of 100-200 fim diameter granular proembryonic masses, 1-2 mm diameter nodular proembryonic masses, dedifferentiated proembryonic masses and a low frequency of free cells. In medium with galactose as the carbon source, the cultures appeared to be similar, except that nodular proembryonic masses of 1-2 mm diameter had lost their integrative appearance. In medium v^th glycerol, free round and elongated cells were observed more frequently than in cultures with other carbon sources. Nevertheless sucrose, galactose and glycerol had no effect on restoration of embryogenic potential of these cultures. Different carbon sources significantly affected the growth and proliferation of the cultures as indicated by their final volume and fresh weight after 2 weeks in culture. There was no significant difference between galactose and sucrose with respect to culture fresh weight; however, galactose and sucrose resulted in significantly higher fresh weight than

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104 glycerol. Volume of the cultures was greatest in medium with galactose and smallest in medium with glycerol (Figure 4-6). 1 — — — I — — — r Sue at Qd Cartxn soLToes Sue at Gd Cartxn sources Figure 4-6. Effects of carbon source on fresh weight and volume of embryogenic nucellar 'Thomas' avocado cultures. The inoculum consisted of mixed, nodular and dedifferentiating proembryonic masses. Inoculum of 0.4 g was inoculated in 40 ml liquid medium. Data were taken after two weeks in culture. Medium composition was MS without PGR with carbon source as treatment: 3% (w/v) sucrose (Sue), 5.4% (w/v) galactose (Git) and 2.8% (v/v) glycerol (Gel). The effect of total nitrogen concentration and ratio of NO^iNHd"^ on the growth and development of 'T362' embryogenic cultures After 2 weeks, there were differences in color and morphology of the cultures (Table 4-4). Globular and early cotyledonary stage somatic embryos developed in three treatments, i.e., 60 mM nitrogen consisting of 75 and 100% NO3" and 30 mM nitrogen consisting of 100% NO3". Only dedifferentiating proembryonic masses were present in the other treatments.

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105 Table 4-4. The effect of total N and YoNOi on color and morphology of avocado 'T362' suspension cultures.^ JN total "Kin ' IN Us percentage i^oior oi cuiiure OI-/ PEMs D^diflferentiatino 60 100 dark yellow + + + 75 yellow + ... ' CA 50 whitish yellow 25 whitish brown + 0 whitish brown + 30 100 yellow + + 75 pale yellow + 50 whitish yellow + 25 whitish brown + 0 whitish brown + ^Inoculum was taken from dedifferentiating proembryonic masses that had been in culture for over 1 year. + = present, = absent There was a significant interaction between nitrogen level and % NO3" with respect to fresh weight gain (Table 4-5). Regression analyses indicated that fresh weight gain responded curvelinearly with % NOs' (Figure 4-9). Final medium pH was significantly affected by % NO3" but not by total nitrogen concentration and interaction between % NO3" and total nitrogen concentration (Table 4-5). Therefore, data for total nitrogen concentration were pooled for regression analysis against % NO3". Final medium pH increased quadratically with increasing % NO3" (Figure 4-7). At 0-30% NO3", the medium pH fell to <4.0, and then increased linearly with increasing % NO3' to 100%. At total nitrogen concentration of 60mM, fresh weight gain increased with increasing % NOs' from 0 to 75% and then decreased with % NOs' though 100%. This curve can be divided into 2 response groups: low response, which was a fijnction of % NO3" from 0 to 50%, and high response, which was a fimction of % NOs' from 50 to 100%. In the low response, fresh weight gain ranged from 1 to 7 fold, while in the high

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106 response, fresh weight gain ranged from 7 to 1 1 fold. The high response group coincided with high pH, while the low response group coincided with low pH. The fresh weight gain peaked at ca. 82.5% NO3" at ca. pH 5.7. A similar pattern occurred when the total nitrogen of the medium was lower, e.g., 30 mM; however, the fresh weight responses against % NO3" were generally lower than with 60 mM. At 30 mM nitrogen, the high response group corresponded with % NOs' from 70-100. This interval also coincided with high final medium pH. At this level of nitrogen, fresh weight increase peaked at ca. 85%, NO3' at pH 6.5. Table 4-5. ANOVA for the effect of nitrogen concentration and % NOs' on proembryonic mass fresh weight gain and medium pH in liquid medium. Source DF Sum of Squares Mean Square F Value Pr>F Fresh weight increase Treatment 9 5.7.47 56.38 38.24 0.0001 Error 33 48.65 1.47 Total 42 556.13 Per cent NOs^ 4 420.42 105.10 71.29 0.0001 N concentration 1 47.17 47.17 31.99 0.0001 Percent*Concentration 4 36.62 9.15 6.21 0.0008 Medium pH Treatment 9 72.96 8.10 64.08 0.0001 Error 34 4.30 0.12 Total 43 77.26 Per cent NO3* 4 70.59 17.64 139.51 0.0001 N concentration 1 0.17 0.17 1.34 0.2547 Percent*Concentration 4 1.74 0.44 3.45 0.0181

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107 16 14 ^ 12 8 ^10 c 1 8 I 4 i6 O N = eOmM N = 30mM 25 50 75 NOs" percentage (%) 100 Figure 4-7. Eflfect of total nitrogen concentration and NOs'/NHt^ ratio on culture fresh weight gain (x 100%) and medium pH. Ratio of N03'/NH4'^ are expressed as NO3' percentage and set as the X axis. The fitted regression lines were represented as follows: fresh weight gain at 60 mM N, Y, = -10.7 + 27.3 x -10.3 x^ + 1. 1 x^ = 0.84; fresh weight gain at 30 mM nitrogen, Y2 = -1.4 + 12.4 x -5.2 x^ + 0.57 x^ R^ = 0.88; medium pH, Y3 = 3.6 + 0.0009 x + 0.0003 x^ R^= 0.91.

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t 108 Somatic Embryo Maturation and Germination Upon transfer to somatic embryo maturation medium, cotyledonary somatic embryos with diameter > 8 nun continued to enlarge to ca. 1-2 cm diameter. Proembryonic masses were present in some of the cultures. After ca. 9-10 months, 2.5% (1/40), 5% (3/60), 5% (2/40) of 'T362', 'Booth 8' and 'Booth T somatic embryos, respectively, developed shoots. No shoot development was observed from about 40 somatic embryos of 'M25864' and 200 somatic embryos of 'Isham'. Upon transfer to light, the somatic embryos turned green, and the shoots elongated (Figure 4-1 G). Shoot Proliferation and Plantlet Regeneration from Shoot-Derived Somatic Embryos Only a single shoot grew from the single axillary bud on each nodal explant and from the apical bud of each shoot tip. Upon transfer to ASP medium, multiplication rates of 6 fold for each 8-week subculture cycle were obtained. The in vitro shoots could be rooted with a frequency of 60%. Rooting generally occurred after 2-4 months in culture. Figure 4-1 H represents proliferating shoots and rooted shoots of somatic embryo origin. Discussion In most of the embryogenic genotypes, avocado somatic embryos developed in liquid medium in the presence of plant growth regulators. These somatic embryos, however, have never been able to develop to maturity in liquid medium as they become necrotic and died. Those somatic embryos also failed to develop further and died on semisolid medium; however, secondary somatic embryos that grew from them developed normally. The development of somatic embryos to maturity occurred with high frequency when proembryonic masses were used as inoculum instead of later stage (cotyledonary) somatic embryos. Somatic embryos of certain mango cultivars, on the other hand, can

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109 initiate development in liquid medium and continue to develop on semisolid medium (DeWald et al., 1989b). It was possible to control the development of avocado somatic embryos by manipulating certain physical parameters, i.e., gelling agent and sucrose concentration, in addition to the genotype effect. Gelling agent affected somatic embryo development in avocado in two ways: 1) a specific level is required for differentiation and 2) a higher level is required for development of opaque instead of hyperhydric somatic embryos. Increasing gelling agent has been reported to reduce hyperhydricity even though it decreases other growth parameters in shoot cultures of Cynara scolymus (Debergh et al., 1981), Gerbera jamesonii, Forsythia intermedia, Oreopanax nymphaeifolium (Debergh, 1983) and Amelachier arborea (Brand, 1993). Increasing gellan gum concentration fi-om 2 to 6 g also increased the reversion of hyperhydric to opaque somatic embryos in mango (Monsalud et al., 1995). Increasing gellan gum concentration increases the gel rigidity/medium hardness (Huang et al., 1995), lowers medium matrix potential (Debergh et al., 1981, Debergh, 1983), and lowers medium water potential (Ghashghaie et al., 1991). Sucrose can affect somatic embryo development in two ways: 1) as a carbon source and 2) as an osmoticum which affects morphogenesis (DeWald et al., 1989b). As a carbon source, as indicated by its effect on biomass accumulation, sucrose is optimum for growth of embryogenic avocado suspensions at concentration of 30-50 g 1"^ (see Chapter 3). The higher sucrose concentration may function as osmoticum that controls morphogenesis (Litz & Conover, 1982). The effect of sucrose on avocado somatic embryo development may be genotype-dependent since 11-13% sucrose resulted in development of globular somatic embryos fi"om proembryonic masses of 'T362' but not from 'Booth 8' and 'Yon' (data not shown). Pliego-Alfaro & Murashige (1988) reported that the percentages of cultures with somatic embryo development fi"om embryogenic

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IW cultures derived from 'Hass' zygotic embryo explants was not significantly affected by sucrose concentration. On somatic embryo development medium that is solidified with 6-7 g 1'^ gellan gum, avocado somatic embryos developed from proembryonic masses as globular or nodular stage, heart stage and later to a mature stage. Only somatic embryos that were opaque and white could enlarge fiarther, developed shoots and turned green under light condition. The morphology of embryogenic suspension cultures changed with time from globular, nodular proembryonic masses and somatic embryos to dedifferentiating proembryonic masses without apparent somatic embryo development, indicating diminishing (or even loss of) embryogenic potential (see Chapter 3). Somatic embryo development could not occur from cultures that had lost their embryogenic potential (unpublished results). By replacing sucrose in the medium with galactose (Kochba et al., 1978; Kochba et al., 1982; Cabasson et al., 1995), lactose (Kochba et al., 1978; Kochba et al., 1982), rafifinose (Kochba et al., 1978) and glycerol (Ben-Hayyim & Neuman, 1983), somatic embryo development could be increased dramatically from embryogenic cultures of citrus. Somatic embryo development could also be recovered from citrus embryogenic cultures that had apparently lost embryogenic potential in sucrose-containing medium by replacing sucrose with galactose (Cabasson et al., 1995), or glycerol (Gavish et al., 1991; Vu et al., 1993). Attempts to obtain somatic embryos from similar avocado cultures by replacing sucrose with galactose or glycerol were unsuccessful. Differential growth of avocado suspension cultures in plant growth media with 3 different carbon sources has also been reported in citrus. In avocado suspensions, galactose medium resulted in greater fresh weight and volume of sedimented cultures, although growth parameters of citrus suspensions were lower with galactose compared to sucrose-containing medium (Cabasson et al., 1995; Kochba et al., 1978). The lower growth rate of avocado suspension cultures in medium containing glycerol compared to those in medium

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Ill containing sucrose is in agreement with results for citrus (Gavish et al., 1991; Vu et al, 1993). Varying the salt composition, especially the nitrogen content of the plant growth medium, affected somatic embryo development from embiyogenic cultures (see Chapter 3). Niedz (1994) reported that the N03":NH4^ ratio affected only the growth of citrus embryogenic cultures, but had no effect on either embryogenic potential or culture morphology. In contrast, the ratio of NOs'iNHt"^ affected both growth and differentiation of an avocado embryogenic culture that had lost its embryogenic potential. Avocado cultures were able to grow in liquid medium with KNO3 as the sole nitrogen source, but were unable to grow in liquid medium with only NH4CI. Optimum growth occurred when both NO3' and NH4'^ were present in the medium in a ratio of ca. 3:1. The ability of plant cells to grow in medium with KNO3 as the only nitrogen source has been reported, even though better growth was obtained when both NO3' and NH4* were present in the medium (Kirby et al., 1987). The low rate or absence of growth of avocado suspension in medium with low nitrate content (low N03":NH4'^ ratios) may be due to NH/ toxicity. NH4CI as the sole nitrogen in the medium was inhibitory to Pinus strobus callus growth due to NHi^ and not CI" (Kaul & Hofl&nan, 1993). The NH4^ toxic effect could be alleviated by incorporating KNO3 in the medium (Kaul & Hoflfinan, 1993). Despite their susceptibility to ammonium ion toxicity, plant cells can be adapted to grow in NH4CI as the sole nitrogen source when acids of the tricarboxylic acid cycle are provided (Kirby et al., 1987) The importance of the type of nitrogen (reduced or oxidized form) for somatic embryogenesis has been classically demonstrated by Halperin & Wetherell (1968). The association of eflBcient somatic embryo development with high N03":NH4* ratios has been suggested previously for avocado on semisolid medium that consisted of B5 salts compared to MS (see Chapter 3). Similar results have been reported for mango, in which B5 medium stimulated higher somatic embryo development than MS or Vi MS (DeWald et

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> 112 aL, 1988) and for a forest tree Ocotea catharimnsis (Moura-Costa et al., 1993). Using dedifferentiating proembryonic masses, avocado somatic embryos could be recovered in medium with high N03":NH4^ ratio while no somatic embryos could be recovered in medium with low NOs'iNEl,"^ ratio. The somatic embryos that developed in liquid medium with a high ratio of N03":Nfi4'^ (1 :0 to 3 : 1), however, failed to enlarge. Maturing avocado somatic embryos were routinely obtained on MS medium with a NOs iNHt^ ratio of 2:1 (see Chapter 4; Pliego-Alfaro & Murashige, 1988; Mooney & Van Staden, 1987). This may indicate a differential requirement of nitrogen ratio, which is related to the stage of development of the somatic embryos. Joy et al. (1996) observed that different amino acids accumulated in somatic embryos of different stages of development. Different amino acids in the medium also encouraged the growth of carrot somatic embryos of different stages of development (Higashi et al., 1996). Avocado somatic embryo development to germination requires ca. 9-10 months. This time span corresponds to the time required from fruit set to fruit maturity, which is 6-12 months, depending on the cultivar (Whiley, 1992). At maturity, avocado seeds are large, 3-7 cm in diameter, whereas mature avocado somatic embryos are only 1.5-2.0 cm diameter. The plant conversion rate from avocado somatic embryos has been low, ca. 05%, and similar to that reported by Pliego-Alfaro and Murashige (1987) and Mooney and Van Staden (1988). The failure of shoot development has been attributed to failure of shoot meristem development in maturing carrot somatic embryos (Nickle & Yeung, 1993). Similar observations were also found from histological studies of somatic embryos of avocados (Pliego-Alfaro & Murashige, 1988; Mooney & Van Staden, 1987). Those histological studies demonstrated that even though there were meristematic regions in the basal and apical region of the somatic embryos, they were much less pronounced than the zygotic embryo counterpart. In carrot, such failure could be corrected by incorporating ABA in the medium (Nickle & Yeung, 1993). In contrast, Pliego-Alfaro & Murashige (1988)

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' 113 found that ABA, cold treatment or GA3 had no effect on shoot development from avocado somatic embryos. Fujiwara and Komamine (1975) found that cytokmin was important for organization of the apical meristem of carrot somatic embryos. Attempts to improve plant conversion by incorporating BA in the maturation medium were unsuccessful (data not shown). The failure of treatments to overcome this developmental anomaly may be because they were appUed too late during development. Barlass and Skene (1983) reported that immature avocado zygotic embryos developed shoots with greater frequency when they were taken from abscised fruit >6weeks old. Apparently the first 6-7 weeks of embryo development is a critical period for somatic embryo development, and somatic embryos of this stage should be pulsed with a shoot-inducing treatment to improve plant conversion. Alternatively, maturation treatments such as culturing somatic embryos on medium with high osmoticum, slow desiccation, ABA treatment or their combination (Etienne et al., 1993; Pence, 1992; Pliego-Alfaro et al., 1995a; b) should also be tried to increase plant conversion. Shoots that develop from somatic embryos, however, could be micropropagated, and plantlets could be regenerated using standard protocols.

