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Citrus embryogenesis in vitro : culture initiation, plant regeneration, and phenotypic characterization

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Title:
Citrus embryogenesis in vitro : culture initiation, plant regeneration, and phenotypic characterization
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Gmitter, F. G ( Frederick George ), 1951-
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English
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xiii, 152 leaves : ill. ; 28 cm.

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Subjects / Keywords:
Callus ( jstor )
Embryogenesis ( jstor )
Embryos ( jstor )
Genetic mutation ( jstor )
Germination ( jstor )
In vitro fertilization ( jstor )
Orange fruits ( jstor )
Ovules ( jstor )
Plants ( jstor )
Seedlings ( jstor )
Citrus -- Breeding ( lcsh )
Dissertations, Academic -- Horticultural Science -- UF
Horticultural Science thesis Ph. D
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1985.
Bibliography:
Bibliography: leaves 139-150.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Frederick George Gmitter, Jr.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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CITRUS EMBRYOGENESIS IN VITRO:
CULTURE INITIATION, PLANT GENERATION,
AND PHENOTYPIC CHARACTERIZATION







BY

FREDERICK GEORGE GMITTER, JR.


















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 1985

















ACKNOWLEDGEMENTS



The following dissertation, though it lists the name of one individual as author, is the product of the energy and concern of several people. Without their encouragement, counsel, and assistance, this manuscript would not exist. I want to extend my sincere appreciation and heartfelt gratitude to the following:



Arlene, my wife, for her unbounded enthusiasm, unselfish support, encouragement, typing, and especially

love, throughout my years as a student;



Katherine and Fred Gmitter, Sr., my parents and the

rest of our family, for childhood encouragement, and

their prayers and pride;



Dr. Gloria Moore, friend and adviser, for the opportunity to explore and to accomplish, for advice and counsel, sharing of knowledge, and invaluable assistance with the preparation of this manuscript;



Dr. Wayne Sherman, for friendship, advice, humor, and

assistance;


ii









Dr. Paul Lyrene, for challenging thoughts and statistical advice;



Dr. Prem Chourey and D. L. Curt Hannah, for their time and effort on my behalf;



Vicki Vaughn and Anne Harper, without whose technical assistance and collective sense of humor this work would not have been possible;



Steve Hiss, for help with the computing of statistics;



Katherine Williams, for diligent preparation of the manuscript;



The many friends that we have met during our years in Gainesville, in the Fruit Crops department (staff and fellow students) and around town, for their kind support, assistance, and sharing of good times. Arlene and I will cherish for life the memories of knowing you all.


















TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS.......................ii

LIST OF TABLES.......................vii

LIST OF FIGURES.......................ix

KEY TO ABBREVIATIONS....................x

AB STRACT..........................xii

CHAPTER

I INTRODUCTION....................1

II LITERATURE REVIEW...................6

Introduction. ................ . 6
Somaclonal Variation................7
Citrus Tissue Culture ...............19
Characterization of Seedling Citrus Plants 27 Conclusion......................30

III EMBRYO PRODUCTION AND ESTABLISHMENT OF EMBRYOGENIC CALLUS CULTURES FROM UNDEVELOPED
CITRUS OVULES....................33

Introduction.....................33
Materials and Methods...............34

General Information on Tissue Culture
Methods....................34
Experiment 1: Production of Embryos and
Embryogenic Callus from Undeveloped
Ovules of Several Citrus Species . 35
Experiment 2: Production of Embryos and
Embryogenic Callus by Undeveloped
Ovules from Immature Fruit of 'Hamlin'
Orange....................36
Experiment 3: Growth and Habituation
of and Embryo Production by 'Hamlin'
Orange Embryogenic Callus .........37




iv










CHAPTER Page

Results.....................38

General Commnents ..............38
Experiment 1: Production of Embryos and
Embryogenic Callus from Undeveloped
Ovules of Several Citrus Species .. 44
Experiment 2: Production of Embryos and
Embryogenic Callus by Undeveloped
Ovules from Immature Fruit of 'Hamnlin'
Orange .. ............ . 50
Experiment 3: Growth and Habituation
of and Embryo Production by 'Hamlin'
Orange Embryogenic Callus.........54

Discussion . *.. .. .. .. ... .. ....58
Conclusion.....................62

IV EMBRYO DEVELOPMENT, GERMINATION, AND PLANT
ESTABLISHMENT ............... . 64

Introduction....................64
Materials and Methods ..............65
Results.....................66
Discussion.....................72
Conclusions....................79

V CHARACTERIZATION OF CITRUS PLANTS REGENERATED
FROM EMBRYOGENIC CULTURES............82

Introduction....................82
Materials and Methods ..............84

General Remarks. ................84
Cytogenetic Characterization of
Regenerated Plants ............85
Electrophoretic Characterization of
Regenerated Plants ............87
Morphological Characterization of
Regenerated Plants ............92

Results and Discussion ..............97

Cytological Evaluation...........97
Electrophoretic Evaluation..........99 Morphological Evaluation ..........103

Growth rate ............ 110
Mean internode length .........118 Petiole LIW ratio ..........121 Leaf blade L/W ratio. .........124

Conclusions...................129


v










CHAPTER Page

vi SUMMARY . . . . . . . . . 131

LITERATURE CITED . . . . . . . . . 139

BIOGRAPHICAL SKETCH . . . . . . . . 151


















































vi

















LIST OF TABLES

Table Page

3.1. Effects of cultivar and culture conditions on
the percentage of undeveloped Citrus ovules
that produced embryos .. .. .. .. .. .. ..45

3.2. Effect of cultivar and culture conditions on
the number of primary embryos produced by
undeveloped ovules from Citrus fruit harvested
8 months after anthesis .............46

3.3. Effect of cultivar and culture conditions on
the efficiency of embryo production by
undeveloped ovules from Citrus fruit harvested
8 months after anthesis..............48

3.4. Effect of cultivar and culture conditions on
secondary proliferation from undeveloped Citrus
ovules ......................49

3.5. Effect of culture conditions on embryo production by undeveloped ovules from 'Hamnlin' orange
fruit harvested 8 weeks after anthesis. ......51

3.6. Effect of media amendments on embryo production
by undeveloped ovules from 'Hamlin' orange
fruit harvested 12 weeks after anthesis . . 53

3.7. Origin and fresh weight increase of 'Hamlin'
orange embryogenic callus lines ..........55

3.8. Fresh weight increase and cotyledonary embryo
production by 'Hamlin' orange embryogenic callus
s ublines .....................56

3.9. Results of subjective visual examination of
callus proliferation and embryo production from
'Hamlin' orange embryogenic callus . . . 57

4.1. Germination and development of roots and shoots
from primary embryos of several Citrus
cultivars....................67





vii










Table Page

4.2. Germination of embryos and survival of plants
from embryogenic cultures of several Citrus
cultivars ....... ................... 69

4.3. Germination of embryos and survival of plants
from embryogenic cultures of several Citrus
cultivars by initial treatment .. ......... ..71

4.4. Effect of initial treatment on regeneration
survival percentages of several Citrus
cultivars ....... ................... 73

5.1. Origin and number of plants in groups used for
studies of phenotypic stability of regenerated
'Hamlin' orange plants .... .............. 86

5.2. Number of Citrus plants regenerated from
primary and secondary embryos of several
cultivars that were evaluated for isozyme
banding patterns, and total number of zymograms evaluated ...... ................ 88

5.3. Electrode and gel buffer systems used for
electrophoretic evaluation of 'Hamlin' orange
plants ........ ..................... 90

5.4. Enzyme stain systems utilized for electrophoretic evaluation of 'Hamlin' orange plants
and number of major bands observed ........ ..93

5.5. Summary of group means, standard deviations,
and coefficients of variation for growth rate, mean internode length, petiole L/W ratio, and
leaf blade L/W ratio of 'Hamlin' orange plants 105

5.6. Summary of F-tests and T-tests comparing growth
rates of the following groups of 'Hamlin'
orange plants ...... ................. ..106

5.7. Summary of F-tests and T-tests comparing mean
internode length of groups of 'Hamlin' orange
plants ......... ..................... .107

5.8. Summary of F-tests and T-tests comparing
petiole L/W ratios of groups of 'Hamlin' orange
plants ......... ..................... .108

5.9. Summary of F-tests and T-tests comparing leaf
blade L/W ratios of groups of 'Hamlin' orange
plants .......... ..................... 109




viii

















LIST OF FIGURES

Figure Page

3.1. Proliferation of cotyledonary embryos and
proembryos from a cultured ovule. .........41

3.2. Initation of embryogenic callus from cultured
ovules ......................43

5.1. Abnormal plant type (thin pointed leaves, short
internodes, low vigor) observed among regenerated Citrus plants. ..............113

5.2. Example of low and normal vigor among regenerated 'Hamlin' plants of the same age and
from the same treatment.............115

5.3. Example of normal and abnormal 'Hamlin'
seedling leaf blade and petiole shape. .....117





























ix
















KEY TO ABBREVIATIONS


ABA: abscisic acid.

ATP: adenosine 5' triphosphate disodium salt.

BA: N-(phenylmethyl)-1 H-purin-6-amine,
(benzyladenine).

CH: casein hydrolysate.

DZ: butanedioic acid mono-(2,2-dimethylhyrazide),
(daminozide).

GA3: gibberellic acid.

H: histidine buffer.

IAA: indole acetic acid.

Kn: kinetin.

LBTC: lithium borate/Tris-citrate buffer.

L/W: leaf blade or petiole length/width ratio.

ME: malt extract.

MIL: mean internode length.

MS: Murashige and Skoog basal medium.

MT: Murashige and Tucker medium for Citrus.

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide. NAA: napthyl acetic acid.

NAD: 3-nicotinamide adenine dinucleotide phosphate
disodium salt.

Na2EDTA: disodium ethylenediaminetetraacetic acid. NBT: nitro blue tetrazolium.



x










PCNE: primary cell of the nucellar embryo.

PMS: phenazine methosulfate.

TB: Tris-borate buffer.

TC: Tris-citrate buffer.

Tris: Tris-(hydroxymethyl)amino methane.

2,4-D: (2,4-dichlorophenoxy)acetic acid.












































xi

















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



CITRUS EMBRYOGENESIS IN VITRO:
CULTURE INITIATION, PLANT REGENERATION,
AND PHENOTYPIC CHARACTERIZATION By

FREDERICK GEORGE GMITTER, JR.

December, 1985

Chairman: Wayne B. Sherman Cochairman: Gloria A. Moore Major Department: Horticultural Science (Fruit Crops)

Citrus cultivar improvement via standard breeding

methods has been difficult, so that alternative approaches have been suggested. Potentially useful variation has arisen among plants of other genera that have undergone a cycle of tissue culture regeneration. This study was undertaken to assess the degree of stability of parental phenotype expression among Citrus plants produced by in vitro embryogenesis. Also, the factors affecting embryo and embryogenic callus production and development, embryo germination, and plant regeneration were studied for several cultivars.

Cultures were initiated with unfertilized ovules placed on various modifications of MT medium. The degree of




xii










response varied with cultivars, as did the most effective treatments for embryo production and callus initiation. Greater levels of response resulted when 'Hamlin' orange ovules were extracted from more mature fruit (5-8 mo) than from immature fruits (2-3 mo). Primary and secondary embryo production was common, but long-term embryogenic callus proliferation was less frequent. Although not always necessary, 2,4-D (0.01 mg 1- ) enhanced embryogenic callus production from 'Hamlin' orange. Callus exhibited habituation to growth substances in the media.

Plant regeneration was limited by failure of embryos to undergo normal morphogenesis and balanced germination, and of plants to survive transfer to the external environment. Of 2,870 embryos initially produced, 733 underwent normal development, and 239 survived regeneration. Culture age did not affect regenerability, but initiation treatment may have influenced final survival rates.

Regenerate and nucellar 'Hamlin' seedlings were

characterized by chromosome number, electrophoretic profile, and vegetative morphology. No chromosome number or isozyme variants were identified. Morphologically variant plants (vigor, internode length, leaf shape) were observed among all groups, but more extreme variants were found among tissue culture groups. Variant phenotypes were observed more frequently in the tissue culture groups, as well. However, no specific culture treatment was identified that consistently generated more variable plants.



xiii

















CHAPTER I

INTRODUCTION



Citrus species are economically important fruit crop plants in the United States and throughout tropical and subtropical regions of the world. Although an array of cultivars exists, most commonly grown varieties have arisen as chance, well-adapted superior seedlings or as mutant forms of already existing cultivars (44). Despite active and vigorous Citrus breeding programs, very few cultivars have been produced via standard plant breeding methods (3,37). A number of factors have made Citrus cultivar improvement a slow and difficult process. Most cultivars are cross-pollinated and highly heterozygous (108). Large individual plant size and extended periods of juvenility characterize most Citrus species. As a result, breeding programs require long-term investments of land, time, and money (108). Several horticulturally superior clones exhibit pollen and/or ovule sterility and thus, are unsuited for use as parents in desired crosses (33). Likewise, numerous examples of cross- and self-incompatibility exist that prevent the potential of certain specific parental combinations from being realized (108).




1






2


However, the greatest impediment to genetic improvement of Citrus cultivars using standard breeding methods has been the unusual reproductive biology of the genus. Most Citrus species produce seeds that are polyembryonic, resulting from the proliferation of somatic embryos of nucellar origin within the embryo sac (33,34). In polyembryonic types, certain cells characterized by a large nucleolus and densely-staining cytoplasm are found in the nucellus of ovules at anthesis; such cells are not present in the nucellus of monoembryonic types (53). These cells are stimulated to divide and differentiate into somatic embryos about the time that the zygotic embryo begins development (50-70 days after pollination) (21,27,53). The nucellar embryos compete with the zygotic embryo for nutrients and space, and frequently, the zygotic embryo perishes (33). Polyembryony is dominant to monoembryony in Citrus, with possibly more than a single gene involved (16,17,87). The end result of polyembryony from the perspective of cultivar improvement is that most seedlings produced by controlled pollination are genetically identical to the maternal parent, and sufficient numbers of sexual progeny are not produced to allow for selection and subsequent genetic improvement. Additionally, unless the morphological characteristics of the parents are considerably different, the sexual and nucellar seedlings produced are visually indistinguishable from each other, limiting further the efficiency of cultivar improvement programs (38,108).






3



Several alternative approaches to the problem of Citrus cultivar improvement have been attempted or suggested. For example, the use of monoembryonic or slightly polyembryonic types as seed parents can maximize the number of zygotic progeny produced (108). The search for nucellar or bud mutant selections is a part of Citrus improvement programs throughout the world (7,22,43,48,98,135). Use has been made of mutagenizing radiation, as well, in attempts aimed at producing improved forms (39,108,129). Other suggested methods that may increase the number of genetic combinations that could be evaluated take advantage of plant tissue culture technology and include zygotic embryo rescue (93), cell selection (5), and somatic hybridization (126,127,130). Numerous reports have been published within the last decade of variant phenotypes and heritable mutations arising among plants of diverse genera that have been produced by tissue culture techniques (30,67,80); Scowcroft and Larkin coined the term "somaclonal variation" to describe this phenomenon (67). Some of the variant plants exhibited improvement of economically important traits and have been, therefore, incorporated into breeding programs and commercial enterprises (40,65,68,72). Thus far, most studies of the phenotypic stability of tissue culture-produced plants have focused on herbaceous, annual species. However, aberrant plant phenotypes were found among Citrus plants regenerated from cultured monoembryonic nucelli (80).






4


Citrus is the most responsive of woody perennial fruit crops to tissue culture manipulations (13). In vitro embryogenesis occurs readily among many Citrus species, directly from cultured nucelli or ovules (fertilized or undeveloped) and indirectly from embryogenic callus or cultured protoplast colonies (11,13,14,15,55,56,64,73,92, 93,112,127,130). Likewise, a number of Citrus species have been propagated by organogenesis directly from various somatic explant sources (2,19,26,52,90) or after callus production (19,91,103). Despite the many reports of in vitro plant production in Citrus, little information on the stability of character expression in regenerated plants has been published (80). The ultimate objectives of the studies described in this dissertation were to produce Citrus plants via various tissue culture methods and to characterize those plants morphologically, electrophoretically, and cytogenetically, for evaluation of the degree of phenotypic stability exhibited by regenerated Citrus plants.

The first chapter to follow is a review of literature pertinent to somaclonal variation in plants, Citrus tissue culture studies (specifically embryogenic cultures), and Citrus plant characterization. The 3 chapters following the Literature Review will deal respectively with research into embryogenic culture induction and development, plant regeneration and survival, and studies of plant phenotypic stability. Important points and conclusions that are particularly relevant to Citrus will be discussed within the






5


individual chapters. The final chapter will summarize and relate the conclusions of this project to the general body of research on sornaclonal variation in plants.

















CHAPTER II

LITERATURE REVIEW



Introduction

The objective of this review of the literature is to examine 3 different areas of plant science that are interrelated in the original research described in this dissertation. First of all, some of the evidence that has accumulated documenting somaclonal variation will be presented, with emphasis placed on factors indirectly associated with the expression of variability among regenerated plants. Secondly, research in the area of Citrus tissue culture (particularly the phenomenon of in vitro embryogenesis) will be described and discussed; the emphasis here will be not only on the methods and mechanics of tissue culture, but also on the suitability of Citrus plant material for studies of somaclonal variation. Furthermore, the potential relevance of somaclonal variation to Citrus from the perspective of cultivar improvement and plant propagation will be discussed. Finally, a brief review of methods used to characterize Citrus plants will be presented, with an emphasis on electrophoresis and morphological evaluations.




6






7


Somaclonal Variation

Genetic alteration of cultured plant cells was reported by several researchers in the 1960s. Torrey reported that callus cultures of Pisum sativum L. exhibited increasing levels of polyploidy and aneuploidy with increased time in culture; the gradual loss of the organogenic capacity of these cultures was correlated with increased cytogenetic abnormality (124). Murashige and Nakano (76) and D'Amato

(20) likewise have reported an increase in chromosomal aberrations of cultured cells with increased time in culture. In their review, Larkin and Scowcroft cite numerous examples of phenotypic variation observed among callus sublines of several different genera for characters such as morphogenetic pattern, growth rate, pigmentation, growth regulator habituation, and metabolite production (67). The instability of cultured cell and callus phenotypes has been known, therefore, for some time.

The first reports on the recovery of variant types

among tissue culture-produced plants were those of Heinz and Mee, who worked with sugar cane (Saccharum sp.) (41,42). Several papers on the variation observed among regenerated plants of other genera have been published since that time, including potato (Solanum tuberosum L.) (105,106,119), tobacco (Nicotiana sp.) (89), and rice (Oryza sativa L.) (83,113). Many of the earlier reports concerned polyploid species or crop plants that are propagated asexually (e.g., sugar cane, tobacco, or potato). Rice, however, being a






8


seed-propagated diploid, did not fall into the above categories. Within the past decade, numerous reports of variant phenotypes appearing among populations of regenerated plants of other seed-propagated diploid species have been published (9,23,24,29,35). Tissue culture-associated variability, therefore, is not limited to polyploid or vegetatively propagated species.

Many of the reports of variant plant phenotypes

resulted from direct observation of the original regenerated plants (41,42,67,68,105,106,119). For variant plants expressing traits of potential genetic or commercial importance to be of use in plant breeding programs, the observed variation must be heritable. (A possible exception to this requirement may be clonally propagated plants, where nonheritable variation could be of use if the trait is expressed in a stable manner following vegetative propagation. However, even with such plant material, the observed variation(s) must be genetic in nature if the desired trait is to be incorporated into new or different genetic backgrounds.) Reports have appeared recently, with increasing frequency, that document the heritability, and therefore the genetic basis, of several somaclonal variant traits among various plant genera. For example, Gengenbach et al. (35) selected fertile, T-toxin resistant maize (Zea mays L.) plants from susceptible, male-sterile material that was put into tissue culture and subjected to sub-lethal selection pressure with the toxin produced by Helminthosporium maydis.






9



Brettell et al. (8) selected similar plant types from cultured maize that had not been challenged with T-toxin. In these reports, the T-toxin resistance and male fertility were shown to be cytoplasmically inherited. Evidence of heritable nuclear gene mutations among various plant genera has also been forthcoming. Evans and Sharp (29,30) have reported the occurrence of 13 nuclear gene mutations among 230 cellus-derived tomato plants (Lycopersicon esculentum L. Mill.). Both recessive and dominant mutations were observed among progeny of the original regenerated plants, and these mutations were demonstrated to be heritable. Allelism of some of the mutations to other known mutant alleles was also demonstrated (30). Sun et al. (113) reported a rice somaclone exhibiting a dwarf growth habit; this trait was shown to be heritable and controlled by a single recessive gene. Rice plants variant for heritable quantitative or cytoplasmically controlled characters were reported as well. These true breeding variants support the idea that at least some of the variation observed among somaclones is, indeed, genetic in nature.

Mutations of alleles from the dominant to the recessive condition cannot be detected directly in the primary regenerates, unless the mutated locus was heterozygous initially. Consequently, the progenies resulting from self-pollination of primary regenerates have been screened to uncover masked recessive mutations. Prat (89) observed unique plant phenotypes among progeny of self-pollinated, primary






10



regenerates of Nicotiana sylvestris Spegaz et Comes that were not present in the original regenerated plants, thus indicating that recessive mutations were present, but masked, in the regenerates. Likewise, Engler and Grogan

(24) reported 3:1 segregations among progeny of selfpollinated regenerated lettuce (Lactuca sativa L. spp. capitata); they interpreted these segregations as an indication of heritable, single gene mutations. Somaclonal variation is not limited to traits under the control of single genes; appropriate use of statistical techniques has revealed that variants for quantitative traits exist, as well. Larkin et al. (66) reported heritable mutations for quantitative traits such as plant height and heading date, as well as dominant and recessive mutations for qualitative traits in regenerated wheat (Triticum. aestivum L.) plants and their progeny. As more attention is paid to genetic analysis of regenerated plant material in the future, it may be expected that more of the variants for quantitatively inherited traits will be demonstrated to be the result of heritable mutations. Any valuable characters demonstrated to be heritable may be incorporated, then, into plant breeding programs.

A question regarding the origin of somaclonal variation is whether recovered variant plant types are the result of genetic variability that is pre-existent within cell populations of the initial explant tissue or of de novo mutations occurring in the in vitro environment. Although






11



reports have been published supporting both of these views, the bulk of the evidence accumulated thus far supports the latter conclusion. For example, when Thomas et al. (119) regenerated potato plants from protoplast-derived callus colonies, they found that different plant types arose from the same individual callus colony. If the recovered variation was pre-existent in explant tissue, then individual calli (originating presumably from single isolated protoplasts) should have produced uniformly normal or mutant plants. Because more than one plant type was produced from an individual callus, they concluded that the mutations that resulted in phenotypic variability must have occurred during the tissue culture process itself. Prat (89) likewise observed different mutations in regenerated N. sylvestris plants arising from an individual protoplast-derived callus; furthermore, normal and mutant types were observed among plants from an individual callus. Engler and Grogan (24) found that most mutant lettuce phenotypes came from protoplast-derived calli that produced phenotypically normal sister clones. Edallo et al. (23) found mutant maize phenotypes arising from a single initial callus. Although the above studies support the de novo origin of phenotypic variability, they do not conclusively disprove the idea that somaclonal variation results from pre-existent genetic heterogeneity. It is conceivable, for example, that the calli from which the variable plant types above were produced were heterogeneous, perhaps originating from more than






12



one isolated protoplast. McCoy et al. (71) have pointed out the possibility, at least in the case of chromosomal instability, that somatic plant tissue may be composed of heterogeneous cell populations when compared with homogeneous germ cell lines. The culture of somatic tissues may, therefore, result in chromosomally aberrant plants that are "reflective of normal chromosome instability" found in whole plant tissues (71). However, the same researchers also reported that the frequency of chromosomal aberration in regenerated plants increased with increased time in culture, thus providing support for the de novo hypothesis. In contrast, Navarro et al. (80) have suggested that the variant Citrus plants they obtained via in vitro somatic embryogenesis from monoembryonic Citrus types were not the result of mutations that occurred during the tissue culture process because the plants produced from individual cultured nucelli were either uniformly normal or aberrant. Therefore, until more conclusive, definitive studies are undertaken, the true nature of the origin of somaclonal variants (i.e., de novo vs. preexistent mutations) will remain uncertain. However, the evidence available at present suggests that, in most instances, somaclonal variation is a result of de novo mutation.

Several review articles have been published that

compile the evidence for and the history of the phenomenon of somaclonal variation; furthermore, speculation on the basic nature of the mechanisms responsible for in vitro






13



induced mutation and/or variation has been abundant (67,85,96). Consequently, the remainder of this review will focus not on basic causes, but rather on specific factors that have been directly or indirectly associated with the expression of variability among plants from tissue culture. These factors can be placed into 2 groups: genetic or tissue culture-related factors. The genetic factors involve the actual genetic background of the material cultured, including ploidy levels and changes thereof. Tissue culture related factors include explant source, media composition, culture age at the time of plant regeneration, and the actual pathway of regeneration (i.e., direct or indirect organogenesis vs. embryogenesis).

Murashige (75) assumed that the appearance of aberrant phenotypes among regenerated plants was the result of either polyploidy or aneuploidy in the regenerated plants. In fact, much of the variation reported has been shown to be related to changes in chromosome number and/or structure. Latunde-Dada and Lucas (69) observed a positive correlation between the degree of tolerance to Verticillium wilt among regenerated alfalfa (Medicago sativa L.) plants and regenerate ploidy level. They concluded that this variation was the result of increased gene dosage and not a mutational event. Prat (89) reported that among initial regenerated N. sylvestris plants, the diploids were phenotypically similar to the parent, but tetraploids were not. However, as pointed out previously, these apparently normal diploids






14



possessed masked recessive mutations. Ogihara (81) noted that ploidy levels of regenerated Haworthia setata plants were associated with some morphological aberrancies, but the expression of other characters was unaffected. Although Reisch and Bingham (95) found that morphological abnormalities were associated with aneuploidy in regenerated alfalfa plants, aberrant phenotypes were found also among diploid and tetraploid regenerates.

Sacristan's (100) studies with Crepis capillaris (L.) Wallr. cell cultures point out that the absence of changes in chromosome number does not preclude the possibility of chromosome rearrangement as a source of variation. Murata and Orton (79) have provided evidence via meiotic analysis of regenerated celery plants (Apium graveolens L. var. 'dulce') of chromosome rearrangement in diploids; perhaps ironically, aneuploidy produced by chromosome breakage and fusion in some regenerates resulted in normal, functionally diploid plants. However, Burk and Chaplin (10) found no chromosome number differences or evidence of meiotic pairing irregularities among morphologically variant, regenerated tobacco plants. The evidence presented in preceding paragraphs on the existence of single gene mutations among regenerated plants points out that although phenotypic variability can certainly result from poly- or aneuploidization, it is not the sole cause, because variations can arise also in cytogenetically normal plants (i.e., with normal chromosome numbers and gross structure).





15



The genetic background of the initial plant material

placed into culture has been shown to result in differences in the degree of variation observed among regenerated plants. More electrophoretic and morphologic variants were found among sugar cane somaclones derived from a chromosomally unstable parent than those derived from a stable line (42). McCoy et al. (71) used 2 oat cultivars to regenerate plants and found that one produced more plants with various cytogenetic abnormalities than the other. Sun et al. (113) used 18 rice cultivars (9 from each of 2 ecogeographic races). They reported that from one race no polyploids were produced, and mutations were found in only 1 of 9 cultivar somaclone families. The other race produced varying numbers of polyploids, and mutations were noted among families from 5 of 9 cultivars. Therefore, the amount of variation observed was related to the ecogeographic origin of the cultivar cultured. Prat (89) used 2 different lines in the work with N. sylvestris: an original line (selfed for 7 generations) and a diploid androgenetic line derived from the original line (5 cycles of pollen culture and 2 selfed generations of one doubled haploid plant). More morphological variation was obtained from the original than from the androgenetic line. Prat speculated that this resulted from differences in mutation rates between the lines. Navarro et al. (80) reported that although aberrant Citrus plants were obtained from cultured nucelli of monoembryonic cultivars, the polyembryonic types they studied






16



produced uniform plants. (The research described in Chapter V of this dissertation indicates that this conclusion may not always be true.) Shepard (106) reported high levels of variability among regenerated 'Russet Burbank' potato plants (a tetraploid cultivar released in the early 1900s), but Wenzel et al. (134) observed no variation among plants regenerated from dihaploid potato material. It was suggested that perhaps the different results were related to differences in the age of the clone used as starting material, genetic differences, or perhaps technique differences. Thomas et al. (119), however, used Wenzel's technique with a young clone ('Manis Bard', released in 1974) and observed significant variability. It seems in this case that the different results are likely related to genetic differences between normal tetraploid and dihaploid material. The above examples illustrate that differences in genetic background, regardless of the nature of those differences, may influence the amount of variation observed among regenerated plants.

Several manipulable parameters of the tissue culture

process have been shown to influence the amount of variability observed. The nature of the explant source is one such parameter. Navarro et al. (80) reported that in vitro shoot tip graft propagation of Citrus produces trees that are true to type, but plants produced from cultured nucelli (of monoembryonic cultivars) were variable. However, it is possible that these differences may be related to the method of regeneration (organogenesis vs. embryogenesis) (132).






17


Swartz et al. (115) reported that Rubus plants (blackberry) produced by in vitro shoot proliferation of axillary meristems were phenotypically invariant in the field, except for

1 sectorial leaf variant. Wakasa (133) observed that the explant source had an effect on the percentage of variant plants obtained from tissue culture-propagated pineapple (Ananas comosus); syncarp or slip explants produced nearly 100% variant plants, but crown explants yielded only 7% variant plants. Numerous reports of variant somaclones derived from protoplasts have been published (24,69,89,105, 119). The general trend seems to be that the more organized the initial explant is (e.g., shoot tips vs. mesophyll protoplasts), the greater will be the phenotypic stability observed among regenerates.

Media components, culture age at the time of plant regeneration, and the method of regeneration are other parameters that may affect the amount of variability observed. Reisch and Bingham (95) found that most of the morphologically variant alfalfa somaclones they obtained came from cultures on ethionine-amended medium. Ogihara

(81) reported that media amended with NAA and kinetin produced more ploidy and karyotypic variation than did the same basal medium amended with IAA, but no media effects on the stability of morphological character expression of Haworthia were observed. Johnson et al. (49) concluded that the presence of 2,4-D in the media did not increase the






18



level of chromosomal aberrancy among regenerated alfalfa plants. Culture age at the time of plant regeneration influenced chromosomal variability among cultured Pisum sativum L. (124) and oats (71); increased time in culture correlated with increased frequency of aberrations. In contrast, the frequency of chromosome number changes in alfalfa (49) and maize (23) regenerates was not increased following increased culture age.