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CHAPTER 5 PROTOPLAST ISOLATION, CULTURE AND SOMATIC EMBRYO REGENERATION OF AVOCADO Introduction Avocado production worldwide has been threatened by root rot disease caused by an Oomycete, Phytophthora cinnamomi Rands, especially in areas with heavy soil and poor drainage (Zentmeyer, 1980; Zentmeyer et al., 1994). Crop production in afifected areas has been dependent on the use of a few tolerant roostocks (Zentmeyer et al., 1994). Conventional breeding for resistance to root-rot disease has been impeded due to low heritability of the tolerance trait (Coffey, 1987), long juvenility period and low fiuit set (ca. 0.001%) (Blanke & Lovatt, 1995), all of which are typical obstacles in breeding perennial fruit crops. Protoplast culture and regeneration-based biotechnology approaches have been demonstrated to be very important tools for complementing conventional breeding (Gmitter et al., 1992; Ochatt et al., 1992). For example, with citrus, following the first report of somatic hybridization between 'Trovita' sweet orange Citrus sinensis and Poncirus trifoliata (Ohgawara et al., 1985), this technique has been successfully utilized to produce somatic hybrid plants from at least 150 parental combinations (Grosser, 1993; Ohgawara et al., 1994; Grosser et aL, 1994; Mourao-Filho, 1995; Louzada «& Grosser, 1994; Ling & Iwamasa, 1995; J. W. Grosser, personal communication) that have been intended for various purposes, i.e., production of allotetraploid hybrids for rootstocks, for crosses with diploids to produce seedless scion cultivars and introgression of useful traits from citrus relatives that are sexually incompatible and for confirming the hybrid origin of certain species. Production of citrus cybrid plants has been achieved through asymmetric 114

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115 donor-recipient protoplast fusion (Vardi et al., 1987), electrofiision (Saito et al.. 1993) and PEG-mediated fusion of protoplasts (Grosser et al., 1996) and could eventually allow the study of cytoplasmic inheritance of certain characters. Direct gene transfer to citrus protoplasts and recovery of transgenic plants has also been described (Kobayashi & Uchimiya, 1989; Vardi et al., 1990; Schell, 1991). Citrus somatic hybridization has been based on the availability of protoplast-to-tree protocols for several species, e.g., C. sinensis (Kobayashi et al., 1985; Grosser & Gmitter, 1990; Vardi & Galun, 1988), C. mitis (Sim et al., 1988), C. aurantium, C. limon, C. paradisi and C. reticulata (Vardi & Galun, 1988). In addition, protocols for protoplast culture and regeneration for citrus relatives have been reported for Microcitrus (Vardi et al., 1986), Murraya paniculata (Jumin & Nito, 1995), Atalantia bilocularis, Hesperethusa crenulata, Glycosmis pentaphylla, Triphasia trifolia, Murraya koenigii (Jumin & Nito, 1996a) and Citrosis schweinjurthii (Jumin & Nito, 1996b). Reports of protoplast culture and regeneration of other ftuit trees have been limited to deciduous woody perennials in the Rosaceae family, e.g., Malus spp., Prunus spp. and Pyrus spp. (Ochatt, 1990; Ochatt, 1993; Ochatt et al. 1992), including a haploid 'Golden Delicious' apple clone (Malus X domestica Borkh.) (Patat-Ochatt et al., 1993), 'Starkrimson' apple (Ding et al., 1995), sour cherry (Prunus cerasus L.) (Ochatt, 1990), colt cherry (Prunus avium X pseudocerasus) (Ochatt, 1990) and pear (Pyrus communis L.) (Ochatt & Power, 1988). Protoplast culture and regeneration has also been reported for woody perennial fruit vines in the family Vitaceae, i.e., Vitis vinifera (Kovalenko & Galkin, 1990), Vitis sp. (Reustle et al., 1995) and in the family Actinidiaceae, i.e., Actinidia deliciosa var. deliciosa 'Hayward' (Oliveira & Pais, 1991), and among some tropical and subtropical fruit species including Passiflora edulis (Manders et al., 1991) and Diospyros kaki (Tao et al., 1991). Somaclonal variants that showed high levels of salt and drought tolerance were recovered from protoplast-derived cultures of Prunus avium X pseudocerasus (Ochatt and Power, 1989) and somaclonal variants of Pyrus communis

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116 that showed differences in rootability have been reported (Ochatt, 1987). Somatic hybrid plants have been obtained following protoplast fusion only between 'Colt' cherry (Prunus avium) and Pyrus communis (Ochatt et al, 1989) despite attempts with other combinations in or between these genera (Ochatt & Patat-Ochatt, 1994) and between Passiflora spp. (Domelas et al., 1995; Otoni et al., 1995). Application of protoplast-based technologies to avocado improvement could have great utility, particularly with respect to breeding for root-rot resistance. Protoplast fusion could be used to overcome sexual and graft incompatibility barriers between avocado and the root-rot resistant small-seeded Persea species in the subgenus Eriodaphne (PliegoAlfaro & Bergh, 1992; Bergh & Lahav, 1996), e.g., P. borbonia, P. cinerascem, P. pachypoda, P. caerulea, etc. (Zentmeyer, 1980). Production of transgenic rootstocks could be accomplished by direct transfer of antifungal genes, i.e., P-glucanase and/or chitinase (Lamb et al., 1992), into protoplasts of existing rootstocks. There have been only been a few reports concerning either protoplast culture or regeneration of avocado. Protoplasts have been isolated from nonmorphogenic avocado callus for studying sunblotch viroid replication (Blickle et al., 1986) and from fruit mesocarp tissue for study of fruit ripening (Percival et al., 1991); however, regeneration from protoplasts derived from these tissues was not described. The purpose of this study was 1) to develop protocols for protoplast isolation from embryogenic suspension cultures of avocado, 2) to define optimum conditions for culture and somatic embryos regeneration from the protoplasts.

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117 Materials and Methods Plant Materials i Plant material used for protoplast isolation consisted of 8-12-day-old avocado embryogenic suspension cultures maintained with a 2-week subculture interval. Details of culture initiation, maintenance, plant growth media and culture conditions are described in Chapters. The embryogenic suspension cultures used in these experiments had been maintained for 1-3 years and their morphologies had changed over time. Initially, the cultures consisted of proembryonic masses that produced secondary somatic embryos repetitively and proembryonic masses. The cultures later consisted primarily of dedifferentiated proembryonic masses ('Esther', 'M25864' and 'Lamb'), mixtures of dedifferentiated, dedifferentiating and highly organized proembryonic masses ('T362', nucellar-derived 'T362', 'Thomas' and nucellar-derived 'Thomas') and highly organized proembryonic masses ('Isham' and nucellar-derived 'Hass'). Protoplast Isolation ... Approximately 0.8-1.2 g of 8-14 day-old avocado embryogenic cultures were incubated in a mixture consisting of 2.5 ml of either 0.7 M MS8P' or 0.7 M MS"8P protoplast culture medium (Table A-2 and A-3) and 1.5 ml enzyme digestion solution (Table A-4) in 60 x 15 mm sterile plastic Petri dishes and sealed with Nescofilm (Grosser & emitter, 1990). ' The digestion mixtures were incubated in darkness at 25°C on a rotary shaker at 50 rpm overnight (15-18 h). They were then passed through a sterile 45 |xm mesh stainless steel screen to remove undigested cell clumps and debris. The filtrates were transferred into sterile 15 ml screw cap centriflige tubes and precipitated by centrifugation at 100 x g

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118 for 5 min in a clinical centrifuge. After the supematants were removed, the protoplast pellets were purified by gradient centrifugation using CPW25S and CPW13M (Table A5b) according to Grosser & Gmitter (1990). The protoplasts at the interphase were collected with Pasteur pipettes and transferred to 15 ml sterile centrifuge tubes and washed once with either 0.7 M MS 8P or MS8P" protoplast medium. The protoplasts were then repelletted and resuspended with 0.7 M protoplast medium to a volume of 20x for fiirther studies. Protoplast yields fi-om several protoplast isolation trials with 'T362', 'Thomas', 'M25864' and 'Esther' were determined using a Fuchs Rosenthal haemocytometer. Protoplast viability was measured using membrane staining fluorescence diacetate (FDA) according to Huang et al. (1986). Approximately 10 ^1 aliquot of a 10 mg-ml'^ stock solution of FDA in acetone was added to 1 ml protoplast suspension. Viable protoplasts were viewed with a fluorescence microscope (Nikon) using 365 nm illumination, and the number of protoplasts was determined by counting with a haemocytometer. Protoplast viability was also determined using the dye exclusion method with Evans blue (Gahan, 1989). Protoplast suspension samples (1-2 ml) were pelleted by centrifugation and resuspended with Evans blue (0.1% w/v) in 0.7 M MS"8P protoplast medium for 5 min, and rinsed and resuspended in fi-esh protoplast medium of the same composition. Protoplast number was determined by counting under a light microscope. Frequency of viable protoplasts consisted of the number of transparent protoplasts divided by the total number of transparent and blue protoplasts. Effect of Protoplast Density and Medium Osmolarity on Growth of Avocado 'T362' Protoplasts Plated in Agarose Disc Type Medium A factorial experiment was designed to determine the optimum treatment among four concentrations of medium osmolarity (0.4, 0.5, 0.6 and 0.7 M, each containing 0.15 M sucrose balanced with mannitol) and three protoplast plating densities (4 x lO', 1 x lO'

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} 119 and 0.25 x lO' protoplasts ml"' medium). The basal media ware MS8P' solidified with 20 g r' agarose type Vn (Sigma) that was dissolved in a microwave oven followed by autoclaving for 15 min at 1.1 kg cm"^ and 121° C. The media were kept liquid at 45° C until use. One half ml of protoplast suspension stock at 10 x of a certain treatment density was mixed with 4.5 ml of medium of known osmolarity and then plated on 60 x 15 mm sterile plastic Petri dishes. There were 9 discs in each Petri dish, each consisting of 2 drops of protoplast-agarose medium. When the agarose medium solidified, it was soaked with 3-4 ml liquid medium of the same composition. The Petri dishes were sealed with Nescofilm and maintained in darkness at 25°C. Plating eflBciency as measured according to the equation [I microcallus x (I microcallus + I nondividing protoplasts)"*] x 100% was determined fi^om 2 diflferent places on a disc (center and periphery) fi-om 2 discs fi^om 3 Petri dishes (3 replications) of each treatment 3 weeks after culturing. The microcallus had various two-dimensional shapes: elliptical, rectangular, square, circular, etc. that made it difficult to accurately measure the area. Therefore, the approximate size of the microcallus was determined fi"om the length and width measurement using a Leitz Diavert inverted microscope. Microcalli that were clearly visible in a single focal plane were measured fi^om one disc or as many as 12 microcalli fi-om 3 replications. The data were analyzed statistically using Proc GLM (SAS Institute, 1992). Plating efficiency data were transformed v^th arch sine transformation for analysis, and nontransformed data were presented graphically.

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120 Eflfect of Nitrogen Source. Medium Osmolaritv and Protoplast Density on the Growth and Development of Avocado 'T362' Protoplasts i n Liq uid Medium A factorial experiment was designed to test the effects of 3 growth parameters of protoplast cultures at 2 levels in liquid growth medium. Nitrogen sources were provided by media MS 8P and MS8P", in which the total nitrogen content of the former was 61.2 mM, with 18.8 mM from NOs' and 42.4 mM from organic NH4'^(glutamine), while the latter had 60.0 mM nitrogen, v^th 39.4 mM from NO3" and 20.6 mM from inorganic Mi/. Medium osmolarity was either 0.6 or 0.4 M (0.15 M sucrose balanced with 0.45 or 0.25 M mannitol, respectively). The protoplast densities were either 0.8 x lO' or 1.6 x lO' protoplasts ml"\ The protoplasts were cultured in 2 ml liquid medium in 60 x 15 mm sterile plastic Petri dishes sealed with Nescofilm and maintained in darkness at 25°C. Growth of protoplasts was determined by counting the number of microcalli or globular somatic embryos in 2 adjacent squares (4 mm^) in the center of a Petri dish after the Petri dish had been swiried to obtain an even distribution of microcalli/globular proembryonic masses. The number of microcalli and globular proembryonic masses were counted from three different areas of a Petri dish (left, center and right), after it was swirled to obtained an even distribution of microcallus/globular proembryonic masses, from 3 Petri dishes per treatment. Observations were made 1 month after culture. The data on the number of microcalli and globular proembryonic masses were presented as percentages and were transformed with arcsine transformation for ANOVA computation (SAS Institute, 1992) and their non-transformed values were presented graphically. The data on the relative number of microcalli/globular somatic embryos were also analyzed for ANOVA and their means and standard errors were presented graphically.

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121 Plating Efficiency of Nucellar-Derived 'T362' Avocado Protoplasts in 0.4 MS'SP and the Effect of Dilution on Somatic Embryo Development. Plating efficiency Protoplasts of 'T362' were cultured in 2 ml of 0.4 M MS 8P with a density of 1x10^ ml ' in 60 X 15 mm plastic Petri dishes, sealed with Nescofilm, and incubated in darkness at 25°C. Their growth and development were observed after 1, 5, 8 and 14 days. Three cultures were sampled at each observation, and were transferred to 15 ml centrifuge tubes and pelleted by centrifugation at 100 g for 3-5 min. Some of the liquid medium was pipetted out, leaving only 0.2, 0.2, 0.3 and 0.5 ml for observation after 1, 5, 8 and 14 days, respectively. The protoplasts and microcalli were categorized into several groups based on aggregation, necrosis and level of division, and were counted using a Fuchs Rosenthal haemocytometer. Data were presented as percentages (means and standard error) of cells/microcallus within each category (see Results, Table 5-6) and plating efficiency was calculated as the sum of the percentage from categories of cells and clusters of cells that underwent division. • ' Effect of time and level of dilution on the development of somatic embryos A factorial experiment was carried out to determine the effect of subculture time and dilution rate of cultured protoplasts on the development of somatic embryos. 'T362' protoplasts were cultured in 2 ml of 0.4 M MS"8P with a density of 1x10^ ml' \ At 14, 21 and 28 days after culture, protoplast-derived cultures were subcultured in 2 ml liquid 0.15 M MS 8P with a density of 1/3-1/96 of the original cultures to make dilutions of 3, 6, 12, 24, 48 and 96-fold, respectively. Protoplast-derived culture morphologies were not the same at the time of subculture. At 14 days after culture, the cultures consisted of mostly globular proembryonic masses ca. 75 ^im diameter. At day 21, the globular proembryonic masses were larger (200-300 |im) and in clumps of 0.5-1.0 mm. At day 28, the proembryonic masses were dedifferentiating and were ca. 200-300 |xm and in clusters of 1 .3-1.5 mm.

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122 After 1 month of culture, 3 replicates (3 Petri dishes) from each dilution treatment and subculture time were randomly sampled and observed. The number of somatic embryos >2 mm diameter were counted from each replicate (1 dish) under a dissecting microscope and each culture was weighed. The data were analyzed as a factorial experiment. Analysis of variances were computed using Proc GLM (SAS Institute, 1992). Means and standard errors were calculated and were plotted in graphs. Effect of Genotype on Protoplast Yield and Culture Development in Liquid 0.4 M MS"8P Medium Protoplasts from several genotypes were isolated and purified as described above. They were cultured in 2 ml of 0.4 M MS"8P medium in 60 x 15 mm plastic Petri dishes with a plating density of 0.8-1.6 protoplasts ml"V The cultures were sealed with Nescofihn and maintained in darkness at 25''C. The morphology of embryogenic cultures at the time of protoplast isolation, their yields and the presence of microcallus after 1 month of culture were recorded. Somatic Embryo Maturation and Germination on Semisolid Medium Globular somatic embryos or proembryonic masses (>1.5 mm diameters) that developed after 1-2 months of culture from 4week-old 'T3 62 'protoplast cultures on 0.4M MS"8P protoplast medium that had been diluted 20-fold and subcultured in 2 ml M 0.15 MS'8P were used as inocula for observing somatic embryo development on semisolid medium. The inocula, each weighing ca 0.01 g, were transferred to SED medium (Table A-1). There were 9 inocula per Petri dish (150 x 20 mm), which were sealed with Parafilm and maintained in darkness at 25°C

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123 After 1-2 months of culture, opaque somatic embryos (>0.8 cm diameter) were transferred individually onto SEMG medium (Table A-1) and subcultured at two-monthintervals thereafter (see Chapter 4 for details). Results The protoplast yield from avocado embryogenic suspension cultures composed of proembryonic masses (PEM) ranged from 2-30 x 10* protoplasts g"^ fresh weight, depending on the genotype (Table 5-1). PEM-derived avocado protoplasts were mostly ca 20 nm diameter, although the range was ca. 15-27 [im diameter with a few (< 0.5-1%) being ca. 50 jam diameter. Starch grain content varied with respect to genotype. For example, 'Thomas' and 'M25864' protoplasts had abundant starch grains, while 'Esther' had relatively fewer and 'T362' and nucellar-derived 'Thomas' had virtually no starch grains. Protoplast viability as determined by FDA and Evans blue tests 24 h after isolation and purification was also genotype-dependent. The FDA test consistently produced a value 20% lower than the Evans blue test. Protoplast viability of ca. 80% (FDA test) was observed from 'T362'and nucellar-derived 'Thomas' (Figure 5-1). I Effect of Protoplast Density and Medium Osmolarity on Growth of Avocado 'T362' Protoplasts Plated on Agarose Disc Type Media ! Microcalli developed from dividing protoplast-derived cells after 21 days in agarose disc media. Plating eflBciency of avocado 'T362' protoplasts 21 days after culture was significantly affected by protoplast plating density, medium osmolarity and their interaction (Table 5-2). Among those factors, plating density appeared to be the most important factor, since its contribution to the variability of the model was 88% as measured by its sum of squares. Figure 5-2 showed that at a plating density of 0.25 x lO' protoplast ml'\ irrespective of medium osmolarity, plating efficiencies were significantly lower than with other plating densities. Plating efficiencies at plating densities 1 and 4 x

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» 124 10* were generally high (35-47%) even though their variations were statistically significantly affected by medium osmolarity. Table 5-1. Protoplast yield per gram fi-esh weight of avocado embryogenic suspension cultures derived fi-om zygotic embryos of four avocado cultivars. Cultivars Yield (Means±Se X 10*) Number of Trials T362 3.50 ±0.85^ • ,4' Thomas 30.38 ±3.73 M25864 7.20 ± 1.21 • .4 ... Esther 33.00 i blue M25864' T362' Thomas' Genotype Figure 5-1. Viability of protoplasts isolated fi'om embryogenic cultures derived fi-om zygotic embryos of different avocado genotypes. The viability tests were conducted after storage of protoplasts on ice for 24 h.

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125 Table 5-2. ANOVA of the effect of medium osmolality and protoplast density on the plating eflSciency, length and width of microcalli derived from 'T362' avocado protoplasts cultured in agarose disc type method, three weeks after culture. Source df Sum of Squares Mean Square F Value Pr>F Plating eflSciencv Osmolarity (0) 3 435.019 145.006 5.64 0.0051 Density(D) 2 8846.277 4423.139 172.03 0.0001 0*D 5 457.427 91.485 3.56 0.0165 Error Ll jOj.OO / Total 32 10041.109 Length of microcallus Osmolarity (0) 3 2154.585 718.195 15.54 0.0001 Density (D) 2 1128.476 564.238 12.21 0.0001 0*D 5 1231.1754 246.235 5.33 0.0002 Error 22 5129.000 46.207 Total 32 9459.844 Width of microcallus Osmolarity 3 1608.619 536.206 13.42 0.0001 Density 2 1018.1524 509.076 12.74 0.0001 0*D 5 892.773 178.555 4.47 0.0010 Error 11 4435.000 39.955 Total 32 7871.967

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126 Medium Osmolarity (M) i Figure 5-2. Eflfect of medium osmolarity and protoplast plating density on plating efficiency of 'T362' avocado protoplasts cultured in agarose disc type method after three weeks. Medium Osmolarity (M) Figure 5-3. Effect of medium osmolarity and protoplast plating density on length of microcalli that developed from 'T362' avocado protoplasts cultured in agarose disc type method after three weeks.