It has been suggested that the variation observed may be influenced by the pathway of regeneration (i.e., direct organogenesis, organogenesis through a callus intermediary, embryogenesis, or protoplast isolation and subsequent whole plant regeneration via organogenesis or embryogenesis). In a sense, this parameter is reflective of explant source because the explant used usually determines the regeneration method employed. Nonetheless, examples of somaclonal variation have been described among plants regenerated by each of these procedures (9,24,29,47,80,89,95,101,105,107, 119). The effect of differences of any of the culture parameters on the degree of variation obtained cannot be generalized; their relative importance and influence will depend ultimately on the specific plant material being cultured.

In conclusion, it has been shown that somaclonal

variation occurs among diploid and polyploid species, and among seed- and vegetatively-propagated plants. Variants have been detected among primary regenerates, and more






19



frequently, among sexual progeny populations produced by primary regenerates. Variation may be associated with gross chromosomal changes (number or structure), and variants may possess heritable genetic mutations of nuclear or cytoplasmic genes. The event that results in alteration of the genome may occur in culture (de nova), but some instances of variation pre-existent in explant sources have been reported, as well. The level of variability obtained may be influenced by the genetic background of the plant material cultured or by various manipulations of tissue culture parameters. Valuable (economic and genetic) mutations have been obtained from somaclones of several plant genera.



Citrus Tissue Culture

The first reports of the use of reproductive structures of Citrus plants as explant sources for in vitro culture experiments were those of Maheshwari and Ranga Swamy (70,94). They attempted to initiate cultures with whole ovaries and ovules from immature flower buds, as well as with fertilized ovules, nucelli excised from fertilized ovules, and adventive nucellar embryos from Citrus microcarpa. Bunge fruit harvested after pollination. Several modifications of White's medium were used in their work. Ovaries and unfertilized ovules were unresponsive, but seedling plants were produced by fertilized ovules cultured on the basal medium devoid of any supplementation. Plants were produced when adventive embryos,, in various stages of






20


development, were cultured on the basal medium amended with casein hydrolysate (CH), but only well-developed cotyledonary embryos underwent development in the absence of CH. Therefore, the inclusion of maternal tissue in the explant resulted in simpler media requirements for embryo development. Excised nucelli gave rise to embryos and a proliferation of macroscopic spherical bodies called "pseudobulbils." Continued proliferation and the production of occasional embryos only occurred on CH-supplemented medium. Sabharwal (99) likewise demonstrated with Citrus reticulata Blanco cv. Nagpuri cultures that when adventive embryos were cultured with the surrounding nucellar tissue, callus and pseudobulbil proliferation predominated. By contrast, when embryos were cultured without nucellar tissue, embryo differentiation and development predominated over callus proliferation. Culturing embryos initially in the dark was beneficial, but prolonged dark culture led to etiolation (70). ME was found to inhibit embryo germination, and 2,4-D supplementation resulted in callus, but not embryo, production.

The reports cited above concluded that pollination was necessary for adventive embryogenesis in vitro because there was no response among explants from fruit harvested prior to anthesis. However, Mitra and Chaturvedi (73) demonstrated that pollination was not a requirement. They cultured whole and dissected ovaries (walls, placenta, ovules) from unpollinated flowers of C. sinensis L. Osbeck, C.





21



aurantifolia (Christm.) Swing., and C. maxima (Burm.) Merrill on modified MS media supplemented with adenine sulphate and GA, or Kn and IAA with or without GA. Embryogenesis occurred among explants from the 2 former species (polyembryonic) but not the latter (monoembryonic). More embryo production arose from ovary walls than placenta or ovules. Unfertilized ovules (from fruit of unpollinated flowers harvested after 120 days) were placed into culture; these ovules also produced embryos and callus proliferation. Button and Bornman (11), Kochba et al. (64), and Kochba and Spiegel-Roy (59) have also reported embryogenesis in vitro from unfertilized, undeveloped ovules of several Citrus species. Starrantino and Russo (112) have cited the absence of the dominant trifoliate leaf marker among plants regenerated from cultured unfertilized ovules (from mature fruit of flowers pollinated by Poncirus trifoliata (L.) Raf.) as evidence of the nucellar origin of such plants. Species and media used were different in the early works and may account for the disparate results. However, it is clear that pollination is not required for in vitro adventive embryogenesis from ovules of numerous Citrus species.

Sabharwal (99) was the first to point out that it was

not the ordinary cells of the nucellus that produce embryos, pseudobulbils, or callus proliferation; rather, it was the proembryo initials that were the source of in vitro embryo production or callus proliferation. Kobayashi et al. (53,54) performed histological studies of developing ovules





22


from fruit of 3 polyembryonic and 4 monoembryonic Citrus types. They coined the phrase "primordium cell of nucellar embryo" (PCNE) to describe certain unique, denselycytoplasmic cells characterized by a large nucleus with a conspicuous nucleolus that were found within the nucellus of the polyembryonic types, as early as anthesis or shortly afterwards (depending on cultivar). These cells initiated division shortly after the first division of the fertilized egg (approximately 50 days after pollination), and subsequently, developed into nucellar embryos. Such embryogenic cells were found also in unfertilized ovules from mature fruit, but most of the nucellar tissue was degenerated (112). No PCNEs were found in monoembryonic Citrus ovules, regardless of the stage of fruit development (54). Tisserat and Murashige (120,121) demonstrated that the chalazal half of monoembryonic C. medica L. ovules suppressed embryogenesis in Daucus carota L. cv. Queen Anne's Lace and C. reticulata cv. Ponkan embryogenic callus. High levels of ethanol production and concentrations of IAA, ABA, and GA several times higher in C. medica than C. reticulata ovules were noted and suggested as the source of embryogenicsuppressive activity.

Although no PCNEs have been found in monoembryonic

Citrus nucelli, some monoembryonic Citrus ovules synthesize embryogenic-suppressive compounds, and some attempts to produce embryos from monoembryonic ovules have not been successful (73,74), adventive embryogenesis in vitro by





23


monoembryonic Citrus types has been reported. Rangan et al. (92,93) were able to induce adventive embryogenesis among nucelli (120 days after pollination) of 3 different monoembryonic types cultured on MS medium supplemented with either ME, or NAA, adenine sulphate, and orange juice. Embryos arose directly from the cultured nucelli, but no proliferation of callus or pseudobulbils was observed. Juarez et al. (50) reported the production of embryos and short-term embryogenic callus proliferation arising from monoembryonic 'Clementine' (C. clementina Hort ex. Tan.) ovules cultured as per Rangan et al. (92). Although in vitro adventive embryogenesis by monoembryonic Citrus nucelli has been demonstrated, the initial cells responsible for embryogenesis or callus proliferation have not been identified.

Ovules from seedless 'Shamouti' orange fruit (C.

sinensis) harvested 1 to 6 weeks after anthesis were used by Kochba et al. (64) to initiate embryogenic callus cultures. The callus lines produced from those cultures have been the subject of extensive studies of the factors that influence the expression of embryogenic capability. Small callus colonies were initiated on MT media (78) supplemented with ME. These colonies were transferred to MT supplemented with IAA and kinetin, which promoted vigorous callus proliferation (59). It was demonstrated that the morphogenetic pattern desired (callus growth or embryo development) could






24



be obtained by appropriate manipulation of the culture medium. Low levels of adenine or ME stimulated embryo development at the expense of callus proliferation. Callus growth was promoted or inhibited, depending on relative kinetin/IAA ratios. Eventually callus growth and embryo production decreased, despite auxin and cytokinin supplementation. However, when transferred to basal MT, the callus proliferated and produced numerous embryos, indicating phytohormone habituation of the callus (60). With continued time in culture, the embryogenic capacity of the callus was diminished again (57).

A series of experiments were undertaken to develop methods that would promote additional proliferation of callus and embryos. The 'Shamouti' callus had been subcultured routinely at 4-5 wk intervals. By extending the interval between subculturing, the embryogenic response of the callus was enhanced (57). Shorter culture intervals favored callus proliferation, and longer intervals promoted embryo formation. Transferring callus to a sucrose medium after a cycle of sucrose deprivation produced a response similar to the effect of increased subculture intervals, but of a lesser degree. The positive effect of sucrose deprivation diminished in subsequent culture cycles, but the effect of extended intervals persisted for several cycles. other factors in addition to sucrose deprivation were implicated, specifically, hormone level alterations. When non-habituated callus was exposed to gamma irradiation, callus





25



proliferation was suppressed, but embryo production was enhanced; irradiation of the media produced the same effect (110). Habituated callus responded in like fashion, but no media effect was observed (60). The enhanced embryogenic response exhibited by callus following extended culture intervals, sucrose deprivation, use of galactose and related sugars as the carbon source in the media (63), or irradiation of callus suggested that hormone levels and ratios may be altered by these manipulations. Using various phytohormones, growth regulators, and inhibitors, Kochba and Spiegel-Roy (61,62) and Moore (74) have shown that auxins and cytokinins suppressed embryogenesis in habituated callus or culture of unfertilized ovules, but inhibitors of auxinor GA-biosynthesis, or low levels of ABA or ethylene enhanced the embryogenic response. Although extensive research on the factors influencing embryogenesis by 'Shamouti' callus has been reported, little information has been published on factors critical to callus culture initiation, except the work of Moore (74). Likewise little information on plant production from these embryogenic cultures has been published.

Button et al. (14) made histological observations of the gross structure and fine cellular structure of habituated 'Shamouti' callus. The callus was composed of loosely attached, small spherical bodies, each of which was found to be either an individual or a cluster of proembryos,






26



resembling nucellar embryos produced in vivo. Examination of individual cells showed them to possess characteristics typical of meristematic cells (dense cytoplasm with numerous mitochondria, lipid bodies, ribosomes, large nuclei with conspicuous nucleoli, etc.). Proembryos originated from single cells that were isolated by thick cell walls. Internal divisions resulted in a multi-cellular embryo that enlarged through the cell wall, which disintegrated. Additionally, other cells within the proembryo were capable of becoming isolated to undergo the same process of embryo development. The callus tissue, in fact, was shown to be composed almost entirely of proliferating proembryos, embryos, and pseudobulbils. (The latter resulted when proembryos developed an epidermal layer and represented an abnormal alternative developmental pathway.) These studies demonstrated the single cell origin of callus-derived embryos.

The regeneration of Citrus plants via several other in vitro methods has been reported. For example, plants have been produced via adventive embryogenesis from callus colonies derived from protoplasts that were isolated from embryogenic callus of 'Shamouti' and other Citrus species and cultivars (12,126,127,130). X-ray irradiation (126,130) and the use of feeder cell layers (126,128) stimulated the proliferation of and embryo production from protoplastderived callus colonies. Citrus plants have been produced






27



via various organogenic methods including shoot proliferation by axillary buds of shoot tips or stem segments (1,2,52), from adventitious buds formed on cultured stem segments (Moore, personal communication, 19), and by organogenesis from short- or long-term leaf or stem callus cultures (19,36,90,91). The most commonly used growth regulator for the initiation of organogenesis has been 6-benzylaminopurine. The potential exists for populations of Citrus plants from one or several different clones to be regenerated by various in vitro methods, thus providing an opportunity for studying factors that may be related to the expression of somaclonal variation. Factors that could be studied include the method of regeneration (embryogenesis vs. organogenesis, and within categories, direct vs. indirect via callus), the effect of 2,4-D in the culture medium, the influence of culture age at the time of plant regeneration, explant source differences, genotype effects, and the effect of protoplast-mediated plant regeneration on phenotypic stability.



Characterization of Seedling Citrus Plants

The difficulty encountered when attempting to identify zygotic Citrus seedlings among progeny of polyembryonic seed parents has been discussed in Chapter I. The use of simply inherited genetic markers could help to solve the problem, but few markers beside the trifoliate leaf trait of Poncirus trifoliata have been characterized in Citrus species or






28



relatives. As a consequence of the need to distinguish zygotic seedlings and the absence of genetic markers, numerous alternative approaches to plant characterization have been attempted. Cotyledon color of seed-borne embryos has been used to a limited extent, as has insecticide sensitivity (dimethoate) (21). Infrared spectroscopy of leaf oils has been used, also, but the time required to analyze hundreds of samples is prohibitive; furthermore, leaf oil spectrographs change with increased plant age (88). Tatum et al. (117) used TLC (thin layer chromatography) of flavonoid and other non-volatile components to distinguish nucellar and zygotic seedlings; again, only limited sample numbers can be handled with this technique. Browning or non-browning of crude shoot homogenates has been shown to be a taxon-specific trait conditioned by a single dominant gene (25,26,28). No variation for this character has been observed within individual Citrus species. The procedure is quick, simple, and inexpensive, and can be used with appropriate specific parental combinations to distinguish zygotic from nucellar progeny.

Vegetative characters of seedling Citrus plants have

been used to identify hybrid progeny (4,38,118). Leaf shape has been found to be among the most useful vegetative characters for zygotic seedling identification (38) or for identifying tetraploid progeny (4). Leaf shapes frequently have been expressed as a length/width ratio (38,118). Leaf blade and petiole size and shape vary within species and may






29



be used to identify zygotic seedlings from appropriate inter- or intra-specific crosses (38,118). Other vegetative traits used to characterize Citrus seedlings include growth habit and vigor, stem characteristics, leaf color and venation, and relative stoma and oil gland size (4,45,38). Many of these characters (e.g., leaf shape) are variable even within individual plants, so experience is necessary before vegetative variants can be consistently identified with accuracy.

The use of isozyme loci as genetic markers in Citrus

provides another method of plant characterization. Isozyme analysis can be a powerful tool, particularly when the genetics of the enzyme system utilized are understood. Isozyme genes are expressed in a codominant manner. Fairly large numbers of individuals can be screened at one time, usually for several different enzyme staining systems. Torres et al. (122,123) and Moore (unpublished) have established the genetic basis and allelic constitution of several isozyme loci in various Citrus species (see Table 5.4 in Chapter V). These enzyme staining systems and others have been used to distinguish nucellar and zygotic seedlings from certain crosses (46,109) and have been suggested as a means to identify somatic hybrids (6).

To conclude, various methods of Citrus seedling characterization have been developed. The methods vary as to their cost, efficiency, and limitations. Although these techniques provide the means to identify variant seedling






30



plants in uniform populations, the actual variant phenotypes have not been correlated with any traits of horticultural or economic significance. These techniques, therefore, only serve as a screen for variants of specific types. Horticulturally valuable variation must await plant maturity and fruit production to be detected.



Conclusion

Phenotypic instability has been reported among tissue culture regenerated plants of several different genera. Reports of polyembryonic Citrus plant regeneration have indicated relative uniformity among individuals; however, somaclonal variants have arisen from embryogenic cultures of monoembryonic Citrus. It is possible that a more detailed evaluation of polyembryonic Citrus regenerates might have detected variant plant phenotypes. Phenotypic instability may be a problem for plant propagation. Likewise, it may obscure the results of attempted genetic transformations of Citrus via molecular techniques. Good estimates of the background level of variation among non-transformed regenerate populations are necessary to assess accurately the consequences of molecular manipulation of the genome among transformed, regenerate plants. But, somaclonal variation may prove to be a potential source of genetic variation useful for genetic studies or cultivar improvement. For example, a clone superior for most economically important characters may be improved if, perhaps, a disease-resistant






31


variant were found among regenerated somaclones. The idea here is that all of the desirable qualities of the parent clone would be retained, with the addition of another valuable trait. Most currently grown Citrus cultivars arose as spontaneous nucellar or bud sport mutations, and were not the result of controlled hybridization. The production of variant somaclones from established cultivars may allow many more of these mutant phenotypes to be screened. A less ambitious but equally valuable hope--the production of unique genetic or breeding lines--may be more likely to be realized.

Citrus is a genus that provides unique opportunities to study factors involved in the expression of somaclonal variation. Numerous routes to plant regeneration are available, with various manipulations of culture parameters possible. Consequently, there are many possible comparisons of the effects of these factors on the level of phenotypic stability observed among regenerated Citrus plants. Furthermore, the production of nucellar seedlings by polyembryonic types provides a control population to compare in vitro and in vivo induced variability. A thorough assessment of the variation resulting from the different regeneration pathways may provide the information necessary to control Citrus phenotypic stability. Uniform or variant populations of plants could be regenerated, then, depending on the specific desired result. One limitation to the use of this technology is the fact that methods of identifying






32



horticulturally valuable variants among juvenile, seedling progeny are lacking. Plants must still be grown to maturity to evaluate economically important characteristics. The appearance of such valuable variants among regenerated Citrus plants has yet to be demonstrated.

















CHAPTER III

EMBRYO PRODUCTION AND ESTABLISHMENT OF EMBRYOGENIC
CALLUS CULTURES FROM UNDEVELOPED CITRUS OVULES


Introduction



The first step in the establishment of populations of regenerated plants was the production of embryos either via direct embryogenesis from cultured undeveloped ovules or from embryogenic callus. This chapter describes experiments designed to study the effect of various factors (growth regulators, light, stage of fruit development at the time of ovule extraction, species or cultivar differences) on direct embryo production and on the initiation and establishment of embryogenic callus. Embryo production, growth rate, and development of hormone habituation in long-term embryogenic callus cultures were also examined. Embryos produced in some of these experiments were used in the studies of embryo development, plant establishment, and phenotypic stability described in Chapters IV and V.











33






34



Materials and Methods



General Information on Tissue Culture Methods

Fruit from open-pollinated flowers on field-grown trees were harvested at various stages of development as described in the following sections. The fruit were washed with a mild detergent solution and rinsed with deionized water. Portions of the fruit and peel were removed so that only the central cube of locule tissue with the ovules attached remained. Undeveloped (unfertilized or abortive) ovules, which could be distinguished from fertilized ovules by their size, were removed with forceps and placed into glass funnels lined with moist filter paper and supported by 125 ml Ehrlemeyer flasks. Ovules were disinfested by pouring solutions of sodium hypochlorite (20% v/v) and ethanol (70% v/v) through the funnel so that the ovules were immersed in each solution for approximately one minute. This was followed by several rinses with autoclaved distilled water at room temperature. Disinfestation, inoculation, and transfer were done under a laminar flow hood.

The basal medium used in all experiments was that of

Murashige and Skoog (77) as modified for Citrus by Murashige and Tucker (MT) (78). The medium was gelled with 0.8% agar, and the pH was adjusted to 5.7. Growth substance treatments were added to the medium prior to autoclaving at 1210 C, at

1.1 kg cm- for 17 minutes. Media were poured into 100 X 15 mm sterile plastic Petri dishes that were sealed






35


with masking tape following inoculation or culture transfer. All cultures were maintained at 270 C with 16 h fluorescent light (76 pmol s- m-2) except were otherwise noted.


Experiment 1: Production of Embryos and Embryogenic Callus

from Undeveloped Ovules of Several Citrus Species

Ovules were isolated from fruit harvested in October, 1983, approximately 8 months after anthesis, from trees of the following species and cultivars: Citrus sinensis cvs. Hamlin, Pell Navel, Pineapple; C. paradisi Macf. cv. Marsh; C. reticulata cv. Owari; C. paradisi x C. reticulata cv. Orlando; C. aurantifolia cv. Key; and C. limon (L.) Burm.f. cv. Bearss. Following disinfestation, the ovules were placed on MT medium amended with either ME (500.0 mg 1-1) or 2,4-D (0.01 mg 1- ) alone or the same concentration of 2,4-D in combination with BA (0.1 mg 1-1) or DZ (0.1 mg 1-1). Media amendments and concentrations were selected because embryogenic cultures from several Citrus types had previously been obtained in this laboratory and others using these materials. Six Petri dishes with 20 ovules each were used for each cultivar x treatment combination. All cultures were maintained under a 16 h photoperiod, except for an additional treatment group for each cultivar on ME medium that was maintained in the dark. All cultures were subcultured on the treatment media every 4-8 weeks.






36



The number of ovules producing embryos directly without an intermediate callus stage, the number of embryos produced, and the number of ovules with proliferating callus were recorded 56 days after culture initiation. Only embryos that had reached cotyledonary or heart-shaped stages of development were counted; these were designated primary embryos. Further observation of cultures for embryo production and callus development were made. Embryos that developed after primary embryos were removed from cultures for germination (see Chapter IV) were designated secondary embryos. It was not possible in all cases to determine whether the secondary embryos originated via direct embryogenesis from the cultured ovules or from the small clumps of embryogenic callus that began to proliferate.



Experiment 2: Production of Embryos and Embryogenic Callus
by Undeveloped Ovules from Immature Fruit of 'Hamlin'
Orange

Undeveloped ovules were isolated from immature 'Hamlin' orange fruit harvested in April and May, 1984, 8 and 12 weeks after anthesis, respectively. In April, 120 ovules (6 plates with 20 ovules each) were cultured on each of the following amended media: 1) 500.0 mg 1- 1 ME (16 h light), 2) 500.0 mg 1- 1 ME (No light), 3) 0.01 mg 1- 1 2,4-D, 4) 2,4-D/0.1 mg 1- 1 BA, and 5) 2,4-D/0.1 mg 1- 1 DZ. The number of responsive ovules (those producing embryos) and the number of embryos produced were recorded 56 days after culture initiation. In May, 1000 ovules (50 plates with






37


20 ovules each) were cultured on each of the following amended media: 1) 500.0 mg 1- ME, 2) 0.01 mg 1-1 2,4-D/0.1 mg 1- BA, and 3) 0.01 mg 1- 2,4-D/0.1 mg 1- DZ; an additional 100 ovules were cultured on MT (unamended basal medium). Embryo production was evaluated 28 days after culture initiation. Cultures were subcultured on the treatment media every 4-8 weeks. The number of plates with embryogenic callus after 240 days was recorded.


Experiment 3: Growth and Habituation of and Embryo
Production by 'Hamlin' Orange Embryogenic Callus

Undeveloped ovules were harvested from 'Hamlin' orange fruit in August, 1982 (about 5 months after anthesis) and cultured on basal medium supplemented with various concentrations and combinations of 2,4-D, BA and DZ (Moore and Gmitter, submitted). Embryogenic callus lines were selected from the following treatments for further study in this project: 1) 0.01 mg 1- 2,4-D/0.01 mg 1- BA, 2) 0.01 mg 1-1 2,4-D/0.1 mg 1-1 BA, and 3) 1.0 mg 1-1 2,4-D/0.1 mg 1-1 BA. The selected cultures were transferred from the initiation media to MT supplemented with 0.01 mg 1- 2,4-D/0.1 mg 11 DZ and subcultured at 4-6 week intervals until March, 1983. On that date, 5 sublines were isolated from treatment 1 above, 3 sublines were isolated from treatment 2, and

2 sublines were isolated from treatment 3. The tissue sector comprising each subline was selected on the basis of morphology and color (white, friable callus; brownish-tan callus; or green proliferating masses of embryos) with the






38



objective of isolating lines of different exnbryogenic capacity. However, all selected sublines produced tissues of various types, so the attempt to isolate lines on this basis was abandoned.

Fresh weight increase of sublines was determined and

recorded after 28 days. The cultures were then divided and transferred to either 0.01 mg 1-1 2,4-D/0.1 mg 1-1 DZ or

0.1 mg 1-1DZ supplemented medium; sublines were designated Al, A2, etc. on the former and Bi, B2, etc. on the latter medium. Fresh weight increases and numbers of embryos produced were recorded after 15 days. At that time, B lines were subcultured on the same medium; one half of the proliferation of the A lines was subcultured on the DZ-containing medium, and the other half was transferred to unsupplemented MT medium (C lines). A subjective visual evaluation of callus proliferation and embryo production was made 28 days after subculture. These lines were transferred every 4-6 weeks to fresh media of the same composition and were the source of plants from embryogenic callus used in the studies described in Chapters IV and V.



Results



General Comments

The following pattern of response was observed in all undeveloped ovule cultures where embryogenesis occurred. The ovules became swollen and acquired a light-green color






39


within 7-14 days after culture initiation. The integuments of the ovules then split and one to several developing primary embryos emerged without prior callus production. Frequently, other less-developed globular structures were produced from within the ovule or from embryos already present (Fig. 3.1). Some of these structures continued normal development, became cotyledonary embryos, and were designated secondary embryos. A creamy-white friable callus arose on occasion from the proliferating embryos. Although no histological studies were done, this callus appeared to be composed of proliferating proembryos and was distinguished from the proliferating secondary embryos only by the relative proportion of developed and undeveloped structures (see Fig. 3.2).

The secondary proliferation of embryos and callus

described above was fairly common, but long-term proliferation was more rare. Much of the initial proliferation ceased after several weeks. Alternatively, in a number of instances embryo differentiation halted, but embryo growth continued to produce "pseudobulbils" (large green or white spherical structures). Pseudobulbil proliferation resulted in callus line termination and prevented plant regeneration from occurring, despite the fact that the pseudobulbils could be induced to form roots (data not presented). On occasion, embryogenic callus proliferations arose from the surface of cultured pseudobulbils. In general, though, pseudobulbil production represented an undesirable,






























Figure 3.1. Proliferation of cotyledonary embryos and
proembryos from a cultured ovule.





41































Figure 3.2. Initiation of embryogenic callus from cultured
ovules.





43 Ae






44



alternate path of embryo development that interfered with the continued proliferation of embryos or embryogenic callus.


Experiment 1: Production of Embryos and Emibryogenic Callus
from Undeveloped Ovules of Several Citrus Species

The percentages of cultured undeveloped ovules that

produced primary embryos are shown in Table 3.1; Table 3.2 lists the number of embryos produced by each cultivar and each treatment within cultivars. The sweet orange cultivars 'Hamlin', 'Pell Navel', and 'Pineapple' were the most responsive types in terms of both percentage of ovules producing embryos and total numbers of embryos produced. 'Marsh' grapefruit, 'Orlando' tangelo, and 'Key' lime were intermediate in their degree of response. 'Bearss' lemon responded only on the malt extract medium in the absence of light and produced very few embryos. 'Owari' satsumna was unresponsive to all treatments.

Sweet orange ovules cultured on 2,4-D/DZ or ME supplemented media in the light were the most responsive in terms of total embryo production. These treatments were superior for all 3 sweet orange cultivars, but the most effective treatments varied among the other cultivars. For example, media supplemented with 2,4-D or 2,4-D/BA were most effective for embryo production from 'Marsh' grapefruit, but either ME treatment was of greater effectiveness than the three 2,4-D supplemented media for embryo production from 'Orlando' tangelo. 'Bearss' lemon responded only when









Table 3.1. Effects of cultivar and culture conditions on the percenage of undeveloped
Citrus ovules that produced embryos. Fruit were harvested approximately 8 months after anthesis. Cultures were scored 56 days after initiation.



Treatment (mg 1- )a
0.01 2,4-D/ 0.01 2,4-D/
Cultivar 500.0 ME 500.0 ME 0.01 2,4-D 0.1 BA 0.1 DZ
Light No light Light Light Light


Hamlin orange 48 35 38 15 42

Pell Navel orange 71 42 50 44 73

Pineapple orange 53 43 54 44 50

Orlando tangelo 20 28 12 7 9

Marsh grapefruit 7 7 17 14 5

Owari satsuma 0 0 0 0 0

Key lime 13 28 11 7 14

Bearss lemon 0 3 0 0 0


aEach cultivar x treatment combination utilized 120 ovules except Orlando (2,4-D = 100 ovules).



En









Table 3.2. Effect of cultivar and culture conditions on the number of primary embryos
produced by undeveloped ovules from Citrus fruit harvested 8 months after
anthesis. Embryos were counted 56 days after culture initiation.



Treatment (mg 10.01 2,4-D/ 0.01 2,4-D/
Cultivar 500.0 ME 500.0 ME 0.01 2,4-D 0.1 BA 0.1 DZ Total
Light No light Light Light Light


Hamlin orange 145 93 97 35 132 502

Pell Navel orange 251 133 172 155 258 969

Pineapple orange 168 98 118 100 148 632

Orlando tangelo 55 69 23 18 29 194

Marsh grapefruit 27 54 115 80 19 295

Owari satsuma 0 0 0 0 0 0

Key lime 52 113 28 15 53 261

Bearss lemon 0 17 0 0 0 17

Total 698 577 553 403 639 2870


a Each cultivar x treatment combination utilized 120 ovules except Orlando (2,4-D = 100 ovules).





47



cultured on ME medium in the absence of light; the same treatment was the most effective for embryo production from 'Key' lime. No single treatment that was most efficient for primary embryo production from the different Citrus types could be identified.

The number of primary embryos produced per ovule cultured, by treatment within cultivar, is given in Table 3.3. The relative ranking of cultivars and genotypes is identical to that observed in Tables 3.1 and 3.2; these values are listed and embryo production expressed thus for comparisons of production efficiency between this experiment and Experiment 2. Table 3.3 also lists the number of embryos produced per responsive ovule. Among the oranges, the treatments producing the greatest numbers of embryos per responsive ovule were 2,4-D/DZ and ME (light), the same treatments that produced the greatest percentages of responsive ovules and consequently the highest total number of embryos produced. The values for embryos per responsive ovule for 'Orlando' tangelo and 'Key' lime were of the same magnitude as the values of the orange cultivars. Although fewer ovules of 'Marsh' grapefruit and 'Bearss' lemon produced embryos (Table 3.1), more embryos were produced per responsive ovule by these cultivars.

The percentage of undeveloped ovules in each cultivar X treatment combination where proliferating callus was present after 56 days is listed in Table 3.4. The oranges were the most responsive cultivars for ovular callus production.













Table 3.3. Effect of cultivar and culture conditions on the efficiency of embryo production by undeveloped ovules from Citrus fruit harvested 8 months after anthesis.
Embryos were counted 56 days after initiation.





Treatment (mg I1 a
Cultivar 500.0 ME 500.0 ME 0.01 2,4-D 0.01 2,4-D/0.1 BA 0.01 2,4-D/0.1 DZ
Licht No light Light Light Licht
Embryos/ Embryos! Embryos/ Embryos/ Embryos! Embryos! Embryos! Embryos! Embryos! Embryos!
ovule responsive ovule responsive ovule responsive ovule responsive ovule responsive
cultured ovule cultured ovule cultured ovule cultured ovule cultured ovule


Hamlin orange 1.21 2.59 0.78 2.19 0.81 2.16 0.29 1.89 1.10 2.64

Pell Navel orange 2.09 2.97 1.11 2.63 1.43 2.87 1.29 2.90 2.15 2.97

Pineapple orange 1.40 2.63 0.82 1.88 0.98 1.82 0.83 1.89 1.23 2.47

Orlando tangelo 0.46 2.29 0.58 2.09 0.23 1.92 0.14 2.13 0.24 2.64

marsh grapefruit 0.23 3.38 0.45 5.40 0.96 5.25 0.67 4.71 0.16 3.17

Owari satsuma 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Key lime 0.43 3.25 0.94 3.32 0.23 2.15 0.13 1.88 0.44 3.12

Bearss lemon 0.00 0.00 0.14 4.25 0.00 0.00 0.00 0.00 0.00 0.00


a Each cultivar x treatment combination used 120 ovules except Orlando (2,4-D 100 ovules).