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The size of microcalli as represented by their lengths and their widths also was significantly affected by the treatments and their interaction (Table 5-2). At plating densities of 0.25 and 1 x lO' protoplasts ml'\ higher medium osmolarity significantly reduced the size of microcalli. At a higher plating density (4 x lO' protoplasts ml"^), medium osmolarity has no effect on the size of microcalli. The length and the width of microcalli responded similarly to the treatments; therefore only microcalli length was presented graphically (Figure 5-3). The largest microcalli (280 urn) occurred with a treatment combination of 1 x lO' protoplasts ml"' and medium osmolarity of 0.4 M, and this was determined as the best treatment combination since it also yielded a high plating efficiency. The difference in plating efficiencies and microcallus size as affected by protoplast plating densities when the protoplasts were cultured at medium osmolarity of 0.4 M are depicted in Figure 5-4. A high plating density (4 x lO' protoplast ml"') with a high plating efficiency resulted in a very high number of microcalli per agarose disc (Figure 5-4 A). Optimal plating density (1 x lO' protoplasts ml"') and a high plating efficiency resulted in a lower number but greater size of microcalli (Figure 5-4 C-D) whereas a high plating density resulted in high plating efficiency. A low plating density of 0.25 x 10^ protoplasts ml"' with a low plating efficiency resulted in few microcalli per disc (Figure 5-4 B). Small microcalli were also observed in the liquid medium surrounding the agarose medium (Figure 5-3 E), indicating that avocado protoplasts could also be grown in liquid medium. Replacement of liquid medium in the treatment combination 0.4 M and 1 x lO' protoplast ml'' with medium of the same composition but with lower osmolarity (0.15 M) at 3 weeks post-culture resulted in the development of organized proembryonic masses (Figure 5-4 F) from 50% of microcalli ca. 3 weeks later, while no organized growth was observed in medium of unaltered osmolarity. Proembryonic masses also developed from

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Figure 5-4. The growth and diflferentiation of embryogenic culture-derived protoplasts of zygotic-derived 'T362' cultured in agarose medium with medium osmolarity of 0.4 M as affected by plating density. An agarose disc containing microcalli from protoplasts cultured with plating density of 4 x lO' (A), 0.25 x 10^ (B) and 1 x lO' protoplasts nil'\ Note the differences in size and density of microcalli in an agarose disc. (D) A higher magnification of a microcallus from C. Note the microcallus showed organization of the cell proliferation. (E) A microcallus that differentiated in liquid medium surrounding the agarose medium. (F) organized proembryonic masses developed from a microcallus after replacing the liquid medium of 0.4 M with 0.12 M liquid medium of the same composition. (G) Organized proembryonic masses from a microcalUus in liquid medium after treatment similar to that of F. (H) Organized proembryonic masses and globular somatic embryos in agarose discs.

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130 Figure 5-5 A culture dish containing proembryonic masses and somatic embryos that differentiated from agarose-embedded protoplasts. Somatic embryos and organized proembryonic masses developed both inside the agarose medium and in the Uquid medium surrounding the agarose after 3 weeks in medium with 0.4 M medium followed by replacement of the Uquid medium with 0. 15 M liquid medium of the same composition for 4-5 weeks.

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131 microcalli in liquid medium (Figure 5-4 G). Numerous somatic embryos and organized proembryonic masses developed in agarose discs ca. 7-9 weeks from initial culture, resulting in rapidly growing cultures (Figure 5-5). Transfer of the 3-week-old agarose discs containing microcalli from the treatment combination 0.4 M and 1 x 10^ protoplast ml"' into liquid MSP medium resulted in the establishment of embryogenic cultures after ca. 4 months; however, when the microcalli were transferred to semisolid MS medium containing 13% sucrose, embryogenic cultures were observed only after three weeks, while no growth was observed on semisolid MS medium containing 3% sucrose. Eflfect of Nitrogen Source. Medium Osmolaritv and Protoplast Density on the Growth and Development of Avocado 'T362' Protoplasts in Liquid Medium The objective of this experiment was to determine the effects of nitrogen source, medium osmolarity and plating density on the growth and development of 'T362' protoplasts in liquid medium. After one month of culture, protoplasts had developed as either unorganized cell masses (microcalli) or organized proembryonic masses. Both morphologies had a diameter of 50-200 nm. Table 5-3 indicates that medium osmolarity, nitrogen source, plating density and the interaction of osmolarity and nitrogen source significantly affected the relative number of microcalli/proembryonic masses that were formed from cultured protoplasts. Even though plating density also had a significant effect on the growth of microcalli, its contribution to the variability of the model as indicated by its sum of squares was only 2.3%, while the other factors, i.e., medium osmolarity and nitrogen source, contributed as much as 39.1% and 39.6%, respectively. Nitrogen source and osmolarity were therefore the critical factors for relative number of microcalli/proembryonic masses formed from protoplasts, while plating density levels tested were not as critical.

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132 Table 5-3. ANOVA of the effect of nitrogen sources, medium osmolarity and protoplast density on number of microcalli and proembryonic masses from avocado 'T362'protoplasts, one month after culture^. Source jjr oUIIl Ul Ol|UalCa Mpfln Sniiare F Value Pr >F Osmolarity (0) 834.26 834.26 205.36 0.0001 Nitrogen (N) 846.09 846.09 208.27 0.0001 Density (D) 49.59 49.59 12.21 0.0131 0*N 326.34 326.34 80.33 0.0001 0*D 0.51 0.51 0.13 0.0131 N*D 4.59 4.59 1.13 0.9023 0*N*D 7.59 7.59 1.87 0.9023 Error 16 65.00 4.06 Corrected Total 23 2133.99 ^ Number of microcalli and proembryonic masses were determined by counting the number of those two morphologies inside 2 squares of 2 mm^ observation fields that were located in the center of the Petri dish. The cultures were swirled to provide an even distribution of microcalli and proembryonic masses. The best treatment combination that resulted in the highest number of microcalli/globular somatic embryos from protoplasts was medium MS"8P with medium osmolarity of 0.4 M (Figure 5-6). The percentages of microcalli that developed from protoplasts were also significantly affected by medium osmolarity, nitrogen source and their interaction. Plating density and its interaction with other factors were not as critical as osmolarity and nitrogen source for microcalli development; however, it was as significant as other factors and their interactions for affecting the percentage of proembryonic masses that developed from protoplasts (Table 5-4). Figure 5-7 shows very clearly that at medium osmolarity of 0.6 M, regardless of the nitrogen source of the medium and plating density, only microcalli developed from protoplasts. At medium osmolarity of 0.4 M, however, proembryonic mass development was dependent upon the source of nitrogen and protoplast density. Irrespective of the plating density, with medium osmolarity of 0.4 M, medium with glutamine (MS'SP) allowed the formation of more proembryonic masses than medium

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133 with NH4NO3 (MS8P"). With a treatment combination of 0.4 M MS"8P, the lower plating density (0.8 x lO' protoplast ml ') allowed the development of more proembryonic masses than the higher plating density (1.6 x lO' protoplast ml''). Since it is easier to adjust the plating density than to alter the nitrogen content in the medium, the combination of medium osmolarity and nitrogen source of 0.4 M MS"8P was adopted as the standard medium for avocado protoplast culture. Table 5-4. ANOVA of the effect of medium osmolarity, nitrogen source and plating density on the percentages of microcalli and somatic embryo development from avocado 'T362' protoplasts, one month after culture^. Source df Sum of Squares Mean Square F Value Pr>F Percent microcalli Osmolarity (0) 1.886 1.886 107.82 0.0001 Nitrogen (N) 1.037 1.037 59.58 0.0001 Density (D) 0.135 0.135 7.78 0.0131 0*N 1.037 1.037 59.58 0.0001 0*D 0.135 0.135 7.78 0.0131 N*D 0.000 0.000 0.02 0.9023 0*N*D 0.000 0.000 0.02 0.9023 Error 16 0.278 0.017 Corrected Total 23 4.499 Percent proembrvonic masses Osmolarity (0) 1.876 1.876 107.82 0.0001 Nitrogen (N) 1.037 1.037 59.58 0.0001 Density (D) 0.135 0.135 7.78 0.0131 0*N 1.037 1.037 59.58 0.0001 0*D 0.135 0.135 7.78 0.0131 N*D 0.000 0.000 0.02 0.9023 0*N*D 0.000 0.000 0.02 0.9023 Error 16 0.278 0.017 Corrected Total 23 4.498 ^The data represented the percentage of microcalli and proembryonic masses. The percentage data were transformed with arcsine transformation for the analysis.

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134 Figure 5-6. Effect of nitrogen source, medium osmolarity and protoplast plating density on the number of microcalli and proembryonic masses that developed from 'T362' avocado protoplasts after one month in culture. MS= MS'8P, MS+ = MS8P", 0.8 indicates 0.8 x lO', 1.6 indicates 1.6 x lO'. Osmolanty: 0.4 0.4 0.4 0.4 0.6 0.6 0.6 0.6 Medium: MSMSMS MS MSMSMS MS Density: 0.8 1.6 0.8 1.6 0.8 1.6 0.8 1.6 Figure 5-7. Effect of nitrogen source, medium osmolarity and protoplast plating density on the percentage of microcalli and proembryonic masses that developed from 'T362' avocado protoplasts after one month in culture. MS= MS'8P, MS+ = MS8P", 0.8 indicates 0.8 x lO', 1.6 indicates 1.6 x 10^

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Plating Efficiency of Avocado 'T362' Protoplasts in 0.4 MS'SP and the Effect of Dilution on Somatic Embryo Development Plating efficiency and development of proembryos from protoplasts An objective of this experiment was to observe the growth and development of protoplasts in the standard avocado protoplast liquid medium in order to measure the response of protoplasts at early stages in culture. The results are presented in Table 5-5. Table 5-5. Percentage of protoplasts that divided on day 1, 5, 8 and 12 after culture^. Day 1 Day5 Day 8 Day 12 (protoplasts ± S.E.) % Singulated protoplasts shriveled/brown 0.0 ±0.0 19.9 ±2.0 24.0 ± 2.0 31.5±3.4 healthy/transparent 48.9 ±7.0 19.2 ±1.6 17.6 ± 1.3 3.4 ± 1.6 1st division (2 cells) 0.0 ±0.0 1.5 ±0.8 3.7 ±2.2 0.7 ±0.7 2nd division (3-4 cells) 0.0 ±0.0 0.0 ±0.0 0.9 ±0.1 0.4 ±0.4 3rd division (5-8 cells) 0.0 ±0.0 0.0 ± 0.0 0.4 ± 0.3 0.4 ± 0.4 proembryos (>9 cells) 0.0 ±0.0 0.0 ± 0.0 0.2 ±0.2 0.4 ± 0.4 Cluster of budded or chained protoplasts sh^iveled^rown 0.0 ±0.0 0.0 ± 0.0 15.1 ±0.4 26.1 ±2.7 healthy/transparent 51.2 ±7.0 56.1 ±3.6 33.6 ±2.6 15.1 ±4.5 1st division (2 cells) 0.0 ±0.0 3.6 ±0.5 3.8 ± 1.0 11.2 ±0.2 2nd division (3-4 cells) 0.0 ± 0.0 0.0 ±0.0 1.6 ±0.3 7.8 ± 1.4 3rd division (5-8 cells) 0.0 ±0.0 0.0 ±0.0 0.5 ±0.3 1.7 ±0.4 proembryos (>9 cells) 0.0 ±0.0 0.0 ±0.0 0.2 ±0.2 1.7± 1.1 Plating Efficiency 0.0 ±0.0 5.1 ±0.6 9.2 ± 1.5 25.0 ± 1.2 ^ Protoplast were cultured in 2 ml liquid medium of 0.4M MS'SP with plating density of 1 X lO' protoplast ml"'.

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136 After one day in culture, newly isolated protoplasts (Figure 5-8 A) began to clump together, forming chains or clusters of 3-16 protoplasts (Figure 5-8 B). Some of these clusters or chains disassociated when the cultures were resuspended for observation with a haemocytometer. Protoplast budding was observed in ca. 51% of the samples (Figure 5-8 B and C). Elongation and the beginning of cell division were also observed in some of the protoplasts/cells. After five days in culture, the protoplasts/cells were grouped into two main categories: singulated protoplasts/cells, and clustered, budded and chained (designated as clustered) protoplasts/cells. Approximately 20% of the singulated protoplasts failed to survive and turned brown, while some (19.2%) remained healthy and transparent; only 1.5% of the protoplasts underwent the first division (Figure 5-8 C). No entirely shriveled and brown clustered protoplasts/cells were observed, and most of them appeared to be alive; however, only ca 3 .6% of the protoplasts/cells underwent the first division. The first cell division was observed in one or more cells from the clustered, chained, and budded cells, indicating the single cell origin of the microcalli or proembryonic masses that develop from protoplasts. The percentage of cells/cluster of cells undergoing their first division 5 days after culture was 5.1%. At day 8, the singulated and the clustered cells also underwent the second and, subsequent divisions (Figure 5-8 D). Clustered cells began to die and turned entirely brown at this time (ca. 15%), and the percentage of singulated protoplasts that failed to survive also increased. After 12 days, the culture morphologies resembled those observed 8 days after culture; however, the plating efficiency was variable. Plating efficiencies at days 5, 8 and 12 after culture were 5, 9 and 25%, respectively (Table 5-5). Microscopic observation revealed that protoplast-derived cell divisions produce cell clusters that are very tightly organized (Figure 5-8 E and F) demonstrated by the roundness of the cell clusters. Continued observation indicated that these cell clusters

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Figure 5-8. Somatic embryogenesis from avocado protoplasts cultured in liquid medium. Embryogenic culture-derived 'T362' protoplasts were cultured in 2 ml liquid medium consisting of 0.4 M MS"8P in 60 x 15 ml Petri dishes after 0-21 days in culture. (A) Freshly isolated protoplasts. (B) Chained protoplast, after 5 days in culture. (C) Budding and a dividing (1'^ division) protoplast after 5 days. (D) Cluster of cells, some of which underwent 2nd-3rd division forming proembryos. (E) Globular proembryos in clusters, after 14 days in culture. (F) A higher magnification of E. (G) Dedifferentiating proembryos after 21 days of culture. (H) globular somatic embryos or proembryonic masses attained size of 1-3 mm after subculture at medium of lower osmolarity (0. 15 M) and low density.

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139 gave rise to globular somatic embryos (Figure 5-8 E & F) which were morphologically different from microcalli (Figure 5-4 D-E). The globular somatic embryos started to dediflferentiate after 21 days of culture (Figure 5-8-G). Dilution experiment One month after subculture at diluted culture densities, these cultures were composed of different sizes of globular somatic embryos and microcalli (Figure 5-8 H). The globular somatic embryos ranged 0.15-30 mm diameter and the microcalli were ca 0.5-1.5 mm diameter. Analysis of variance indicated that subculture age, dilution rate and their interaction significantly affected the number of somatic embryos and the fresh weight per Petri dish (Table 5-6). , Table 5-6. ANOVA of the effect of subculture age and dilution rate on culture fresh weight and number of somatic embryos that developed from nucellus-derived 'T362' protoplasts, one month after culture. Source DF Sum of Squares Mean Square F Value Pr>F Fresh weight Age 5 0.045 0.022 13.87 0.0032 Dilution rate 2 0.246 0.049 6.28 0.0001 Age*Dilution rate 10 0.179 0.018 5.04 0.0001 Error 67 0.227 0.004 Corrected Total 84 0.713 Number of somatic embrvos >2 mm Age 5 519.369 259.685 111.63 0.0001 Dilution rate 2 123.568 24.714 10.62 0.0001 Age*Dilution rate 10 326.086 32.608 14.02 0.0001 Error 67 155.867 2.326 Corrected Total 84 1187.812

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140 Figure 5-9. EflFect of subculture age and dilution rate in medium of low osmolarity (0.15 M MS'SP) on the formation of large (> 2 mm diameter) somatic embryos. Figure 5-10. EflFect of subculture age and dilution rate in medium of low osmolarity (0. 15 M MS'SP) on fresh weight accumulation of protoplast-derived proembryos after one month of culture. I

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141 Subculture age was very critical for development of somatic embryos ^2 mm diameter. When protoplast cultures were subcultured with diluted density at day 15, dilution rates of 6-48-fold resulted in the formation of 6-12 globular somatic embryos per plate. When the protoplast-derived cultures were subcultured at diluted density at days 21 and 28, high dilution rates (49-96-fold) were required for the development of somatic embryos with diameter >2 mm, yet at low frequency (maximum 5 somatic embryos per plate) (Figure 59); however, abundant somatic embryos ^2 mm were observed in addition to disorganized proembryonic masses. Figure 5-11 indicated that the fresh weight of cultures decreased with respect to dilution rate, when cultures were subcultured at day 15. A similar trend was also observed for subculture at day 21, except that fresh weight of cultures was higher than that of cultures subcultured at day 14 with dilution rates of 6-fold and higher. With subculture at day 28 the dilution rate had virtually no effect on culture fresh weight. At high dilution rates (12-96 fold), the fresh weight of cultures that were subcultured at day 28 was much higher than that of cultures subcultured at earlier ages. Nevertheless, the fresh weights of cultures that were subcultured at day 28 were as much as the highest fresh weight that could be obtained by other cultures that were subcultured earlier and at lower dilution rate, i.e., ca. 2.5-3.3 gram per Petri dish. ; Effect of Proembryonic Mass Morphology on Protoplast Yields and Responses in Liquid 0.4 M MS'8P Medium ' I Table 5-7 has summarized the variation in growth and development responses of avocado protoplasts isolated from proembryonic masses of different morphologies and genotypes in standard liquid avocado protoplast medium (0.4 M MS'8P) with standard plating density (I x lO' protoplasts ml '). When the proembryonic masses used for protoplast isolation were from newly established suspension cultures at which time the proembryonic mass was largely nodular ('Booth 8', 'Isham', 'Hass'), low protoplast yields

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142 were obtained. This type of protoplast did not divide and was necrotic after 3-4 weeks. High protoplast yields were recovered from suspension cultures that consisted of a mixture of nodular proembryonic masses and disorganized proembryonic masses Table 5-7. Relationship among genotypes, their embryogenic characteristics, protoplast yield and response after one month in liquid medium 0.4 M MS"8P, at plating density of 0.8-1.2 x lO' protoplast ml'\ Cultivar/ Type of Embryogenic Morphology of Protoplast Response: Genotype of Mother Culture* PEMs at Time of Yield' microcaUi or Tree Protoplast Isolation*" PEMs or Both 'Booth 8' late dedifferentiating nodular low none 'Esther' early dedifferentiating disorganized high microcalli 'M25864' early dedifferentiating disorganized high microcaUi 'Isham' no dedifferentiating nodular low none 'Thomas' (zygotic) intermediate , mixture high microcalli/PEMs 'Thomas' (zygotic) intermediate disorganized high microcalli/no growth 'Thomas' (nucellar) late dedifferentiating mixture high microcalli/PEMs 'Thomas' (nucellar) late dedifferentiating disorganized high no growth 'Hass' late dedifferentiating nodular low no growth 'Hass' late dedifferentiating mixture high microcalli 'Lamb' late dedifferentiating nodular low 'Lamb' late dedifferentiating disorganized high microcalli 'T362' late dedifferentiating mixture high microcalli/PEMs * Type of embryogenic culture was determined from newly initiated embryogenic culture in liquid medium or from newly initiated embryogenic culture on semi solid medium. *" Morphologies of cultures at time of protoplast isolation may have changed from their initial morphologies. 'High yield if the volume of pelleted protoplast after gradient centrifugation was >0.01 ml, low yield if the volume of pelleted protoplast after gradient centrifiigation was 0.010.03 ml. Approximately 1 g fresh weight of proembryonic masses was used for protoplast isolation, "Protoplast culture was not attempted. i •

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143 i i Figure 5-11. Somatic embryo development from protoplasts derived cultures as affected by culture method. (A) Numerous but small somatic embryos developed when the entire content of protoplast culture disc was poured onto a dish with SED medium. (B) Large and maturing somatic embryos developed when organized proembryonic masses were used as inoculum in a similar manner to that described for somatic embryo development (Chapter 4). The scale in A is twice of B.