Table 3.4. Effect of cultivar and culture conditions on secondary proliferation from
undeveloped Citrus ovules. Fruit were harvested 8 months after anthesis.
Cultures were scored 56 days after initiation.



Treatment (mg 1)a
0.01 2,4-D/ 0.01 2,4-D7
Cultivar 500.0 ME 500.0 ME 0.01 2,4-D 0.1 BA 0.1 DZ
Light No light Light Light Light


Hamlin orange 25b 25 25b 15b 29b

Pell Navel orange 47 44 35 38 50

Pineapple orange 39 41 47 22b 29b

Orlando tangelo 17b 26 14b 16 17

Marsh grapefruit 1 5 5 4 1

Owari satsuma 7 5 5 3 5

Key lime 8b 13b 11 5 10b

Bearss lemon 1 4b 0 0 0


a Each cultivar x treatment combination used 120 ovules except Orlando (2,4-D = 100 ovules).
bThese combinations produced enbryogenic callus lines that persisted at least 180 days.






50



'Orlando' tangelo was intermediate in the percentage of ovules producing callus. 'Key' lime and 'Bearss' lemon had few responsive ovules, but those that did respond produced embryogenic callus. 'Marsh' grapefruit and 'Owari' satsuma produced lttle callus and that which was produced was not embryogenic. ovules in several cultivar X treatment combinations were decreased in number and lost to an outbreak of contamination in the culture room prior to further evaluation. As a result, comparisons of numbers of embryogenic callus lines that could be established from the various treatments could not be made. However, established embryogenic callus lines that continued to proliferate were obtained from several of the Citrus cultivars. Those genotype x treatment combinations marked with a "b" in Table 3.4 produced embryogenic callus that persisted at least 180 days.



Experiment 2: Production of Embryos and Embryogenic Callus
by Undeveloped Ovules from Immature Fruit of 'Hamlin'
Orange

Table 3.5 describes primary embryo production under various culture conditions by undeveloped ovules from immature 'Hamlin' orange fruit harvested approximately

8 weeks after anthesis. The response level of ovules from fruit of this age was much lower than that of ovules from more mature fruit, regardless of initiation treatment (See Results: Experiment 1). The most effective treatment for embryo production by ovules at this stage of development was






51





Table 3.5. Effect of culture conditions on embryo production by undeveloped ovules from 'Hamlin' orange fruit harvested 8 weeks after anthesis. Embryos
were counted 56 days after culture initiation.



b Number Number
Treatment (mg 1 Percent Number embryos/ embryos/
responsive embryos ovule responsive ovules produced cultured ovule


500.0 ME 4.2 6 0.05 1.20
Light

500.0 ME 7.5 14 0.12 1.56
No light

0.01 2,4-D 0.8 1 0.01 1.00
Light

0.01 2.4-D/0.1 BA 0.0 0 0.00 0.00
Light

0.01 2.4-D/0.1 DZ 1.7 3 0.03 1.50
Light

MT 3.3 2 0.03 1.00
Light


a Each culture treatment utilized 120 ovules, except 60 ovules were used in the MT treatment. b Responsive ovules are those from which embryos were produced.





52



the ME/no light regime (0.12 embryos/ovule cultured). Exubryogenesis was suppressed by the addition of 2,4-D to the medium. When the response level of ovules from such fruit was compared with the greatest efficiency of embryo production from 'Hamlin' ovules at 8 months after anthesis (1.21 embryos/ovule cultured on ME/light regime), it was noted that the older ovules were approximately ten times more productive. The numbers of embryos produced per responsive ovule also decreased in this experiment, when compared with the results of Experiment 1. The production of secondary embryos or embryogenic callus was not observed in this experiment.

Embryo production under various culture conditions by undeveloped ovules from immature 'Hamlin' orange fruit harvested 12 weeks after anthesis is described in Table 3.6. Embryos were produced on all media, but the response level was again less than that of 8-month ovules (See Results: Experiment 1). Treatment differences were not apparent for the percent of ovules responding, but there were differences in efficiency of embryo production (0.20 vs. 0.06 embryos/ ovule cultured for ME vs. MT) and in the number of embryos produced per responsive ovule among treatments (2.46 vs.

1.20 for ME vs. MT). No 2,4-D induced suppression of embryogenesis was observed among 12-week ovules. None of the ME-initiated cultures produced proliferating embryogenic callus 240 days after culture initiation, but 31 of






53



Table 3.6. Effect of media amendments on embryo production by undeveloped ovules from 'Hamlin' orange
fruit harvested 12 weeks after anthesis.
Cultures were held under a 16 h photoperiod and
were evaluated 28 days after initiation.



-1 Number Number
Treatment (mg 1 Percent Number embryos/ embryo/
responsive embryos ovule responsive ovules produced cultured ovule


500.0 ME 7.9 (960) b 187 0.20 2.46

0.01 2.4-D/0.1 BA 7.3 (960) 130 0.14 1.86

0.01 2.4-D/0.1 DZ 6.8 (900) 113 0.13 1.85

MT 5.0 (100) 6 0.06 1.20


a Responsive ovules are those from which embryos were produced.
b Number in parentheses is the number of ovules evaluated.






54



48 plates supplemented with 2,4-D/BA and 27 of 40 plates supplemented with 2,4-D/DZ did contain active, proliferating embryogenic callus at 240 days.


Experiment 3: Growth and Habituation of and Embryo
Production by 'Hamlin' Orange Ernbryogenic Callus

The origin of 11 selected embryogenic callus lines and the fresh weight increase of these lines in grams and expressed as a percentage of initial explant weight is shown in Table 3.7. Callus lines initiated on MT medium supplemented with 1.0 mg 1-1 2,4-D/0.1 mg 1-1 BA displayed the greatest percentage increase in fresh weight. There was no clear pattern observed among the other callus lines for rate of proliferation.

Table 3.8 lists fresh weight increase and the number of cotyledonary embryos produced in callus sublines cultured on 2,4-D/DZ or DZ supplemented media. Although some differences were observed among sublines in the rate of fresh weight increase, the number of embryos produced by the different sublines varied little. Differences observed among lines initially for the magnitude of fresh weight increase (Table 3.7) did not persist through subsequent observations (Table 3.8).

Table 3.9 describes the result of a subjective visual examination of callus subline proliferation and embryo production on 3 different media. No further quantitative information was collected on callus proliferation. However,









Table 3.7. origin and fresh weight increase of 'Hamlin' orange embryogenic callus lines.
Cultures were subcultured following initiation on MT medium supplemented with
0.01 mg 1- 2,4-D/0.1 mg 1- DZ. Fresh weight increase was determined after
28 days.



Initiation medium Initial explant Fresh weight increase
Line No. 2,4-D/BA (mg 1- 1 weight (g) g


1 0.01/0.01 0.183 2.019 1103

2 0.01/0.01 0.194 1.908 984

3 0.01/0.01 0.352 2.384 677

4 0.01/0.01 0.171 0.643 376

5 0.01/0.01 0.143 1.080 755

6 0.01/0.01 0.248 1.095 441

7 0.01/0.1 0.401 2.218 553

8 0.01/0.1 0.278 2.774 998

9 0.01/0.1 0.417 2.248 539

10 1.0/0.1 0.017 0.941 5535

11 1.0/0.1 0.019 1.217 6405

wl
En






56



Table 3.8. Fresh weight increase and cotyledonary embryo
production by 'Hamlin' orange embryogenic callus
sublines. Callus was cultured on MT medium
supplemented with 0.01 mg 1 1 2,4-D/0.1 mg 1 1

DZ (A) or 0.1 mg 1- DZ alone (B). Cultures
were evaluated after 15 days.



Subline Initial explant Fresh weight increase No. embryos
weight g


Al 0.149 0.441 296 13
A2 0.060 0.501 850 12
A3 0.326 0.705 216 16
A4 0.084 0.631 751 14
A5 Lost to contamination
A6 0.226 0.742 328 13
A7 0.286 0.849 297 15
A8 0.140 0.537 384 14
A9 0.394 0.700 178 8
A10 0.129 0.956 741 12
All 0.147 0.827 563 16
Mean =13.30

Bl 0.216 0.828 383 15
B2 0.058 0.537 926 9
B3 0.125 0.732 586 11
B4 Lost to contamination
B5 0.159 1.105 695 11
B6 0.271 0.679 251 7
B7 0.239 1.218 510 15
B8 0.131 1.079 824 14
B9 Lost to contamination
B10 Lost to contamination
Bli 0.184 0.617 335 12
Mean =11.75









Table 3.9. Results of subjective visual examination of callus proliferation and embryo
production from 'Hamlin' orange embryogenic callus. Callus was cultured on
either MT Medium (C) or on the same medium amended with 0.01 mg 1-1
2,4-D/0.1 mg 1-1 DZ (A) or 0.1 mg 1- DZ (B). Cultures were evaluated 28 days
after subculturing.



Callus Embryo Callus Embryo Callus Embryo
proliferation production proliferation production proliferation production


Subline A B C

1 +a ++ .... + +
2 + + .... + +
3 ++ ++ ++ + ++ ++
4 ++b + .... ++ ++
5 .... ++ + ....
6 + + ++ + ....
7 + ++ ++ + ++ ++
8 + + .... ++ +
9 + + .... + +
10 ++ +++ .... ++ ++
11 + ++ ++ ++ ....

aIncreased numbers of pluses (+) indicated greater responses.

bDashes indicate sublines lost to contamination.




(-T






58



callus proliferation and embryo production were observed on all media, including MT, indicating hormone habituation.

Habituation of callus that was initiated and proliferated on 2,4-D-containing media was accomplished on MT. One habituated callus line (C-8) that was particularly prolific was used to regenerate a number of plants utilized in the studies described in Chapters IV and V. Growth and embryo production gradually decreased in a number of lines, and several lines were lost to contamination. A nondifferentiating, rapidly proliferating callus arose from a number of different embryogenic callus lines, including C-8. This callus has been used to initiate suspension cultures and has been converted back to an embryogenic form following extended periods (4-6 months) between subcultures.



Discussion

The objectives of the experiments described in this

chapter were to identify factors that influence the production of embryos and embryogenic callus from undeveloped Citrus ovules and to produce embryos from which plants could be regenerated for use in additional studies. Conclusions regarding embryo and callus production from such cultures can be drawn from the results. The most important component of primary embryo production was the percentage of cultured ovules that responded and produced embryos. The cultivars and treatments within cultivars with the greatest percentage of responsive ovules corresponded to the cultivars and






59


treatments that produced the greatest total number of embryos. The other component of embryo production was the number of embryos produced per responsive ovule. However, this factor was a minor contributing element to primary embryo production. For example, in Experiment 1, 'Marsh' grapefruit and 'Bearss' lemon produced fewer embryos because of a much lower percentage of responsive ovules, even though they produced the greatest numbers of embryos per responsive ovule.

Differences among cultivars for total number of primary embryos produced were observed in the first experiment. Embryo production ranged from high (oranges) to intermediate (tangelo, grapefruit and lime) to low (lemon) or none (satsuma). Likewise, differences for the most effective culture treatment for embryo production were observed among cultivars. The variable response of different Citrus species and cultivars has been observed previously (64,74). Citrus sinensis is the only species represented by more than one cultivar in this experiment. It is of interest that, although cultivar differences among the oranges in overall response were apparent, the most effective treatments were identical for all orange cultivars, regardless of the parameter considered. Moore observed intraspecific cultivar differences for overall response among other Citrus species other than C. sinensis (74).

The absence of light suppressed embryo and callus production from the orange ovules but stimulated these





60



phenomena in ovules from the other cultivars. As pointed out previously, 'Bearss' lemon produced embryos and callus only in the absence of light. Although a 16-hour photoperiod has been standard for Citrus embryogenic culture initiation (111), the results of this research suggest that light or its absence may play a critical role in the initiation of embryogenic cultures. However, continued subculturing in darkness became detrimental to embryo production and development and callus proliferation; cultures maintained in darkness produced fewer normal and more abnormal embryos, more and larger pseudobulbils, a watery non-differentiating callus growth, and nondifferentiating integumentary callus that interfered with embryogenic callus isolation. So, although dark culture enhanced initial embryo production, it was important to expose initiated cultures to light as soon as possible (no later than 1 month after culture initiation) to encourage normal embryo development and callus proliferation.

The stage of fruit development at which undeveloped

ovules were excised had a dramatic effect on the embryogenic response of 'Hamlin' orange. Fruit harvested 8 weeks after anthesis yielded ovules that were the least responsive of any of those cultured in the various experiments with this cultivar. The number of embryos produced per responsive ovule from such fruit was less than that of 12-week or 8-month ovules, as well. Spiegel-Roy and Vardi (111) have suggested that fruits harvested 4 weeks after anthesis are






61


optimal for embryo and callus production, depending on cultivar. Although no ovules of that age were cultured in this research, the results obtained suggested that more response may be expected from ovules taken from more mature fruit. In addition, 2,4-D inhibited embryogenesis in ovules from 8-week fruit, but not in ovules from 12-week fruit (Results: Experiment 2). Embryogenesis by 'Ponkan' mandarin (C. recticulata) nucellus cultures was inhibited by concentrations of 0.1 mg 1- or greater (119,120). However, the addition of 2,4-D to the culture medium was required for embryogenesis from C. limon ovules (111). In the research with 'Hamlin' orange, 2,4-D inhibition of embryo production was observed among 8-week ovules only. No requirement for 2,4-D was demonstrated by any of the cultivars used, including 'Bearss' lemon (C. limon) ovules.

Embryogenic callus production was also affected by the stage of fruit development at the time of ovule isolation, and by cultural conditions. Embryogenic callus was generated from cultured 12-week ovules on 2,4-D/BA or 2,4-D/DZ supplemented media (but not on MT or ME); from 5-month ovules on 2,4-D, 2,4-D/BA, or 2,4-D/DZ; and from 8-month ovules on all media. No embryogenic callus resulted from cultured 8-week 'Hamlin' ovules on any media. In contrast, Vardi et al. (131) reported that embryogenic callus arose in from 2 to 24% of the ovules from 7 Citrus cultivars that were cultured on ME supplemented medium 2-6 weeks after






62



anthesis. Moore (74) cultured undeveloped ovules from mature fruit of 17 Citrus cultivars on ME supplemented medium. Embryos and short-term embryo proliferation were produced by 15 of the cultivars, but no persistent embryogenic callus was obtained. The results of the experiments described in this chapter suggest that 2,4-D at low concentrations (0.01 mg 1-1 ) with or without BA or DZ (0.1 mg 1-1) enhance embryogenic callus production by undeveloped 'Hamlin' ovules compared with the standard ME-supplemented medium. Unfortunately, no conclusions may be drawn regarding media effect on long-term callus production by the other cultivars studied because of losses to contamination. However, embryogenic callus could be successfully initiated and maintained on media containing 2,4-D, in many cases, contrary to report of Citrus callus sensitivity to 2,4-D (111,120,121).



Conclusions

1. The most important component of total primary embryo

production was the percent of ovules that were responsive; the number of embryos produced per responsive

ovule was of secondary importance.

2. Differences among cultivars for total embryo production

and for most effective treatment for embryo production

or embryogenic callus initiation were observed.

3. The absence of light inhibited embryo and callus

production among orange ovules, stimulated those





63



responses among other cultivars, and proved essential

for 'Bearss' lemon response. Continued subculturing in

the absence of light was detrimental to further normal

development.

4. Stage of fruit development at the time of ovule isolation affected embryo and embryogenic callus production from undeveloped 'Hamlin' ovules. Generally,

ovules from more mature fruit were the most responsive. 5. Embryogenic callus was initiated with relative ease but

rarely persisted. The establishment of long-term 'Hamlin' embryogenic callus lines was enhanced by

2,4-D, but it was not always necessary. Callus

initiated and proliferated on media amended with growth

regulators became habituated to unamended medium.

6. Sufficient numbers of embryos were produced from ovule

and callus cultures for studies of plant regeneration

and phenotypic stability of regenerated plants.

















CHAPTER IV

EMBRYO DEVELOPMENT, GERMINATION, AND PLANT ESTABLISHMENT


Introduction

Although in vitro somatic embryogenesis has been observed in numerous plant genera, information on the survival of plants regenerated from embryogenic cultures has rarely been published. Citrus is unique among economically important woody perennial plants because, as discussed in the Literature Review (Chapter II) and demonstrated in Chapter III, many species of the genus are capable of in vivo and in vitro somatic embryogenesis. Numerous schemes have been proposed to utilize the embryogenic capability of Citrus in plant improvement programs. Additionally, the potential of mass in vitro propagation of Citrus has come under scrutiny in Florida because of a replant shortage following severe freezes and an outbreak of Citrus canker (Xanthomonas campestri pv. citri). Consequently, there has been interest in the efficiency of the regeneration process, in the degree of plant survival, and in the phenotypic stability of regenerated plants. The objectives of the research detailed in this chapter were to regenerate plants from the Citrus embryos produced in the studies described in



64





65



Chapter III, and to observe plant development from the embryonic stage to establishment in soil, paying particular attention to factors that limit the efficiency of the system. Phenotypic stability of regenerated plants will be explored in Chapter V.



Materials and Methods

Embryo production from cultured unfertilized ovules and embryogenic callus was described in Chapter III. When primary embryos had reached a sufficient size (5-10 mm long) and stage of development (possessing cotyledons and root and shoot primorida), they were transferred to MT medium supplemented with 1.0 mg 1- 1 GA 3 to induce germination. The number of embryos that exhibited either root or shoot growth and the number of germinated embryos was recorded 3-4 weeks after transfer to the germination medium. The criterion for germination was balanced elongation of both root and shoot structures. When root length reached 2.5-5.0 cm and some amount of shoot elongation had occurred, the germinated embryos were transferred to pots containing a commercial soil mix composed of sphagnum peat moss, horticultural grade vermiculite and perlite, composted pine bark, and washed granite sand. Humidity was maintained at a high level initially by covering transplants in pots with polyethylene bags. The bags were cut open every few days with increasing size cuts to result in a gradual decrease in humidity levels and to produce acclimated plants. Transplants were held in





66



the growth chamber initially, under the same conditions of temperature and light as the cultured embryos and were taken to the greenhouse after acclimation, usually within 4 weeks. Plants were fertilized with a standard water-soluble 20-2020 fertilizer 4 weeks after transfer to soil. Surviving plants were counted after 12 weeks. A second collection of embryos was taken from the 'Hamlin' ovule cultures (Chapter III, Experiment 1) 8 weeks after the primary embryo harvest; these were designated secondary embryos. Embryos originating from long-term 'Hamlin' embryogenic callus lines which had been maintained for over 18 months (Chapter III, Experiment 3) were isolated and used to compare germination and survival rates of embryos from long-term callus cultures with that of primary and secondary embryos.



Results

Germination (both root and shoot elongation) and root and shoot development by primary embryos of several Citrus cultivars is described in Table 4.1. A total of 733 primary embryos were placed on the germination medium. The number of embryos producing roots was 645 (88%), and 464 (63%) of the embryos produced shoots. The number of embryos that germinated was 419 (57%). There were 226 embryos that developed roots but not shoots; likewise there were 45 embryos that produced shoots but not roots.

The root development response of the cultivars ranged from 69% of 'Key' lime embryos to 97% (97 of 100) of 'Marsh'





67



Table 4.1. Germination and development of roots and shoots
from primary embryos of several Citrus cultivars
cultured on MT medium supplemented with 1.0 mg

11 GA 3.



Embryos: number (percentage)
Producing Producing Producing Cultivar Cultured roots shoots both


Hamnlin orange 174 149 (86) 101 (58) 84 (48)

Pell Navel orange 230 210 (91) 142 (62) 131 (57) Pineapple orange 96 86 (90) 62 (65) 57 (59)

Marsh grapefruit 100 97 (97) 80 (80) 78 (78)

Orlando tangelo 79 63 (80) 54 (68) 45 (57)

Key lime 45 31 (69) 21 (47) 20 (44)

Bearss lemon 9 9(100) 4 (44) 4 (44)


Total 733 645 (88) 464 (63) 419 (57)





68



grapefruit and 100% (9 of 9) of 'Bearss' lemon embryos. The shoot elongation response ranged from 44% (4 of 9) of 'Bearss' lemon embryos to 80% (80 of 100) of 'Marsh' grapefruit embryos. The percentage of embryo germination (both root and shoot development) ranged from 44% of 'Key' lime and 'Bearss' lemon embryos to 78% of 'Marsh' grapefruit embryos.

Embryo developmental abnormalities were observed, such as pluricotyly, multiple shoot meristem proliferation, embryo fusion, fasciation, and pseudobulbils. These abnormalities often inhibited the normal course of plant regeneration. For example, although 'Hamlin' orange produced 502 primary embryos (Table 3.1), only 174 (35%) of these embryos exhibited sufficiently normal development to warrant transfer to the germination medium (Tables 4.1, 4.2). Cultivar differences were evident for the percentage of embryos that were judged to be sufficiently normal. These values ranged from 15% for 'Pineapple' embryos to 53% for 'Bearss' lemon embryos (Table 4.2). The number and percentage of embryos that germinated when placed on GAsupplemented medium and the number and percentage of germinated embryos that survived transfer to soil and acclimatization to become established plants are also listed in Table 4.2. Germination has been discussed above. The percentage of germinated embryos that survived the transfer and acclimatization step of the regeneration process was 60% or greater for all cultivars except 'Pineapple' orange and









Table 4.2. Germination of embryos and survival of plants from embryogenic cultures of
several Citrus cultivars.


Embryos placed Germinated Established Regeneration
on GA 3 a embryos b plants c survAval
Genotype No. % No. % No. % %


Pell Navel orange 230 24 131 57 79 60 34
Pineapple orange 96 15 57 59 21 37 22
Marsh grapefruit 100 34 78 78 36 46 36
Orlando tangelo 79 41 45 57 27 60 34
Key lime 45 17 20 44 17 85 38
Bears lemon 9 53 4 44 3 75 33
Hamlin orange
Primary embryos 174 35 84 48 56 67 32
Secondary embryos 184 105 57 69 66 38
Callus-derived embryos 123 79 64 48 61 39

Totals
Primary embryos
(all genotypes) 733 26 419 57 239 57 33
Secondary and callusderived embryos
(Hamlin) 307 184 60 117 63 38

Grand total 1040 603 58 356 59 34

a (Number of embryos placed on GA 3 medium/total number of primary embryos) x 100.

b (Number of germinated embryos/number of embryos placed on GA 3 medium) x 100.

c (Number of established plants/number of germinated embryos) x 100.

d (Number of established plants/number of embryos placed on GA 3 medium) x 100. 110






70



'Marsh' grapefruit. The regeneration survival percentage (percentage of primary embryos placed on germination medium that developed into established plants) for each cultivar is shown in Table 4.2. The survival percentages for individual cultivars clustered about the 34% total survival percentage and ranged from 32-38%; 'Pineapple' orange was the exception with 22% survival.

The regeneration survival percentages of primary, secondary, and embryogenic callus-derived embryos of 'Hamlin' orange were compared in Table 4.2. The data suggest that there were no differences among the 3 groups. The difference between the survival percentages of primary embryos of all cultivars and 'Hamlin' secondary and callus derived embryos was small (33% vs 39%, respectively). on the whole, 1 of 3 embryos placed on the germination medium survived the regeneration process. overall, approximately 1 of 12 primary embryos produced originally from cultured ovules became an established plant (239 of 2870).

The initiation treatment used to produce embryos may have had some influence on plant survival (Table 4.3). Embryos produced from either ME/dark or 2,4-D/DZ treatments underwent successful regeneration and establishment with greater frequency than embryos arising from other treatments. No clear differences among treatments were observed for the percentages of primary embryos that were judged visually to be developmentally normal (and placed on





71



Table 4.3. Germination of embryos and survival of plants
from embryogenic cultures of several Citrus
cultivars by initial treatment. Embryos and
plants from all cultivars were included in each
initial treatment group.



Embryos
placed Germinated Established Regeneration
Initial on GA a embryos b plants c sr~a
treatment No. 4a No. % No. %c sv~


ME (light) 174 25 94 54 49 52 28

ME (dark) 146 25 87 60 62 71 43

2,4-D 143 26 84 59 37 44 26

2,4-D/BA 130 32 68 52 27 40 21

2,4-D/DZ 140 22 86 61 64 74 46


a (Number of embryos placed on GA 3medium/total number of primary embryos) x 100.
b (Number of germinated embryos/number of embryos placed on GA medium) x 100.

c(Number of established plants/number of germinated embryos) x 100.
d (Number of established plants/number of embryos placed on GA medium) x 100.






72



GA-supplemented medium) or that germinated. However, the percentage of germinated embryos that survived transfer to soil and acclimatization was greater for the ME/dark and 2,4-D/DZ than the other treatments. The values in Table 4.3 were generated by summing over all cultivars; however, either ME/dark or 2,4-D/DZ treatments produced the group of embryos with the greatest regeneration survival percentage for 5 or 6 cultivars (Table 4.4). The treatments most effective for embryo production from cultivars were frequently the treatments with the highest rate of regenerate survival (Table 3.1, 4.4).



Discussion

Several hundred embryos were produced by ovule cultures of most cultivars in Experiment 1, Chapter III. The number of surviving plants regenerated from these embryos was significantly lower, however. The first requirement for plant regeneration was the development of a normal, viable embryo. A number of developmental abnormalities were observed among the embryos produced from all cultivars and on all media. The presence of these abnormal embryos limited the total number of plants that could be produced. Although no data were presented, the relative proportion of abnormal embryos in 'Hamlin' callus or secondary proliferation appeared to be the same as that in 'Hamlin' primary populations. Culture age, therefore, did not appear to greatly affect normal embryo production or development.








Table 4.4. Effect of initial treatment on regeneration survival percentages of several
Citrus cultivars.



Regeneration survival percentage a

Initial treatment
Cultivar ME/Light ME/Dark 2,4-D 2,4-D/BA 2,4-D/DZ


Hamlin orange 32 48 9 15 43

Pell Navel orange 27 17 38 19 54

Pineapple orange 16 29 17 --b 57

Marsh grapefruit 75 40 29 35 40

Orlando tangelo 43 44 13 17 -Key lime 14 67 -- -- 20



a(Number established plants/number embryos placed on GA 3 medium) x 100. b Dashes indicate that no embryos were transferred to germination medium from these combinations.





74



Embryo development, from the microscopic, undifferentiated form to the macroscopic, cotyledonary stage was the first efficiency-limiting step of the Citrus plant regeneration system. Approximately 3 out of 4 embryos produced did not successfully complete the step.

The second requirement for successful plant regeneration was balanced germination of the embryo (i.e. proportional elongation of both root and shoot structures). Most of the embryos that were placed on the germination medium underwent normal root elongation, but overall, fewer of the embryos underwent normal shoot development. Some embryos exhibited root development only and rarely survived transfer to soil because their root systems degenerated before shoot elongation occurred. Likewise, there were embryos that exhibited shoot development but not root development, although the number in this category was lower than the former. Repeated subculturing on the germination medium did not induce shoot or root development, respectively, among the recalcitrant embryos. Histological studies of nongerminating embryos were not performed. Such studies might have determined whether the unresponsiveness of nongerminating embryos was related to anatomical abnormalities of meristematic regions, or whether other factors (perhaps suboptimal growth regulator components or concentrations, or genetic factors) were involved with the suppression of germination. Ultimately, the failure of embryos to successfully complete the second step of the regeneration process





75



(balanced germination) eliminated 43% of the embryos that had survived the first step.

The ability of germinated embryos to survive transfer

from the tissue culture environment to soil and the external environment was the third requirement for successful regeneration. Although many embryos germinated, the balance between root and shoot growth was not always favorable for plant survival. Transplants with normal roots but minimal shoot growth usually degenerated in soil before commencement of shoot elongation, presumably because of the inability of the shoot to support the energy demand for continued growth and development. Likewise, transplants with normal or excessive shoot development but minimal root growth rarely survived, possibly because the roots could not supply nutrients and water that were directly available to the shoots previously from the culture medium. In general, only those embryos that had balanced root and shoot growth survived the transfer to soil. once plants were actively growing in soil, successful acclimatization to lower humidity and greater light intensity was accomplished by gradual exposure to both. Greater plant mortality resulted from the transfer of germinated embryos to soil than from acclimatization. When plant death did occur in this phase of the regeneration process, it usually resulted from degeneration in the absence of growth after germination and transplanting or from soil-borne pathogens that attacked






76



inactive or weakly-growing plants, and not from adverse reactions to increased light and decreased humidity levels.

The data listed in Table 4.3 suggested that the initiation treatment used to produce embryos may have had some influence on plant survival. Specifically, embryos produced from the ME/dark or 2,4-D/DZ treatments had greater regeneration survival percentages than embryos from the other treatments. As pointed out in the Results (Chapter IV), the influence of initiation treatment on regenerate survival was not manifested through differences in embryo development and germination; rather, the greatest differences were observed at the transfer and acclimatization step. The mechanism by which initial treatment influences transplant survival percentage is unclear although it may be related to embryo production capacity because the treatments most effective for embryo production were generally the treatments with the highest rate of regenerate survival. The effect of initiation treatment on regeneration survival percentage was not cultivar specific; ME/dark- or 2,4-D/DZ-produced embryos had the greatest regeneration survival percentage for 5 of

6 cultivars.

Cultivar differences were observed for total embryo

production (Chapter III), the percentage of embryos developing normally, the percentage of embryos that underwent balanced germination, and the percentage of plants that survived transfer to soil and acclimatization. However, overall there were no great differences among cultivars for






77



regeneration survival percentage, with the exception of 'Pineapple' orange.

It might be expected that the regeneration survival percentage of secondary embryos or embryos from long-term callus cultures would be lower than that of primary embryos because of accumulated polyploidy or aneuploidy resulting from increased culture age. McCoy et al. (71) reported that the frequency of cytogenetically abnormal regenerated plants of Avena sativa increased with culture age. Long-term Citrus embryogenic callus has been reported to remain diploid, except when initiated on media containing 2,4-D (131). Some of the primary and secondary embryos and all of the long-term cultures that were used for plant regeneration studies were exposed to 2,4-D at some point during the culture process. However, no differences in survival rates were observed among the 3 groups of 'Hamnlin' orange embryos. As with embryo development, culture age had no major influence on regeneration survival percentage.