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144 ('T362', mixture of disorganized and nodular PEMs of 'Thomas' from zygotic or nucellar origin). These protoplasts developed into microcalli and globular somatic embryos after one month of culture. Consistently high yields were obtained when completely dedifferentiated proembryonic masses were used for protoplast isolation. These protoplasts, however, only developed as microcalli and had lost their morphogenic potential (disorganized proembryonic masses from 'Lamb'), sometimes budding and forming chains of dividing cells without fiirther division (disorganized 'Thomas' proembryonic masses). i Somatic Embryo Development. Maturation and Germination In attempting to obtain good quality somatic embryos that would develop normally to germination, the method that was developed for citrus (Grosser & Gmitter, 1990) was tried. Accordingly, the entire culture (from 1 culture dish) that consisted of enlarged nodular proembryonic masses (1-2 mm) and smaller proembryonic masses was poured onto semisolid SED medium in a 110 x 20 mm Petri dish. After 1 month of culture, a mixture of small (:^0.5 mm diameter), opaque and hyperhydric somatic embryos developed and covered almost the entire surface of the SED medium (Figure 5-11 A). These somatic embryos did not enlarge ftirther and did not germinate after transfer to germination medium. Alternatively, only the large (1-2 mm) protoplast-derived nodular proembryonic masses and dedifferentiating proembryonic masses were used as inocula on semisolid medium. After 1-2 months of culture, proembryonic masses as well as somatic embryos of different sizes, different stages of development and of varying hyperhydricity developed from the inoculum (Figure 5-1 1 B). Only opaque somatic embryos >0.8 cm diameter were transferred individually to somatic embryo germination medium. The somatic embryos enlarged to ca 1.0-1.5 cm diameter. After transfer onto germination medium, ca. 60% of the cultures produced

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145 secondary somatic embryos and/or disorganized proembryonic masses, whereas the remainder continued to enlarge. After 4 successive transfers, no shoot development was observed from ca. 80 somatic embryos. Discussion Avocado protoplasts could be isolated and purified with consistently high yields from embyogenic suspension cultures consisting of dedifferentiating proembyonic masses using a protocol that has been used routinely for citrus (Grosser & Gmitter, 1990), and for grape and mango (J. W. Grosser, personal communication). Avocado protoplast yields consisting of 3-30 x 10* protoplasts g"' fresh weight were as high as the yields of other species, e.g., citrus (Grosser and Gmitter, 1990; Vardi et al., 1987), pear (Ochatt & Power, 1988), sour cherry (Ochatt, 1990), peach (Matsuta et al., 1986; Mills & Hammerschlag, 1994) and Coffea arabica (Grezes et al., 1994). Protoplasts isolated from embryogenic 'T362' avocado cultures developed directly into proembryos and subsequently into nodular proembryonic masses and later heart stage and more mature stage somatic embryos when the physical and chemical cultural conditions were optimized. This direct pathway of somatic embryo regeneration from protoplasts derived from embryogenic cultures has been reported for somatic hybrids of citrus (Grosser & Gmitter, 1990), including Citrus sinensis (Kobayashi et al., 1985; Niedz, 1993), Citrus mitis (Sim et al., 1988), Citrus madurensis (Ling et al., 1989), Citrus unshiu (Ling et al., 1990) and grapevine (Reustle et al., 1995). Indirect somatic embryo development from embryogenic culture-derived protoplasts has been reported for citrus and citrus relatives (Kunitake et al., 1991; Jumin & Nito, 1995, 1996a, b), and involves regeneration after protoplast-derived microcallus has been transferred to medium containing plant growth regulators. No plant growth regulators are required for direct somatic embryogenesis from avocado protoplast cultures derived from embryogenic

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146 cultures. This indirect pathway of embryogenesis reported for citrus is probably related to the morphogenic potential of the embryogenic cultures from which the protoplasts were isolated. In those studies, the protoplasts were isolated from embryogenic cultures estabUshed from seedling stem mstead of nucellus or ovular tissues, and it was not clear if those embryogenic cultures consisted of organized or disorganized proembryonic masses. Somatic embryo development was not prolific from these cultures (Jumin & Nito, 1995; 1996a; b). Differentiation via organogenesis has also been reported from protoplasts, and is typical of woody fi^iit species regenerated from leaf mesophyll protoplasts, e.g., pear, Pyrus spp., (Ochatt, 1993b), stone fruits {Prunus spp.) (Ochatt, 1993c) and apple (Mains X domestica) (Patat-Ochatt, 1994). The regeneration pathway of a protoplast seems to be determined by the competence of the source tissue, unplying the importance of choice of source tissue. The early stages of development of proembryos, proembryonic masses and somatic embryos from avocado embryogenic protoplasts was affected by cultural parameters that included physical and chemical factors and by genetic and epigenetic characteristics of the protoplast sources. Plating density and medium osmolarity have been reported to be two of the most critical physical parameters for citrus protoplast culture and significantly affect plating eflBciency and somatic embryo development (Kobayashi et al., 1985). While optimal plating density of avocado protoplasts at ca lO' ml"' is similar to that reported for other species, i.e., citrus (Vardi et al., 1975; Vardi & Galun, 1988; Grosser & Gmitter, 1990), the optimum medium osmolarity of 0.4 M is low compared to the standard 0.6-0.7 M used for other perennial species, i.e., citrus (Grosser & Gmitter, 1990; Hidaka & Kajiura, 1988; Niedz, 1993; Sim et al., 1988; Ling et al., 1990) and grape (Reustle et al., 1995). When protoplasts were cultured at high medium osmolarity (0.6-0.7 M), the osmolarity needed to be reduced progressively, while no weaning was required for avocado and citrus (Kobayashi et al., 1985) cultured at 0.4 M. The beneficial effect of

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> 147 low medium osmolarity for efficient somatic embryo development has been reported in citrus by Kobayashi et al. (1985). Omission of NH4NO3 from the basal medium has been reported to be beneficial for microcallus development from mesophyll protoplasts of Populus (Russell & McCown, 1986, 1988) and oi Pyrus sp. (Ochatt, 1992; 1993b; Ochatt & Caso, 1986). Grosser (1994) suggested that high NH4NO3 concentration in the medium (as in MS) could be toxic to protoplasts; however, glutamine at 42.4 has been routinely used as one of the nitrogen sources in citrus protoplast culture medium (Grosser & Gmitter, 1990). Whether the increased frequency of somatic embryo formation from 'T362' avocado protoplasts following replacement of NH4NO3 by glutamine is related to the ratio of reduced nitrogen in the medium or to the presence of the organic form of ammonia is not clear. Experiments concerning the effects of the ratio and concentration of inorganic, reduced and oxidized nitrogen indicated that medium with a low ratio or even without reduced nitrogen were beneficial for somatic embryo development from dedifferentiated proembryonic masses (see Chapter 3). In contrast, Higashi et al. (1996) reported that embryogenic carrot cell clusters required reduced nitrogen for somatic embryo development, and organic nitrogen, i.e., glutamine, strongly affected the development of mature somatic embryos. Development of globular somatic embryos or proembryonic masses of 1-2 mm diameter required subculture of protoplast-derived cultures into lower densities and into medium with low osmolarity (0.15 M). Reduction of density of cells derived from citrus protoplasts resulted in somatic embryo development and caused Kobayashi et al. (1985) to suggest that embryogenic potential might be repressed when cells are cultured at high cell density. Grosser and Gmitter (1990) suggested that cultures with high protoplast plating efficiency should be diluted; otherwise no somatic embryos could be regenerated. Proembryos and somatic embryos that developed from avocado protoplasts were unable to develop to maturity and germinate after transfer to semisolid medium, unlike

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> 148 citrus (Grosser & Gmitter, 1990; Kobayashi et al., 1985; Hidaka & Kajiura, 1988; Sim et al., 1988; Niedtz, 1993). As a result, the proembryonic masses or somatic embryos that developed from avocado protoplasts were used as inocula to obtain good quality white, opaque cotyledonary somatic embryos of ^ 0.8 cm diameter which enlarged to 1.0-15 cm diameter on medium with low osmolarity. This approach has been used to develop good quality somatic embryos from embryogenic suspension cultures of avocado (Chapter 4). The low frequency germination of protoplast-derived mature somatic embryos is probably related to the low plant regeneration from avocado somatic embryos (see Chapter 4; Pliego-Alfaro & Murashige; 1988, Mooney & Van Staden, 1987). In conclusion, a protocol for simple and efiScient isolation, culture and regeneration of mature somatic embryos from protoplasts isolated from avocado embryogenic suspension cultures has been developed for the first time. This protocol could open opportunities for avocado improvement by somatic cell genetics. For example somatic hybridization between avocado and root rot resistant small seeded Persea species could be attempted and direct gene transfer protocols could be developed.

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CHAPTER 6 AVOCADO SHOOT CULTURE AND PLANTLET DEVELOPMENT AND NET PHOTOSYNTHESIS IN A NON-ELEVATED AND ELEVATED CO2 ENVIRONMENT Introduction In vitro propagation of avocados (Persea americana Mill.) from seedling shoot tip and nodal expiants has been described with limited success, including some shoot elongation of existing buds, limited shoot proliferation and formation of scaly leaves (Schroeder, 1979, 1980; Young, 1983; Vega-Solarzano, 1989; Gonzalez-Rosas & Salazar-Garcia, 1984; Harty, 1985; Barringer, 1996). Shoot elongation and formation of expanding leaves were also reported from nodal stem segments of 1 -year-old P. schiedeana Nees (Gonzalez-Rosas et al., 1985). Schall (1987) and Cooper (1987) described culture of avocado seedling shoot tips, and subsequently rooted and acclimatized plantlets. Attempts by Pliego-Alfaro & Murashige (1988) to micropropagate adult trees from shoot tips using in vitro serial grafting were unsuccessfiil. Limited shoot multiplication that declined with each subculture and low rooting frequency were reported when shoots were derived from heavily pruned grafted scions derived from mature phase avocado (Pliego-Alfaro et al., 1987). A protocol for rooting of juvenile shoots in vitro has been developed (Pliego-Alfaro, 1988). In addition to their low multiplication rate, survival of shoots in vitro was reported to be ca 80% when Murashige &. Skoog (1962) (MS) formulation was used as the basal medium (Schall, 1988). On MS medium, avocado shoots developed scaly leaves, leaf tip bum and shoot die back, depending on the genotype. These symptoms were similar to ammonia toxicity symptoms that have been described by Lovatt (1988). On an SM 149 -\ '^-^y .

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150 medium supplemented with activated charcoal, necrosis of the stems in contact with medium has also been observed (Witjaksono, unpublished data). Despite success in propagating juvenile phase avocado by shoot tip culture, detailed studies regarding acclimatization of in v/7ro-grown plantlets have not been reported (Schall, 1987; Cooper, 1987). Exposing in vitro-gcovm plantlets and seedlings to higher irradiance, low nutrient concentration, and elevated atmospheric CO2 concentration have been demonstrated to increase net photosynthesis and biomass production (Kozai, 1991) with concommitant increased acclimatization rate of plantlets (Kozai, 1991; Laforge et al., 1991). The photosynthesis of avocado shoots or plantlets in vitro has not been reported. The same protocols described for shoot multiplication of juvenile phase avocado are ineffective for mature phase avocado selections, especially for Phytophthora root rottolerant rootstocks; however these protocols could be useful for multiplying breeding lines and germplasm, e.g., multiplication of shoots from immature embryos isolated from abscised fruitlets that resulted from controlled avocado crosses (Skene & Barlass, 1983) and multiplication of shoots from somatic hybrids (Grosser & Gmitter, 1990; Ochatt, 1990). Multiplication of shoots that developed from germinating avocado somatic embryos would be very critical, especially when the germinating somatic embryos are derived from somatic hybridization, genetic transformation or from nucellar explants, since the plant conversion frequency is very low (Pliego-Alfaro & Murashige, 1988; Mooney & Van Staden, 1987; see Chapter 4). The purpose of the studies described below was to develop a shoot multiplication protocol that would permit high multiplication and survival rates of in vitro-grown plantlets and to compare the photosynthetic rate of in v/7ro-grown shoots and plantlets grown in different atmospheric CO2 concentrations.

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151 Materials and Methods General Procedures The pH of all media was adjusted to 5.7-5.8 with either 0. 1-1.0 N KOH or HCl, and 8 g 1"' TC agar (Carolina Biological Supply Co., 2700 York Rd, Burlington NC 27215-3398) was added thereafter unless specified otherwise. The plant growth medium was melted by autoclaving at 12rC at 1.1 kg cm"^ for 5 min, and dispensed in 25 ml aliquots into 150 x 25 mm glass test tubes. The test tubes were capped with polypropylene closures and autoclaved at 12 TC at 1.1 kg cm"^ for 15 min and cooled as slants unless stated otherwise. ' After explant inoculation, the tubes were capped with autoclaved 12 x 12 cm Suncaps® [clear plastic film with a 10 mm gas permeable fiher (Sigma Chemical Co., St. Louis, MO)] and secured with rubber bands. The cultures were maintained in an incubator at 25 + 2°C with an irradiance of 100-120 ^mol m"^ s'^ and a 16 h photoperiod provided by Gro-Lux lamps, regular spectrum (Osram Sylvania Inc., Danvers, MA 01923). I Plant Materials Shoots derived from juvenile phase proliferating shoot cultures of 'Guaram 13' avocado were provided by Dr. Fernando Pliego-Alfaro, Departamento de Biologia Vegetal, Universidad de Malaga, Malaga, Spain. The shoot cultures were maintained on plant growth medium consisting of (in mg l '): NH4NO3, 719.4; KNO3, 1816.4; NaNOs, 85; Ca(N03)2 4 H2O, 944.0; MgS04 7 H2O, 245.3; KH2PO4, 136.0; KCl, 372; myoinositol, 100; MS micronutrients and vitamins; and 4.44 viM BA and sucrose, 30.000. The medium was solidified with 6 g 1* agar (Sigma Chemical Co., St. Louis, MO). The shoot cultures were thereafter maintained in 8-week subculture cycles on a plant growth medium

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152 containing MS major salts devoid of NH4NO3, MS minor salts supplemented with modified LS organic addenda and 4.44 uM BA, thereafter referred as Shoot Multiplication (SM) medium (Table A-1). Effect of KNO^ on Avocado Shoot Proliferation Five levels of KNO3 as the sole inorganic nitrogen source in the medium were tested (20, 30, 40, 50 and 60 mM). Otherwise, the composition of the medium was the same as SM medium. There were 10 tubes (replicates) per treatment. The explants were 'Guaram 13' nodal stem segments 1-1.5 cm in length vAih 1-4 axillary buds that had been maintained on SM medium for 8 weeks. After 8 weeks, shoot number, shoot length and number of leaves were recorded for plants for data analysis. Effects of Total Nitrogen Concentration. NO^'iNH^^ Ratio and Atmospheric CO7 Concentration on Growth of Shoot Cultures The total nitrogen concentration and ratio of NOa'iNHj* were as follows: 20 mM of NOs'iNHi^ with a ratio of 1:0, 20 mM of NOs^NH,^ with a ratio of 3:1, 40 mM of N03":NH4^ with a ratio of 3 : 1 and 60 mM of N03":NH4'^ with a ratio of 3 : 1 . The nitrogen was supplied as KNO3 and NH4NO3. The remaining composition of the plant growth medium was similar to SM medium. Cultures were grown either in an incubator with ambient atmospheric CO2 (350 ^imol mol"') or in a plexyglass box with an elevated CO2 concentration. Irradiance in the incubator and in the box were 120-150 ^imol m'^ s"' and 150-170 \xmo\ m'^ s\ respectively, provided by Gro-Lux lamps, regular spectrum (Osram Sylvania Inc., Danvers, MA 01923) with a 16 h photoperiod. The box was constructed of 0.55 cm-thick plexyglass v^th an outer dimension of 107.2 (length) x 50.6 (width) x 21.7 (height) cm. The CO2 was supplied to the box via tygon tubing from a tank of 2% (v:v) CO2 in nitrogen. The gas passed through distilled water before entering the box with a flow rate of 1 1.66-12.56 cm^ min"'.

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The explants for the experiment consisted of 'Guaram 13' nodal stem segments of 1-1.5 cm with 1^ axillary buds from 2-month-old shoot cultures maintained on SM medium. There were 10 tubes (replicates) per treatment. After 8 weeks, the number of shoots (<1 cm and ^1 cm length), length of shoots ^1 cm, number of small leaves (2-4 mm width) and the number of expanded leaves (>4 nmi width) were determined for shoot cultures maintained in the incubator. After 10 weeks of culture, growth variable data similar to those determined at 8 weeks were collected from all cultures in both growth environments. Net photosynthesis and dry matter content were determined for five randomly sampled cultures from each treatment in each atmospheric CO2 environment (see Photosynthesis Measurements for detail). Effect of Ambient CO2 on Plantlet Development Rooting of shoots was done using a two step method, consisting of induction and development (Pliego-Alfaro, 1988). Induction involved the use of explant shoots (1.5-2 cm long with 1-3 leaf primordia and non-expanded leaves) that were excised from proliferating 'Guaram 13' shoot cultures maintained on SM medium for 2 months. Individual shoots (10-15) were cultured for 3 days in Magenta™ GA7 vessels (Magenta Corporation, Chicago, IL) containing 50-60 ml Root Induction (RI) medium (Table A-1) and capped with sterile transparent Suncaps® (Sigma Chemical Co., St. Louis, MO). The cultures were maintained in an incubator with ambient CO2 concentration. After 3 days on RI medium, shoots were individually transferred into 150 x 25 mm test tubes containing ca. 25 ml semi-solid Root Development (RD) medium (Table A-1). The medium was cooled with the tubes remaining upright. There were 60 shoots; 30 shoots were maintained in a growth chamber at ambient atmospheric CO2 and 30 shoots were maintained in the box with elevated atmospheric CO2. After 9 weeks, net

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photosynthesis was determined for 10 randomly selected cultures from each growth environment, and growth data were collected. Photosynthesis Measurements j Net photosynthesis was measured for: 1) Proliferating shoot cultures, their shoot cuttings with subtended callus and plantlets and plantlet-derived shoot cuttings . Preliminary experiments indicated that proliferating shoot cultures on which basal callus developed had a negative net photosynthesis, indicating net respiration. Likewise, shoots from which basal callus had been removed showed positive net photosynthesis. To confirm these observations that net negative photosynthesis value of the intact proliferating shoot cultures might have been due to respiring callus masses, net photosynthesis was determined for proliferating shoot cultures (on medium with NOs'iNlT ratio of 3:1 at 40 mM) maintained in ambient and elevated CO2 concentrations (4 replicates each). Subsequently, all shoots in proliferating shoot cultures were removed from the basal callus which was subcultured separately. The dissected shoots from a proUferating culture were subcultured for net photosynthesis measurement in a single tube containing fresh medium of the same composition. Net photosynthesis was also determined for the subtended callus that had been removed. Stem, leaf and callus dry weights (24-48 h, 60° C) and leaf area (measured with a Portable Area Meter, Model LI -3 00, Lambda Instruments Corporation, Lincoln, NE) were recorded. 1 Dissection of shoots from proliferating cultures may cause wound respiration. To determine if dissection had a significant effect on respiration (and therefore net photosynthesis) of the shoot cuttings, an experiment was conducted using plantlets that developed under elevated CO2 concentration. Net photosynthesis was measured for four intact plantlets. Subsequently, the plantlets were decapitated 1-2 mm above the root

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155 zone. The resulting shoot cuttings were subcultured individually on RD medium for net photosynthesis measurement. Leaf area, stem and leaf dry weight were recorded. 2^ Shoots derived from proliferating shoot cultures from nitrogen concentration, NO^'iNHa"^ ratio and atmospheric CO2 concentration experiments . Ten weeks after the nodal stem segments were cultured, shoots had proliferated and basal callus had developed where proliferating shoots were in contact with the plant growth medium. Five cultures were randomly sampled from the N03':NH4^ ratio and atmospheric CO2 concentration experiments. Two to four shoots (2-5 cm length) were removed from each proliferating shoot culture without damaging the leaves and were subcultured onto fresh medium with 40 mM nitrogen with a NOs'iNHLt"^ ratio of 3 : 1 for photosynthesis measurement. Leaf area and fresh and dry weights of the stems and leaves were recorded from the subcultured shoots. Dry matter content (%) of the shoots was calculated using the formula: 100% [(fresh weight dry weight): dry weight] x 100%. 3) Plantlets that developed in ambient and elevated CO2 environment . Ten replicates (test tubes) were randomly sampled from rooted shoot cultures that developed in ambient and elevated CO2 environments. Net photosynthesis was determined from each tube. Shoot length, number of leaves, leaf area, number of roots and root length and shoot, leaf, root and callus dry weights were recorded. Net photosynthesis was measured with an ADC, LCA-3 portable gas exchange system under shade with irradiance ca 295-486 |j,mol m"^ s"' at 30°C. The culture tubes were each fitted with a two-holed rubber stopper into which copper tubing had been inserted. The copper tubings served as inlet and outlet ports. The stoppers were sealed to the glass tubes with Parafilm. Air (containing 350 CO2 ^il 1'') was pumped through rubber tubing into the inlet port at a flow rate of 400 ml min"\ Air pressure inside the culture tubes was presumably sufficient to allow mixing of air. Air flowed from the outlet port through rubber tubing to the infrared gas analyzer and the CO2 depletion rate in the tube was determined. Net photosynthesis was expressed in terms of leaf area or dry weight.

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1 Statistical Analyses All statistical tests were performed using SAS software for IBM-compatible personal computers, version 6.08 for Windows (SAS Institute, 1992) or SigmaPlot™ (Jandel Scientific, San Rafael, CA). The following statistical analyses were performed: Effect of KNO^ concentration on shoot growth in the incubator . The effects of KNO3 concentration on shoot growth responses were determmed by regression analysis. Effect of NO^ -jNHa ^ ratio on shoot growth responses in the incubator after 8 weeks . The effect of two different NOs'iNHt^ ratios (1:0 and 3:1) on shoot growth responses was compared using standard t-tests. Effect of N concentration on shoot growth responses in the incubator after 8 weeks . The growth responses of shoots to different N concentrations (N03':NH4'^ ratio was 3:1) were determined by regression analysis. Effects of atmospheric CO2 and NOa ': NH i '^ ratio on shoot growth responses and photosynthesis after 10 weeks . Interactions between atmospheric CO2 concentrations and nitrogen ratios (at 20 mM total N in the medium) on shoot growth responses and net photosynthesis were determined by ANOVA. If there were no interactions, data were pooled across CO2 concentrations to test nitrogen ratio effects and data were pooled across nitrogen ratios to test CO2 effects. A standard t-test was used to compare growth and photosynthetic responses between a medium containing 20 mM with a N03 :NH4^ ratio of 1 :0 and a medium providing the same total N concentration with a NOs^NHt^ ratio of 3:1. Growth and photosynthetic responses of shoots in an ambient CO2 environment were compared to those of shoots grown in an elevated CO2 concentration with a standard t-test. Effects of atmospheric CO? and N concentration on shoot growth responses and photosynthesis after 10 weeks . Interactions between atmospheric CO2 concentration and i i -A

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157 nitrogen concentration (20, 40 or 60 mM total nitrogen with a N03 :NH4'^ ratio of 3:1) on shoot growth and net photosynthesis were determined by ANOVA. If there were no interactions, data were pooled across CO2 concentrations to test nitrogen effects and data were pooled across nitrogen concentrations to test CO2 effects. The effects of total nitrogen concentration on shoot growth and net photosynthesis were determined by linear regression analysis. Growth and photosynthetic responses of shoots in an ambient CO2 environment were compared to those of shoots grown under elevated CO2 conditions with a standard t-test. Effect of atmospheric CO? on plantlet development after 9 weeks . The effect of atmospheric CO2 on the growth and development of plantlets was determined by standard t-test. Resuhs General Morphology of the Cultures In addition to single or multiple shoot elongation from the preexisting axillary buds in the nodal explants, soft brown and or compact green calli developed at the cut end of each nodal stem segment propagule that was in contact vAth medium. After 8-10 weeks, callus covered the entire explant and covered the surface of the medium in some cultures. Effect of KNO2 on Growth Responses of Shoot Cultures The total number of shoots per culture was not significantly affected by KNO3 concentration in the medium (P > 0.05); however, the proportion of healthy or necrotic shoots was significantly different with different KNO3 concentrations. The number of healthy shoots decreased curvelinearly with increasing KNO3 concentration (Figure 6-1A), whereas the number of necrotic shoots increased curvelinearly with increasing KNO3

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> 158 concentration (Figure 6-1-B). At 20 mM KNO3, no necrotic shoots were observed. The length of healthy shoots decreased curvelinearly as KNO3 concentration decreased (Figure 6-1-C), while the length of necrotic shoots increased with increasing KNO3 concentration (Figure 6-1-D). The total length of shoots was not significantly affected by KNO3 concentration (P > 0.05). Figure 6-1. In vitro shoot growth of 'Guaram 13' avocado in response to varying concentrations of KNO3 (N) as the sole inorganic nitrogen source in the medium. The growth variables and their regression lines are: A) number of healthy shoots, Y = -1.04 + 0.39 N 0.0095 + 0.000058 N^ r^ = 0.35; B) number of necrotic shoots, Y = -0.76 + 0.02 N 0.00036 r^ = 0.47; C) healthy shoot length, Y = -1.49 + 0.29 N 0.0071 + 0.000048 N^ r^ = 0.28; D) necrotic shoot length, Y = -0.3 + 0.0014 N 0.00054 r^ = 0.50.

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159 The number of scaly leaves, number of small leaves, number of expanded leaves, number of abscised leaves and total number of leaves decreased curvelinearly with increasing KNO3 concentration (Figure 6-2-A-E). For all growth variables, the coefficient of correlations (r^) of the regression lines were low, ranging from 0.28 to 0.56. I I I I I I I I L 20 30 40 50 60 KNO3 concentration (mM) Figure 6-2. In vitro leaf growth of 'Guaram 13' avocado shoots in response to varying concentrations of KNO3 (N) as the sole inorganic nitrogen source in the medium. The growth variables and their regression lines are: A) number of scaly leaves, Y = -24.46 + 2.89 N 0.07 + 0.00051 N^ r^ = 0.27; B) number of small leaves, Y = -17.84 + 0.5 N + 0.0035 r^ = 0.35; C) number of expanding leaves, Y = 23.76 + 2.39 N 0.06 + 0.0005 N^ r^ = 0.31; D) number of abscised leaves, Y = -25 + 2. 19 N 0.05 + 0.00043 N^ r^ = 0.28; E) total number of leaves, Y = 32.5 + 4.96 N 0. 14 + 0.001 1, r^ = 0.56.

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Effect of N0V:NEi4^ Ratio on Shoot Growth Response of Cultures in the Inc ubator after 8 Weeks I .Eight weeks after culture, a 3:1 NOs^NH** ratio resulted in more shoots >1 cm (P < 0.03), and greater total shoot length (P < 0.02) than did a N03":NH4^ ratio of 1:0. The number of shoots >1 cm long and the average shoot length were not significantly different between the two N ratio treatments. Neither treatment significantly afiected the number of expanded leaves; however, when the N03":NH4'^ ratio was 3:1, significantly more small leaves (P < 0.01) and a greater total number of leaves (P < 0.07) were observed than for the other treatment (Table 6-1). Table 6-1. The effect of NOs iNIlt^ ratio at 20 mM on the growth of 'Guaram 13' avocado shoot cultures maintained in an incubator, eight weeks after culture. Growth variables N03':NH4^ ratio 1;0 3:1 (Mean + SE) (Mean ± SE) P^ Number of shootsl cm 1.10 ±0.28 3.30 ±2.58 0.03 Total number of shoots 2.49 ±0.61 7.54 ± 1.83 0.02 Average shoot length 1.88 ±0.38 2.05 ±0.32 0.73 Number small leaves(2-4 mm width) 0.22 ±0.15 2.80 ±0.79 0.01 Number of expanding leaves (>4 mm width) 2.67 ±1.18 4.30 ± 1.26 0.36 Total number of leaves 2.89 ± 1.30 7.10 ± 1.80 0.07 ^ Mean separation by standard t-test I Effect of N Concentration on Shoot Growth Responses in an Incubator after 8 Weeks No increase in average shoot length was detected as nitrogen levels increased fi^om 20 to 60 mM (P > 0.05). The average length of the shoots > 1 cm increased linearly with increasing total nitrogen concentration, even though the r^ value was low (0. 14) (Figure 63 A). There were positive curvelinear correlations between number of shoots >1 cm, total

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161 shoot length, total number of leaves, number of expanding leaves and nitrogen concentrations (Figure 6-3 B-E). The nitrogen concentration of 40 mM was optimum for shoot culture growth with respect to variables such as number of shoots >1 cm, total shoot length, total number leaves and number of expanding leaves. ! Figure 6-3. In vitro growth of 'Guaram 13' avocado shoots in response to varying concentrations of nitrogen (N) in the form of 3 NO3" : 1 NH,^. The growth variables and their regression lines are as follows: A) number of shoots>l cm, Y = -3.2 + 0.43 N + 0.005 r^ = 0.22; B) average shoot length, Y = 1.68 +0.02 N, r^ =0. 14; C) total shoot length, Y = -7.45 + 0.97 N + 0.61 r^ = 0.20; D) number of expanding leaves, Y = -27.1 + 1.7 N + 0.02 r^ = 0.57; E) total number of leaves, Y = -5.8 + 2. 1 N + 0.002 r^ = 0.56. i

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162 Effects of Atmospheric COj and NO.':NH^^ Ratio on Shoot Grow th and Photosynthesis after 10 Weeks 1 There were no significant interactions between atmospheric CO2 concentration and N03':NH4^ ratio for any of the growth variables measured. Shoot cultures grown on medium with a N03':NH4* ratio of 3:1 had a significantly higher number of shoots > 1 cm and a higher number of expanding leaves than those grown on medium with N03":NH4'^ ratio of 1:0 (Table 6-2). The NOs^NHt"^ ratio had no effect on number of shoots < 1 cm, total number of shoots, average shoot length, number of small leaves and total number of leaves (Table 6-2). Cultures that were maintained in an elevated CO2 environment produced significantly more shoots, shoots < 1 cm length (but not > 1 cm length), and leaves (both small and expanding) than plants grown in an ambient CO2 environment (Table 6-3); however, average shoot length was not significantly affected by the Table 6-2. Effect of medium NOs^NUt* ratio at 20 mM on the growth variables and net photosynthesis of avocado shoot cultures after ten weeks in culture. Growth variable N03":NH4'^ ratio 1:0 3:1 (Mean ± SE) (Mean ± SE) Number of shootsl cm 2.60 ± 0.28 5.32 ±0.98 0 0142 Total number of shoots 7.60 ± 1.36 9.26+1.25 0 3736 Average shoot length 1.83 ±0.12 2.13 ±0.21 0 2255 Number small leaves(2-4 mm width) 10.30 ± 1.90 12.79 ±3.60 0 5454 Number of expanding leaves (>4 mm width) 6.25 ±1.14 12.89 ±2.02 0 0078 Total number of leaves 16.55 ±2.25 25.68 ±4.61 0 0862 Net photosynthesis rate (urnol CO2 m"^ s'^^ 1 17.43 ±4.58 13.98 ±2.28 0 5120 Dry matter content (%) 16.26 ± 1.67 21.67 ±3.94 0 2298 ^ Mean separation by standard t-test Data across atmospheric CO2 concentration treatments were pooled for the analysis, since their interaction with nitrogen ratio was not significant (p>0.05).

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163 atmospheric CO2 concentration (Table 6-2) or the medium NOa'.l^'^ ratio (Table 6-3). Net photosynthesis of the shoots was not afifected by medium NOa'iNHt^ ratios (Table 62). Net photosynthesis of shoot cuttings from cultures grown in elevated CO2 was significantly lower than that of cultures grown in ambient CO2 (Table 6-3). The dry matter content was not significantly affected by atmospheric CO2, N03":NH4* ratios, or their interaction (P > 0.05); however, there was a tendency for shoot cultures grown in elevated CO2 to accumulate more dry matter than those in an ambient CO2 (P < 0.06) (Table 6-3). Table 6-3. Effect of atmospheric CO2 environment on the growth variables and net photosynthesis of avocado shoot cultures after ten weeks in culture". Growth variable Atmospheric CO 2 concentration Elevated Ambient (Mean ± SE) (Mean ± SE) P" Number of shootsl cm ' 4.74 ± 0.93 3.15 ±0.53 0.1490 Total number of shoots 10.79 ± 1.50 6.15 ±0.86 0.0121 Average shoot length 1.79 ±0.10 2.16 ±0.21 0.1296 Number small leaves(2-4 mm width) 17.16 ±3.60 6.15 ±0.81 0.0073 Number of expanding leaves (>4 mm width) 12.16 ± 1.87 6.95 ± 1.50 0.0370 Total number of leaves 29.32 ± 4.08 13.10±2.14 0.0015 Net photosynthesis rate ([imol CO2 m'^ s'') 10.61 ±2.45 20.80 ±3.89 0.0423 Dry matter content (%) 23.15 ±3.92 14.77 ±0.84 0.0637 " Mean separation by standard t-test ^ Data across NOs iNUt"^ ratio were pooled for the analysis, since their interaction with atmospheric CO2 concentration was not significant (p>0.05).

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1 164 Effects of Atmospheric CO; and N Concentration on Shoot Growth Responses and * Photosynthesis after 10 Weeks j l After 10 weeks of culture, there were no interactions between nitrogen level and atmospheric CO2 concentration for any of the variables measured (P > 0.05). Unlike observations 8 weeks after culture, nitrogen level had no significant effect on any variable measured 10 weeks after culture, except for dry matter content. Shoot dry matter content was negatively and curvelinearly related to nitrogen concentration, although the r^ value was low (0.18) (Figure 6-4). Cultures maintained in an elevated CO2 concentration had higher values for all growth variables measured than those maintained in an ambient CO2 concentration, except for average shoot length (Table 6-4) (Figure 6-5). Net photosynthesis of shoots removed fi"om cultures maintained in elevated atmospheric CO2 was significantly lower than with shoots maintained in an ambient atmospheric CO2 environment (11.111.8 versus 16.5 ± 1 .6 nmol CO2 m"^ s'') (Table 6-4). 50 40 30 20 A A 10 1 » 1 1 20 40 60 Total nitrogen (mM) Figure 6-4. Dry matter content of avocado shoots in vitro in response to varying concentration of total nitrogen (N) in the form of 3 NO3" : 1 NHt"^, after 10 weeks in culture. The regression line is represented with Y = 30.8 0.54 N + 0.0043 N^, r^ = 0. 18. Data across treatments of atmospheric CO2 concentrations were pooled for the analysis since their interaction with total nitrogen concentration were not significant (P > 0.05).

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165 The Net Photosynthesis of Proliferating Shoot Cultures. Microcuttings with Subtended Callus and Plantlets and Their Plantlet-Derived Shoot Cuttings As determined in a preliminary experiment, intact proliferating shoot cultures had negative net photosynthesis, indicating respiration, regardless of the atmospheric CO2 concentration. Shoot cuttings that were removed from proliferating shoot cultures, however, had positive net photosynthesis value. The calli from the shoot cultures had high net respiration rates (Table 6-5). The net photosynthetic rate of plantlets grown in an elevated CO2 environment was not significantly different from that of the plantlet-derived shoot cuttings (P > 0.05) (Table 6-5), indicating no significant effect of cutting on their net photosynthesis. Table 6-4. The effect of atmospheric CO2 concentration on the growth variables and net photosynthetic rate of avocado shoot cultures after ten weeks in culture''. Growth Variable Atmospheric CO2 concentration Elevated Ambient pz (Mean + SE) (Mean ± SE) Number of shootsl cm 6.41 ±0.70 4.53 ±0.42 0. 0259 Total number of shoots 10.83 ±0.98 7.60 ± 0.66 0. 0090 Average shoot length 2.31 ±0.22 2.70 ±0.17 0. 1562 Number small leaves(2-4 mm width) 15.52 ±2.43 9.28 + 0.95 0. 0219 Number of expanding leaves (>4 mm width) 20.38 ± 1.80 12.20 ± 1.33 0. 0006 Total number of leaves 35.90 ±3.27 21.48 ±2.12 0. 0006 Net photosynthesis rate (pmol CO2 m"^ s"') 11.13 ±1.84 16.51 ± 1.60 0. 0358 Dry matter content (%) 19.16 ±2.82 15.08 ±0.66 0. 1790 Data across total nitrogen concentration were pooled for this analysis since their interaction with atmospheric CO2 concentration was not significant (p>0.05). ^ Mean separation by standard t-test

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166 ABCD EFGH Figure 6-5. Shoot proliferation of 'Guaram 13' avocado on media of different N content under elevated and ambient CO2 concentration, after ten weeks. Interveinal chlorosis is apparent on leaves of shoots cultured under elevated CO2 concentration. A-D, shoot cultured under elevated CO2 and E-H, shoot under ambient CO2 environment. A and E, at 20 mM N of 1 NOs' : 0 T
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167 123 45 6789 10 Elevated CO2 j Ambient Figure 6-6. Plantlets of 'Guaram 13' avocado grown under elevated and ambient CO2 concentration, after nine weeks. The first 5 plantlets fi-om left were cultured under elevated CO2 concentration; the last five plantlets were cultured under ambient CO2 concentration. Interveinal chlorosis is apparent on leaves of the plantlets cultured under elevated CO2 concentration.