Kochba et al. (58) studied the effects of various

cultural manipulations on embryo germination and found that GA 3and adenine sulphate stimulated root initiation and development. Spiegel-Roy and Vardi (111) developed a procedure for embryo development and plant regeneration for Citrus that includes several transfers to solid or liquid media supplemented variously with sucrose, galactose, adenine, GA 3, and malt extract. After plantlets reach a certain size they are cultured on paper bridges in tubes for





78



further development prior to transfer to soil. Preliminary experiments in this laboratory indicated that GA-supplemented medium was sufficient to stimulate germination, and that plant survival was greater when germinated embryos were transferred more rapidly to soil. The use of the procedures described by Spiegel-Roy and Vardi might have increased the number of plants produced, but the methods actually used in this research were much less complicated and laborious. No comparison of methods was possible, however, because data on plant survival were not published in the cited reports.

Although embryogenic cultures of Citrus were established with relative ease and substantial numbers of embryos were produced by these cultures, the majority of the embryos developed abnormally and did not become established plants. Despite this fact, it was possible to produce a sufficient number of plants for the studies of phenotypic stability detailed in Chapter V. However, use of this regeneration system as a means of commercial propagation of Citrus seems impractical at this time. The greatest limitation thus far has been obtaining normal embryo development and balanced germination. Further research is needed in the area of appropriate physical and chemical manipulations of the system to increase the percentage of normally developed germinating embryos. Likewise, the procedures for transfer to soil and plant acclimatization may be improved, perhaps, by altering the soil mix, using fungicidal soil treatments, or using different methods for humidity control. The





79



alternative to pursuit of the suggested research objectives would be to increase the number of explains cultured to generate the necessary number of normal, viable embryos to produce the desired number of regenerated plants.



Conclusions

1. The most critical step of the plant regeneration

process was the development of viable, normal embryos.

Three of every 4 embryos initiated did not undergo

normal morphogenesis, and this was the greatest limitation to plant production. Other critical steps were

stimulation of balanced germination of embryos and

successful transfer from the in vitro environment to

the external environment.

2. One of every 12 embryos initially produced was successfully established as a regenerated plant. One of every 3 embryos placed on the germination medium survived the

regeneration process. This level of efficiency is too low for practical application of the system to commercial propagation of Citrus.

3. Plant regeneration from secondary embryos or from

embryos derived from long-term embryogenic callus of

'Hamlin' orange was as efficient as from primary

'Hamlin' embryos. Culture age did not have any influence on the efficiency of plant regeneration.

4. Large differences among cultivars were observed for

initial embryo production, percentage of embryos that





80



developed normally, percentage of embryos that

germinated, and the percentage of germinated embryos

that survived transfer to soil and acclimatization.

However, in overall regeneration survival percentage

(i.e. percentage of embryos placed on the germination

medium that became established plants), no cultivar

differences were observed, except for the less-responsive 'Pineapple' orange.

5. Embryos produced by ME/dark and 2,4-D/DZ initiation

treatments had the greatest regeneration survival percentages. No differences were observed among treatment groups for normal embryo development or

germination. The greatest difference among treatment

groups was observed for the percentage of germinated embryos that survived transplanting and acclimatization.

6. Future research should focus on the effects of manipulation of physical and chemical parameters of the

tissue culture system to increase the percentage of

responsive ovules, the percentage of embryos that

develop normally, and the percentage of germinating

embryos. Histological studies would be of use in

determining whether germination failure is related to

anatomical abnormalities. Experiments on soil pathogen

and humidity control, and soil mix components may lead

to development of a procedure that increases the





81



percentage of germinated embryos that survive transfer to soil and acclimatization.
















CHAPTER V

CHARACTERIZATION OF CITRUS PLANTS
REGENERATED FROM EMBRYOGENIC CULTURES


Introduction

The difficulties encountered when standard plant

breeding methods are employed for Citrus cultivar improvement were discussed in Chapter I, along with the need for alternative approaches to cultivar improvement. The reports of variant plant types arising among several genera after in vitro regeneration or propagation have been presented and discussed in Chapter II. The potential of somaclonal variation as an alternative method of Citrus germplasm enhancement has also been discussed, as has the need for information on stability of regenerated plants for genetic transformation research. The results of a series of studies, designed to characterize Citrus plants regenerated from embryogenic cultures and to determine if evidence of variability among regenerated plants exists, are recorded in this chapter.

The results of the studies performed on the regenerated plants were compared with those obtained from identical studies of a population of nucellar seedlings to determine the extent of variability among the products of in vivo and



82





83



in vitro enbryogenesis. Although relative uniformity of nucellar seedlings has been observed, variant forms have been reported among nucellar populations. For example, aneuploid (82) and polyploid (4,45) seedlings have been identified. Nucellar seedlings have arisen that differed from the parental type in tree growth habit, crop yield, or fruit characteristics (size, shape, color, or quality)

(32). Qualitative isozyme differences have been observed among plants of the same cultivar and among nucellar seedlings arising from the same seed (46). As a consequence of possible nucellar seedling variability and a desire to utilize the nucellar population as a control group in phenotypic stability studies, a substantial number of seedlings (100) was characterized in the same manner as the plants produced by tissue culture methods.

An attempt was made to determine the effect of various culture parameters on the frequency and degree of variation among regenerated plants. The groups of plants produced in the work described earlier had various histories. Specifically, plants were regenerated in the presence or absence of 2,4-D (a mutagenic agent), from primary embryos (direct embryogenesis), from secondary embryos or embryogenic callus-derived embryos (indirect embryogenesis). The influence of culture age and embryo source on phenotypic stability of regenerated Citrus plants was examined among the several plant populations available.





84



Cytogenetic stability of plants was evaluated by

mitotic chromosome counts made with root tips, to determine if any aneuploid or polyploid plants were produced. Electrophoretic evaluation of plants was undertaken to determine if any variation at the level of specific, chemically defined genetic loci could be detected. Finally, vegetative characteristics of regenerated plants were quantified and statistically analyzed to examine the stability of regenerates for gross morphological traits. The variability observed among populations of regenerated plants, for all characters, was compared with the variability of the nucellar seedling population.



Materials and Methods



General Remarks

The studies of phenotypic stability of regenerated

Citrus plants were limited to 'Hamlin' orange because of the availability of plants derived from primary and secondary embryos and from embryogenic callus that had been maintained for 18 months prior to embryo isolation and plant regeneration. The conditions of plant establishment were described in Chapter IV. Acclimated plants were placed in a shaded greenhouse for several weeks prior to placement in an unshaded greenhouse. Plants were transplanted into pots (15 cm diameter) in the soil mix described previously, and





85



maintained under standard practices of irrigation, fertilization, and pest control.

A total of 11 groups of plants were produced from embryogenic cultures for the studies described in this chapter. The origin and number of these plants are listed in Table 5.1. A control group of 100 nucellar seedlings was randomly selected from a population of 350 seedlings produced by G.A. Moore. 'Hamlin' orange flowers were emasculated and hand pollinated in March, 1983 with pollen of Poncirus trifoliata. Seeds were collected and planted in October, 1983. Sexual hybrids were eliminated on the basis of expression of the dominant trifoliate leaf trait; the remaining seedlings were presumed to be nucellar. The seedlings were grown in the unshaded greenhouse in the same soil mix as the other plants. Plants produced from embryogenic cultures were not evaluated electrophoretically until they had undergone at least one growth flush in the unshaded greenhouse. Morphological evaluations of all plants were delayed until at least 2 common growth flushes had occurred.



Cytogenetic Characterization of Regenerated Plants

Mitotic chromosome counts of 34 nucellar seedlings and 51 plants randomly selected from all tissue culture populations were made to assess the cytogenetic stability of regenerated 'Hamlin' orange plants. Root tips were harvested from actively growing plants between 0800 and 1000





86






Table 5.1. Origin and number of plants in groups used for
studies of phenotypic stability of regenerated
'Hamlin' orange plants.



Group designation Origin Number plants


Primary embryos 67

P1 ME/Light 25

P2 ME/Dark 16

P3 2,4-D 5

P4 2,4-D/BA 3

P5 2,4-D/DZ 18

Secondary embryos 60

Sl ME/Light 19

S2 ME/Dark 6

S3 2,4-D 9

S4 2,4-D/BA 13

S5 2,4-D/DZ 13

C Embryogenic callus 66

N Nucellar Seedlings 100





87



and placed in a pretreatment saturated solution of 8-hydroxyquinoline and maltose (4:1, approximately) for 4-5 hours. Tips were removed from the pretreatment and fixed for up to 7 days in a solution of 95% ethanol and glacial acetic acid (3:1). Long-term storage was in 70% ethanol at -150 C. Following hydrolysis in 5N HCl at room temperature for approximately 15 minutes, tips were rinsed with tap water and placed in Feulgen stain for 15 minutes (or until pink staining was visible). The root tips were then placed on glass slides with a drop of modified carbol fuchsin stain (51) and covered with glass cover slips. Careful application of heat and pressure yielded root tip preparations that allowed mitotic chromosome counts.



Electrophoretic Characterization of Regenerated Plants

The isozyme banding patterns of all 'Hamlin' orange

plants listed in Table 5.1 were evaluated for 11 different enzyme activity staining systems. Some of the plants regenerated from primary and secondary embryos of other cultivars were also examined electrophoretically (see Table 5.2). All 'Hamlin' orange plants were examined at least one time for each enzyme staining system. More frequently, several samples per plant were taken over time, and several zymograms for each stain were evaluated. Zymograms were evaluated for number and relative position of major bands.




Full Text
26
resembling nucellar embryos produced iri vivo. Examination
of individual cells showed them to possess characteristics
typical of meristematic cells (dense cytoplasm with numerous
mitochondria, lipid bodies, ribosomes, large nuclei with
conspicuous nucleoli, etc.). Proembryos originated from
single cells that were isolated by thick cell walls.
Internal divisions resulted in a multi-cellular embryo that
enlarged through the cell wall, which disintegrated.
Additionally, other cells within the proembryo were capable
of becoming isolated to undergo the same process of embryo
development. The callus tissue, in fact, was shown to be
composed almost entirely of proliferating proembryos,
embryos, and pseudobulbils. (The latter resulted when
proembryos developed an epidermal layer and represented an
abnormal alternative developmental pathway.) These studies
demonstrated the single cell origin of callus-derived
embryos.
The regeneration of Citrus plants via several other in
vitro methods has been reported. For example, plants have
been produced via adventive embryogenesis from callus
colonies derived from protoplasts that were isolated from
embryogenic callus of 'Shamouti' and other Citrus species
and cultivars (12,126,127,130). X-ray irradiation (126,130)
and the use of feeder cell layers (126,128) stimulated the
proliferation of and embryo production from protoplast-
derived callus colonies. Citrus plants have been produced


21
aurantifolia (Christm.) Swing., and C. maxima (Burm.)
Merrill on modified MS media supplemented with adenine
sulphate and GA, or Kn and IAA with or without GA. Embryo-
genesis occurred among explants from the 2 former species
(polyembryonic) but not the latter (monoembryonic). More
embryo production arose from ovary walls than placenta or
ovules. Unfertilized ovules (from fruit of unpollinated
flowers harvested after 120 days) were placed into culture;
these ovules also produced embryos and callus proliferation.
Button and Bornman (11), Kochba et al. (64), and Kochba and
Spiegel-Roy (59) have also reported embryogenesis in vitro
from unfertilized, undeveloped ovules of several Citrus
species. Starrantino and Russo (112) have cited the absence
of the dominant trifoliate leaf marker among plants regener
ated from cultured unfertilized ovules (from mature fruit of
flowers pollinated by Poncirus trifoliata (L.) Raf.) as
evidence of the nucellar origin of such plants. Species and
media used were different in the early works and may account
for the disparate results. However, it is clear that
pollination is not required for in vitro adventive embryo-
genesis from ovules of numerous Citrus species.
Sabharwal (99) was the first to point out that it was
not the ordinary cells of the nucellus that produce embryos,
pseudobulbils, or callus proliferation; rather, it was the
proembryo initials that were the source of _in vitro embryo
production or callus proliferation. Kobayashi et al.
(53,54) performed histological studies of developing ovules


148
103. Sauton, A., A Mouras, and A. Lutz. 1982. Plant
regeneration from citrus root meristems. J. Hort.
Sci., 57: 227-231.
104. Scandalios, J.G. 1969. Genetic control of multiple
molecular forms of enzyme in plants: A review.
Biochem. Genet. _3; 37-79.
105. Secor. G.A., and J.F. Shepard. 1981. Variability of
protoplast-derived potato clones. Crop Sci.,
21: 102-105.
106. Shepard, J.F., D. Bidney, and E. Shahin. 1980.
Potato protoplasts in crop improvement. Science,
28: 17-24.
107. Sibi, M., M. Biglary, and Y. Demarly. 1984. Increase
in the rate of recombinants in tomato (Lycopersicon
esculentum L.) after in vitro regeneration. Theor.
Appl. Genet., 68: 317-321.
108. Soost, R.K., and J.W. Cameron. 1975. Citrus. In J.
Janick and J.N. Moore (eds.), Advances in Fruit
Breeding, Purdue University Press, West Lafayette,
Indiana, pp. 507-540.
109. Soost, R.K., T.E. Williams, and A.M. Torres. 1980.
Identification of nucellar and zygotic seedlings of
Citrus with leaf isozymes. HortScience, 15: 728-729.
110. Spiegel-Roy, P., and J. Kochba. 1973. Stimulation of
differentiation in orange (Citrus sinensis) ovular
callus in relation to irradiation of the media. Rad.
Bot. 1_3 : 97-103.
111. Spiegel-Roy, P., and A. Vardi. 1984. Citrus. In
P.V. Ammirato, D.A. Evans, W.R. Sharp, Y. Yamada
(eds.), Handbook of Plant Cell Culture, Vol. 3,
Macmillan, New York, N.Y., pp. 355-372.
112. Starrantino, A., and F. Russo. 1980. Seedlings from
undeveloped ovules of ripe fruits of polyembryonic
citrus cultivars. HortScience, 15: 296-297.
113. Sun, Z., C. Zhao, K. Zheng, X. Qi, and Y. Fu. 1983.
Somaclonal genetics of rice, Oryza sativa L. Theor.
Appl. Genet., 67: 67-73.
Sunderland, N. 1977. Nuclear cytology. In H.E.
Street (ed.), Plant Cell and Tissue Culture, Vol. 2,
Univ. of Calif"! Press, Berkeley, Calif., pp. 177-206.
114.


level of chromosomal aberrancy among regenerated alfalfa
plants. Culture age at the time of plant regeneration
influenced chromosomal variability among cultured Pisum
18
sativum L. (124) and oats (71); increased time in culture
correlated with increased frequency of aberrations. In
contrast, the frequency of chromosome number changes in
alfalfa (49) and maize (23) regenerates was not increased
following increased culture age.
It has been suggested that the variation observed may
be influenced by the pathway of regeneration (i.e., direct
organogenesis, organogenesis through a callus intermediary,
embryogenesis, or protoplast isolation and subsequent whole
plant regeneration via organogenesis or embryogenesis). In
a sense, this parameter is reflective of explant source
because the explant used usually determines the regeneration
method employed. Nonetheless, examples of somaclonal
variation have been described among plants regenerated by
each of these procedures (9,24,29,47,80,89,95,101,105,107,
119). The effect of differences of any of the culture
parameters on the degree of variation obtained cannot be
generalized; their relative importance and influence will
depend ultimately on the specific plant material being
cultured.
In conclusion, it has been shown that somaclonal
variation occurs among diploid and polyploid species, and
among seed- and vegetatively-propagated plants. Variants
have been detected among primary regenerates, and more


88
Table 5.2. Number of Citrus plants regenerated from primary
and secondary embryos of several cultivars that
were evaluated for isozyme banding patterns, and
total number of zymograms evaluated.
Cultivar
No. plants
No. Zymograms
Pell Navel orange
50
289
Pineapple orange
2
16
Marsh grapefruit
29
161
Key lime
66
428
Bearss lemon
7
34
Total
154
928


24
be obtained by appropriate manipulation of the culture
medium. Low levels of adenine or ME stimulated embryo
development at the expense of callus proliferation. Callus
growth was promoted or inhibited, depending on relative
kinetin/IAA ratios. Eventually callus growth and embryo
production decreased, despite auxin and cytokinin supple
mentation. However, when transferred to basal MT, the
callus proliferated and produced numerous embryos, indicat
ing phytohormone habituation of the callus (60). With
continued time in culture, the embryogenic capacity of the
callus was diminished again (57).
A series of experiments were undertaken to develop
methods that would promote additional proliferation of
callus and embryos. The 'Shamouti' callus had been sub
cultured routinely at 4-5 wk intervals. By extending the
interval between subculturing, the embryogenic response of
the callus was enhanced (57). Shorter culture intervals
favored callus proliferation, and longer intervals promoted
embryo formation. Transferring callus to a sucrose medium
after a cycle of sucrose deprivation produced a response
similar to the effect of increased subculture intervals, but
of a lesser degree. The positive effect of sucrose depriva
tion diminished in subsequent culture cycles, but the effect
of extended intervals persisted for several cycles. Other
factors in addition to sucrose deprivation were implicated,
specifically, hormone level alterations. When non-habit-
uated callus was exposed to gamma irradiation, callus


35
with masking tape following inoculation or culture transfer.
All cultures were maintained at 27 C with 16 h fluorescent
-1 -2
light (76 ymol s m ) except were otherwise noted.
Experiment 1: Production of Embryos and Embryogenic Callus
from Undeveloped Ovules of Several Citrus Species
Ovules were isolated from fruit harvested in October,
1983, approximately 8 months after anthesis, from trees of
the following species and cultivars: Citrus sinensis cvs.
Hamlin, Pell Navel, Pineapple; C. paradisi Macf. cv. Marsh;
C. reticulata cv. Owari; C. paradisi x C. reticulata cv.
Orlando; C. aurantifolia cv. Key; and C. limn (L.) Burm.f.
cv. Bearss. Following disinfestation, the ovules were
placed on MT medium amended with either ME (500.0 mg 1 or
2,4-D (0.01 mg 1 alone or the same concentration of 2,4-D
in combination with BA (0.1 mg 1 *) or DZ (0.1 mg l-1).
Media amendments and concentrations were selected because
embryogenic cultures from several Citrus types had pre
viously been obtained in this laboratory and others using
these materials. Six Petri dishes with 20 ovules each were
used for each cultivar x treatment combination. All cul
tures were maintained under a 16 h photoperiod, except for
an additional treatment group for each cultivar on ME medium
that was maintained in the dark. All cultures were subcul
tured on the treatment media every 4-8 weeks.


92
malic acid (pHed to 7.0 with NaOH), 10 ml 1.0 M Tris
HC1 (pH 8.0), and 70 ml H2O;
11) PER = peroxidase (Donor:hydrogen peroxide oxidore-
ductase, E.C.1.11.1.7) 250 mg p-phenylenediamine,
50 mg MnSO^, 5 ml 1.0 M sodium acetate (pHed to 4.7
with glacial acetic acid), 30 ml 95% ethanol, 65 ml
H2<0, and 0.5 ml 30% H202.
Enzyme systems have been listed according to buffer system
used, in Table 5.4. The number of loci and major bands
visualized from 'Hamlin' orange leaf extracts and the
genotype of 'Hamlin' at these loci are listed in Table 5.4
also. LAP was visualized with greater clarity when the TC
buffer system was used rather than H. The greatest clarity
of SDH zymograms was achieved when the TB buffer system was
used, so reruns of plant materials for PER, GOT, or G6PD
stains occasionally were performed with TB to allow better
resolution of SDH zymograms. The 11 enzyme activity stain
systems selected for this work were chosen on the basis of
demonstrated zymogram stability among individual plants of
various ages from each of several Citrus cultivars (Moore,
unpublished.
Morphological Characterization of Regenerated Plants
Obvious differences among regenerated plants for gross
plant morphology were observed. Four specific plant charac
ters were measured in November and December, 1984 for
statistical evaluation of these differences. The objectives


CHAPTER III
EMBRYO PRODUCTION AND ESTABLISHMENT OF EMBRYOGENIC
CALLUS CULTURES FROM UNDEVELOPED CITRUS OVULES
Introduction
The first step in the establishment of populations of
regenerated plants was the production of embryos either via
direct embryogenesis from cultured undeveloped ovules or
from embryogenic callus. This chapter describes experiments
designed to study the effect of various factors (growth
regulators, light, stage of fruit development at the time of
ovule extraction, species or cultivar differences) on direct
embryo production and on the initiation and establishment of
embryogenic callus. Embryo production, growth rate, and
development of hormone habituation in long-term embryogenic
callus cultures were also examined. Embryos produced in
some of these experiments were used in the studies of embryo
development, plant establishment, and phenotypic stability
described in Chapters IV and V.
33


2
However, the greatest impediment to genetic improvement
of Citrus cultivars using standard breeding methods has been
the unusual reproductive biology of the genus. Most Citrus
species produce seeds that are polyembryonic, resulting from
the proliferation of somatic embryos of nucellar origin
within the embryo sac (33,34). In polyembryonic types,
certain cells characterized by a large nucleolus and
densely-staining cytoplasm are found in the nucellus of
ovules at anthesis; such cells are not present in the
nucellus of monoembryonic types (53). These cells are
stimulated to divide and differentiate into somatic embryos
about the time that the zygotic embryo begins development
(50-70 days after pollination) (21,27,53). The nucellar
embryos compete with the zygotic embryo for nutrients and
space, and frequently, the zygotic embryo perishes (33).
Polyembryony is dominant to monoembryony in Citrus, with
possibly more than a single gene involved (16,17,87). The
end result of polyembryony from the perspective of cultivar
improvement is that most seedlings produced by controlled
pollination are genetically identical to the maternal
parent, and sufficient numbers of sexual progeny are not
produced to allow for selection and subsequent genetic
improvement. Additionally, unless the morphological char
acteristics of the parents are considerably different, the
sexual and nucellar seedlings produced are visually indis
tinguishable from each other, limiting further the effi
ciency of cultivar improvement programs (38,108).


62
anthesis. Moore (74) cultured undeveloped ovules from
mature fruit of 17 Citrus cultivars on ME supplemented
medium. Embryos and short-term embryo proliferation were
produced by 15 of the cultivars, but no persistent embryo-
genic callus was obtained. The results of the experiments
described in this chapter suggest that 2,4-D at low concen
trations (0.01 mg 1 l) with or without BA or DZ (0.1 mg 1
enhance embryogenic callus production by undeveloped
'Hamlin' ovules compared with the standard ME-supplemented
medium. Unfortunately, no conclusions may be drawn regard
ing media effect on long-term callus production by the other
cultivars studied because of losses to contamination.
However, embryogenic callus could be successfully initiated
and maintained on media containing 2,4-D, in many cases,
contrary to report of Citrus callus sensitivity to 2,4-D
(111,120,121).
Conclusions
1. The most important component of total primary embryo
production was the percent of ovules that were respon
sive; the number of embryos produced per responsive
ovule was of secondary importance.
2. Differences among cultivars for total embryo production
and for most effective treatment for embryo production
or embryogenic callus initiation were observed.
The absence of light inhibited embryo and callus
production among orange ovules, stimulated those
3.


28
relatives. As a consequence of the need to distinguish
zygotic seedlings and the absence of genetic markers,
numerous alternative approaches to plant characterization
have been attempted. Cotyledon color of seed-borne embryos
has been used to a limited extent, as has insecticide
sensitivity (dimethoate) (21). Infrared spectroscopy of
leaf oils has been used, also, but the time required to
analyze hundreds of samples is prohibitive; furthermore,
leaf oil spectrographs change with increased plant age (88).
Tatum et al. (117) used TLC (thin layer chromatography) of
flavonoid and other non-volatile components to distinguish
nucellar and zygotic seedlings; again, only limited sample
numbers can be handled with this technique. Browning or
non-browning of crude shoot homogenates has been shown to be
a taxon-specific trait conditioned by a single dominant gene
(25,26,28). No variation for this character has been
observed within individual Citrus species. The procedure is
quick, simple, and inexpensive, and can be used with appro
priate specific parental combinations to distinguish zygotic
from nucellar progeny.
Vegetative characters of seedling Citrus plants have
been used to identify hybrid progeny (4,38,118). Leaf shape
has been found to be among the most useful vegetative
characters for zygotic seedling identification (38) or for
identifying tetraploid progeny (4). Leaf shapes frequently
have been expressed as a length/width ratio (38,118). Leaf
blade and petiole size and shape vary within species and may


11
reports have been published supporting both of these views,
the bulk of the evidence accumulated thus far supports the
latter conclusion. For example, when Thomas et al. (119)
regenerated potato plants from protoplast-derived callus
colonies, they found that different plant types arose from
the same individual callus colony. If the recovered
variation was pre-existent in explant tissue, then individ
ual calli (originating presumably from single isolated
protoplasts) should have produced uniformly normal or mutant
plants. Because more than one plant type was produced from
an individual callus, they concluded that the mutations that
resulted in phenotypic variability must have occurred during
the tissue culture process itself. Prat (89) likewise
observed different mutations in regenerated N. sylvestris
plants arising from an individual protoplast-derived callus;
furthermore, normal and mutant types were observed among
plants from an individual callus. Engler and Grogan (24)
found that most mutant lettuce phenotypes came from
protoplast-derived calli that produced phenotypically normal
sister clones. Edallo et al. (23) found mutant maize
phenotypes arising from a single initial callus. Although
the above studies support the de novo origin of phenotypic
variability, they do not conclusively disprove the idea that
somaclonal variation results from pre-existent genetic
heterogeneity. It is conceivable, for example, that the
calli from which the variable plant types above were pro
duced were heterogeneous, perhaps originating from more than


102
resulted in the examination of a minimum of 14 and a possi
ble maximum of 15-20 genetic loci from among the thousands
of loci making up the Citrus genome. The probability of
mutations occurring in this small fraction of the total
genomic complement of DNA was very low. Electrophoretic
evaluation, therefore, afforded a narrow window through
which changes of the nuclear genome could be studied. It
would have been considered good evidence of gene mutation
had isozyme variants been found. The stability of electro
phoretic profiles was significant, but it was not taken to
imply that mutations had not occurred elsewhere in the
genome. Further, mutations may have occurred at these loci
that did not result in visible electrophoretic variation.
For example, it is possible to change a non-critical nucleo
tide in a codon without changing the corresponding amino
acid, or to change an amino acid without altering protein
charge or conformation (and thus mobility). More fre
quently, though, such changes would be detectable. Ulti
mately, however, the newly developing techniques for using
restriction fragment length polymorphisms as genetic markers
may provide a more effective way to screen for mutations at
many more loci and to characterize large numbers of plants
at the molecular level than does gel electrophoresis
(31,97,116).
The electrophoretic evaluation of plants was of value
in this study because the uniformity of isozyme profiles



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19
frequently, among sexual progeny populations produced by
primary regenerates. Variation may be associated with gross
chromosomal changes (number or structure), and variants may
possess heritable genetic mutations of nuclear or cyto
plasmic genes. The event that results in alteration of the
genome may occur in culture (de novo), but some instances of
variation pre-existent in explant sources have been
reported, as well. The level of variability obtained may be
influenced by the genetic background of the plant material
cultured or by various manipulations of tissue culture
parameters. Valuable (economic and genetic) mutations have
been obtained from somaclones of several plant genera.
Citrus Tissue Culture
The first reports of the use of reproductive structures
of Citrus plants as explant sources for in vitro culture
experiments were those of Maheshwari and Ranga Swamy
(70,94). They attempted to initiate cultures with whole
ovaries and ovules from immature flower buds, as well as
with fertilized ovules, nucelli excised from fertilized
ovules, and adventive nucellar embryos from Citrus
microcarpa Bunge fruit harvested after pollination. Several
modifications of White's medium were used in their work.
Ovaries and unfertilized ovules were unresponsive, but
seedling plants were produced by fertilized ovules cultured
on the basal medium devoid of any supplementation. Plants
were produced when adventive embryos, in various stages of


86
Table 5.1. Origin and number of plants in groups used for
studies of phenotypic stability of regenerated
'Hamlin' orange plants.
Group designation
Origin
Number plants
Primary embryos
67
PI
ME/Light
25
P2
ME/Dark
16
P3
2,4-D
5
P4
2,4-D/BA
3
P5
2,4-D/DZ
18
Secondary embryos
60
SI
ME/Light
19
S2
ME/Dark
6
S3
2,4-D
9
S4
2,4-D/BA
13
S5
2,4-D/DZ
13
C
Embryogenic callus
66
N
Nucellar Seedlings
100


78
further development prior to transfer to soil. Preliminary
experiments in this laboratory indicated that GA-supple-
mented medium was sufficient to stimulate germination, and
that plant survival was greater when germinated embryos were
transferred more rapidly to soil. The use of the procedures
described by Spiegel-Roy and Vardi might have increased the
number of plants produced, but the methods actually used in
this research were much less complicated and laborious. No
comparison of methods was possible, however, because data on
plant survival were not published in the cited reports.
Although embryogenic cultures of Citrus were estab
lished with relative ease and substantial numbers of embryos
were produced by these cultures, the majority of the embryos
developed abnormally and did not become established plants.
Despite this fact, it was possible to produce a sufficient
number of plants for the studies of phenotypic stability
detailed in Chapter V. However, use of this regeneration
system as a means of commercial propagation of Citrus seems
impractical at this time. The greatest limitation thus far
has been obtaining normal embryo development and balanced
germination. Further research is needed in the area of
appropriate physical and chemical manipulations of the
system to increase the percentage of normally developed
germinating embryos. Likewise, the procedures for transfer
to soil and plant acclimatization may be improved, perhaps,
by altering the soil mix, using fungicidal soil treatments,
or using different methods for humidity control. The


Table 4.4. Effect of initial treatment on regeneration survival percentages of several
Citrus cultivars.
Regeneration survival percentage3
Initial treatment
Cultivar
ME/Light
ME/Dark
2,4-D
2,4-D/BA
2,4-D/DZ
Hamlin orange
32
48
9
15
43
Pell Navel orange
27
17
38
19
54
Pineapple orange
16
29
17
__b
57
Marsh grapefruit
75
40
29
35
40
Orlando tngelo
43
44
13
17

Key lime
14
67


20
a(Number established
plants/number
embryos placed
on GA^ medium)
x 100.
^Dashes indicate that
combinations.
no embryos were transferred
to germination
medium from
these