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168 Table 6-5 Net photosynthesis (nmol CO2 g'^ s"*) of intact avocado proliferating shoot cultures, their subcultured microcuttings and subtended callus in two atmospheric CO2 concentrations, and intact plantlets and plantlet-derived shoots in an elevated CO2 concentration before and after cutting^. Type of culture Atmospheric CO2 concentration Intact culture Subcultured Callus Shoot cutting (Mean ± SE) (Mean + SE) (Mean ± SE) Shoot Plantlet Elevated Ambient Elevated -0.20 + 0.14 -0.27 ± 0.24 0.40 ± 0.09 0.15 ±0.07 0.38 + 0.13 0.39 ± 0.09 -0.18 ±0.05 -0.21 ±0.02 'Data represent means ± SEs of 4 replicates, after 10 weeks of culture. Effect of Growth Environment on Plantlet Development 9 Weeks after Culture Roots were visible from shoots after 3 weeks. After 9 weeks of culture, 90% of the shoots developed roots. In some of the cultures, callus also developed at the base of the shoot cuttings. Plantlets that developed in an elevated CO2 environment grew more vigorously than those in the ambient CO2 environment; they had thicker and longer stems, more and thicker roots and more leaves, although the older expanded leaves developed interveinal chlorosis (Figure 6-6). Shoot length and total leaf area of plantlets that developed in elevated CO2 concentration were significantly greater than of plantlets in ambient CO2 (Table 6-6). Number of leaves, average leaf area, number of roots, average root length and dry matter content were not significantly affected by atmospheric CO2 concentration; however, root, stem, callus and leaf dry weights were significantly higher for plantlets that developed in an elevated atmospheric CO2 environment than in a nonelevated CO2 environment. The net photosynthetic rate on a leaf area basis of plantlets grown in an elevated CO2 environment (19.3 ± 4.8 ^mol CO2 m'^ s'') tended to be lower than that of plantlets in

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169 an ambient CO2 environment (31.3 ± 7.24 ^lmol CO2 m*^ s'), when measured in an ambient atmospheric CO2 environment (Table 6-6). Table 6-6 'Guaram 13' avocado plantlet development in two atmospheric CO2 environments, nine weeks after culture. Growth variable Atmospheric CO2 concentration Elevated Ambient Shoot length (cm) j Number of leaves i Leaf area total (cm ) Average leaf area (cm^) Number of root Average root length (cm) Root dry weight (g) Stem dry weight (g) Callus dry weight (g) Leaf dry weight (g) Net photosynthesis rate (nmol CO2 m'^ s'') Dry matter content (%) ^ Mean separation by standard t-test (Mean + SE) (Mean ± SE) 4.13 ±0.28 2.74 ±0.21 0.0008 12.75 ±0.80 9.54 ± 1.67 0.1048 13.76+1.76 6.88 ±1.01 0.0034 1.09 ±0.14 0.93 ±0.18 0.4595 6.0 ±0.65 4.64 ± 0.62 0.1449 4.74 ± 0.42 4.42 ±0.54 0.6437 0.02 ±0.00 0.01 ±0.00 0.0062 0.02 ± 0.00 0.01 ±0.00 0.0148 0.05 ±0.01 0.03 ±0.01 0.0150 0.07 ±0.01 0.03 ± 0.00 0.0011 19.34 ±4.84 31.35 ±7.24 0.1900 18.84 ±3.65 16.94 ± 2.67 0.2849 Discussion Murashige & Skoog (1962) salt-based media have been successfully employed for in vitro propagation of plants, including herbaceous plants such as tropical foliage (Miller & Murashige, 1976), vegetable crops (Seckinger, 1991), woody plants, i.e., Rosa spp. (Ma et al., 1996) and tropical and subtropical Suit trees (Litz & Jaiswal, 1991) that are mostly woody perennials. MS-based media have been shown to be unsatisfactory for shoot culture of some woody species such as Dipterocarpus intricatus (Linington, 1991), Pistachio vera (Barghchi & Alderson, 1985), Kalmia latifolia (Lloyd & McCown, 1980),

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: 170 I , and avocado (Pliego-Alfaro et aL, 1987). Therefore, several modifications of MS formulation have been attempted. Reduction of the major and or minor salts concentration to 30-50% of the original concentration improved rooting of juvenile avocado shoots (Pliego-Alfaro, 1988), but not the multiplication of adult shoot cultures (Pliego-Alfaro et aL., 1987). Omission of NH4NO3 fi-om the standard medium formulation was reported to improve the survival rate of juvenile phase shoot cultures to 100%, although the multiplication rate was still limited (Witjaksono, 1991). In the current study, the multiplication rate of avocado shoot cultures was not enhanced by increasing the KNO3 concentration as the sole inorganic nitrogen source. Shoot necrosis, leaf abscission and reduction of leaf and shoot production were observed with KNO3 concentrations higher than 20 mM; however, incorporation of 25% inorganic ammonium to total inorganic nitrogen in the medium significantly improved the multiplication rate and the health of the cultures as indicated by the production of healthier and more leaves. I The optimum nitrogen concentration for shoot multiplication, determined at 8 weeks after culture, was 40 mM with a 3:1 NOs^NHj^ ratio. The theoretical multiplication rate under these conditions would be 13 -fold for each 8 week subculture cycle, and 100,000 plantlets could be theoretically produced fi^om one nodal stem in one year. This multiplication rate is higher than the multiplication rate of 100 fold per 4 month subculture cycle that was reported for P. indica (Nel et al.., 1982), 3 -fold per month cycle for 'Hass' and 'Hopkin' non clonal avocado (Cooper, 1987) and 77,000 plants per year for P. palustris (Kane et al... 1989). | The shoot proliferation occurs by axillary branching. This is consistent with previously reported results for avocado (Schall, 1987; Cooper, 1987), P. schiedaena (Gonzales-Rosas et al.., 1985), P. palustris (Kane et al.., 1989) and P. indica (Nel et al.., 1985; Campos & Pais, 1996). ' ^ -

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171 i . " Due to the high respiration rate of the basal callus in avocado shoot cultures, intact proliferating shoot cultures show net positive respiration, although subcultured shoots show net positive photosynthesis. Net photosynthesis was measured using shoots that were separated from callus in proliferating shoot cultures. This was justified since dissection of the plantlets did not significantly affect net photosynthesis of the dissected shoots. Growmg avocado shoot cultures or plantlets in an elevated CO2 concentration resulted in higher biomass production as indicated by a greater number of total leaves and number of shoots and a greater plant dry weight than cultures grown in an ambient CO2 environment. Greater biomass production due to higher CO2 levels was also reported for in vitro plantlets of banana (Navarro et al.., 1994), potato (Solanum tuberosum) (Kozai et al.., 1988a), Cymbidium (Kozai et al.., 1988b), carnation (Dicmthus caryophyllus L) (Kozai & Iwanami, 1988), strawberry {Fragaria x ananassa Duch), raspberry Rubus idaeus L.) and asparagus {Asparagus officinalis L.) (Laforge et al., 1991). Similar results were also found with ex vitro studies of banana (Schaflfer et al, 1996), mangosteen (Garcinia mangostana L.) (Downton et al., 1991), acclimatized plantlets of strawberry (Desjardins et al., 1987) and acclimatized in vitro propagated, non-rooted grape shoots (Laksoetal., 1986). | Net photosynthesis of avocado shoots maintained in a growth chamber without CO2 enrichment is in the range that has been observed under field conditions for avocado (14-20 nmol CO2 m"^ s"') (Whiley & Schaffer, 1994); however, the net photosynthetic rates (3 1 ± 7 ^mol CO2 m"^ s'^) of plantlets grown in an ambient CO2 environment were considerably higher than those reported in the literature. Consistent with other experiments, long-term exposure to an elevated CO2 environment significantly reduced net photosynthesis of the avocado shoots and plantlets. For example, Navarro et al. (1994) found that photosynthetic fixation of in vitro-grovm banana plants at ambient CO2 concentration decreased as CO2 concentration of the growth environment increased.

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172 Similar results were also reported by Schaffer et aL (1996) working with banana in an environmentally controlled glasshouse. The decrease in photosynthetic efficiency of plants grown in an elevated CO2 atmosphere does not explain the high biomass production under such conditions. Several studies have demonstrated that the actual photosynthetic rates of plants adapted to high CO2, when measured in their elevated CO2 environment, were higher than the rate of plants grown at ambient CO2 concentration (Dube & Vidaver, 1992; Kozai & Iwanami, 1988; Kozai et al., 1988a; Kozai et al., 1988b). Schaffer et al. (1996) demonstrated that net photosynthesis of banana plants increased with increasing CO2 concentration in the cuvettes, but plants that were exposed to elevated CO2 were less photosynthetically efficient than those exposed to ambient CO2 environment. The higher actual photosynthetic rate in an elevated CO2 environment was due to increased CO2 in the environment available for fixation (Schaffer et al., 1996). The higher actual photosynthetic rates in the elevated CO2 environment presumably resulted in the increased biomass for those plants compared to plants grown at ambient CO2 (Schaffer et al., 1996). As a summary, a protocol for enhancing multiplication of healthy shoot cultures of juvenile phase 'Guaram 13' avocado has been developed. Avocado shoots and plantlets in vitro are photosynthetically active regardless of the availability of sucrose in the medium. Under an elevated compared to a non-elevated CO2 environment, the mixotrophic avocado shoots and plantlets grew better, even though their photosynthetic efficiencies were lower. Plantlets (i.e., rooted shoots) have higher photosynthetic efficiencies than do shoot culture-derived shoot cuttings in vitro. The practical implications of these observations are as follows: 1) plantlets developed under elevated CO2 would provide vigorous planting materials with photosynthetic efficiency comparable to plants ex vitro, 2) shoot cuttings fi-om in vitro shoot cultures are photosynthetically active, indicating the potential for combining acclimatization and rooting ex vitro; however a rooting step in

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173 vitro would be a better method for acclimatization since plantlets have higher photosynthesis eflBciencies than shoot culture-derived shoot cuttings.

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CHAPTER? PROTOPLAST FUSION BETWEEN AVOCADO AND ITS RELATIVES, JNCLUDJNGNECTANDRA CORIACEA AND PERSEA SPP. I Introduction I The major production problem of avocado is root-rot disease caused by Phytophthora cinnamomi. This avocado disease causes serious damage worldwide, especially in areas with heavy soil and poor drainage (Gustafson, 1976; Whiley, 1992). In California alone the losses due to this disease have been estimated to be $6,000,000 annually (Coffey, 1987). Breeding rootstock with resistance to this disease has been slow due to the absence of resistance traits in Persea canericana and other sexually compatible species in the subgenus Persea (Pliego-Alfaro & Bergh, 1992). Resistance traits, however, are available in most of the small-fruited Persea species within the subgenus Eriodaphne of the genus Persea (Kopp, 1966), e.g., P. borbonia, P. cinerascens, P. pachypoda, etc. and in other related genera of the Lauraceae, i.e., Nectandra, Phoebe and Ocotea (Zentmeyer, 1980). Unfortunately, these resistant small seeded species are both sexually and graft incompatible with Persea americana and other large-fruited Persea spp. in the subgenus Persea (Bergh, 1975; Pliego-Alfaro & Bergh, 1992; Bergh & Lahav, 1996). An alternative way that has been proposed to bridge the incompatibility barrier is by protoplast fusion (Bergh & EUstrand, 1986; Pliego-Alfaro & Bergh, 1992). Protoplast fusion has been successfully used to produce somatic hybrids with pest and disease resistance and stress tolerance characteristics of one of the parents and include potato and tomato, Brassica, eggplant and tobacco and their relatives (summarized in Bajaj, 1994). The somatic hybrids produced from the fusion of eggplant with its distant relatives grew poorly due to poor rooting. The plants were also highly sterile; however, 174

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somatic hybrids produced from eggplant with its more closely related species such as S. aethiopicum showed vigorous growth and high yields (Sihachakr et al., 1994). This indicates that somatic hybrids involving distantly related species may not have direct utility, although they can be used in breeding programs. The potential of somatic hybridi2ation for fruit crop improvement has been demonstrated largely with citrus (Grosser & Gmitter, 1990; Louzada & Grosser, 1994) and with pear (Ochatt et al., 1989) and passion fruit (Otoni et al., 1995; Domelas et al., 1995). Somatic hybrid plants have been obtained following protoplast fusion between sexually and graft incompatible 'Colt' cherry (Prunus avium x pseudocerasus) and wild pear (Pyrus communis) (Ochatt et al, 1989). The hybrids were graft compatible to both parents (Ochatt & Patat-Ochatt, 1994). Somatic hybrid plants from sexually incompatible genera of Rutaceae species, i.e., between C. sinensis and Atalantia ceylanica (Louzada et aL, 1993) and between C sinensis and Severinia disticha (Grosser et al., 1988) have been reported. Somatic hybrids among Rutaceae species that are sexually incompatible and diflHcult to graft onto each other, i.e., C. sinensis with Clausena lansium (Lour.) Skeels has also been obtained, but it did not survive (Louzada & Grosser, 1994). The purpose of this work was to develop a protocol for protoplast fusion between avocado and the root-rot resistant Nectandra and small-fruited Persea species. Materials & Methods Plant Materials Nectandra coriacea mature fruits as indicated by the dark skin color were collected from Everglades area. The fleshy part of the fruits (carpels) were removed and the seeds were washed thoroughly with running tap water. The seeds were air dried, placed in plastic bags and stored in a refrigerator until use. Mature seeds of P. borbonia, I

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> 176 P. pachypoda and P. cinerascens were collected from the South Coast Experiment Station of the University of California and were supplied by Dr. John A. Menge of the University of California, Riverside. They were stored in a refrigerator until use. All these species were used as sources of leaf mesophyll protoplasts. Seeds were germinated in vitro. The seed coats were removed and the embryos were washed with tap water to remove any seed coat remnants. They were surface disinfested in a 20% (v/v) solution of commercial bleach containing 10-20 drops 1"^ of Tween 20® for 10-20 min and rinsed with 2 changes of sterile, deionized water. The embryos were cultured on a basal plant growth medium consisting of MS salts, 0.4 mg 1'* thiamin HCl, 100 mg f' myo inositol, 30 g 1* sucrose and solidified with 2 g f' Gel Gro gellan gum. The pH of the medium was adjusted to 5.7-5.8 vwth KOH or NaOH prior to addition of gelling agent. The medium was dispensed in 100 x 15 mm sterile plastic Petri dishes in 20 ml aliquots. There were 8-12 embryos per Petri dish.. The Petri dishes were sealed with Parafilm® and maintained in darkness at 25°C. Seeds began to germinate after 3-5 days. After 10-15 days, seedlings had > 2 cm long roots and > 0.5 cm long shoots were transferred individually onto filter bridges in liquid plant growth medium contained in test tubes. Plant growth medium consisted of MS salts without NH4NO3, 1 mg 1'^ thiamine-HCl, 100 mg 1"^ myo-inositol, 30 g f' sucrose and 400 mg 1"^ glutamine. The pH was adjusted to 5.7-5.8 and medium was distributed in 20 ml quantities to 25 x 150 mm test tubes, each of which contained a filter paper support (Whatman # 2). The tubes were capped with kaputs (polypropylene closures) and autoclaved at 1.1 kg cm"^ and 12rc for 15 min. After culturing the seedlings into the nutrient tubes, the kaputs were replaced with Suncaps® (Sigma Chemical, St. Louis, MO). The cultures were maintained under irradiance of 50-60 ^mol photon m"^ s"' for 16 h. The liquid medium was replaced at 2 month intervals at which time the leaves were removed for protoplast isolation and the shoots were cut back to ca. 0.5 cm above the cotyledons.

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177 Protoplast Isolation Young, non-expanded leaves of 0.3-1 cm width usually were removed from the in vitro-grovm seedlings and placed in liquid medium-wetted sterile glass Petri dishes. There were usually 1-3 leaves that could be extracted from each seedling and usually 20-30 seedlings were necessary to yield one gram of leaf tissue. The leaves were feathered with a sharp blade parallel to the mid vein. The leaf tissue (ca. 500 mg) was transferred into a 50 ml Erlenmeyer flask containing 5 ml enzyme mixture (Table A-4) and 6 ml 0.7 M MS' 8P (Table A-2) and secured with aluminum foil and Nescofibn. The digestion mixture was incubated in darkness at 25° C for 16 h on a rotary shaker at 75-100 rpm. The protoplasts were purified as described (see Chapter 5; Grosser & Gmitter, 1990). Embryogenic cuhures of 'T362', 'Thomas' zygotic embryo-derived cuhure and 'Hass' that was no longer embryogenic were used as a source of avocado protoplasts. Avocado protoplasts were produced as described in Chapter 5. The resulting protoplasts were resuspended in 0.7 MS"8P at 20 X their pelleted volume. Protoplast Fusion Protoplast fusions were performed using the PEG method (Grosser & Gmitter, 1990). Avocado protoplasts and leaf mesophyll protoplasts were mixed in a ratio of 1:2 volume which yielded a protoplast ratio of ca. 1:4. Protoplast fusion was induced in sterile plastic Petri dishes (60 x 15 mm). Into each fusion dish, 2 drops of protoplast mixture were added, and 2 drops of PEG solution (Table A-6) were added immediately to the center of the droplets. Twelve min later, two drops of elution solution (Solution A + B with A:B ratio of 9:1) were added, with one drop at opposite sides of the liquid pool containing the aggregated protoplast mixture and PEG. After another 12 min, 12-15 drops of fresh 0.7 M MS'8P medium were added to the periphery of the liquid droplet containing fusing protoplasts. After 5 min, the PEG, elution solution and the medium that

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7 178 formed the droplet were removed carefully with a Pasteur pipette and immediately replaced with 12-15 drops of fresh 0.7 M MS 8P medium. This washing step was done carefully 3 X with 10 min intervals so as not to remove protoplasts in the center of the dish. I Two sets of fusion experiments were done: 1) between zygotic-derived embryogenic 'T362' avocado protoplasts and leaf mesophyll protoplasts oi Nectandra coriacea and between embryogenic nucellar-derived 'Thomas' protoplasts and leaf mesophyll protoplasts of P. borbonia, 2) between embryogenic protoplasts of either 'Thomas' zygotic embryo-derived cultures or 'Hass' that had lost embryogenic potential with leaf mesophyll protoplasts of P. pachypoda. These fijsion combinations were: 'Hass' + P. pachypoda, 'Hass' + 'Thomas' zygotic, 'Thomas' zygotic + P. pachypoda, and mixture of protoplasts without fusion of 'Hass' and 'Thomas' zygotic, 'Thomas' zygotic and P. pachypoda, and individual protoplasts of 'Hass', 'Thomas' zygotic and P. pachypoda. Protoplast culture j Protoplasts were cultured directly in the fusion dish. After the final wash, 3 ml of 0.4 M MS"8P medium were added to each Petri dish and was carefully spread to form a shallow liquid cover over the entire Petri dish. The dishes were sealed with Nescofilm® and incubated in darkness at 25°C. After 2-5 weeks by which time microcallus or proembryonic masses of 100-200 nm had formed, the cultures were subcultured at a diluted density of 20-40-fold in 3 ml of 0.15 M MS 8P medium. After 1 month more, somatic embryos and proembryonic masses of 1-2 mm diameter were visible. These somatic embryos and proembryonic masses were transferred to semisolid somatic embryo development medium.