23
monoembryonic Citrus types has been reported. Rangan et al.
(92,93) were able to induce adventive embryogenesis among
nucelli (120 days after pollination) of 3 different mono-
embryonic types cultured on MS medium supplemented with
either ME, or NAA, adenine sulphate, and orange juice.
Embryos arose directly from the cultured nucelli, but no
proliferation of callus or pseudobulbils was observed.
Juarez et al. (50) reported the production of embryos and
short-term embryogenic callus proliferation arising from
monoembryonic 'Clementine' (C. Clementina Hort ex. Tan.)
ovules cultured as per Rangan et al. (92). Although in
vitro adventive embryogenesis by monoembryonic Citrus
nucelli has been demonstrated, the initial cells responsible
for embryogenesis or callus proliferation have not been
identified.
Ovules from seedless 'Shamouti' orange fruit (C.
sinensis) harvested 1 to 6 weeks after anthesis were used by
Kochba et al. (64) to initiate embryogenic callus cultures.
The callus lines produced from those cultures have been the
subject of extensive studies of the factors that influence
the expression of embryogenic capability. Small callus
colonies were initiated on MT media (78) supplemented with
ME. These colonies were transferred to MT supplemented with
IAA and kinetin, which promoted vigorous callus prolifera
tion (59). It was demonstrated that the morphogenetic
pattern desired (callus growth or embryo development) could


Table 3.9. Results of subjective visual examination of callus proliferation and embryo
production from 'Hamlin' orange embryogenic callus. Callus was cultured on
either MT Medium (C) or on the same medium amended with 0.01 mg I-*-
-1 -1
2,4-D/0.1 mg 1 DZ (A) or 0.1 mg 1 DZ (B). Cultures were evaluated 28 days
after subculturing.
Callus
proliferation
Embryo
production
Callus
proliferation
Embryo
production
Callus
proliferation
Embryo
production
Subline A
B
C
1
+a
++
__ __
_ _
+
+
2
+
+
--

+
+
3
++
++
++
+
++
++
4
++b
+


++
++
5

++
+


6
+
+
++
+


7
+
++
++
+
++
++
8
+
+


++
+
9
+
+


+
+
10
++
+++


++
++
11
+
++
++
++

aIncreased numbers of pluses (+) indicated greater responses.
^Dashes indicate sublines lost to contamination.
Ln


CHAPTER II
LITERATURE REVIEW
Introduction
The objective of this review of the literature is to
examine 3 different areas of plant science that are inter
related in the original research described in this disserta
tion. First of all, some of the evidence that has
accumulated documenting somaclonal variation will be pre
sented, with emphasis placed on factors indirectly associ
ated with the expression of variability among regenerated
plants. Secondly, research in the area of Citrus tissue
culture (particularly the phenomenon of iri vitro embryo-
genesis) will be described and discussed; the emphasis here
will be not only on the methods and mechanics of tissue
culture, but also on the suitability of Citrus plant mate
rial for studies of somaclonal variation. Furthermore, the
potential relevance of somaclonal variation to Citrus from
the perspective of cultivar improvement and plant propa
gation will be discussed. Finally, a brief review of
methods used to characterize Citrus plants will be pre
sented, with an emphasis on electrophoresis and morpho
logical evaluations.
6




98
Navarro et al. (80) reported that all aberrant plants from
monoembryonic 'Clementine' nucellar cultures were diploid.
In this study, diploid chromosome counts (2n=2x=18)
were made for 30 of 34 nucellar seedlings and 45 of
51 'Hamlin' orange plants produced by tissue culture. Poor
preparation or absence of mitotic figures prevented
chromosome counts from being made on the other 10 plants
studied. No aneuploid or polyploid plants were found among
either nucellar seedlings or tissue culture plants. The
number of plants characterized was too low and the propor
tions of the populations studied were not large enough to
state that chromosome number stability in all populations
was absolute. Differences of plant morphology were observed
among plants that had been determined to be diploid, so
these differences were not the result of changes in chromo
some number. The evidence gathered thus far suggests that
regenerated 'Hamlin' orange plants (from the various culture
regimen and embryo sources) maintained the diploid number as
did 'Hamlin' nucellar seedlings, and that there was no rela
tionship between observed morphological aberrancy and
chromosome number. The tenative nature of these conclusions
and the need for more extensive cytogenetic evaluation of
the individual plant groups must be emphasized.
Recommendation for such additional studies include
complete mitotic characterization of all 'Hamlin' plants
produced in this project. Also, chromosome counts of
embryogenic callus cells and embryos in various stages of


LIST OF FIGURES
Figure Page
3.1. Proliferation of cotyledonary embryos and
proembryos from a cultured ovule 41
3.2. Initation of embryogenic callus from cultured
ovules 4 3
5.1. Abnormal plant type (thin pointed leaves, short
internodes, low vigor) observed among regen
erated Citrus plants 113
5.2. Example of low and normal vigor among regen
erated 'Hamlin' plants of the same age and
from the same treatment 115
5.3. Example of normal and abnormal 'Hamlin'
seedling leaf blade and petiole shape 117
IX


Table 3.3
. Effect of cultivar and culture conditions on the efficiency of embryo produc
tion by undeveloped ovules from Citrus fruit harvested 8 months after anthesi
Embryos were counted 56 days after initiation.
Treatment (mg 1 ^)a
Cultivar 500.0 ME 500.0 ME 0.01 2,4-D 0.01 2,4-D/0.1 BA 0.01 2,4-D/0.1 DZ
Light No light Light Light Light
Embryos/ Embryos/ Embryos/ Embryos/ Embryos/ Embryos/ Embryos/ Embryos/ Embryos/ Embryos/
ovule
cultured
responsive
ovule
ovule
cultured
responsive
ovule
ovule
cultured
responsive
ovule
ovule
cultured
responsive
ovule
ovule
cultured
responsive
ovule
Hamlin orange
1.21
2.59
0.78
2.19
0.81
2.16
0.29
1.89
1.10
2.64
Pell Navel orange
2.09
2.97
1.11
2.63
1.43
2.87
1.29
2.90
2.15
2.97
Pineapple orange
1.40
2.63
0.82
1.88
0.98
1.82
0.83
1.89
1.23
2.47
Orlando tngelo
0.46
2.29
0.58
2.09
0.23
1.92
0.14
2.13
0.24
2.64
Marsh grapefruit
0.23
3.38
0.45
5.40
0.96
5.25
0.67
4.71
0.16
3.17
Owari satsuma
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Key lime
0.43
3.25
0.94
3.32
0.23
2.15
0.13
1.88
0.44
3.12
Bearss lemon
0.00
0.00
0.14
4.25
0.00
0.00
0.00
0.00
0.00
0.00
aEach cultivar x
treatment
combination
used 120
ovules except Orlando (2,4-D =
100 ovules).


122
mean was not found to be different from the ME or callus
group means, but the mean of the callus group was determined
to be greater than that of the ME group (because of pairwise
comparisons). To summarize, embryo source had a significant
effect on group mean differences for petiole L/W ratios, but
the different growth regulator components in the initiation
media did not.
The tissue culture groups had greater petiole L/W
ratios than the nucellar population, so it was not surpris
ing to note that several individual plants from tissue
culture had values that exceeded the maximum number observed
in the nucellar population. However, 2 plants (Pl-06,
Pl-20) from the ME or primary embryo group were noted to
have values less than the minimum observed in the nucellar
population. Conclusions regarding variability of groups for
petiole ratios were not made on the basis of minimum-maximum
comparisons. However, the range of values for nucellar
seedlings was exceeded by unique individual plants from at
least 2 groups (ME or primary embryos). More plants were
found with values greater than the mean than less. It must
be noted that not all leaves on any statistically extreme
plant were of extreme shape. In fact, a range of petiole
shapes was observed on each plant, but generally, those
leaves selected for measurement from stem mid-regions were
typical of the shape observed on the majority of leaves on
any particular plant.


79
alternative to pursuit of the suggested research objectives
would be to increase the number of explants cultured to
generate the necessary number of normal, viable embryos to
produce the desired number of regenerated plants.
Conclusions
1. The most critical step of the plant regeneration
process was the development of viable, normal embryos.
Three of every 4 embryos initiated did not undergo
normal morphogenesis, and this was the greatest limita
tion to plant production. Other critical steps were
stimulation of balanced germination of embryos and
successful transfer from the in vitro environment to
the external environment.
2. One of every 12 embryos initially produced was success
fully established as a regenerated plant. One of every
3 embryos placed on the germination medium survived the
regeneration process. This level of efficiency is too
low for practical application of the system to commer
cial propogation of Citrus.
3. Plant regeneration from secondary embryos or from
embryos derived from long-term embryogenic callus of
'Hamlin' orange was as efficient as from primary
'Hamlin' embryos. Culture age did not have any influ
ence on the efficiency of plant regeneration.
Large differences among cultivars were observed for
initial embryo production, percentage of embryos that
4.


43


PCNE:
PMS:
TB:
TC:
Tris:
2,4-D:
primary cell of the nucellar embryo,
phenazine methosulfate.
Tris-borate buffer.
Tris-citrate buffer.
Tris-(hydroxymethyl)amino methane.
(2,4-dichlorophenoxy)acetic acid.
xi


30
plants in uniform populations, the actual variant phenotypes
have not been correlated with any traits of horticultural or
economic significance. These techniques, therefore, only
serve as a screen for variants of specific types. Horti-
culturally valuable variation must await plant maturity and
fruit production to be detected.
Conclusion
Phenotypic instability has been reported among tissue
culture regenerated plants of several different genera.
Reports of polyembryonic Citrus plant regeneration have
indicated relative uniformity among individuals; however,
somaclonal variants have arisen from embryogenic cultures of
monoembryonic Citrus. It is possible that a more detailed
evaluation of polyembryonic Citrus regenerates might have
detected variant plant phenotypes. Phenotypic instability
may be a problem for plant propagation. Likewise, it may
obscure the results of attempted genetic transformations of
Citrus via molecular techniques. Good estimates of the
background level of variation among non-transformed regener
ate populations are necessary to assess accurately the
consequences of molecular manipulation of the genome among
transformed, regenerate plants. But, somaclonal variation
may prove to be a potential source of genetic variation
useful for genetic studies or cultivar improvement. For
example, a clone superior for most economically important
characters may be improved if, perhaps, a disease-resistant


104
relative vigor (growth rate expressed as cm mo "S mean
internode length, and leaf petiole and blade shapes
(expressed as L/W ratios). Groups means (x), variances
2
(s ), and coefficients of variation (C.V.) were calculated
to assess and to compare the amount of variability observed
visually in individual groups for these 4 vegetative char
acteristics. These statistics have been summarized in
Table 5.5. Comparison of C.V.'s (which are relative mea
sures of variability within groups) indicated that the
greatest level of variation was for growth rate. The leaf
L/W ratio was generally the least variable of the character
istics studied. The mean and variance values listed in
Table 5.5 were used to perform t-tests (to compare means)
and F-tests (to compare variances), the results of which
have been listed in Table 5.6 (growth rate), Table 5.7 (mean
internode length), Table 5.8 (petiole L/W ratio), and Table
5.9 (blade L/W ratio). The result of these analyses are
discussed for each trait below.
In each of the plant populations, and for each char
acter, no discrete categories were apparent; rather, the
values of plants in each group were continuously distrib
uted, so deciding which plants were not normal was not
always a clear choice. It was decided, therefore, to
describe the variation observed in each group of plants by
comparison with variation observed in the nucellar seedling
population, rather than to select arbitrary values to
delineate the normal and aberrant regions of the range of


147
91. Raj Bhansali, R., and H.C. Arya. 1979. Organogenesis
in Citrus limetiodes Tanaka (sweet lime) callus
culture. Phytomorph., 28: 97-100.
92. Rangan, T.S., T. Murashige, and W.P. Bitters. 1968.
In vitro initiation of nucellar embryos in mono-
embryonic Citrus. HortScience, 3^: 226-227.
93. Rangan, T.S., T. Murashige, and W.P. Bitters. 1969.
In vitro studies of zygotic and nucellar embryogenesis
in citrus. Proc. First Int. Citrus Symp.,
1: 225-229.
94. Ranga Swamy, N.S. 1961. Experimental studies on
female reproductive structures of Citrus microcarpa
Bunge. Phytomorph., 11: 109-127.
95. Reisch, B., and E.T. Bingham. 1981. Plants from
ethionine-resistant alfalfa tissue cultures: Varia
tion in growth and morphological characteristics.
Crop Sci., 21: 783-788.
96. Reisch, B., 1983. Genetic variability in regenerated
plants. In D.A. Evans, W.R. Sharp, P.V. Ammirato, Y.
Yamada (eds.), Handbook of Plant Cell Culture,
Volume 1: Techniques for Propagation and Breeding.
Macmillan, New York, N.Y., pp. 748-769.
97. Rivin, C.J., E.A. Zimmer, C.A. Cullis, and V. Walbot.
1983. Evaluation of genomic variability at the
nucleic acid level. Plant Mol. Biol. Rep., 1: 9-16.
98. Russo, F. 1981. Bud variations in citrus cultivars
in Italy. Proc. Int. Soc. Citriculture, pp. 91-94.
99. Sabharwal, P.S. 1983. In vitro culture of ovules,
nucelli, and embryos of Citrus reticulata var.
Nagpuri. In P. Maheswari and N.S. Ranga Swamy (eds.),
Plant Tissue and Organ CultureA Symposium, Int. Soc.
Plant Morphologists, Delhi, India, pp. 265-274.
100. Sacristan, M.D. 1971. Karyotypic changes in callus
cultures from haploid and diploid plants of Crepis
capillaris (L.) Wallr. Chromosoma, 33: 273-283.
101. Sacristan, M.D. 1982. Resistance responses to Phoma
lingam of plants regenerated from selected cell and
embryogenic cultures of haploid Brassica napus.
Theor. Appl. Genet., 61: 193-200.
102. SAS User's Guide. 1979 Edition. J.T. Helwig and K.A.
Council (eds.), SAS Institute, Inc., pp. 425-426.


25
proliferation was suppressed, but embryo production was
enhanced; irradiation of the media produced the same effect
(110). Habituated callus responded in like fashion, but no
media effect was observed (60). The enhanced embryogenic
response exhibited by callus following extended culture
intervals, sucrose deprivation, use of galactose and related
sugars as the carbon source in the media (63) or irra
diation of callus suggested that hormone levels and ratios
may be altered by these manipulations. Using various
phytohormones, growth regulators, and inhibitors, Kochba and
Spiegel-Roy (61,62) and Moore (74) have shown that auxins
and cytokinins suppressed embryogenesis in habituated callus
or culture of unfertilized ovules, but inhibitors of auxin-
or GA-biosynthesis, or low levels of ABA or ethylene
enhanced the embryogenic response. Although extensive
research on the factors influencing embryogenesis by
'Shamouti' callus has been reported, little information has
been published on factors critical to callus culture ini
tiation, except the work of Moore (74). Likewise little
information on plant production from these embryogenic
cultures has been published.
Button et al. (14) made histological observations of
the gross structure and fine cellular structure of habit
uated 'Shamouti' callus. The callus was composed of loosely
attached, small spherical bodies, each of which was found to
be either an individual or a cluster of proembryos,


44
alternate path of embryo development that interfered with
the continued proliferation of embryos or embryogenic
callus.
Experiment 1: Production of Embryos and Embryogenic Callus
from Undeveloped Ovules of Several Citrus Species
The percentages of cultured undeveloped ovules that
produced primary embryos are shown in Table 3.1; Table 3.2
lists the number of embryos produced by each cultivar and
each treatment within cultivars. The sweet orange cultivars
'Hamlin', 'Pell Navel', and 'Pineapple' were the most
responsive types in terms of both percentage of ovules
producing embryos and total numbers of embryos produced.
'Marsh' grapefruit, 'Orlando' tngelo, and 'Key' lime were
intermediate in their degree of response. 'Bearss' lemon
responded only on the malt extract medium in the absence of
light and produced very few embryos. 'Owari' satsuma was
unresponsive to all treatments.
Sweet orange ovules cultured on 2,4-D/DZ or ME supple
mented media in the light were the most responsive in terms
of total embryo production. These treatments were superior
for all 3 sweet orange cultivars, but the most effective
treatments varied among the other cultivars. For example,
media supplemented with 2,4-D or 2,4-D/BA were most effec
tive for embryo production from 'Marsh' grapefruit, but
either ME treatment was of greater effectiveness than the
three 2,4-D supplemented media for embryo production from
'Orlando' tngelo. 'Bearss' lemon responded only when


146
79. Murata, M., and T.J. Orton. 1983. Chromosome struc
tural changes in cultured celery cells. In Vitro 19;
83-89.
80. Navarro, L., J.M. Ortiz, and J. Juarez. 1985. Aber
rant citrus plants obtained by somatic embryogenesis of
nucelli cultured in vitro. HortScience, 20: 214-15.
81. Ogihara, Y. 1981. Tissue culture in Haworthia.
Part 4: Genetic characterization of plants regenerated
from callus. Theor. Appl. Genet., 60: 353-363.
82. Ohta, Y., and K. Furusato. 1957. Polyploidy and
heteroploidy in Citrus seedlings. Seiken Ziho,
8: 37-39.
83. Oono, K. 1978. High frequency mutations in rice
plants regenerated from seed callus, p. 52 (abstract).
Fourth Int. Congr. Plant Tissue Cell Culture, n.p.,
Calgary, Canada.
84. Orton, T.J. 1980. Chromosomal variability in tissue
cultures and regenerated plants of Hordeum. Theor.
Appl. Genet., 56: 101-112.
85. Orton, T.J. 1983. Experimental approaches to the
study of somaclonal variation. Plant Mol. Biol. Rep.,
1: 67-76.
86. Orton, T.J. 1984. Somaclonal variation: Theoretical
and practical considerations. In J.P. Gustafson (ed.),
Gene Manipulation in Plant Improvement, 16th Stadler
Genetics Symposium, n.p., pp. 427-468.
87. Parlevliet, J.E., and J.W. Cameron. 1959. Evidence on
the inheritance of nucellar embryony in citrus. J.
Amer. Soc. Hort. Sci., 74: 252-259.
88. Pieringer, A.P., and G.J. Edwards. 1964. Identifica
tion of nucellar and zygotic citrus seedlings by
infrared spectorscopy. J. Amer. Soc. Hort. Sci.,
86_: 226-234.
89. Prat. D. 1983. Genetic variability induced in
Nicotiana sylvetris by protoplast culture. Theor.
Appl, Genet., 64: 223-230.
Raj Bhansali, R., and H.C. Arya. 1978. Tissue culture
propagation of citrus trees. In P.R. Cary (ed.), Proc.
Int. Soc. Citriculture, Sydney, Australia, pp. 135-140.
90.


59
treatments that produced the greatest total number of
embryos. The other component of embryo production was the
number of embryos produced per responsive ovule. However,
this factor was a minor contributing element to primary
embryo production. For example, in Experiment 1, 'Marsh'
grapefruit and 'Bearss' lemon produced fewer embryos because
of a much lower percentage of responsive ovules, even though
they produced the greatest numbers of embryos per responsive
ovule.
Differences among cultivars for total number of primary
embryos produced were observed in the first experiment.
Embryo production ranged from high (oranges) to intermediate
(tngelo, grapefruit and lime) to low (lemon) or none
(satsuma). Likewise, differences for the most effective
culture treatment for embryo production were observed among
cultivars. The variable response of different Citrus
species and cultivars has been observed previously (64,74).
Citrus sinensis is the only species represented by more than
one cultivar in this experiment. It is of interest that,
although cultivar differences among the oranges in overall
response were apparent, the most effective treatments were
identical for all orange cultivars, regardless of the
parameter considered. Moore observed intraspecific cultivar
differences for overall response among other Citrus species
other than C. sinensis (74).
The absence of light suppressed embryo and callus
production from the orange ovules but stimulated these


38
objective of isolating lines of different embryogenic
capacity. However, all selected sublines produced tissues
of various types, so the attempt to isolate lines on this
basis was abandoned.
Fresh weight increase of sublines was determined and
recorded after 28 days. The cultures were then divided and
transferred to either 0.01 mg 1 1 2,4-D/0.1 mg l-1 DZ or
0.1 mg 1 1 DZ supplemented medium; sublines were designated
Al, A2, etc. on the former and Bl, B2, etc. on the latter
medium. Fresh weight increases and numbers of embryos
produced were recorded after 15 days. At that time, B lines
were subcultured on the same medium; one half of the prolif
eration of the A lines was subcultured on the DZ-containing
medium, and the other half was transferred to unsupplemented
MT medium (C lines). A subjective visual evaluation of
callus proliferation and embryo production was made 28 days
after subculture. These lines were transferred every
4-6 weeks to fresh media of the same composition and were
the source of plants from embryogenic callus used in the
studies described in Chapters IV and V.
Results
General Comments
The following pattern of response was observed in all
undeveloped ovule cultures where embryogenesis occurred.
The ovules became swollen and acquired a light-green color


58
callus proliferation and embryo production were observed on
all media, including MT, indicating hormone habituation.
Habituation of callus that was initiated and pro
liferated on 2,4-D-containing media was accomplished on MT.
One habituated callus line (C-8) that was particularly
prolific was used to regenerate a number of plants utilized
in the studies described in Chapters IV and V. Growth and
embryo production gradually decreased in a number of lines,
and several lines were lost to contamination. A non
differentiating, rapidly proliferating callus arose from a
number of different embryogenic callus lines, including C-8.
This callus has been used to initiate suspension cultures
and has been converted back to an embryogenic form following
extended periods (4-6 months) between subcultures.
Discussion
The objectives of the experiments described in this
chapter were to identify factors that influence the produc
tion of embryos and embryogenic callus from undeveloped
Citrus ovules and to produce embryos from which plants could
be regenerated for use in additional studies. Conclusions
regarding embryo and callus production from such cultures
can be drawn from the results. The most important component
of primary embryo production was the percentage of cultured
ovules that responded and produced embryos. The cultivars
and treatments within cultivars with the greatest percentage
of responsive ovules corresponded to the cultivars and


CHAPTER I
INTRODUCTION
Citrus species are economically important fruit crop
plants in the United States and throughout tropical and
subtropical regions of the world. Although an array of
cultivars exists, most commonly grown varieties have arisen
as chance, well-adapted superior seedlings or as mutant
forms of already existing cultivars (44). Despite active
and vigorous Citrus breeding programs, very few cultivars
have been produced via standard plant breeding methods
(3,37). A number of factors have made Citrus cultivar
improvement a slow and difficult process. Most cultivars
are cross-pollinated and highly heterozygous (108). Large
individual plant size and extended periods of juvenility
characterize most Citrus species. As a result, breeding
programs require long-term investments of land, time, and
money (108). Several horticulturally superior clones
exhibit pollen and/or ovule sterility and thus, are unsuited
for use as parents in desired crosses (33). Likewise,
numerous examples of cross- and self-incompatibility exist
that prevent the potential of certain specific parental
combinations from being realized (108).
1


72
GA-supplemented medium) or that germinated. However, the
percentage of germinated embryos that survived transfer to
soil and acclimatization was greater for the ME/dark and
2,4-D/DZ than the other treatments. The values in Table 4.3
were generated by summing over all cultivars; however,
either ME/dark or 2,4-D/DZ treatments produced the group of
embryos with the greatest regeneration survival percentage
for 5 or 6 cultivars (Table 4.4). The treatments most
effective for embryo production from cultivars were fre
quently the treatments with the highest rate of regenerate
survival (Table 3.1, 4.4).
Discussion
Several hundred embryos were produced by ovule cultures
of most cultivars in Experiment 1, Chapter III. The number
of surviving plants regenerated from these embryos was
significantly lower, however. The first requirement for
plant regeneration was the development of a normal, viable
embryo. A number of developmental abnormalities were
observed among the embryos produced from all cultivars and
on all media. The presence of these abnormal embryos
limited the total number of plants that could be produced.
Although no data were presented, the relative proportion of
abnormal embryos in 'Hamlin' callus or secondary prolifer
ation appeared to be the same as that in 'Hamlin' primary
populations. Culture age, therefore, did not appear to
greatly affect normal embryo production or development.


14
possessed masked recessive mutations. Ogihara (81) noted
that ploidy levels of regenerated Haworthia setata plants
were associated with some morphological aberrancies, but the
expression of other characters was unaffected. Although
Reisch and Bingham (95) found that morphological abnor
malities were associated with aneuploidy in regenerated
alfalfa plants, aberrant phenotypes were found also among
diploid and tetraploid regenerates.
Sacristan's (100) studies with Crepis capillaris (L.)
Wallr. cell cultures point out that the absence of changes
in chromosome number does not preclude the possibility of
chromosome rearrangement as a source of variation. Murata
and Orton (79) have provided evidence via meiotic analysis
of regenerated celery plants (Apium graveolens L. var.
'dulce') of chromosome rearrangement in diploids; perhaps
ironically, aneuploidy produced by chromosome breakage and
fusion in some regenerates resulted in normal, functionally
diploid plants. However, Burk and Chaplin (10) found no
chromosome number differences or evidence of meiotic pairing
irregularities among morphologically variant, regenerated
tobacco plants. The evidence presented in preceding para
graphs on the existence of single gene mutations among
regenerated plants points out that although phenotypic
variability can certainly result from poly- or aneuploidi-
zation, it is not the sole cause, because variations can
arise also in cytogenetically normal plants (i.e., with
normal chromosome numbers and gross structure).


130
zymograms confirmed the nucellar origin of regenerated
plants and nucellar seedlings.
3. Morphologically variant plant types were observed among
plants from tissue culture and nucellar seedlings, but
more extreme variation was found among the tissue
culture groups. This suggested that although variabil
ity may have existed among initial cell populations
within explants, in vitro embryogenesis was less
stringent and allowed more variation to be expressed
than in vivo embryogenesis.
4. No specific tissue culture treatment was identified
that consistently generated more variable plants.
However, culture age differences were found to have a
much greater effect on phenotypic stability than
differences of initiation media.
5. There were limitations associated with each approach to
plant characterization. As a consequence, additional
cytogenetic and morphological evaluations will be
undertaken. Specifically, more mitotic counts and
meiotic analysis will be performed, and mature plant
and fruit characteristics will be evaluated.


87
and placed in a pretreatment saturated solution of
8-hydroxyquinoline and maltose (4:1, approximately) for
4-5 hours. Tips were removed from the pretreatment and
fixed for up to 7 days in a solution of 95% ethanol and
glacial acetic acid (3:1). Long-term storage was in 70%
ethanol at -15 C. Following hydrolysis in 5N HC1 at room
temperature for approximately 15 minutes, tips were rinsed
with tap water and placed in Feulgen stain for 15 minutes
(or until pink staining was visible). The root tips were
then placed on glass slides with a drop of modified carbol
fuchsin stain (51) and covered with glass cover slips.
Careful application of heat and pressure yielded root tip
preparations that allowed mitotic chromosome counts.
Electrophoretic Characterization of Regenerated Plants
The isozyme banding patterns of all 'Hamlin' orange
plants listed in Table 5.1 were evaluated for 11 different
enzyme activity staining systems. Some of the plants
regenerated from primary and secondary embryos of other
cultivars were also examined electrophoretically (see
Table 5.2). All 'Hamlin' orange plants were examined at
least one time for each enzyme staining system. More
frequently, several samples per plant were taken over time,
and several zymograms for each stain were evaluated.
Zymograms were evaluated for number and relative position of
major bands.


83
in vitro enbryogenesis. Although relative uniformity of
nucellar seedlings has been observed, variant forms have
been reported among nucellar populations. For example,
aneuploid (82) and polyploid (4,45) seedlings have been
identified. Nucellar seedlings have arisen that differed
from the parental type in tree growth habit, crop yield, or
fruit characteristics (size, shape, color, or quality)
(32). Qualitative isozyme differences have been observed
among plants of the same cultivar and among nucellar seed
lings arising from the same seed (46). As a consequence of
possible nucellar seedling variability and a desire to
utilize the nucellar population as a control group in
phenotypic stability studies, a substantial number of
seedlings (100) was characterized in the same manner as the
plants produced by tissue culture methods.
An attempt was made to determine the effect of various
culture parameters on the frequency and degree of variation
among regenerated plants. The groups of plants produced in
the work described earlier had various histories. Specifi
cally, plants were regenerated in the presence or absence of
2,4-D (a mutagenic agent), from primary embryos (direct
embryogenesis), from secondary embryos or embryogenic
callus-derived embryos (indirect embryogenesis). The
influence of culture age and embryo source on phenotypic
stability of regenerated Citrus plants was examined among
the several plant populations available.


50
'Orlando' tngelo was intermediate in the percentage of
ovules producing callus. 'Key' lime and 'Bearss' lemon had
few responsive ovules, but those that did respond produced
embryogenic callus. 'Marsh' grapefruit and 'Owari' satsuma
produced lttle callus and that which was produced was not
embryogenic. Ovules in several cultivar X treatment com
binations were decreased in number and lost to an outbreak
of contamination in the culture room prior to further
evaluation. As a result, comparisons of numbers of embryo
genic callus lines that could be established from the
various treatments could not be made. However, established
embryogenic callus lines that continued to proliferate were
obtained from several of the Citrus cultivars. Those
genotype x treatment combinations marked with a "b" in
Table 3.4 produced embryogenic callus that persisted at
least 180 days.
Experiment 2: Production of Embryos and Embryogenic Callus
by Undeveloped Ovules from Immature Fruit of 'Hamlin'
Orange
Table 3.5 describes primary embryo production under
various culture conditions by undeveloped ovules from
immature 'Hamlin' orange fruit harvested approximately
8 weeks after anthesis. The response level of ovules from
fruit of this age was much lower than that of ovules from
more mature fruit, regardless of initiation treatment (See
Results: Experiment 1). The most effective treatment for
embryo production by ovules at this stage of development was


143
45. Hutchison, D.J., and H.C. Barrett. 1981. Tetraploid
frequency in nucellar seedlings from single trees of
Carrizo and Troyer citrus hybrids. Proc. Int. Soc.
Citriculture, pp. 27-29.
46. Iglesias, L., H. Lima, and J.P. Simon. 1974. Iso
enzyme identification of zygotic and nucellar seedlings
in Citrus. J. Hered., 65; 81-84.
47. Irvine, J.E. 1984. The frequency of marker changes in
sugarcane plants regenerated from callus culture.
Plant Cell Tissue Organ Culture, 3^: 201-209.
48. Iwamasa, M., and M. Nishiura. 1981. Recent citrus
mutant selections in Japan. Proc. Int. Soc. Citri
culture pp. 96-99.
49. Johnson, L.B., D.L. Stuteville, S.E. Schlarbaum, and
D.Z. Skinner. 1984. Variation in phenotype and
chromosome number in alfalfa protoclones regenerated
from non-mutagenized calli. Crop Sci., 24: 948-951.
50. Juarez, J., L. Navarro, and J.L. Guardiola. 1976.
Obtaining nucellar plants of various cultivars of
Clementine by in vitro culture of the nucellus.
Fruits, 12: 751-761. (Translated from French.)
51. Kao, K.N. 1975. A nuclear staining method for plant
protoplasts. In O.L. Gamborg and L.R. Wetter (eds.).
Plant Tissue Culture Methods, Natl. Res. Council of
Canada, Saskatoon, pp. 60-62.
52. Kitto, S.L., and M.J. Young. 1981. In vitro propa
gation of Carrizo citrange. HortScience,
16: 305-306.
53. Kobayashi, S., I. Ikeda, and M. Nakatani. 1978.
Studies on the nucellar embryogenesis in citrus. I.
Formation of the nucellar embryo and development of
ovule (in Japanese). Bull. Fruit Tree Res. Stn. E.,
2: 9-24.
54. Kobayashi, S., I. Ikeda, and M. Nakatani. 1981. Role
of the primordium cell in nucellar embryogenesis in
citrus. Proc Int. Soc. Citriculture, pp. 44-48.
55. Kobayashi, S., I. Ikeda, and M. Nakatani. 1982.
Studies on nucellar embryogenesis in citrus. III. On
the differences in the ability to form embryoids in in
vitro culture of ovules from poly- and mono-embryonic
cultivars (in Japanese). Bull. Fruit Tree Res. Stn.
E., 4: 21-27.


135
regenerated plants. Several of the plants produced will be
maintained until flowering for meiotic analysis to look for
evidence of chromosome structural changes. None of the
factors studied (2,4-D, culture age, or direct vs. indirect
embryogenesis) appeared to have any influence on cytogenetic
stability, as determined by chromosome count.
At least 12 chemically-defined genetic loci were
examined electrophoretically for changes in banding pat
terns, in all nucellar and tissue culture plants. No
detectable mutations were found. Electrophoresis may not
necessarily be an effective method of screening plant
populations for variant types because of the limited number
of loci sampled. However, the uniformity of the banding
patterns that was observed provides support for the idea
that all of the plants produced in this work (in vivo and in
vitro) were of nucellar origin. As with the cytogenetic
analysis, no relationships between the system parameters
(2,4-D, age, etc.) and electrophoretic variation were
observed.
In contrast to the apparent cytogenetic and electro
phoretic stability, morphologically variant plants were
found among all populations studied, but the extremity and
frequency of variation was generally greater in the tissue
culture populations than among the nucellar seedlings. The
genetic nature of these variants has not been determined
because of the limitations to genetic studies associated
with perennial, cross-pollinated plants. The traits that


124
subgroups, indicating that none of the various initiation
treatments had any effect on variability of petiole shape.
Leaf blade L/W ratio
Leaf blade L/w ratios were calculated to quantify and
compare blade shapes and variances of groups. The relation
ship between leaf blade shape and L/W ratios was as
described previously for petiole shape. When plants were
grouped according to initiation treatment, the mean values
of the tissue culture groups were not significantly differ
ent from each other, but all were greater than the mean
value of the nucellar seedlings (Table 5.9). When plants
were groups by embryo source, the secondary and callus
groups were not different from each other, and both were
greater than the primary group. All of the tissue culture
group means were greater than the mean value of the nucellar
population. The tendency was for tissue culture plants to
have longer, thinner leaf blades and petioles. Growth
regulator components in the initiation treatment media did
not affect blade ratios, but embryo source was associated
with differences between means. Comparisons of mean values
of subgroups within main groups revealed no significant
differences, indicating that none of the initiation treat
ments had any effect on subgroup means.
As with petiole ratios, there were more plants with
greater than lesser values, reflecting the tendency toward
longer, thinner blades among tissue culture plants. Only


20
development, were cultured on the basal medium amended with
casein hydrolysate (CH), but only well-developed cotyledon
ary embryos underwent development in the absence of CH.
Therefore, the inclusion of maternal tissue in the explant
resulted in simpler media requirements for embryo develop
ment. Excised nucelli gave rise to embryos and a prolif
eration of macroscopic spherical bodies called
"pseudobulbils." Continued proliferation and the production
of occasional embryos only occurred on CH-supplemented
medium. Sabharwal (99) likewise demonstrated with Citrus
reticulata Blanco cv. Nagpuri cultures that when adventive
embryos were cultured with the surrounding nucellar tissue,
callus and pseudobulbil proliferation predominated. By
contrast, when embryos were cultured without nucellar
tissue, embryo differentiation and development predominated
over callus proliferation. Culturing embryos initially in
the dark was beneficial, but prolonged dark culture led to
etiolation (70). ME was found to inhibit embryo germina
tion, and 2,4-D supplementation resulted in callus, but not
embryo, production.
The reports cited above concluded that pollination was
necessary for adventive embryogenesis in vitro because there
was no response among explants from fruit harvested prior to
anthesis. However, Mitra and Chaturvedi (73) demonstrated
that pollination was not a requirement. They cultured whole
and dissected ovaries (walls, placenta, ovules) from
unpollinated flowers of C. sinensis L. Osbeck, C.


4
Citrus is the most responsive of woody perennial fruit
crops to tissue culture manipulations (13) In vitro
embryogenesis occurs readily among many Citrus species,
directly from cultured nucelli or ovules (fertilized or
undeveloped) and indirectly from embryogenic callus or
cultured protoplast colonies (11,13,14,15,55,56,64,73,92,
93,112,127,130). Likewise, a number of Citrus species have
been propagated by organogenesis directly from various
somatic explant sources (2,19,26,52,90) or after callus
production (19,91,103). Despite the many reports of in
vitro plant production in Citrus, little information on the
stability of character expression in regenerated plants has
been published (80) The ultimate objectives of the studies
described in this dissertation were to produce Citrus plants
via various tissue culture methods and to characterize those
plants morphologically, electrophoretically, and cytogeneti
cally, for evaluation of the degree of phenotypic stability
exhibited by regenerated Citrus plants.
The first chapter to follow is a review of literature
pertinent to somaclonal variation in plants, Citrus tissue
culture studies (specifically embryogenic cultures), and
Citrus plant characterization. The 3 chapters following the
Literature Review will deal respectively with research into
embryogenic culture induction and development, plant regen
eration and survival, and studies of plant phenotypic
stability. Important points and conclusions that are
particularly relevant to Citrus will be discussed within the


Table 5.5. Summary of group means, variances, and coefficients of variation for growth
rate, mean internode length, petiole L/W ratio, and leaf blade L/W ratio of
'Hamlin' orange plants.
Growth rate Mean internode
Plant
(cm-mo1)
lenoth (cm)
Petiole
L/W ra
tic
Blade
L/W ratio
Source
n
X
s2
CV
X
s2
CV
X
2
s
cv
X
s2
CV
Primary
embryos
67
3.679
1.529
33.6
2.039
0.115
16.6
3.752
0.703
21.3
1.855
0.018
7.2
Secondary
embryos
60
2.605
0.822
36.1
1.377
0.111
24.2
4.612
0.553
16.1
1.988
0.130
18.2
ME medium
66
3.240
1.795
41.4
1.767
0.247
28.2
4.042
0.895
23.4
1.935
0.132
18.8
2,4-D media
61
3.097
1.203
35.4
1.682
0.195
26.2
4.284
0.706
19.6
1.899
0.013
6.0
Embryogenic
callus
66
2.629
1.512
43.5
1.483
0.154
26.5
4.493
0.612
17.4
1.942
0.022
7.7
Nucellar
seedlings
100
3.172
0.524
22.8
2.137
0.100
14.8
3.556
0.522
20.3
1.776
0.012
6.1


Table 5.9. Summary of F-tests and t-tests comparing leaf blade L/W ratios of groups
of 'Hamlin' orange plants. Abbreviations used for groups are as listed in the
title of Table 5.6.
Comparison
A vs. B
F
df
P>Fa
A:Bb
t
df
P> t C £
L:Bb
1
2
7.35
59 + 66
0.0001
-
-2.71
73.3
0.0084
-
1
C
1.25
65+66
0.3685
=
-3.56
131
0.0005
-
1
N
1.52
66 + 99
0.0598
=
4.21
165
0.0001
+
2
C
5.88
59 + 65
0.0001
+
0.93
77.0
0.3560
=
2
N
11.15
59 + 99
0.0001
+
4.44
65.4
0.0001
+
C
N
1.90
65 + 99
0.0040
+
7.81
109.4
0.0001
+
ME
2,4-D
10.10
65+60
0.0001
+
0.77
78.7
0.4437
=
ME
C
5.97
65+65
0.0001
+
-0.14
86.2
0.8920
=
ME
N
11.31
65+99
0.0001
+
3.46
72.6
0.0009
+
2,4-D
C
1.69
65 + 60
0.0408
-
-1.83
121.1
0.0703
=
2,4-D
N
1.12
60 + 99
0.6086
=
6.86
159
0.0001
+
Probability of
a greater
F value.
bIf
A>B, then +;
if A then -,
if A=B, then
= ^
c
Probability of a greater absolute value of t.
109


123
F-tests performed to compare group variances for
petiole L/W ratios revealed no statistically significant
differences among groups in all pair-wise comparisons,
except the ME and nucellar comparison in which the former
group was shown to have a significantly greater variance.
As noted above, the ME group was the source of the 2 plants
with aberrant petiole shapes. The tissue culture plants had
greater mean petiole ratios than the nucellar seedlings, but
the variances were not statistically different. It would
seem, therefore, that the phenotypic expression of genes
controlling petiole shape may have been influenced in some
fashion by tissue culture treatment. Another possible
explanation for mean differences may have been the fact that
the plant groups were not initiated simultaneously or grown
in identical environments at all times. Differences in
plant age or previous environmental conditions may have been
acting on the current expression of petiole shape. In any
event, the effects of embryo source or culture treatment on
petiole ratios were manifested in the individual populations
as groups, and no increased variation resulted. This
conclusion was evidenced by the fact that mean values of the
tissue culture groups were greater than the nucellar popu
lation, but group variances were not significantly different
from the nucellar variance, with one exception. Addition
ally, comparisons of subgroups within main groups as
described previously revealed no significant differences
between mean petiole ratios or variances of the various


17
Swartz et al. (115) reported that Rubus plants (blackberry)
produced by in vitro shoot proliferation of axillary meri-
stems were phenotypically invariant in the field, except for
1 sectorial leaf variant. Wakasa (133) observed that the
explant source had an effect on the percentage of variant
plants obtained from tissue culture-propagated pineapple
(Ananas comosus); syncarp or slip explants produced nearly
100% variant plants, but crown explants yielded only 7%
variant plants. Numerous reports of variant somaclones
derived from protoplasts have been published (24,69,89,105,
119). The general trend seems to be that the more organized
the initial explant is (e.g., shoot tips vs. mesophyll
protoplasts), the greater will be the phenotypic stability
observed among regenerates.
Media components, culture age at the time of plant
regeneration, and the method of regeneration are other
parameters that may affect the amount of variability
observed. Reisch and Bingham (95) found that most of the
morphologically variant alfalfa somaclones they obtained
came from cultures on ethionine-amended medium. Ogihara
(81) reported that media amended with NAA and kinetin
produced more ploidy and karyotypic variation than did the
same basal medium amended with IAA, but no media effects on
the stability of morphological character expression of
Haworthia were observed. Johnson et al. (49) concluded that
the presence of 2,4-D in the media did not increase the


27
via various organogenic methods including shoot prolif
eration by axillary buds of shoot tips or stem segments
(1,2,52), from adventitious buds formed on cultured stem
segments (Moore, personal communication, 19), and by
organogenesis from short- or long-term leaf or stem callus
cultures (19,36,90,91). The most commonly used growth
regulator for the initiation of organogenesis has been
6-benzylaminopurine. The potential exists for populations
of Citrus plants from one or several different clones to be
regenerated by various in vitro methods, thus providing an
opportunity for studying factors that may be related to the
expression of somaclonal variation. Factors that could be
studied include the method of regeneration (embryogenesis
vs. organogenesis, and within categories, direct vs.
indirect via callus), the effect of 2,4-D in the culture
medium, the influence of culture age at the time of plant
regeneration, explant source differences, genotype effects,
and the effect of protoplast-mediated plant regeneration on
phenotypic stability.
Characterization of Seedling Citrus Plants
The difficulty encountered when attempting to identify
zygotic Citrus seedlings among progeny of polyembryonic seed
parents has been discussed in Chapter I. The use of simply
inherited genetic markers could help to solve the problem,
but few markers beside the trifoliate leaf trait of Poncirus
trifoliata have been characterized in Citrus species or


29
be used to identify zygotic seedlings from appropriate
inter- or intra-specific crosses (38,118). Other vegetative
traits used to characterize Citrus seedlings include growth
habit and vigor, stem characteristics, leaf color and
venation, and relative stoma and oil gland size (4,45,38).
Many of these characters (e.g., leaf shape) are variable
even within individual plants, so experience is necessary
before vegetative variants can be consistently identified
with accuracy.
The use of isozyme loci as genetic markers in Citrus
provides another method of plant characterization. Isozyme
analysis can be a powerful tool, particularly when the
genetics of the enzyme system utilized are understood.
Isozyme genes are expressed in a codominant manner. Fairly
large numbers of individuals can be screened at one time,
usually for several different enzyme staining systems.
Torres et al. (122,123) and Moore (unpublished) have estab
lished the genetic basis and allelic constitution of several
isozyme loci in various Citrus species (see Table 5.4 in
Chapter V). These enzyme staining systems and others have
been used to distinguish nucellar and zygotic seedlings from
certain crosses (46,109) and have been suggested as a means
to identify somatic hybrids (6) .
To conclude, various methods of Citrus seedling charac
terization have been developed. The methods vary as to
their cost, efficiency, and limitations. Although these
techniques provide the means to identify variant seedling


51
Table 3.5. Effect of culture conditions on embryo produc
tion by undeveloped ovules from 'Hamlin' orange
fruit harvested 8 weeks after anthesis. Embryos
were counted 56 days after culture initiation.
Treatment (mg 1 ^)a
Percent
responsive
ovules
Number
embryos
produced
Number Number
embryos/ embryos/
ovule responsive
cultured ovule
500.0 ME
Light
4.2
6
0.05
1.20
500.0 ME
No light
7.5
14
0.12
1.56
0.01 2,4-D
Light
0.8
1
0.01
1.00
0.01 2.4-D/0.1 BA
Light
0.0
0
0.00
0.00
0.01 2.4-D/0.1 DZ
Light
1.7
3
0.03
1.50
MT
Light
3.3
2
0.03
1.00
aEach culture treatment utilized 120 ovules, except 60
ovules were used in the MT treatment.
Responsive ovules are those from which embryos were
produced.


66
the growth chamber initially, under the same conditions of
temperature and light as the cultured embryos and were taken
to the greenhouse after acclimation, usually within 4 weeks.
Plants were fertilized with a standard water-soluble 20-20-
20 fertilizer 4 weeks after transfer to soil. Surviving
plants were counted after 12 weeks. A second collection of
embryos was taken from the 'Hamlin' ovule cultures (Chap
ter III, Experiment 1) 8 weeks after the primary embryo
harvest; these were designated secondary embryos. Embryos
originating from long-term 'Hamlin' embryogenic callus lines
which had been maintained for over 18 months (Chapter III,
Experiment 3) were isolated and used to compare germination
and survival rates of embryos from long-term callus cultures
with that of primary and secondary embryos.
Results
Germination (both root and shoot elongation) and root
and shoot development by primary embryos of several Citrus
cultivars is described in Table 4.1. A total of 733 primary
embryos were placed on the germination medium. The number
of embryos producing roots was 645 (88%) and 464 (63%) of
the embryos produced shoots. The number of embryos that
germinated was 419 (57%). There were 226 embryos that
developed roots but not shoots; likewise there were
45 embryos that produced shoots but not roots.
The root development response of the cultivars ranged
from 69% of 'Key' lime embryos to 97% (97 of 100) of 'Marsh'


100
Citrus cultivars (Table 5.2) revealed uniform banding
patterns among plants of each cultivar for each enzyme
staining system. SDH, PER, ME, and LAP zymograms were
observed, on rare occasions, with an additional band that
ran slower than the normal, major bands. Additionally, a
few LAP and IDH zymograms were developed on which a major
band was not present. These aberrant zymograms resulted
from some cause other than mutation, however, because
several repeat runs using different leaf samples taken at
various times from these same plants resulted in consis
tently uniform and normal banding patterns. No plants were
found that consistently produced a unique banding pattern
(band number or electrophoretic mobility). The uniform
visible expression of specific chemically defined genetic
loci indicated that no electrophoretically detectable
mutations had occurred at these loci.
Other researchers have reported variability in total
protein and isozyme banding patterns in Citrus. Navarro
found that morphologically abnormal 'Clementine' mandarin
plants (monoembryonic) produced from nucellar cultures had
altered total leaf protein profiles after polyacrylamide gel
electrophoresis (80). No such evaluations were made in this
study, but others have found that it is difficult to
distinguish individual bands when crude extracts of Citrus
leaf tissue are run on gels and stained for total protein
(K. Cline, personal communication). Vardi reported that 5
of 6 'Shamouti' orange plants (5 diploids and 1 tetraploid)


152
Mr. Gmitter was admitted to the graduate program of the
Fruit Crops Department of the University of Florida.
Doctoral studies commenced in 1982, under the direction of
G.A. Moore and W. B. Sherman. He was a graduate assistant
in the program of G.A. Moore from January, 1982 until June,
1985. During this time he has served as President of the
Fruit Crops Graduate Students (1982-1983) and has gained
recognition for academic and research activities in citri-
culture by being awarded the Hughes Foundation and Ward
awards in 1983 and 1984. Following completion of doctoral
studies, Mr. Gmitter will be employed at the Citrus Research
and Education Center of the University of Florida (Lake
Alfred, FL) to work in the area of Citrus cultivar improve
ment.


96
above that were produced by tissue culture techniques.
These 5 groups were also compared with each other. The
results of the F-tests comparing group variances made it
possible to draw conclusions concerning the effects of 2,4-D
in the culture initiation medium and culture age at the time
of embryo isolation (primary vs. secondary vs. callus-
derived embryos) on the variability of character expression
in the groups described above. T-tests were performed along
with F-tests to allow comparisons of group means.
The second level of data organization and analysis was
as follows, using the group numbers listed in Table 5.1.
Within the group of plants from primary embryos, groups Pi
and P2 were compared to determine whether light had any
effect on group means or variances for character expression.
Groups P4 and P5 were compared individually with group P3 to
determine whether BA or DZ had any effect as additions to
the 2,4-D medium on group means or variances for character
expression. Then, the plants of groups PI and P2 were
considered together and compared with the combined plants of
groups P3, P4, and P5 to determine the effect of 2,4-D on
group means and variances of plants derived from primary
embryos for character expression. The same sequence of
comparisons were made for the same purposes with the groups
of plants produced from secondary embryos.
The objectives of these statistical studies of morpho
logical character expression have been listed below:


63
responses among other cultivars, and proved essential
for 'Bearss' lemon response. Continued subculturing in
the absence of light was detrimental to further normal
development.
4. Stage of fruit development at the time of ovule iso
lation affected embryo and embryogenic callus produc
tion from undeveloped 'Hamlin' ovules. Generally,
ovules from more mature fruit were the most responsive.
5. Embryogenic callus was initiated with relative ease but
rarely persisted. The establishment of long-term
'Hamlin' embryogenic callus lines was enhanced by
2,4-D, but it was not always necessary. Callus
initiated and proliferated on media amended with growth
regulators became habituated to unamended medium.
Sufficient numbers of embryos were produced from ovule
and callus cultures for studies of plant regeneration
and phenotypic stability of regenerated plants.
6.


141
21. DeLange, J.H., and A.P. Vincent. 1977. Citrus breed
ing: new techniques in stimulation of hybrid
production and identification of zygotic embryos and
seedlings. Proc. Int. Soc. Citriculture, 2: 589-595.
22. Devaux, R. 1981. New cultivars of Clementine mandarin
in. Morocco. Proc. Int. Soc. Citriculture, pp. 101-102.
23. Edallo, S., C. Zucchinali, M. Perenzin, and F.
Salamini. 1981. Chromosomal variation and frequency
of spontaneous mutation associated with in vitro
culture and plant regeneration in maize. Maydica,
2_6: 39-56.
24. Engler, D.E., and R.G. Grogan. 1984. Variation in
lettuce plants regenerated from protoplasts. J.
Hered., 75: 426-430.
25. Esen, A., R.W. Scora, R.K. Soost. 1975. A simple and
rapid screening procedure for identification of zygotic
Citrus seedlings among crosses of certain taxa. J.
Amer. Soc. Hort. Sci., 100: 558-561.
26. Esen, A., and R.K. Soost. 1974. Polyphenoloxidase-
catalyzed browning of young shoot extracts of Citrus
taxa. J. Amer. Soc. Hort. Sci., 99: 484-489.
27. Esen, A., and R.K. Soost. 1977. Adventive embryo-
genesis in citrus and its relation to pollination and
fertilization. Amer. J. Bot., 64: 607-614.
28. Esen, A., and R.K. Soost. 1977. Separation of
nucellar and zygotic citrus seedlings by use of
polyphenol oxidase-catalyzed browning. Proc. Int. Soc.
Citriculture, 2: 616-618.
29. Evans, D.A., and W.R. Sharp. 1983. Single gene
mutations in tomato plants regenerated from tissue
culture. Science, 221: 949-951.
30. Evans, D.A., W.R. Sharp, and H.P. Medina-Filho. 1984.
Somaclonal and gametoclonal variation. Amer. J. Bot.,
759-774.
31. Flavell, R.B. 1982. Recognition and modifications of
crop plant genotypes using techniques of molecular
biology. In I.K. Vasil, W.R. Scowcroft, and K.J. Frey
(eds.), Plant Improvement and Somatic Cell Genetics,
Academic Press, New York, pp. 277-291.
Frost, H.B., J.W. Cameron, and R.K. Soost. 1957.
Diversity among nucellar-seedling lines of Satsuma
mandarin and differences from the parental old line.
Hilgardia, 27: 201-222.
32.


133
evidence suggesting that initiation treatment differences
had influenced the regeneration survival percentage by
increasing the number of germinated embryos that were
successfully transfered to soil, but not through any effect
on development or germination. Additional studies may
further document this effect and perhaps exploit it to
maximize plant production from this embryogenic system.
Citrus species provide good plant material for studies
of somaclonal variation. Little information has been
published on phenotypic stability of regenerated woody
perennial plants, although an abundance exists for herba
ceous annuals. Citrus plants can be regenerated by several
different methods and under a variety of conditions, includ
ing in vivo nucellar embryogenesis. Consequently, numerous
comparisons are available to answer questions regarding the
effect of media components, method of regeneration, explant
source, etc. on phenotypic variation. Comparisons of plants
produced in in vivo and in vitro may shed light on the
question of de vovo vs. pre-existent genetic variation.
Furthermore, the difficulty of improving Citrus via standard
plant breeding methods and the potential of somaclonal
variation as an additional source of genetic variability for
cultivar improvement makes some investigation of the phenom
enon in Citrus imperative. However, although success has
been achieved in other areas of Citrus plant regeneration,
more work is necessary because reports of regeneration from
protoplasts or from adventitious shoots (from cultured stem


34
Materials and Methods
General Information on Tissue Culture Methods
Fruit from open-pollinated flowers on field-grown trees
were harvested at various stages of development as described
in the following sections. The fruit were washed with a
mild detergent solution and rinsed with deionized water.
Portions of the fruit and peel were removed so that only the
central cube of locule tissue with the ovules attached
remained. Undeveloped (unfertilized or abortive) ovules,
which could be distinguished from fertilized ovules by their
size, were removed with forceps and placed into glass
funnels lined with moist filter paper and supported by
125 ml Ehrlemeyer flasks. Ovules were disinfested by
pouring solutions of sodium hypochlorite (20% v/v) and
ethanol (70% v/v) through the funnel so that the ovules were
immersed in each solution for approximately one minute.
This was followed by several rinses with autoclaved dis
tilled water at room temperature. Disinfestation, inocu
lation, and transfer were done under a laminar flow hood.
The basal medium used in all experiments was that of
Murashige and Skoog (77) as modified for Citrus by Murashige
and Tucker (MT) (78). The medium was gelled with 0.8% agar,
and the pH was adjusted to 5.7. Growth substance treatments
were added to the medium prior to autoclaving at 121 C, at
-2
1.1 kg cm for 17 minutes. Media were poured into
100 X 15 mm sterile plastic Petri dishes that were sealed


125
1 plant was found among the tissue culture groups that had a
lesser mean blade ratio than the minimum value of nucellar
seedlings, but several were found in each group with values
greater than the maximum nucellar ratio. Only 6 plants were
noted among the tissue culture groups with leaves that were
less pointed than normal (see Figure 5.2); however, these
plants did not all have L/W ratios that exceeded the normal
range. It was not always possible to identify aberrant leaf
shapes statistically. Only 1 such plant was observed in the
nucellar population. Therefore, on the basis of a subjec
tive visual evaluation of leaf blade shape, the tissue
culture groups had a greater proportion of variant types
than the nucellar population.
The coefficients of variation calculated for leaf blade
L/W ratios were the lowest observed among the traits
studied, with the exceptions of the secondary embryo or ME
groups (Table 5.5). F-tests of subgroups within main groups
indicated that there were not significant variance differ
ences, except that the variance of the group of plants from
secondary embryos produced on ME (light) was greater than
that of the secondary/ME (no light) group. This difference
was expressed when all ME/secondary plants were compared
with all 2,4-D/secondary plants, as well as the main group
level (see below). Again, subgroup means were not found to
be significantly different, despite variance differences.
F-tests performed with main group variances confirmed
the conclusions drawn above; the secondary group had the


101
regenerated from protoplast colonies had peroxidase zymo
grams that did not differ from nucellar 'Shamouti' plants,
but 1 plant did exhibit a variant banding pattern (125).
Iglesias reported peroxidase variants among plants of the
same cultivar and nucellar seedlings from a common seed
(46). The 'Hamlin' peroxidase zymograms studied in this
research had occasional variable bands that appeared incon
sistently, even among several samples from individual
plants. Therefore, analysis in this enzyme staining system
was confined to the single locus previously found to be
invariant within and among plants, and then no variants were
found. These other researchers viewing isozyme bands
without knowledge of their genetic behavior may have been
observing variability due to developmental or environmental
factors rather than genetic changes.
When enzyme systems selected on the basis of intra-
clonal and environmental stability were used, electophoretic
phenotypes (zymograms) were stable and consistent, in
contrast to gross vegetative seeding phenotype which
resulted from interacting genetic and environmental factors.
The expression of only one of a few specific genetic loci
was examined at one time with electrophoretic analysis,
without the confounding effects of pleiotropy or environ
ment. However, certain limitations of isozyme analysis must
be kept in mind when interpreting the results reported in
this chapter. Screening plants with these 11 enzyme systems


7
Somaclonal Variation
Genetic alteration of cultured plant cells was reported
by several researchers in the 1960s. Torrey reported that
callus cultures of Pisum sativum L. exhibited increasing
levels of polyploidy and aneuploidy with increased time in
culture; the gradual loss of the organogenic capacity of
these cultures was correlated with increased cytogenetic
abnormality (124). Murashige and Nakano (76) and D'Amato
(20) likewise have reported an increase in chromosomal
aberrations of cultured cells with increased time in cul
ture. In their review, Larkin and Scowcroft cite numerous
examples of phenotypic variation observed among callus
sublines of several different genera for characters such as
morphogenetic pattern, growth rate, pigmentation, growth
regulator habituation, and metabolite production (67). The
instability of cultured cell and callus phenotypes has been
known, therefore, for some time.
The first reports on the recovery of variant types
among tissue culture-produced plants were those of Heinz and
Mee, who worked with sugar cane (Saccharum sp.) (41,42).
Several papers on the variation observed among regenerated
plants of other genera have been published since that time,
including potato (Solanum tuberosum L.) (105,106,119) ,
tobacco (Nicotiana sp.) (89), and rice (Oryza sativa L.)
(83,113). Many of the earlier reports concerned polyploid
species or crop plants that are propagated asexually (e.g.,
sugar cane, tobacco, or potato). Rice, however, being a


113


140
11. Button, J., and C.H. Bornman. 1971. Development of
nucellar plants from unpollinated and unfertilized
ovules of the Washington navel orange in vitro. J.S.
African Botany, 37; 127-134.
12. Button, J., and C.E.J. Botha. 1975. Enzymatic macera
tion of Citrus callus and the regeneration of plants
from single cells. J. Exp. Bot., 26: 723-729.
13. Button, J., and J. Kochba. 1977. Tissue culture in
the citrus industry. In J. Reinert and V.P.S. Bajaj
(eds.), Applied and Fundamental Aspects in Plant Cell,
Tissue, and Organ Culture, Springer-Verlag, Berlin,
Heidelberg, New York, pp. 70-92.
14. Button, J., J. Kochba, and C.H. Bornman. 1974. Fine
structure of and embryoid development from embryogenic
ovular callus of 'Shamouti' orange (Citrus sinensis
Osb.). J. Exp. Bot., 25: 446-457.
15. Button, J., and F.H.J. Rijkenberg. 1977. The effect
of subculture interval on organogenesis in callus
cultures of Citrus sinensis. Acta Hort., 78: 225-236.
16. Cameron, J.W., and H.B. Frost. 1968. Genetics,
breeding, and nucellar embryony. Iji W. Reuther, L.D.
Batchelor, H.J. Webber (eds.). The Citrus Industry,
Vol. 2, University of California, Berkeley,
California., pp. 325-370.
17. Cameron, J.W., and R.K. Soost. 1979. Sexual and
nucellar embryony in F^ hybrids and advanced crosses of
Citrus with Poncirus. 1J. Amer. Soc. Hort. Sci.,
104: 408-410.
18.Cardy, B.J., C.W. Stuber, and M.M. Goodman. 1981.
Techniques for starch gel electrophoresis of enzymes
from maize (Zea mays L.). Institute of Statistics
Mimeograph Series, No. 1317, North Carolina State
Univ., Raleigh.
19.Chaturvedi, H.C., and G.C. Mitra. 1974. Clonal
propagation of citrus from somatic cell cultures.
HortScience, 9: 118-120.
D'Amato, F. 1978. Chromosome number variation in
cultured cells and regenerated plants. In T.A. Thorpe
(ed.), Frontiers of Plant Tissue Culture, International
Assoc. Plant Tissue Culture, Calgary, pp. 287-295.
20.