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179 For fiisions involving cultures that had lost their embryogenic potential, ca. 6-8 dishes were cultured for each fusion treatment. Subculture to a lov^^er density in medium with lower osmoticum or the same osmoticum was conducted 40 days after fusion due to the slow growth of the cultures. For cultures that formed microcalli or proembryonic masses prolifically, they were subcultured with densities of 6 to 20-fold dilution. For cultures that did not grow vigorously, several culture dishes were pooled before replacement with fresh medium. j For culture volume determination, 3 dishes representing prolific cultures and 6 to 8 cultures from non-prolific cultures were randomly selected. Sampling of proembryonic masses for DNA analysis was done by randomly selecting 2-3 dishes (at the subculture 40 days after fusion) from each dish. The proembryonic masses were pooled for each fusion combination for DNA isolation. Somatic Embryo Development and Plant Regeneration Details of the method for stimulating somatic embryo development and plant regeneration from fusion experiment are described in Chapter 4. Somatic embryos and proembryonic masses of 1-2 mm diameter were used as inocula for somatic embryo development. ! Hybrid Confirmation Using RAPD Sampling of Culture for PCR-RAPD Analysis From the fusion experiment involving and N. coriacea, one somatic embryo converted into a plantlet that were subsequently micropropagated. Leaves from this materials were sampled for RAPD analysis and compared with leaves sampled from shoot cultures that originated from somatic embryo developed from zygotic-derived 'T362' avocado embryogenic cultures. Leaves of in vitro seedling of N. coriacea and protoplast-

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180 derived proembryonic masses of zygotic-derived 'T362' were sampled and served as parents for the putative hybrid. Proembryonic masses that developed from fusion between nucellar-derived 'Thomas' and P. borbonia protoplasts were sampled ca. 1 month after subculture in diluted density. From the second fusion experiment, proembryonic masses that developed after subculture in diluted density and lower osmoticum were sampled. DNA Isolation ' DNA was isolated using the SDS method modified according to Mourao-Filho (1995). Tissues consisting of leaves, proembryonic masses or somatic embryos, each ca. 100 mg, were ground in liquid nitrogen in sterile 1.5 ml Eppendorf tubes using forceps. Extraction buffer containing 100 mM Tris-HCl pH 8.0, 50 mM NazEDTA (pH 8.0), 500 mM NaCl, 10 mM p mercaptoehtanol and 3% SDS were added to the samples in 700 nl quantity and the mixture was macerated for a few more min. After all samples were prepared, the tubes were incubated for 10 min at 65° C in a thermocycler (PTC 100, Programmable Thermo Cycler, MJ Research Inc.). The samples were then centrifuged for 10 min at 12,000 rpm. The supematants were transferred to clean tubes and to each tube an equal vol of phenol was added and then vortexed for a few sec and then centrifiiged at 12,000 rpm for 10 min. The aqueous, upper phase was transferred to a clean tube and an equal vol of chloroform:isoamyl alcohol (24: 1) was added. The tubes were vortexed for a few sec and centrifuged at 12000 rpm for 10 min. The aqueous phase was transferred to a clean tube and mixed with 0.6 vol of isopropanol and 0. 1 vol of 7.5 M ammonium acetate to precipitate the DNA. The tubes were inverted back and forth very gently for 2 -3 min and then centrifuged at 12000 rpm for 5 min. The supernatant was decanted and the pellet was washed with 500 nl 70% ethanol and then centrifuged at 12000 rpm for 2 min. The ethanol was decanted and the tubes were blotted carefully with Kimwipes and then dried at 65° C in a thermocycler for 5 min. The pellets were dissolved in 50 |il deionized, sterile i

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181 H2O and stored at -20° C before use. The DNA concentration was quantified by spectrophotometry at X = 260 nm, and was then diluted to ca. 50 ng Alternatively, DNA was isolated fi-om samples (derived fi-om fusions involving avocado cultures that had lost embryogenic potential) using the Phytopure Plant DNA Isolation Kit fi-om Nucleon Bioscience (a division of Scotlab Limited, United Kingdom) (Lee & Nicholson, 1997). c 1 Polvmerase Chain Reaction fPCRVRandom Amplified Polvmorphic DNA rRAPD') PCR-RAPD was performed using a DNA Thermal Cycler (Perkin Elmer Cetus) with the following DNA amplification program: 45 cycles of 94° C for 1 min, 36° C for 1 min, 72.2° C for 1 min and followed by soak at 4° C. Each PCR tube contained 25 ^1 reaction mixture consisting of 15.15 ^1 H2O, StoufFel buffer containing 100 mM Tris-HCl, 100 mM KCl, pH 8.3 (Perkin Elmer), 25 mM MgCb, 1 unit Ampli® Taq polymerase (Stoufifel fragment), 400 ^M dNTPs, primer and 10-50 ng genomic DNA The primers tested included A18, F13 and J16 (Operon Technology, Alameda, CA) The DNA was separated in 2% agarose gel electrophoresis in TEA buffer with electrical potential of 200 mV for 5 min followed by 160 mV for 80 min. The gel was stained with 5 ppm ethidium bromide for 30 min for observation and photography using UV light. Results Protoplast Isolation from Leaf Tissue High protoplast yields were obtained from dark green, young and incompletely expanded leaves of Nectandra coriacea (Table 7-1). Protoplasts from this preparation were small, with ca. 10-20 ^m diameter. Protoplasts were released only when the leaf tissue was agitated at 100 rpm.

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182 Persea species generally did not provide good yields of mesophyll protoplasts; however, good yields of P. pachypoda protoplasts were obtained when soft and tender expanded leaves were digested. The protoplasts of P. pachypoda were ca. 20 -30 nm diameter. Persea borbonia and P. cinerascem provided low yields, i.e., ca. 0.02 ml pelleted protoplasts (ca. 1-3 x lO' protoplasts per gram fresh tissue). Persea cinerascens protoplasts were released prolifically during enzyme incubation, but only a few protoplasts could be recovered after centrifugation. Table 7-1 . Leaf mesophyll protoplast jaeld from in vitro seedlings of Nectandra and Persea sp. Species No. Trials Yields ' ' " Nectandra coriacea 3 . 1 1.3 X lO' protoplast g'' Persea borbonia 3 low, ca. 0.02 ml pellets Persea pachypoda 2 3x10'* Persea cinerascens 3 ! low, ca. 0.02 ml pellets Fusion between Avocado 'T362' and Nectandra coriacea and 'Thomas' Nucellar Initial fusion experiments involving equal volume of mesophyll protoplasts of 'T362' avocado and N. coriacea resulted in fiision frequencies of 1-2%. Alternatively, protoplasts were mixed in a proportion of 1 :2 vol of avocado: N. coriacea prior to fiision. In this proportion, the fiision frequency reached 10-20%. The fiised protoplasts could be identified by the presence of chloroplasts and starch granules (Figure 7-1 A). Approximately 600 somatic embryos developed from 4 fiision attempts between avocado 'T362' and Nectandra coriacea. Some somatic embryos (ca. 3-5 mm diameter) germinated precociously, and did not form shoots. Most of the embryos that developed

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183 Figure 7-1. Protoplast fusion between zygotic embryo-derived 'T362' avocado and Nectandra coriacea, the somatic embryos, plantlet regeneration and shoot proliferation. (A) A heterokaryon. Note the chloroplasts (dark) and starch granules (white) intermixed in the fused protoplast. (B) Large somatic embryos from fiision experiment. (C) A plantlet regenerated from a fusion experiment. (D) Proliferating shoots of D.

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184 were larger (1.5-2 cm diameter) (Figure 7-1 B) than the somatic embryos that were regenerated from protoplasts without fiision (Figure 5-11 B). Some somatic embryos formed secondary proembryonic masses at their bases. Approximately 11 months after fusion, one somatic embryo converted successfiilly into a plantlet (Figure 7-1 C). The shoot was removed and proliferated on ASP medium (Table A-1). On this medium, the nodal stem segments and shoot tips produced multiple shoots that did not elongate. Shoot elongation and leaf expansion were obtained after transfer of shoots from medium with 4.44 jiM BA to medium with 0.4 nM BA (Figure 7-1 D). The shoots produced only a few roots with 20% rooting frequency after 8 to 12 weeks and this was accompanied by massive callus formation at the base of the shoot cuttings. The plantlets did not survive acclimatization. RAPD analysis of this putative hybrid shoots did not confirm a somatic hybrid origin (Figure 7-2). RAPD analysis of proembryonic masses that developed from fiision between nucellar-derived 'Thomas' avocado with P. borbonia protoplasts did not confirm somatic hybrid origin (data not shown). j Fusion between Avocado with either avocado or P. pachypoda The growth and development of protoplasts from different sources were different. The leaf mesophyll protoplasts of P. pachypoda did not divide and became necrotic and disintegrated after 40 days. Protoplasts derived from 'Thomas' zygotic budded but did not divide, whereas 'Hass' protoplasts formed microcalli. When the protoplasts were mixed without fiision, they behaved in the same manner. When the protoplasts were fiised, there was improvement of their growth and differentiation. The fiised 'Thomas' (with P. pachypoda) protoplasts formed microcalli, while 'Thomas' zygotic derived and P. pachypoda protoplasts without fiision failed to divide. Similarly, fiision of 'Hass' with either P. pachypoda or 'Thomas' zygotic-derived protoplasts resulted in development of I

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185 1234 56789 10 11 12 M Figure 7-2. RAPD banding patterns of leaf from a somatic embryo that developed from protoplasts fusion between embryogenic cultures of a zygotic-derived avocado line 'T362' and Nectandra coriacea, and its parental sources. Lane 1, 5 and 9 = leaves of shoots derived from a somatic embryo from zygotic-derived 'T362'; lane 2 6 and 10 = leaves of shoot derived from a somatic embryo from fiision between 'T362' and A'', coriacea; lane 3, 7, 11 = leaves oiN. coriacea; lane 4, 8 and 12 = protoplast-derived proembryonic masses of 'T362'. Lane 1-4 used primer A18; lane 5-8 used primer F13; and lane 9-12 used primer J16.

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186 M1234 56 7M Primer Al 8 Primer F13 Primer J16 Figure 7-3. RAPD banding patterns of proembryonic masses of avocados and their fusion with other avocado and P. pachypoda. Lane M = marker, 1= zygotic 'Thomas', 2 = zygotic 'Thomas' + P. pachypoda, 3 = 'Hass' + zygotic 'Thomas' (at protoplasts/microcalli stage, before subculture at diluted densities), 4 = 'Hass' + zygotic 'Thomas', 5 = 'Hass', 6 = 'Hass' + P. pachypoda, 1 ^^P. pachypoda.

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> 187 M o 3 s C/l O 1 i Q O o I 1 o i 2 o ^ O O O o o o O o O 00 o o O I o U I o ^ j3 to ^ CO J2 "S S 5 IP 2 fc o, "ti W3 > a d wo 1 OIS s 1 1 1 o no no mi( no o o O o O o V o O O o o o o o o 1 [lus ilus Has. lus. en Pi microc X)JOIUI o PEMs' microc PEMs OO IT) O — o c 1 c/l cS O 2 Q ^ H ^ S S ^ .2 -2 .2 CO CO
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m microcalli and proembryonic masses, whereas without fiision *Hass' protoplasts developed only microcallus. These diflferences in growth and development were also reflected in the volumes of the cultures (Table 7-2). Development following subculture at lower culture density in medium of lower osmolarity improved culturability following the fusion treatment. While no proembryonic masses could be recovered from 'Hass' protoplasts, proembryonic masses could be recovered following the fusion treatment. The proembryonic masses that developed after fusion showed differences according to fusion combination, i.e., 'Hass' + 'Thomas' zygotic resulted in the highest number of proembryonic masses followed by 'Hass' + P. pachypoda, and the lowest PEM development was with 'Thomas' zygotic + P. pachypoda. RAPD analysis indicated no DNA complementation from proembryonic masses from the fusion experiments. DNA banding patterns resembled those of the most vigorous parents (Figure 7-3). For example, the DNA banding pattern of 'Thomas' zygotic + P. pachypoda fusion was that of 'Thomas', while DNA of 'Hass' + 'Thomas' zygotic was that of 'Hass' (Table 7-2). Discussion Protoplasts could be isolated from seedling leaves of Nectandra and Persea species that are resistant to Phytophthora root-rot. While the protoplast yields were considerably higher for Nectandra, the use of very young leaves required many seedlings and protoplast yields were not very high. The use of very young leaves may have caused the small size of the protoplasts. The isolation of protoplasts from the Persea species was more difficult and yields were inconsistent. The successful isolation of Persea leaf mesophyll protoplasts was dependent on appropriate source tissue and agitation of digestion mixtures that is higher than the standard 50 rpm reported by Grosser and

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189 Gmitter, 1990; however, agitation at 125 rpm instead of 3-50 rpm was required to release protoplasts from leaves of in vitro propagated peach (Prunus persica) plants (Mills & Hanunerschlag, 1994). The shoots that were proliferated from somatic embryos derived from fusion experiments were not of hybrid origin. Nonetheless, the shoots showed increased sensitivity to BA. The ploidy of this material awaits confirmation. The diflBculty to root of such shoots may not be related to protoplast fijsion procedure but may be genotype dependent, since somatic embryo-derived shoots from the same embryogenic culture line also demonstrated similar rooting behavior. The use of embryogenic cultures that have diminished their embryogenic potentials for one of the fusion parents in combination with a leaf parent has been employed successfully to produce somatic hybrids with citrus (Grosser & Gmitter, 1990). According to Grosser and Gmitter (1990) the process of fusion itself may trigger embryogenic competence. Using a similar approach, avocado cultures that had lost their embryogenic potentials were fused with leaf mesophyll protoplasts of Persea spp. No somatic embryos were recovered even though improvement of protoplast growth and differentiation were observed following fusion. The failure to obtain somatic hybrids in these fusion experiments may not necessarily reflect incompatibility between the parents since large numbers of somatic embryos did not develop to maturity. Another limitation of this study is that the number of fusions was limited by the availability of non-avocado parent protoplasts. Alternative methods for isolating non-avocado protoplasts for fusion experiments, e.g., callus, suspension cultures, and leaves from continuously proliferating shoot cultures need to be explored. In addition, fusion should also be attempted for avocado with other species belonging to different genera in the family Lauraceae that have fruit size and shape similar to avocado such as Endiandra and Beilschmieidia (Schroeder, 1995). Borys et al. (1993) reported that pulp of Beilschmieidia annoy (Blake) Kosterm., has taste and texture better

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190 than or similar to avocado. Unfortunately, the graft and sexual compatibility of this species and its resistance to root-rot have not been studied conclusively (Schroeder, 1995). More studies involving fusion between avocado and related species need to be done in order to assess the viability of protoplast fusion for combining avocado with incompatible species. j

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CHAPTERS SUMMARY AND CONCLUSIONS Summary In this study, several in vitro protocols for avocado have been either improved or newly developed. These include initiation and establishment of embryogenic cultures of various genotypes and elite selections, somatic embryo development, shoot proliferation of juvenile materials, protoplast isolation, culture and regeneration and protoplast fusion between avocado and related species. Embryogenic cultures have been induced from immature zygotic embryos from 12 avocado cultivars and from nucellar explants of four avocado cultivars. This is the first report of the recovery of embryogenic nucellar cultures of avocado. The strategy involved explanting onto a sterile, semisolid induction medium consisting of modified B5 macroelements, MS microelements and organics (Appendix 1), and which was supplemented with the plant growth regulator picloram. Embryogenic cultures, which consisted of proembryonic masses, were maintained either on semisolid or in liquid proliferation medium consisting of MS major salts and other addenda (MSP) (Appendix 1), and supplemented with picloram. This is the first report on establishment of embryogenic suspension cultures of avocado. Filter sterilization of the liquid growth medium significantly improved the growth of the culture. Somatic embryo development was initiated using small proembryonic masses or singulated somatic embryos (diameter <1.8 mm) as an inoculum on maturation medium. Somatic embryo development was genotype-dependent and affected by osmolarity sucrose i I -. . 191

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> 192 and gelling agent concentration. The composition of optimized somatic embryo development medium was similar to the proliferation medium (without picloram) and solidified with 6 g/1 Gel-Gro™ gellan gum. Somatic embryos developed fi-om globular to heart to cotyledonary stage somatic embryos, which were white, opaque and round with diameter ca. 0.8-1.2 cm. After subculture of cotyledonary somatic embryos onto medium supplemented with 4.5 BA and solidified with gellan gum for 2-3 subcultures of 2-3 months duration, the somatic embryos were ca. 1-2 cm diameter. Mature somatic embryos were subcultured on germination medium which was similar to maturation medium in composition, but supplemented with 2.89 nM GA3. After 8-12 months combined on maturation and germination medium, a low frequency (ca. 5%) of somatic embryos developed shoots with or without roots. Somatic embryogenesis has also been initiated from nucellar explants of 'Thomas', 'Hass', 'Lamb' and 'T362' on the same induction medium. Somatic embryo maturation occurred only with 'Thomas'; however, although somatic embryos germinated, there was no shoot formation Using 'Guaram 13' as a model, protocols for shoot proliferation of juvenile origin has been improved by manipulating the total nitrogen content and the ratio of N03":NH4'^ of the plant growth medium. Nodal or shoot tip explants of 1 cm have a survival rate of 100% and proliferated at the rate of 12-fold at each 8 week interval when cultured on medium containing modified MS salts (nitrogen content was reduced to 40 mM, with NOs'iNHi* ratio of 3:1). Avocado shoots rooted at the rate of 100% after 4-8 weeks using a previously published protocol. The in vitro shoots and plantlets of avocado photosynthesized at a rate comparable to in vivo avocado plants, regardless of the presence of sugar in the medium. This indicated the viability of in vz/ro-grown avocado shoots and plantlets. This is the first report on the photosynthetic ability of in vitro-grovm avocado shoots and plantlets.