126
greatest variance when plants were grouped by embryo source,
and the ME group had the greatest variance when plants were
grouped by initiation treatment. The primary plant group
was not different from the callus or nucellar groups, but
the callus group was determined to be significantly more
variable than the nucellar seedlings. The probability of a
greater F value for the primary and nucellar comparison was
0.06 (Table 5.9); therefore, the primary group variance was
significantly greater than that of nucellar seedlings at the
10% level. When plants were grouped by initiation treat
ment, the callus group was found to be less variable than
the ME group but more variable than the 2,4-D or nucellar
groups; the latter groups were not significantly different
from each other. Because several plants regenerated from
secondary embryos on ME (light) had extreme values for blade
ratio, and the variances of these groups were thus
increased, both embryo source and media growth components
were seen to have influenced the variability of leaf shape.
However, there was no clear indication with the other
characters studied that any of the specific initiation
treatments as embryo sources (e.g. secondary embryos from
ME-light) were associated with such distinctly different
levels of variation. Whether the increased variation in
this specific subgroup was truly a treatment effect or
simply a statistical aberration has not been decided, but
the evidence from other characters suggests that the latter
alternative is a possibility. Regardless of the resolution


32
horticulturally valuable variants among juvenile, seedling
progeny are lacking. Plants must still be grown to maturity
to evaluate economically important characteristics. The
appearance of such valuable variants among regenerated
Citrus plants has yet to be demonstrated.


74
Embryo development, from the microscopic, undifferentiated
form to the macroscopic, cotyledonary stage was the first
efficiency-limiting step of the Citrus plant regeneration
system. Approximately 3 out of 4 embryos produced did not
successfully complete the step.
The second requirement for successful plant regenera
tion was balanced germination of the embryo (i.e. propor
tional elongation of both root and shoot structures). Most
of the embryos that were placed on the germination medium
underwent normal root elongation, but overall, fewer of the
embryos underwent normal shoot development. Some embryos
exhibited root development only and rarely survived transfer
to soil because their root systems degenerated before shoot
elongation occurred. Likewise, there were embryos that
exhibited shoot development but not root development,
although the number in this category was lower than the
former. Repeated subculturing on the germination medium did
not induce shoot or root development, respectively, among
the recalcitrant embryos. Histological studies of non
germinating embryos were not performed. Such studies might
have determined whether the unresponsiveness of non
germinating embryos was related to anatomical abnormalities
of meristematic regions, or whether other factors (perhaps
suboptimal growth regulator components or concentrations, or
genetic factors) were involved with the suppression of
germination. Ultimately, the failure of embryos to success
fully complete the second step of the regeneration process


Table 3.1. Effects of cultivar and culture conditions on the percenage of undeveloped
Citrus ovules that produced embryos. Fruit were harvested approximately
8 months after anthesis. Cultures were scored 56 days after initiation.
Cultivar
Treatment (mg
l'V
500.0 ME
Light
500.0 ME
No light
0.01 2,4-D
Light
0.01 2,4-D/
0.1 BA
Light
0.01 2,4-D/
0.1 DZ
Light
Hamlin orange
48
35
38
15
42
Pell Navel orange
71
42
50
44
73
Pineapple orange
53
43
54
44
50
Orlando tngelo
20
28
12
7
9
Marsh grapefruit
7
7
17
14
5
Owari satsuma
0
0
0
0
0
Key lime
13
28
11
7
14
Bearss lemon
0
3
0
0
0
aEach cultivar x treatment combination utilized 120 ovules except Orlando
(2,4-D = 100 ovules).
un


94
of these analyses were the identification of individuals
beyond the normal range of values, and comparisons of
variability among groups for these characters. Plant vigor
was expressed as plant height (cm)/age (months since trans
fer to soil). The denominator for vigor of nucellar
seedlings was 14 months, for plants from primary embryos was
10 months, for plants from secondary embryos was 7 months,
and for plants derived from embryogenic callus was 10 or
7 months (depending on the date of embryo isolation and
subsequent transfer to soil). Mean internode length (MIL)
was determined by measuring 3 internodes per plant to
identify extreme plants and to determine whether vigor
differences among plants were related to differences of mean
internode length. Leaf petiole and blade length/width
ratios were calculated for each plant (3 leaves per plant
measured along the mid-rib and perpendicular to the mid-rib
at the widest point on the blade and petiole) to screen the
populations for leaf shape variants. Low ratio values were
indicative of a more rounded leaf shape, and high ratio
values were indicative of a more narrow and elongate shape.
Plants were grown in the greenhouse until a synchronous
growth flush had occurred among all plants to minimize
environmental influences on leaf petiole or blade shape or
internode size. The leaves and internodes measured were
selected from the mid-region of the most recent flush of
shoot growth so consistent comparisons of plants for these
characters could be made. Leaf blade and petiole L/W ratios


Figure 3.2.
Initiation of embryogenic callus from cultured
ovules.


Dr. Paul Lyrene, for challenging thoughts and statis
tical advice;
Dr. Prem Chourey and D. L. Curt Hannah, for their time
and effort on my behalf;
Vicki Vaughn and Anne Harper, without whose technical
assistance and collective sense of humor this work
would not have been possible;
Steve Hiss, for help with the computing of statistics;
Katherine Williams, for diligent preparation of the
manuscript;
The many friends that we have met during our years in
Gainesville, in the Fruit Crops department (staff and
fellow students) and around town, for their kind
support, assistance, and sharing of good times. Arlene
and I will cherish for life the memories of knowing you
all.
in


81
percentage of germinated embryos that survive transfer
to soil and acclimatization.


61
optimal for embryo and callus production, depending on
cultivar. Although no ovules of that age were cultured in
this research, the results obtained suggested that more
response may be expected from ovules taken from more mature
fruit. In addition, 2,4-D inhibited embryogenesis in ovules
from 8-week fruit, but not in ovules from 12-week fruit
(Results: Experiment 2). Embryogenesis by 'Ponkan'
mandarin (C. recticulata) nucellus cultures was inhibited by
concentrations of 0.1 mg 1 ^ or greater (119,120). However,
the addition of 2,4-D to the culture medium was required for
embryogenesis from C. limn ovules (111). In the research
with 'Hamlin' orange, 2,4-D inhibition of embryo production
was observed among 8-week ovules only. No requirement for
2,4-D was demonstrated by any of the cultivars used, includ
ing 'Bearss' lemon (C. limn) ovules.
Embryogenic callus production was also affected by the
stage of fruit development at the time of ovule isolation,
and by cultural conditions. Embryogenic callus was gen
erated from cultured 12-week ovules on 2,4-D/BA or 2,4-D/DZ
supplemented media (but not on MT or ME); from 5-month
ovules on 2,4-D, 2,4-D/BA, or 2,4-D/DZ; and from 8-month
ovules on all media. No embryogenic callus resulted from
cultured 8-week 'Hamlin' ovules on any media. In contrast,
Vardi et al. (131) reported that embryogenic callus arose in
from 2 to 24% of the ovules from 7 Citrus cultivars that
were cultured on ME supplemented medium 2-6 weeks after


5
individual chapters. The final chapter will summarize and
relate the conclusions of this project to the general body
of research on somaclonal variation in plants.


132
old at the time of regeneration. Investigations of factors
that influence the level of embryo and/or callus production
are necessary on an individual cultivar basis to optimize
the regeneration system for specific varieties. Particular
emphasis should be placed on the enhancement of initiation
processes. The failure of certain polyembryonic types
(e.g., 'Owari' satsuma) to respond under the culture con
ditions described may be a challenge to the curious.
Successful research in this area would expand the range of
responsive Citrus cultivars. Fine-tuning of the tissue
culture system would most likely result in the ability to
produce more embryos with greater ease from a wider range of
genotypes.
The majority of the embryos initially produced did not
survive the regeneration process to become whole, growing
plants. Many embryos developed abnormally in culture or
were incapable of normal germination. Several that did
germinate failed to survive the transfer from the controlled
in vitro environment to the variable and challenging exter
nal environment. The age of the cultures from which the
plants were regenerated and cultivar differences did not
effect regenerability. As with culture initiation and
embryo production, fine-tuning of the tissue culture system
may allow greater numbers of embryos to follow the normal
course of development and germination. Improved methods of
transferring germinated embryos to the external environment
would result in greater plant production. There was some


65
Chapter III, and to observe plant development from the
embryonic stage to establishment in soil, paying particular
attention to factors that limit the efficiency of the
system. Phenotypic stability of regenerated plants will be
explored in Chapter V.
Materials and Methods
Embryo production from cultured unfertilized ovules and
embryogenic callus was described in Chapter III. When
primary embryos had reached a sufficient size (5-10 mm long)
and stage of development (possessing cotyledons and root and
shoot primorida), they were transferred to MT medium
supplemented with 1.0 mg 1 1 GA^ to induce germination. The
number of embryos that exhibited either root or shoot growth
and the number of germinated embryos was recorded 3-4 weeks
after transfer to the germination medium. The criterion for
germination was balanced elongation of both root and shoot
structures. When root length reached 2.5-5.0 cm and some
amount of shoot elongation had occurred, the germinated
embryos were transferred to pots containing a commercial
soil mix composed of sphagnum peat moss, horticultural grade
vermiculite and perlite, composted pine bark, and washed
granite sand. Humidity was maintained at a high level
initially by covering transplants in pots with polyethylene
bags. The bags were cut open every few days with increasing
size cuts to result in a gradual decrease in humidity levels
and to produce acclimated plants. Transplants were held in


Table 5.3. Electrode and gel buffer systems used for electrophoretic evaluation of
'Hamlin' orange plants.
System
Electrode buffer9
Gel buffer9
Lithium borate/
Tris-citrate (104)
LBTC
0.016 M LiOH
0.192 M boric acid
0.0016 M LiOH
0.019 M boric acid
0.007 M citric acid
0.046 M Tris
Histidine (18)
H
0.065 M histidine
0.02 M citric acid
pH 5.7 with citric acid
0.009 M histidine
0.003 M citric acid
pH 5.7 with citric acid
Tris-borate
TB
0.038 M Tris
0.002 M citric acid
pH 8.6
0.03 M boric acid
pH 8.5
Tris-citrate
TC
0.05 M Tris
0.016 M citric acid
pH 7.0 with citric acid
0.017 M Tris
0.005 M citric acid
pH 7.0 with citric acid
a10 mg NADP added to buffer in anodal tank and to gel buffers of all systems to enhance
the clarity of stains that contained NADP.


54
48 plates supplemented with 2,4-D/BA and 27 of 40 plates
supplemented with 2,4-D/DZ did contain active, proliferating
embryogenic callus at 240 days.
Experiment 3: Growth and Habituation of and Embryo
Production by 'Hamlin' Orange Embryogenic Callus
The origin of 11 selected embryogenic callus lines and
the fresh weight increase of these lines in grams and
expressed as a percentage of initial explant weight is shown
in Table 3.7. Callus lines initiated on MT medium supple
mented with 1.0 mg 1 1 2,4-D/0.1 mg 1 1 BA displayed the
greatest percentage increase in fresh weight. There was no
clear pattern observed among the other callus lines for rate
of proliferation.
Table 3.8 lists fresh weight increase and the number of
cotyledonary embryos produced in callus sublines cultured on
2,4-D/DZ or DZ supplemented media. Although some differ
ences were observed among sublines in the rate of fresh
weight increase, the number of embryos produced by the
different sublines varied little. Differences observed
among lines initially for the magnitude of fresh weight
increase (Table 3.7) did not persist through subsequent
observations (Table 3.8).
Table 3.9 describes the result of a subjective visual
examination of callus subline proliferation and embryo
production on 3 different media. No further quantitative
information was collected on callus proliferation. However,


136
were analyzed (vigor, internode length, and leaf shape) are
presumably polygenic in nature. There is a greater possi
bility of mutations occurring for traits conditioned by
several genes than for single gene traits because of the
greater number of loci associated with polygenic traits.
The variation observed for these traits suggests that
perhaps one or several mutations may have taken place at
loci influencing the expression of said traits. Likewise,
it is possible that the dwarf growth habit or altered leaf
shapes observed on occasion are due to single gene mutations
at a critical locus. Unfortunately, genetic studies of
these variants were precluded by time limitations.
Comparisons of morphological variability among groups
afforded the only real opportunity to determine whether
2,4-D, culture age, or embyrogenic pathway had any effect on
the level of variation expressed among regenerate popu
lations. It was found that 2,4-D in the culture media had
no influence on phenotypic stability. Whether the variants
observed resulted from pre-existent or de novo mutations was
difficult to determine. Future work in this area should
follow individual plants from their origin to evaluation to
determine if uniform or variable plant types arise from any
given ovule or subsequent callus colony. However, the fact
that a greater degree of variability was observed among the
tissue culture populations suggests that either the variants
observed resulted from de novo mutational events, or the in


149
115.Swartz, H.J., G.J. Galletta, and R.H. Zimmerman.
1983. Field performance and phenotypic stability of
tissue culture-propagated thornless blackberries. J_.
Amer. Soc. Hort. Sci., 108: 285-290.
116. Tanksley, S.D. 1983. Molecular markers in plant
breeding. Plant Mol. Biol. Rep., _1: 3-8.
117. Tatum, J.H., R.E. Berry, and C.J. Hearn. 1977.
Separation of nucellar and zygotic citrus seedlings by
their flavonoids and other non-volatile components.
Proc. Int. Soc. Citriculture, 2: 614-616.
118. Teich, A.H., and P. Spiegel-Roy. 1972. Differ
entiation between nucellar and zygotic citrus
seedlings by leaf shape. Theor. Appl. Genet.,
42: 314-315.
119. Thomas, E., S.W.J. Bright, J. Franklin, V.A.
Lancaster, B.J. Miflin, and R. Gibson. 1982. Varia
tion amongst protoplast-derived potato plants (Solanum
tuberosum cv. 'Maris Bard'). Theor. Appl. Genet.,
62: 65-68.
120. Tisserat, B., and T. Murashige. 1977a. Probable
identity of substances in citrus that repress asexual
embryogenesis. In Vitro, 13: 785-789.
121. Tisserat, B., and T. Murashige. 1977b. Repression of
asexual embryogenesis in vitro by some plant growth
regulators. In Vitro, 13: 799-805.
122. Torres, A.M., R.K. Soost, and U. Diedenhofen. 1978.
Leaf isozymes as genetic markers in citrus Am. J.
Bot., 65: 869-881.
123. Torres, A.M., R.K. Soost, and T. Mau-Lastovicka.
1982. Citrus isozymes. J. Hered., 73: 335-339.
124. Torrey, J.G. 1967. Morphogenesis in relation to
chromosomal constitution in long-term plant tissue
cultures. Physiol. Plant, 20: 265-275.
125. Vallejos, C.E. 1983. Enzyme activity staining. In
S.D. Tanksley and T.J. Orton (eds.), Isozymes in Plant
Genetics and Breeding, Part A, Elsevier, Amsterdam,
pp. 469-516.
126. Vardi, A. 1977. Isolation of protoplasts in citrus.
Proc. Int. Soc. Citriculture, 2: 575-578.


150
127. Vardi, A. 1981. Protoplast derived plants from
different citrus species and cultivars. Proc. Int.
Soc. Citriculture, pp. 149-152.
128. Vardi, A., and D. Raveh. 1976. Cross-feeder experi
ments between tobacco and orange protoplasts. Z.
Pflanzenphysiol., 78; 350-359.
129. Vardi, A., and P. Spiegel-Roy. 1978. Citrus breeding,
taxonomy and the species problem, ^n P.R. Cary (ed.),
Proc. Int. Soc. Citriculture, Sydney, Australia, pp.
51-57.
130. Vardi, A., P. Spiegel-Roy, and E. Galun. 1975.
Citrus cell culture: Isolation of protoplasts,
plating densities, effect of mutagens, and regen
eration of embryos. Plant Sci. Lett., 4^: 231-236.
131. Vardi, A., P. Spiegel-Roy, and E. Galun. 1982. Plant
regeneration from Citrus protoplasts: Variability in
methodological requirements among cultivars and
species. Theor. Appl. Genet., 62: 171-176.
132. Vasil, I.K. V. Vasil, C. Lu, P. Ozias-Akins, Z. Haydu,
and D. Wang. 1982. Somatic embryogenesis in cereals
and grasses. In E.D. Earle and Y. Demarly (eds.),
Variability in Plants Regenerated from Tissue Culture,
Praeger, New York, pp. 3-21.
133. Wakasa, K. 1979. Variation in the plants differenti
ated from the tissue culture of the pineapple. Japan
J. Breed., 29: 13-22.
134. Wenzel, G., O. Scheider, T. Przewozny, S.K. Sopory,
and G. Melchers. 1979. Comparison of single cell
culture derived Solanum tuberosum L. plants and a model
for their application in breeding. Theor. Appl.
Genet., 55: 49-55.
135. Zhang, W.C. 1981. Thirty years achievements in
citrus varietal improvement in China. Proc. Int. Soc.
Citriculture, pp. 51-55.


Table Page
4.2. Germination of embryos and survival of plants
from embryogenic cultures of several Citrus
cultivars 69
4.3. Germination of embryos and survival of plants
from embryogenic cultures of several Citrus
cultivars by initial treatment 71
4.4. Effect of initial treatment on regeneration
survival percentages of several Citrus
cultivars 73
5.1. Origin and number of plants in groups used for
studies of phenotypic stability of regenerated
'Hamlin' orange plants 86
5.2. Number of Citrus plants regenerated from
primary and secondary embryos of several
cultivars that were evaluated for isozyme
banding patterns, and total number of zymo
grams evaluated 88
5.3. Electrode and gel buffer systems used for
electrophoretic evaluation of 'Hamlin' orange
plants 90
5.4. Enzyme stain systems utilized for electro
phoretic evaluation of 'Hamlin' orange plants
and number of major bands observed 93
5.5. Summary of group means, standard deviations,
and coefficients of variation for growth rate,
mean internode length, petiole L/W ratio, and
leaf blade L/W ratio of 'Hamlin' orange plants 105
5.6. Summary of F-tests and T-tests comparing growth
rates of the following groups of 'Hamlin'
orange plants 106
5.7. Summary of F-tests and T-tests comparing mean
internode length of groups of 'Hamlin' orange
plants 107
5.8. Summary of F-tests and T-tests comparing
petiole L/W ratios of groups of 'Hamlin' orange
plants 108
5.9. Summary of F-tests and T-tests comparing leaf
blade L/W ratios of groups of 'Hamlin' orange
plants 109
Vlll


8
seed-propagated diploid, did not fall into the above cate
gories. Within the past decade, numerous reports of variant
phenotypes appearing among populations of regenerated plants
of other seed-propagated diploid species have been published
(9,23,24,29,35). Tissue culture-associated variability,
therefore, is not limited to polyploid or vegetatively
propagated species.
Many of the reports of variant plant phenotypes
resulted from direct observation of the original regenerated
plants (41,42,67,68,105,106,119). For variant plants
expressing traits of potential genetic or commercial impor
tance to be of use in plant breeding programs, the observed
variation must be heritable. (A possible exception to this
requirement may be clonally propagated plants, where non-
heritable variation could be of use if the trait is
expressed in a stable manner following vegetative propaga
tion. However, even with such plant material, the observed
variation(s) must be genetic in nature if the desired trait
is to be incorporated into new or different genetic back
grounds.) Reports have appeared recently, with increasing
frequency, that document the heritability, and therefore the
genetic basis, of several somaclonal variant traits among
various plant genera. For example, Gengenbach et al. (35)
selected fertile, T-toxin resistant maize (Zea mays L.)
plants from susceptible, male-sterile material that was put
into tissue culture and subjected to sub-lethal selection
pressure with the toxin produced by Helminthosporium maydis.


16
produced uniform plants. (The research described in Chap
ter V of this dissertation indicates that this conclusion
may not always be true.) Shepard (106) reported high levels
of variability among regenerated 'Russet Burbank' potato
plants (a tetraploid cultivar released in the early 1900s),
but Wenzel et al. (134) observed no variation among plants
regenerated from dihaploid potato material. It was sug
gested that perhaps the different results were related to
differences in the age of the clone used as starting mate
rial, genetic differences, or perhaps technique differences.
Thomas et al. (119), however, used Wenzel's technique with a
young clone ('Maris Bard', released in 1974) and observed
significant variability. It seems in this case that the
different results are likely related to genetic differences
between normal tetraploid and dihaploid material. The above
examples illustrate that differences in genetic background,
regardless of the nature of those differences, may influence
the amount of variation observed among regenerated plants.
Several manipulable parameters of the tissue culture
process have been shown to influence the amount of variabil
ity observed. The nature of the explant source is one such
parameter. Navarro et al. (80) reported that in vitro shoot
tip graft propagation of Citrus produces trees that are true
to type, but plants produced from cultured nucelli (of
monoembryonic cultivars) were variable. However, it is
possible that these differences may be related to the method
of regeneration (organogenesis vs. embryogenesis) (132).


80
developed normally, percentage of embryos that
germinated, and the percentage of germinated embryos
that survived transfer to soil and acclimatization.
However, in overall regeneration survival percentage
(i.e. percentage of embryos placed on the germination
medium that became established plants), no cultivar
differences were observed, except for the less-respon
sive 'Pineapple' orange.
5. Embryos produced by ME/dark and 2,4-D/DZ initiation
treatments had the greatest regeneration survival
percentages. No differences were observed among
treatment groups for normal embryo development or
germination. The greatest difference among treatment
groups was observed for the percentage of germinated
embryos that survived transplanting and acclimatiza
tion.
6. Future research should focus on the effects of manipu
lation of physical and chemical parameters of the
tissue culture system to increase the percentage of
responsive ovules, the percentage of embryos that
develop normally, and the percentage of germinating
embryos. Histological studies would be of use in
determining whether germination failure is related to
anatomical abnormalities. Experiments on soil pathogen
and humidity control, and soil mix components may lead
to development of a procedure that increases the


110
values. It was assumed that populations with greater
variances were composed of a higher frequency of variant
types with more extreme values and a lower frequency of the
normal type, and that the effects of environmental compo
nents of variance were equal for all groups. Consequently,
the comparisons of variances (F-tests) were interpreted as
measures of phenotypic stability.
Growth rate
The conclusions of the series of t-tests listed in
Table 5.6 designed to compare major group means for growth
rate were as follows. Plants from primary embryos were the
most vigorous, followed by nucellar seedlings. The mean
growth rate of plants from secondary embryos was not signif
icantly different from that of plants from embryogenic
callus. Both groups of plants were less vigorous than
plants from primary embryos or nucellar seedlings. When
plants were divided into groups according to the presence of
ME or 2,4-D in the initiation media, it was found that the
differences in vigor observed between plants from primary or
secondary embryos were averaged out because all group means
were determined to be not significantly different, except
that, as above, plants derived from embryogenic callus were
less vigorous than nucellar seedlings. Therefore, the
composition of the culture initiation media had no effect on
relative plant vigor, but the first embryos produced from
cultured undeveloped ovules on any media were more vigorous


31
variant were found among regenerated somaclones. The idea
here is that all of the desirable qualities of the parent
clone would be retained, with the addition of another
valuable trait. Most currently grown Citrus cultivars arose
as spontaneous nucellar or bud sport mutations, and were not
the result of controlled hybridization. The production of
variant somaclones from established cultivars may allow many
more of these mutant phenotypes to be screened. A less
ambitious but equally valuable hope--the production of
unique genetic or breeding linesmay be more likely to be
realized.
Citrus is a genus that provides unique opportunities to
study factors involved in the expression of somaclonal
variation. Numerous routes to plant regeneration are
available, with various manipulations of culture parameters
possible. Consequently, there are many possible comparisons
of the effects of these factors on the level of phenotypic
stability observed among regenerated Citrus plants. Fur
thermore, the production of nucellar seedlings by poly-
embryonic types provides a control population to compare in
vitro and in vivo induced variability. A thorough assess
ment of the variation resulting from the different regen
eration pathways may provide the information necessary to
control Citrus phenotypic stability. Uniform or variant
populations of plants could be regenerated, then, depending
on the specific desired result. One limitation to the use
of this technology is the fact that methods of identifying


Table 5.8. Summary of F-tests and t-tests comparing petiole L/W ratios of groups of
'Hamlin' orange plants. Abbreviations used for groups are as listed in the
title of Table 5.6.
Comparison
A vs. B
F
df
P>Fa
A:Bb
t
df
P> t C P
i:Bb
1
2
1.27
66+59
0.3504
=
-6.08
125
0.0001
-
1
C
1.15
66 + 65
0.5806
=
-5.27
131
0.0001
-
1
N
1.35
66+99
0.1779
=
1.61
165
0.1097
=
2
C
1.11
65+59
0.6919
=
0.87
124
0.3841
=
2
N
1.06
59+99
0.7871
=
8.85
158
0.0001
+
C
N
1.17
65 + 99
0.4663
=
7.91
164
0.0001
+
ME
2,4-D
1.27
65+60
0.3526
=
-1.52
125
0.1309
=
ME
C
1.46
65+65
0.1286
=
-2.98
130
0.0034
-
ME
N
1.72
65+99
0.0151
+
3.54
113.6
0.0006
+
2,4-D
C
1.15
60 + 65
0.5744
=
-1.45
125
0.1499
=
2,4-D
N
1.35
60 + 99
0.1817
=
5.83
159
0.0001
+
Probability of
a greater
F value.
bIf
A>B, then +;
if A then -,
if A=B, then

Q
Probability of
a greater
absolute
value of
t.
108


produced would benefit to both the propagator and the
geneticist.


CHAPTER
Page
VI SUMMARY 131
LITERATURE CITED 139
BIOGRAPHICAL SKETCH 151
vi


77
regeneration survival percentage, with the exception of
'Pineapple' orange.
It might be expected that the regeneration survival
percentage of secondary embryos or embryos from long-term
callus cultures would be lower than that of primary embryos
because of accumulated polyploidy or aneuploidy resulting
from increased culture age. McCoy et al. (71) reported that
the frequency of cytogenetically abnormal regenerated plants
of Avena sativa increased with culture age. Long-term
Citrus embryogenic callus has been reported to remain
diploid, except when initiated on media containing 2,4-D
(131). Some of the primary and secondary embryos and all of
the long-term cultures that were used for plant regeneration
studies were exposed to 2,4-D at some point during the
culture process. However, no differences in survival rates
were observed among the 3 groups of 'Hamlin' orange embryos.
As with embryo development, culture age had no major influ
ence on regeneration survival percentage.
Kochba et al. (58) studied the effects of various
cultural manipulations on embryo germination and found that
GA^ and adenine sulphate stimulated root initiation and
development. Spiegel-Roy and Vardi (111) developed a
procedure for embryo development and plant regeneration for
Citrus that includes several transfers to solid or liquid
media supplemented variously with sucrose, galactose,
adenine, GA^, and malt extract. After plantlets reach a
certain size they are cultured on paper bridges in tubes for


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.
. Sherman, Chairman
Professor
Fruit Crops Department
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.
GL TVohcR^2
Gloria A. Moore, Cochairman
Assistant Professor
Fruit Crops Department
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.
Agronomy/Plant Pathology
Department


60
phenomena in ovules from the other cultivars. As pointed
out previously, 'Bearss' lemon produced embryos and callus
only in the absence of light. Although a 16-hour photo
period has been standard for Citrus embryogenic culture
initiation (111), the results of this research suggest that
light or its absence may play a critical role in the
initiation of embryogenic cultures. However, continued
subculturing in darkness became detrimental to embryo
production and development and callus proliferation;
cultures maintained in darkness produced fewer normal and
more abnormal embryos, more and larger pseudobulbils, a
watery non-differentiating callus growth, and non
differentiating integumentary callus that interfered with
embryogenic callus isolation. So, although dark culture
enhanced initial embryo production, it was important to
expose initiated cultures to light as soon as possible (no
later than 1 month after culture initiation) to encourage
normal embryo development and callus proliferation.
The stage of fruit development at which undeveloped
ovules were excised had a dramatic effect on the embryogenic
response of 'Hamlin' orange. Fruit harvested 8 weeks after
anthesis yielded ovules that were the least responsive of
any of those cultured in the various experiments with this
cultivar. The number of embryos produced per responsive
ovule from such fruit was less than that of 12-week or
8-month ovules, as well. Spiegel-Roy and Vardi (111) have
suggested that fruits harvested 4 weeks after anthesis are


Table 4.2. Germination of embryos and survival of plants from embryogenic cultures of
several Citrus cultivars.
Embryos
placed
Germinated
Established
Regeneration
Genotype
on GA-
embryos.
plants
survival
No.
%
No.
%
No.
%c
%3
Pell Navel orange
230
24
131
57
79
60
34
Pineapple orange
96
15
57
59
21
37
22
Marsh grapefruit
100
34
78
78
36
46
36
Orlando tngelo
79
41
45
57
27
60
34
Key lime
45
17
20
44
17
85
38
Bearss lemon
Hamlin orange
9
53
4
44
3
75
33
Primary embryos
174
35
84
48
56
67
32
Secondary embryos
184

105
57
69
66
38
Callus-derived embryos
123

79
64
48
61
39
Totals
Primary embryos
(all genotypes)
Secondary and callus-
733
26
419
57
239
57
33
derived embryos
(Hamlin)
307

184
60
117
63
38
Grand total
1040

603
58
356
59
34
£
(Number of embryos placed
on GA3
medium/total number of
primary embryos)
x 100.
(Number of germinated embryos/number of
embryos placed <
on GA^ medium) x
100.
0
(Number of established plants/number of
germinated
embryos) x 100.
^(Number of established plants/number of embryos placed on GA^ medium) x 100.
<£>


85
maintained under standard practices of irrigation, ferti
lization, and pest control.
A total of 11 groups of plants were produced from
embryogenic cultures for the studies described in this
chapter. The origin and number of these plants are listed
in Table 5.1. A control group of 100 nucellar seedlings was
randomly selected from a population of 350 seedlings pro
duced by G.A. Moore. 'Hamlin' orange flowers were emascu
lated and hand pollinated in March, 1983 with pollen of
Poncirus trifoliata. Seeds were collected and planted in
October, 1983. Sexual hybrids were eliminated on the basis
of expression of the dominant trifoliate leaf trait; the
remaining seedlings were presumed to be nucellar. The
seedlings were grown in the unshaded greenhouse in the same
soil mix as the other plants. Plants produced from embryo
genic cultures were not evaluated electrophoretically until
they had undergone at least one growth flush in the unshaded
greenhouse. Morphological evaluations of all plants were
delayed until at least 2 common growth flushes had occurred.
Cytogenetic Characterization of Regenerated Plants
Mitotic chromosome counts of 34 nucellar seedlings and
51 plants randomly selected from all tissue culture popula
tions were made to assess the cytogenetic stability of
regenerated 'Hamlin' orange plants. Root tips were har
vested from actively growing plants between 0800 and 1000


9
Brettell et al. (8) selected similar plant types from
cultured maize that had not been challenged with T-toxin.
In these reports, the T-toxin resistance and male fertility
were shown to be cytoplasmically inherited. Evidence of
heritable nuclear gene mutations among various plant genera
has also been forthcoming. Evans and Sharp (29,30) have
reported the occurrence of 13 nuclear gene mutations among
230 cellus-derived tomato plants (Lycopersicon esculentum L.
Mill.). Both recessive and dominant mutations were observed
among progeny of the original regenerated plants, and these
mutations were demonstrated to be heritable. Allelism of
some of the mutations to other known mutant alleles was also
demonstrated (30). Sun et al. (113) reported a rice soma-
clone exhibiting a dwarf growth habit; this trait was shown
to be heritable and controlled by a single recessive gene.
Rice plants variant for heritable quantitative or cytoplas
mically controlled characters were reported as well. These
true breeding variants support the idea that at least some
of the variation observed among somaclones is, indeed,
genetic in nature.
Mutations of alleles from the dominant to the recessive
condition cannot be detected directly in the primary regen
erates, unless the mutated locus was heterozygous initially.
Consequently, the progenies resulting from self-pollination
of primary regenerates have been screened to uncover masked
recessive mutations. Prat (89) observed unique plant
phenotypes among progeny of self-pollinated, primary


70
'Marsh' grapefruit. The regeneration survival percentage
(percentage of primary embryos placed on germination medium
that developed into established plants) for each cultivar is
shown in Table 4.2. The survival percentages for individual
cultivars clustered about the 34% total survival percentage
and ranged from 32-38%; 'Pineapple' orange was the exception
with 22% survival.
The regeneration survival percentages of primary,
secondary, and embryogenic callus-derived embryos of
'Hamlin' orange were compared in Table 4.2. The data
suggest that there were no differences among the 3 groups.
The difference between the survival percentages of primary
embryos of all cultivars and 'Hamlin' secondary and callus
derived embryos was small (33% vs 39%, respectively). On
the whole, 1 of 3 embryos placed on the germination medium
survived the regeneration process. Overall, approximately 1
of 12 primary embryos produced originally from cultured
ovules became an established plant (239 of 2870).
The initiation treatment used to produce embryos may
have had some influence on plant survival (Table 4.3).
Embryos produced from either ME/dark or 2,4-D/DZ treatments
underwent successful regeneration and establishment with
greater frequency than embryos arising from other treat
ments. No clear differences among treatments were observed
for the percentages of primary embryos that were judged
visually to be developmentally normal (and placed on


Table 3.4. Effect of cultivar and culture conditions on secondary proliferation from
undeveloped Citrus ovules. Fruit were harvested 8 months after anthesis.
Cultures were scored 56 days after initiation.
Cultivar
Treatment (mg 1
-i,*.
500.0 ME
Light
500.0 ME
No light
0.01 2,4-D
Light
0.01 2,4-D/
0.1 BA
Light
0.01 2,4-D/
0.1 DZ
Light
Hamlin orange
2 5b
25
2 5b
15b
29b
Pell Navel orange
47
44
35
38
50
Pineapple orange
39
41
47
22b
29b
Orlando tngelo
17b
26
14b
16
17
Marsh grapefruit
1
5
5
4
1
Owari satsuma
7
5
5
3
5
Key lime
8b
13b
11
5
10b
Bearss lemon
1
4b
0
0
0
aEach cultivar x treatment combination used 120 ovules except Orlando
(2,4-D = 100 ovules).
bThese combinations produced enbryogenic callus lines that persisted at least 180 days.
te*
*3


Figure 3.1. Proliferation of cotyledonary embryos and
proembryos from a cultured ovule.