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.193 Avocado protoplasts were isolated using the citrus protoplast isolation protocol (Appendix 4) from 8-14 day-old embryogenic suspension cultures that had been subcultured at 2-week: intervals. Protoplast yields of ca. 1 x 10*-3 x lO' have been routinely obtained depending on the genotype and morphology of the culture. Culture condition for growth, initiation and maturation of somatic embryos from protoplasts have been optimized. Under optimum conditions, protoplasts differentiated directly into proembryos and subsequently into somatic embryos. Alternatively microcallus formed prior to somatic embryo formation, and was dependent upon genotype and morphology of the source tissue for protoplast isolation. This is the first report of somatic embryo regeneration from avocado protoplasts. ' Leaf mesophyll protoplasts have been isolated from expanding tender leaves from in v/7ro-grown seedlings of Nectandra coriacea, Persea borbonia, and P. pachypoda using protoplast isolation protocols developed for citrus leaves (Grosser & Chandler, 1987) with slight modifications. Protoplast yields varied with respect to leaf quality and can reach 3 x lO' protoplasts per gram of leaf tissue. This is the first report of leaf mesophyll protoplast isolation from these plants Protoplasts from avocado embryogenic culture line 'T362' have been fiised with mesophyll protoplasts of Nectandra coriacea using PEG-mediated chemical fiision (Grosser & Gmitter, 1990). Fusion frequencies reached ca. 13% when protoplasts of avocado and Nectandra coriacea were mixed in a ratio of 1:3-1:4. Large numbers of somatic embryos have been regenerated from several fiision experiments involving avocado and Nectandra coriacea, however, only one somatic embryo converted (one out of 600 somatic embryos) into a plantlet. The shoot was proliferated in vitro and was compared with plantlets derived from a 'T362' somatic embryo. RAPD analysis did not confirm that this plantlet was a somatic hybrid. Protoplasts have also been fiised between avocado lines 'Thomas nucellus', 'Thomas zygotic' and 'Hass' that have lost their embryogenic potential and P. pachypoda and P. borbonia, although somatic embryos have

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not been recovered. RAPD analysis of proembryonic masses that developed from fusion experiments demonstrated no DNA complementation of the parents. Protoplast fusion treatment itself improved the growth and differentiation of protoplasts. Conclusion ; I i . • A complete plant regeneration protocol for avocado through various interconnected tissue culture pathways based on somatic embryogenesis from either embryogenic cultures or protoplasts followed by shoot proliferation through axillary branching and rooting of the shoots has been improved or developed. These advances have several significant impUcations: j | . 1. Application of somatic embryogenesis from nucellus explants of rootstock cultivars such as 'Thomas' or 'Duke 7' has potential for clonal propagation at a lower cost compared to the conventional 'etiolation' technique that is currently used. 2. The viability of protoplast fusion as a method for developing PRR-resistant rootstocks for avocado remains unproven. This is due to the limited number of fusion that have been performed. Higher numbers of fusions, more diverse fiision combinations, the use of embryogenic cultures that had not lost their embryogenic potential, the use of alternative protoplast sources from wild Persea species and electrofusion ought to be evaluated before a final conclusion regarding the viability of the fusion method for avocado improvement can be reached. [ 3. Avocado embryogenic cultures or embryogenic protoplasts could serve as target tissues of option for genetic transformation to generate genetic variability for breeding purposes. Agrobacterium-mediated transformation of 'Thomas zygotic' embryogenic culture with GUS and NPT 11 genes has been

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195 confirmed (Cruz-Hernandez et al, 1998). Single gene transformation of current rootstocks, e.g., 'Thomas', with antifungal genes or transformation of scion cultivars, e. g., 'Hass' with ripening genes for controlling fiiiit ripening would also be attractive. The realization of all these potentials for avocado improvement, however, require the improvement of plant conversion or shoot development fi-om the somatic embryos.

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APPENDIX AVOCADO TISSUE CULTURE MEDIA, PROTOPLASTS ISOLATION AND FUSION SOLUTIONS i i

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199 Table A-2. Avocado protoplast culture medium MS'8P. Component Amount of Stock (Ml) Final Concentration in Medium MS Major Salt Stock (devoid NH4NO3) MS Minor Salt Stock Thiamine-HCl Stock myo-lnositol 8P Multivitamin Stock A 8P Multivitamin Stock B 8? Sugar and Sugar Alcohol Stock 8P Organic Acid Stock Liquid Coconut Endosperm Malt Extract Glutamine Casein Hydrolysate Sucrose Mannitol^ 100 see Appendix 10 see Appendix 10 1 mg 100 mgr' 2 ^ see Appendix 1 see Appendix 10 see Appendix 20 ' see Appendix 0.2% 1.000 gl"* 3.100 gl"' 0.250 g 51.3 gr^(0.15M) 100.1 gr^(0.55M) Filter sterilize with 0.2 jim pore opening This medium is a modification of BH3 medium (Grosser & Gmitter, 1990) in which the inorganic salts and vitamin are replaced with inorganic salts of MS without ammonium nitrate and 1 mg thiamine HCl. MS8P' uses standard MS major salts and no glutamine is added to medium. ^ Only mannitol concentration is altered for different medium osmolarity, while sucrose concentration in the medium stays the same. For 0.6 M MS"8P use 81.99 g 1'^ mannitol, for 0.4 M MS 8P use 45.5 g 1'^ mannitol, for 0. 15 M MS'8P use no mannitol.

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> 200 Table A-3. Stock solutions of 8P organic addenda (Kao & Michayluk,1975) as modified by Grosser and Gmitter (1990) and their final concentration in protoplast culture medium. , , Component Stock Solution (g/ 100 ml) Final Concentration in the Medium (mg 1"^) Sugar and Sugar Alcohol ( 1 OOX) j Fructose 2.5 250 Ribose 2.5 ' Xylose 2.5 i 250 Mannose 2.5 . •! 250 Rhamnose 2.5 ' 250 Cellobiose . 2.5 ! 250 Galactose 2.5 250 Mannitol 2.5 250 Oreanic Acid (SOX) i 1 Sodium pyruvate 0.1 i 20 Citric acid no 1 u.z 1 Malic acid U.z 1 40 rumanc aciu n 0 ' 40 Muhivitamin A Calcium pantothenate 0.05 1 1 Ascorbic acid 0.1 2 Choline chloride 0.05 ' 1 p-Aminobenzoic acid 0.001 0.02 Folic acid 0.02 0.4 Riboflavin 0.01 0.2 Biotin 0.001 0.01 Muhivitamin B Retinol (Vit. A) 0.001 0.01 Cholecalciferol (Vit. D3)** 0.001 1 0.01 Cyanocobalamine (Vit B^) 0.002 0.02 ** insoluble in water, soluble in ethanol

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Table A-4. Enzyme Solution for Protoplast Isolation. Component dtocK m /\IIIUUlll 111 Final 1 no ml Lw mi H\J nil OUIUIIUII P oncentrati on jvianniioi S 12 O J. {J 0.7 M CaCb 14.4 g 1 ml Stock 24.5 mM NaHzPO* 0.44 g 1 ml stock 0.92 mM M.E.S* 4.8 g 1 ml stock 6.25 mM Cellulase Onozuka KS''^ 0.40 g 1% (w/v) Macerase RIO"^ 0.40 g 1% (w/v) Pectolyase Y-23'' 0.08 g 0.2% (w/v) ***Adjust pH to 5.6-6.0 with KOH ***Filter sterilize with 0.2 pm unit From Grosser and Chandler (1987) " Karlan Chemical, Santa Rosa, California ^ Can be replaced with product from Kinki Yakult, Japan ^ Can be replaced with Macerase® from Calbiochem®, La JoUa, California Table A-5. CPW salts Stock Solutions and Their Final Concentration in the Solution. Component Amount in 100 ml Stock Solution (g) Final Concentration (mg r^) CPW Salts Stock V KH2PO4 0.272 27.2 KNO3 1.00 100 MgS04 7H2O 2.50 250 KI 0.0016 0.16 CUSO4 5H2O 0.000025 0.00025 CPW Salt Stock 2 CaCb 2H2O 1.5 150 From Frearson et al. (1973) " 2.5 mg.r' Fe2 (S04)3 6 H2O was omitted from original formulation.

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Table A-6. Amount of CPW Stock Solution and Osmoticum per 100 ml Gradient Centrifugation Solution. 202 COMPONENT CPW13MAN CPW25SUC CPW35SUC CPW Stock 1 CPW Stock 2 Sucrose Mannitol 1 ml 1 ml 13 g 1 ml 1 ml 25 g 1 ml 1ml 35 g pH were adjusted to 5.8 with KOH. Solutions were filter sterilized with 0.2 lam pore opening. Store in cold. Table A-7. Protoplast Fusion Solution. Component Amount in 100 ml Concentration PEG Solution Polyethylene Glycol ( 1 500 MW, Aldrich) 40 g CaCl2 2H2O Glucose ***Adjust pH to 6.0 with KOH 0.97 g 5.41 g 40% (w/v) 66 mM 0.3 M Solution A Glucose CaCb 2H2O Dimethylsulfoxide (DMSO) ***Adjust pH to 6.0 with KOH 7.2 g 0.97 g 10 ml 0.4 M 66 mM 10%(v/v) Solution B Glycine *** Adjust pH to 10. 5 with KOH pellets 2.25 g 0.3 M From Menczel et al. (1981) as modified by Grosser and Gmitter (1990) and Grosser (1994). All solutions were filter sterilized with millipore 0.2 ^m unit.

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225 Vardi A & Galun E (1988) Recent advances in protoplasts culture of horticultural crops: Citrus. Sci Hort 37:217-230 Vardi A & Galun E (1989) Isolation and culture of Citrus protoplasts In: Bajaj YPS (ed) Biotechnology in Agriculture and Forestry, Plant Protoplasts and Genetic Engineering I, Vol 8. SpringerVerlag, Berlin, pp. 147-159 Vardi A, Bleichman S & Aviv D (1990) Genetic transformation of Citrus protoplasts and regeneration of transgenic plants. Plant Sci. 69:199-206 Vasil IK (1983) Isolation and culture of protoplasts of grasses. In: Giles KL (ed) International Review of Cytology Suppl 16: Plant Protoplasts. Academic Press, New York. pp. 79-88 ^ ^ Vasil DC & Vasil V (1980b) Isolation and culture of protoplasts. In: Giles KL (ed) International Review of Cytology Suppl IIB: Plant Protoplasts and Genetic Engineering. Academic Press, New York. pp. 1-19 Vasil V & Vasil IK (1982) The ontogeny of somatic embryos of Pennisetum americanum (L.) K. Schum., I. In cultured immature embryos. Bot Gaz 143:454-465 Vasil V & Vasil IK (1984) Isolation and culture of embryogenic protoplasts of cereal and grasses. In: Vasil IK (ed) Cell Culture and Somatic Cell Genetics of Plants Vol 1: Laboratory Procedures and their Application. Academic Press, New York. pp. 398^04 I Vega-Solarzano D E (1989) Propagation of in vitro rootstock of avocado. Cal Avoc Soc Yrb 73:149-151 Vieitez AM (1995) Somatic embryogenesis in Camellia spp. In: Jain S, Gupta P & Newton R (eds) Somatic Embryogenesis in Woody Plants Vol. 2, Angiosperms. Kluwer Academic Publishers, Dordrecht, pp 235-274 Vieitez FJ (1995) Somatic embryogenesis in chesnut. In: Jain S, Gupta P & Newton R (eds) Somatic Embryogenesis in Woody Plants Vol. 2, Angiosperms. Kluwer Academic Publishers, Dordrecht, pp 375^08 Vu J, Niedz RP & Yelenosky G (1993) Glycerol stimulation of chlorophyll synthesis, embryogenesis, and carboxylation and sucrose metabolism enzymes in nucellar callus of 'Hamlin' sweet orange. Plant Cell Tiss Org Cult 33:75-80 Wann SR (1988) Somatic embryogenesis in woody species. Hort Rev 10:153-181

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226 Wallin A, Nyman A & Svensson M (1995) In: Jain S, Gupta P & Newton R (eds) Somatic Embryogenesis in Woody Plants Vol. 2, Angiosperms. Kluwer Academic Publisher, Dordrecht, pp. 445-460 1 Wang PJ & Hu CY (1984) In vitro cloning of the deciduous timber tree Sassafras randaiense. Z Pflanzenphysiol 113:331-335 Williams LO (1976) The botany of avocado and its relatives. In: Sauls JW, Phillips RL & Jackson LK (eds) Proc First Int Trop Fruit Short Course: The Avocado. Fruit Crops Department, Florida Cooperative Extension Services Institute of Food and ' Agricultural Sciences, University of Florida, Gainesville, pp. 9-1 5 Williams LO (1977) The avocado, a synopsis of the genus Persea, subg. Persea. Econ Bot 31:315-320 i I ; Williams S, Friedrich L, Dincher S, Carozzi N, Kessmann H, Ward E & Ryals J (1992) Chemical regulation of Bacillus thuringiensis 5-endotoxin expression in transgenic plants. Bio/Technology 10:540-543 Williams EG & Maheswaran G (1986) Somatic embryogenesis: factors influencing coordinated behavior of cells as an embryogenic group. Ann Bot 57:443-462 Whiley AW & Schaflfer B (1994) Avocado. In: Schaffer B & Andersen PC (eds). Handbook of Environmental Physiology of Fruit Crops. Vol II. Sub-tropical and Tropical Crops. pp:3-35 Withers LA (1992) In vitro conservation. In: Hammerschlag FA & Litz RE (eds.) Biotechnology of Perennial Fruit Crops. CAB International, Wallingword. UK. pp. 169-202 Witjaksono (1991) The tissue culture medium for 'Pinkerton' avocado (Persea americana Mill.) . Proceeding of the Seminar and National Congress on Biology X. Indonesian Biological Society and lUC Life Science, IPB, Bogor, Indonesia. p.41 1-417 (in Indonesian with English abstract) II Yamamoto M & Kobayashi S (1995) A cybrid plant produced by electroflision between Citrus unshiu (satsuma mandarin) and C. sinensis (sweet orange). Plant Tiss Cult Lett 12:131-137 ' Young MJ (1983) Avocado callus and bud culture. Proc Fla State Hort Soc 96:181-182 Zentmeyer GA (1980) Phytophthora cinnamomi and the Diseases It Causes. The American Phytopathological Society, St. Paul, MN

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227 Zentmeyer GA, Thorn WA & Bums RM (1963) The Duke avocado. Cal Avoc Soc Yrb 47:28-36 II Zentmeyer GA & Thorn WA (1956) Resistance of the Duke variety of avocado to Phytophthrora root rot. Cal Avoc Soc Yrb 40: 169-173 Zentmeyer GA, Menge JA & Ohr HD (1994) Phytophthora root-rot. In: Ploetz RC, Zentmeyer GA, Nishijima WT, Rohrbach KG & Ohr HD (eds.) Compendium of Tropical Fruit Diseases. APS Press, St. Paul MN, pp. 77-79 Zentmeyer GA & Schroeder CA (1953/1954) Test of Persea species for resistance to Phytophthora cinnamomi. Cal Avoc Soc Yrb 38:163-164 Zentmeyer GA & Schroeder CA (1955) Further evidence of resistance to phytophthora rootrot of avocado. Cal Avoc Soc Yrb 39:84-86 Zimmerman JL (1993) Somatic embryogenesis: a model for early development in higher plants. Plant CeU 5: 141 1-1423 •! I Ziv M (1991) Vitrification: morphological and physiological disorders of in vitro plants. In: Debergh PC & Zimmerman RH (eds). Micropropagation. Kluwer Academic Publishers, Dordrecht, pp. 45-69

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BIOGRAPHICAL SKETCH Witjaksono was born on 8 October 1961 in Madiun, East Java Province, Indonesia, the son of the late Sunoto and his mother, Dian Tjahjani. He grew up in Madiun and attended schools in that city. After graduating from high school in 1980, he pursued his studies at Bogor Agricultural University (IPB), in Bogor, Indonesia, and earned a bachelor level degree, "Sarjana Pertanian," in agronomy in 1985. During his final year at IPB, he received a scholarship and a commitment to work following the completion of his degree from the Indonesian Institute of Sciences (LIPI). After his graduation, he worked at the Center for Research and Development in Biology of LIPI. As a result of a competitive examination two years later, he received an overseas fellowship from the Indonesian government. With that fellowship he was admitted into the Department of Botany and Plant Sciences of the University of California at Riverside in order to study plant tissue culture techniques. He received his Master of Science degree in 1989. After working at LIPI for several years, he was again awarded an overseas fellowship to continue his graduate education. In 1992 he started his doctoral program in the Department of Horticultural Sciences, University of Florida. He spent two semesters in Gainesville taking classes and then began his research at the Tropical Research and Education Center, Homestead. He is married to Dyah Kaniasari and blessed with two lovely sons, 5-year-oId Lintang Adyuta Sutawika and 2-year-old Edgar Buwana Sutawika. 228

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Richard E. Litz, Chair 0 Professor of Horticultural Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. rae W. Grosser ^rofessor of Horticultural Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Dennis J. Gray ^ Professor of Horticultond^ience I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fiilly adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. iW^ e. ^ Chael E. Kane ^sociate Professor of Horticultural Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is folly adequate, in scope and quality, as a dissertation for the degree of Doctor of Philo§ophy. 'miogopny. Randy C. Ploetz <^ — ^ Professor of Plant Pathology

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This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fiilfiUment of the requirements for the degree of Doctor of Philosophy. December, 1997 )ean. College of Agriculture Dean, Graduate School


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