84
Cytogenetic stability of plants was evaluated by
mitotic chromosome counts made with root tips, to determine
if any aneuploid or polyploid plants were produced.
Electrophoretic evaluation of plants was undertaken to
determine if any variation at the level of specific,
chemically defined genetic loci could be detected. Finally,
vegetative characteristics of regenerated plants were
quantified and statistically analyzed to examine the stabil
ity of regenerates for gross morphological traits. The
variability observed among populations of regenerated
plants, for all characters, was compared with the variabil
ity of the nucellar seedling population.
Materials and Methods
General Remarks
The studies of phenotypic stability of regenerated
Citrus plants were limited to 'Hamlin' orange because of the
availability of plants derived from primary and secondary
embryos and from embryogenic callus that had been maintained
for 18 months prior to embryo isolation and plant regenera
tion. The conditions of plant establishment were described
in Chapter IV. Acclimated plants were placed in a shaded
greenhouse for several weeks prior to placement in an
unshaded greenhouse. Plants were transplanted into pots
(15 cm diameter) in the soil mix described previously, and


134
internode segments or organogenic callus) have indicated a
great range of responsiveness depending on species or
cultivar, and culture conditions. Unreported preliminary
work by this investigator in the area of organogenic plant
regeneration of 'Hamlin' orange using previously published
techniques yielded very few successful cultures. Culture
conditions suited to the species (or cultivar) cultured must
be developed to improve the efficiency of this regeneration
system so that the questions described above can be
approached.
The purpose of the studies of phenotypic stability of
'Hamlin' orange plants was to identify cytogenetic, elec
trophoretic, or morphologic variants among regenerated
plants and nucellar seedlings. Comparisons of the level of
variation observed within individual groups allowed deter
minations of the effect of 2,4-D in the culture media,
culture age at the time of plant regeneration, indirect vs.
direct embryogenesis (i.e., with or without a callus stage),
and in vitro vs. _in vivo adventive embryogenesis on pheno
typic stability. All plants for which chromosome counts
were made, including morphological variants, possessed the
diploid number of chromosomes. Counts should be made on the
remainder of the populations because a low frequency of
altered chromosome number requires the analysis of more
plants to detect variants. Chromosome counts should be made
of the callus tissue, as well, to determine whether any more
aberrancy exists among cells in culture than among


response varied with cultivars, as did the most effective
treatments for embryo production and callus initiation.
Greater levels of response resulted when 'Hamlin' orange
ovules were extracted from more mature fruit (5-8 mo) than
from immature fruits (2-3 mo). Primary and secondary embryo
production was common, but long-term embryogenic callus
proliferation was less frequent. Although not always
necessary, 2,4-D (0.01 mg 1 ^) enhanced embryogenic callus
production from 'Hamlin' orange. Callus exhibited
habituation to growth substances in the media.
Plant regeneration was limited by failure of embryos to
undergo normal morphogenesis and balanced germination, and
of plants to survive transfer to the external environment.
Of 2,870 embryos initially produced, 733 underwent normal
development, and 239 survived regeneration. Culture age did
not affect regenerability, but initiation treatment may have
influenced final survival rates.
Regenerate and nucellar 'Hamlin' seedlings were
characterized by chromosome number, electrophoretic profile,
and vegetative morphology. No chromosome number or isozyme
variants were identified. Morphologically variant plants
(vigor, internode length, leaf shape) were observed among
all groups, but more extreme variants were found among
tissue culture groups. Variant phenotypes were observed
more frequently in the tissue culture groups, as well.
However, no specific culture treatment was identified that
consistently generated more variable plants.
Xlll


CHAPTER VI
SUMMARY
Plants were produced from in vitro embryogenic cultures
of several Citrus species. The initiation of cultures,
subsequent development and germination of embryos leading to
plant production, and the phenotypic stability of the
regenerated plants were studied. The results of those
studies have been described and discussed in the preceding
chapters. The objectives of this chapter are to summarize
briefly the results, with an emphasis on areas where addi
tional research might prove fruitful, and to relate the
conclusions regarding the degree of phenotypic stability
observed among regenerated Citrus plants to the general
knowledge of somaclonal variation in plants.
It was possible to initiate embryogenesis (direct and
indirect) from undeveloped ovules of several Citrus
cultivars and species with varying levels of success,
depending on cultivar and media, light or dark culture, and
fruit age at the time of ovule extraction. Embryogenic
callus was produced frequently, but long-term proliferation
was rare. Media amended with 2,4-D were shown sometimes to
enhance the production of long-term callus lines. Plants
were produced from callus lines that were at least 18 months
131


93
Table 5.4. Enzyme stain systems utilized for electro
phoretic evaluation of 'Hamlin' orange plants
and number of major bands observed.
Stain
Buffer
system
No. major
bands
No. loci
'Hamlin'
Genotype
ACP
TC
3
7
7
GOT
LBTC, TB
2
2b
SS, MM
GPI
H
3
lb
FS
G6PD
LBTC, TB
2
Ia
?d
IDH
TC
3
lb
MI
LAP
H, TC
3
lb
FS
MDH
H
2
2b
FF, FF
ME
TC
2
2C
II, ?
PER
LBTC, TB
1
Ia
FF
PGD
H
6
p
7
SDH
LBTC, TB
4
Ia
MS
£
Moore, unpublished data.
^Torres et al. (123)
Q
R.K. Soost, personal communication.
Band fixed in all Citrus types examined.


ACKNOWLEDGEMENTS
The following dissertation, though it lists the name of
one individual as author, is the product of the energy and
concern of several people. Without their encouragement,
counsel, and assistance, this manuscript would not exist. I
want to extend my sincere appreciation and heartfelt grati
tude to the following:
Arlene, my wife, for her unbounded enthusiasm, unself
ish support, encouragement, typing, and especially
love, throughout my years as a student;
Katherine and Fred Gmitter, Sr., my parents and the
rest of our family, for childhood encouragement, and
their prayers and pride;
Dr. Gloria Moore, friend and adviser, for the oppor
tunity to explore and to accomplish, for advice and
counsel, sharing of knowledge, and invaluable assis
tance with the preparation of this manuscript;
Dr. Wayne Sherman, for friendship, advice, humor, and
assistance;
11


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF TABLES vii
LIST OF FIGURES ix
KEY TO ABBREVIATIONS X
ABSTRACT xii
CHAPTER
I INTRODUCTION 1
II LITERATURE REVIEW 6
Introduction 6
Somaclonal Variation 7
Citrus Tissue Culture 19
Characterization of Seedling Citrus Plants . 27
Conclusion 30
III EMBRYO PRODUCTION AND ESTABLISHMENT OF
EMBRYOGENIC CALLUS CULTURES FROM UNDEVELOPED
CITRUS OVULES 33
Introduction 33
Materials and Methods 34
General Information on Tissue Culture
Methods 34
Experiment 1: Production of Embryos and
Embryogenic Callus from Undeveloped
Ovules of Several Citrus Species ... 35
Experiment 2: Production of Embryos and
Embryogenic Callus by Undeveloped
Ovules from Immature Fruit of 'Hamlin'
Orange 36
Experiment 3: Growth and Habituation
of and Embryo Production by 'Hamlin'
Orange Embryogenic Callus 37
IV


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.
~2 e
L.C. Hannah
Professor
Vegetable Crops Department
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.
Professor
Fruit Crops Department
This dissertation was submitted to the Graduate Faculty of
the College of Agriculture and to the Graduate School, and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.
December, 1985
Dean, Graduate School


47
cultured on ME medium in the absence of light; the same
treatment was the most effective for embryo production from
'Key' lime. No single treatment that was most efficient for
primary embryo production from the different Citrus types
could be identified.
The number of primary embryos produced per ovule
cultured, by treatment within cultivar, is given in
Table 3.3. The relative ranking of cultivars and genotypes
is identical to that observed in Tables 3.1 and 3.2; these
values are listed and embryo production expressed thus for
comparisons of production efficiency between this experiment
and Experiment 2. Table 3.3 also lists the number of
embryos produced per responsive ovule. Among the oranges,
the treatments producing the greatest numbers of embryos per
responsive ovule were 2,4-D/DZ and ME (light), the same
treatments that produced the greatest percentages of respon
sive ovules and consequently the highest total number of
embryos produced. The values for embryos per responsive
ovule for 'Orlando' tngelo and 'Key' lime were of the same
magnitude as the values of the orange cultivars. Although
fewer ovules of 'Marsh' grapefruit and 'Bearss' lemon
produced embryos (Table 3.1), more embryos were produced per
responsive ovule by these cultivars.
The percentage of undeveloped ovules in each cultivar X
treatment combination where proliferating callus was present
after 56 days is listed in Table 3.4. The oranges were the
most responsive cultivars for ovular callus production.


56
Table 3.8. Fresh weight increase and cotyledonary embryo
production by 'Hamlin' orange embryogenic callus
sublines. Callus was cultured on MT medium
supplemented with 0.01 mg 1 2,4-D/0.1 mg 1
DZ (A) or 0.1 mg 1 1 DZ alone (B). Cultures
were evaluated after 15 days.
Subline
Initial explant
weight
Fresh weight increase No.
g %
embryos
A1
0.149
0.441
296
13
A2
0.060
0.501
850
12
A3
0.326
0.705
216
16
A4
0.084
0.631
751
14
A5
Lost
to contamination
A6
0.226
0.742
328
13
A7
0.286
0.849
297
15
A8
0.140
0.537
384
14
A9
0.394
0.700
178
8
A10
0.129
0.956
741
12
All
0.147
0.827
563
16
Mean
= 13.
30
B1
0.216
0.828
383
15
B2
0.058
0.537
926
9
B3
0.125
0.732
586
11
B4
Lost
to contamination
B5
0.159
1.105
695
11
B6
0.271
0.679
251
7
B7
0.239
1.218
510
15
B8
0.131
1.079
824
14
B9
Lost
to contamination
BIO
Lost
to contamination
Bll
0.184
0.617
335
12
Mean
= 11.
75


13
induced mutation and/or variation has been abundant
(67,85,96). Consequently, the remainder of this review will
focus not on basic causes, but rather on specific factors
that have been directly or indirectly associated with the
expression of variability among plants from tissue culture.
These factors can be placed into 2 groups: genetic or
tissue culture-related factors. The genetic factors involve
the actual genetic background of the material cultured,
including ploidy levels and changes thereof. Tissue culture
related factors include explant source, media composition,
culture age at the time of plant regeneration, and the
actual pathway of regeneration (i.e., direct or indirect
organogenesis vs. embryogenesis).
Murashige (75) assumed that the appearance of aberrant
phenotypes among regenerated plants was the result of either
polyploidy or aneuploidy in the regenerated plants. In
fact, much of the variation reported has been shown to be
related to changes in chromosome number and/or structure.
Latunde-Dada and Lucas (69) observed a positive correlation
between the degree of tolerance to Verticillium wilt among
regenerated alfalfa (Medicago sativa L.) plants and regener
ate ploidy level. They concluded that this variation was
the result of increased gene dosage and not a mutational
event. Prat (89) reported that among initial regenerated N.
sylvestris plants, the diploids were phenotypically similar
to the parent, but tetraploids were not. However, as
pointed out previously, these apparently normal diploids


CITRUS EMBRYOGENESIS IN VITRO:
CULTURE INITIATION, PLANT REGENERATION,
AND PHENOTYPIC CHARACTERIZATION
BY
FREDERICK GEORGE GMITTER, JR.
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
1985


121
revealed no significant differences. The MIL of secondary
embryo derived plants from 2,4-D initiation medium was
greater than the MILs of secondary 2,4-D/BA- or 2,4-D/DZ-
initiated plant groups. No differences were revealed among
other subgroup means. The relative equality of subgroup
means and variances provided greater statistical validity to
the conclusions drawn from comparisons of main group
variances.
Petiole L/W ratio
Petiole L/W ratios were calculated to quantify and
compare the petiole shapes that were observed. Greater
ratios were indicative of petioles that were either normally
winged but abnormally long, wingless but of normal length,
or both wingless and longer (thus generating the greatest
L/W ratio). Lesser ratios were indicative of petioles of
normal length but with wide wings, of shorter length but
normally winged, or both shorter length and wider wings.
Differences between mean petiole L/W ratios were noted when
plants were grouped according to embryo sources (Table 5.7).
The mean ratios of the secondary embryo and callus-derived
groups were significantly greater than primary embryo or
nucellar seedling group means. However, no significant
differences between the means of each of the pairs above
were revealed. When plants were grouped according to
initiation treatment, all tissue culture groups had greater
petiole ratios than the nucellar seedlings. The 2,4-D group


128
groups, may be genetic variants; also, in vitro embryo-
genesis produced variant types with greater frequency than
in vivo somatic embryogenesis. Determination of the genetic
mechanism(s) responsible for morphological variation was not
possible with this juvenile Citrus plant material.
Culture age at the time of embryo isolation affected
mean values of all parameters studied. Growth substances in
the initiation media, however, did not influence character
means and had no effect on the observed variability of
regenerated plants, except for leaf blade shape (which may
have been a statistical anomaly). The age of cultures from
which plants were regenerated had significant effects on
observed variation, but no overall general trend was
obvious. No tissue culture treatment consistently produced
more variable plants for all traits studied. Generally, all
tissue culture groups were significantly more variable
phenotypically than the nucellar seedlings (except for
petiole L/W ratio). The statistical studies confirmed the
subjective visual observation of greater variability among
the tissue culture plants compared with nucellar seedlings.
Although morphological variation was observed and
statistically described, certain aspects of the evaluation
of juvenile plants must be qualified. The differences noted
were observed among juvenile plants, and although plants had
undergone several growth flushes before observations were
made, the persistence of morphological differences as the
plants mature has yet to be seen. Additionally, the


BIOGRAPHICAL SKETCH
Frederick George Gmitter, Jr., was born February 2,
1951, in Hazleton, Pennsylvania. He was educated in the
public school system of Linden, New Jersey, where he was
graduated with honors in 1969.
Mr. Gmitter attended Rutgers University in New
Brunswick, New Jersey, from 1969 until 1972. In 1972, he
met Arlene Krawczyk; they were married in 1973. He worked
at several positions until 1977, when he re-entered Rutgers
to earn a Bachelor of Arts degree in English that was
awarded in 1978. One year was spent as a non-matriculating
student at Cook College prior to admission, in 1979, to the
graduate program of the Department of Horticulture and
Forestry of Rutgers. He worked and studied under L.F. Hough
and was awarded a Master of Science degree in 1982, for
research involving the influence of plant growth regulators
on vegetative and reproductive growth of seedling fruit
trees. Gmitter was awarded the N.F. Childers Award for
Outstanding Student in Pomology in 1978. While at Rutgers,
he served as field evaluator for the fruit breeding program
(1979-1981), field assistant in Pomology Teaching Plots
(1979), research assistant (1980-1981), and course
coordinator for Systematic Pomology (1981).
151


52
the ME/no light regime (0.12 embryos/ovule cultured).
Embryogenesis was suppressed by the addition of 2,4-D to the
medium. When the response level of ovules from such fruit
was compared with the greatest efficiency of embryo
production from 'Hamlin' ovules at 8 months after anthesis
(1.21 embryos/ovule cultured on ME/light regime), it was
noted that the older ovules were approximately ten times
more productive. The numbers of embryos produced per
responsive ovule also decreased in this experiment, when
compared with the results of Experiment 1. The production
of secondary embryos or embryogenic callus was not observed
in this experiment.
Embryo production under various culture conditions by
undeveloped ovules from immature 'Hamlin' orange fruit
harvested 12 weeks after anthesis is described in Table 3.6.
Embryos were produced on all media, but the response level
was again less than that of 8-month ovules (See Results:
Experiment 1). Treatment differences were not apparent for
the percent of ovules responding, but there were differences
in efficiency of embryo production (0.20 vs. 0.06 embryos/
i
ovule cultured for ME vs. MT) and in the number of embryos
produced per responsive ovule among treatments (2.46 vs.
1.20 for ME vs. MT). No 2,4-D induced suppression of
embryogenesis was observed among 12-week ovules. None of
the ME-initiated cultures produced proliferating embryogenic
callus 240 days after culture initiation, but 31 of


142
33. Frost, H.B., and R.K. Soost. 1968. Seed reproduction:
Development of gametes and embryos. In W. Reuther,
L.D. Batchelor, and H.J. Webber (eds.), The Citrus
Industry, Vol. 2, University of California, Berkeley,
Calif., pp. 290-324.
34. Furusato, K., Y. Ohta, and K. Ishibusi. 1957. Studies
on polyembryony in Citrus. Sieken Ziho, 40-48.
35. Gengenbach, B.G., C.E. Green, and C.M. Donovan. 1977.
Inheritance of selected pathotoxin resistance in maize
plants regenerated from cell cultures. Proc. Nat.
Acad. Sci. (USA), 74: 5113-5117.
36. Grinblat, U. 1972. Differentiation of citrus stems in
vitro. J. Amer. Soc. Hort. Sci., 97: 599-603.
37. Hearn, C.J. 1973. Development of scion cultivars of
citrus in Florida. Proc. Fla. State Hort. Soc.,
8£: 84-88.
38. Hearn, C.J. 1977. Recognition of zygotic seedlings in
certain orange crosses by vegetative characters. Proc.
Int. Soc. Citriculture, 611-614.
39. Hearn, C.J. 1985. Development of seedless grapefruit
cultivars through budwood irradiation. (Abst.)
HortScience, 20: 562.
40. Heinz, D.J. M. Krishnamurthi, L.G. Nickell, and A.
Maretzki. 1977. Cell, tissue, and organ culture in
sugarcane improvement. In J. Reinert and Y.P.S. Bajaj
(eds.), Applied and Fundamental Aspects in Plant Cell,
Tissue, and Organ Culture, Springer-Verlag, Berlin,
Heidelberg, New York, pp. 3-17.
41. Heinz, D.J., and G.W. P. Mee. 1969. Plant differ
entiation form callus tissue of Saccharum species.
Crop Sci. 346-348.
42. Heinz, D.J., and G.W.P. Mee 1971. Morphologic,
cytogenetic, and enzymatic variation in Saccharum
species hybrid clones derived from callus tissue. Am.
J. Bot., J58: 257-262.
43. Hensz, R.A. 1981. Bud mutations in citrus cultivars
in Texas. Proc. Int. Soc. Citriculture, pp. 89-91.
44. Hodgson, R.W. 1968. Horticultural varieties of
citrus. In W. Reuther, H.J. Webber, and L.D. Batchelor
(eds.). The Citrus Industry, Vol. 1, University of
California, Berkeley, Calif., pp. 431-591.


95
were selected for evaluation because they have been shown to
be consistent, despite environmental influences on leaf size
(38) .
Mean values and variances were calculated for the
following groups of plants for these 4 characters:
1) the nucellar seedling population;
2) plants from embryos from embryogenic callus;
3) plants from primary embryos from all initiation
treatments;
4) plants from secondary embryos from all initiation
treatments;
5) plants from ME medium (primary and secondary
embryos);
6) plants from 2,4-D media (primary and secondary
embryos).
Means and variances were also calculated for each of the
individual groups listed in Table 5.1. Comparisons of group
means and variances were made in a pairwise fashion.
F-tests were performed to compare group variances, and
Student's t-test was used to compare means. Satterthwaite's
approximation for degrees of freedom was used when variances
were unequal (102). All calculations and statistical tests
were performed by computer according to SAS procedures
(102).
Two levels of data organization were analyzed. First,
the nucellar seedling population was used as the standard of
comparison in F-tests with the 5 groups of plants listed


10
regenerates of Nicotiana sylvestris Spegaz et Comes that
were not present in the original regenerated plants, thus
indicating that recessive mutations were present, but
masked, in the regenerates. Likewise, Engler and Grogan
(24) reported 3:1 segregations among progeny of self-
pollinated regenerated lettuce (Lactuca sativa L. spp.
capitata); they interpreted these segregations as an indi
cation of heritable, single gene mutations. Somaclonal
variation is not limited to traits under the control of
single genes; appropriate use of statistical techniques has
revealed that variants for quantitative traits exist, as
well. Larkin et al. (66) reported heritable mutations for
quantitative traits such as plant height and heading date,
as well as dominant and recessive mutations for qualitative
traits in regenerated wheat (Triticum aestivum L.) plants
and their progeny. As more attention is paid to genetic
analysis of regenerated plant material in the future, it may
be expected that more of the variants for quantitatively
inherited traits will be demonstrated to be the result of
heritable mutations. Any valuable characters demonstrated
to be heritable may be incorporated, then, into plant
breeding programs.
A question regarding the origin of somaclonal variation
is whether recovered variant plant types are the result of
genetic variability that is pre-existent within cell popu
lations of the initial explant tissue or of de novo muta
tions occurring in the in vitro environment. Although


37
20 ovules each) were cultured on each of the following
amended media: 1) 500.0 mg l-1 ME, 2) 0.01 mg I-1
2,4-D/0.1 mg 1_1 BA, and 3) 0.01 mg l-1 2,4-D/0.1 mg l-1 DZ;
an additional 100 ovules were cultured on MT (unamended
basal medium). Embryo production was evaluated 28 days
after culture initiation. Cultures were subcultured on the
treatment media every 4-8 weeks. The number of plates with
embryogenic callus after 240 days was recorded.
Experiment 3: Growth and Habituation of and Embryo
Production by 'Hamlin' Orange Embryogenic Callus
Undeveloped ovules were harvested from 'Hamlin' orange
fruit in August, 1982 (about 5 months after anthesis) and
cultured on basal medium supplemented with various concen
trations and combinations of 2,4-D, BA and DZ (Moore and
Gmitter, submitted). Embryogenic callus lines were selected
from the following treatments for further study in this
project: 1) 0.01 mg 1 ^ 2,4-D/0.01 mg 1 ^ BA, 2) 0.01 mg
l-1 2,4-D/0.1 mg l-1 BA, and 3) 1.0 mg l-1 2,4-D/0.1 mg l"1
BA. The selected cultures were transferred from the initia
tion media to MT supplemented with 0.01 mg 1 1 2,4-D/0.1 mg
1 1 DZ and subcultured at 4-6 week intervals until March,
1983. On that date, 5 sublines were isolated from treat
ment 1 above, 3 sublines were isolated from treatment 2, and
2 sublines were isolated from treatment 3. The tissue
sector comprising each subline was selected on the basis of
morphology and color (white, friable callus; brownish-tan
callus; or green proliferating masses of embryos) with the


99
development should be made. Such counts could be used to
determine if chromosome number variants exist in undiffer
entiated cell populations or among developing embryos, and
whether such variants are eliminated during regeneration due
to incompetence or the inability to compete successfully
with cells or embryos with normal chromosome numbers.
However, the information that may be garnered from mitotic
analysis is limited to changes in chromosome number.
Meiotic analysis, on the other hand, has the potential to
reveal chromosome structural arrangements (inversions,
deletions, translocations, etc.). Such structural changes
have been observed among cultured cells and regenerated
plants of several genera (79,84,85,114). Many of the
regenerated 'Hamlin' plants will be maintained until
flowering to allow for such meiotic studies.
Electrophoretic Evaluation
The second approach to plant characterization was to
screen all plants for mutations at specific, chemically
defined loci. Each of the 100 'Hamlin' orange nucellar
seedlings was evaluated at least once for each of the
11 enzyme staining systems for a minimum of 1100 nucellar
zymograms observed. A total of 2642 zymograms were eval
uated for the 'Hamlin' orange plants produced from all
embryogenic cultures. Zymogram uniformity was observed with
each enzyme system for all plants with the exceptions noted
below. Evaluation of 928 zymograms of 5 other polyembryonic


68
grapefruit and 100% (9 of 9) of 'Bearss' lemon embryos. The
shoot elongation response ranged from 44% (4 of 9) of
'Bearss' lemon embryos to 80% (80 of 100) of 'Marsh' grape
fruit embryos. The percentage of embryo germination (both
root and shoot development) ranged from 44% of 'Key' lime
and 'Bearss' lemon embryos to 78% of 'Marsh' grapefruit
embryos.
Embryo developmental abnormalities were observed, such
as pluricotyly, multiple shoot meristem proliferation,
embryo fusion, fasciation, and pseudobulbils. These abnor
malities often inhibited the normal course of plant regener
ation. For example, although 'Hamlin' orange produced
502 primary embryos (Table 3.1), only 174 (35%) of these
embryos exhibited sufficiently normal development to warrant
transfer to the germination medium (Tables 4.1, 4.2).
Cultivar differences were evident for the percentage of
embryos that were judged to be sufficiently normal. These
values ranged from 15% for 'Pineapple' embryos to 53% for
'Bearss' lemon embryos (Table 4.2). The number and per
centage of embryos that germinated when placed on GA-
supplemented medium and the number and percentage of
germinated embryos that survived transfer to soil and
acclimatization to become established plants are also listed
in Table 4.2. Germination has been discussed above. The
percentage of germinated embryos that survived the transfer
and acclimatization step of the regeneration process was 60%
or greater for all cultivars except 'Pineapple' orange and


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
CITRUS EMBRYOGENESIS IN VITRO:
CULTURE INITIATION, PLANT REGENERATION,
AND PHENOTYPIC CHARACTERIZATION
By
FREDERICK GEORGE GMITTER, JR.
December, 1985
Chairman: Wayne B. Sherman
Cochairman: Gloria A. Moore
Major Department: Horticultural Science (Fruit Crops)
Citrus cultivar improvement via standard breeding
methods has been difficult, so that alternative approaches
have been suggested. Potentially useful variation has
arisen among plants of other genera that have undergone a
cycle of tissue culture regeneration. This study was
undertaken to assess the degree of stability of parental
phenotype expression among Citrus plants produced by in
vitro embryogenesis. Also, the factors affecting embryo and
embryogenic callus production and development, embryo
germination, and plant regeneration were studied for several
cultivars.
Cultures were initiated with unfertilized ovules placed
on various modifications of MT medium. The degree of
Xll


12
one isolated protoplast. McCoy et al. (71) have pointed out
the possibility, at least in the case of chromosomal insta
bility, that somatic plant tissue may be composed of hetero
geneous cell populations when compared with homogeneous germ
cell lines. The culture of somatic tissues may, therefore,
result in chromosomally aberrant plants that are "reflective
of normal chromosome instability" found in whole plant
tissues (71) However, the same researchers also reported
that the frequency of chromosomal aberration in regenerated
plants increased with increased time in culture, thus
providing support for the de novo hypothesis. In contrast,
Navarro et al. (80) have suggested that the variant Citrus
plants they obtained via in vitro somatic embryogenesis from
monoembryonic Citrus types were not the result of mutations
that occurred during the tissue culture process because the
plants produced from individual cultured nucelli were either
uniformly normal or aberrant. Therefore, until more conclu
sive, definitive studies are undertaken, the true nature of
the origin of somaclonal variants (i.e., de novo vs. pre
existent mutations) will remain uncertain. However, the
evidence available at present suggests that, in most
instances, somaclonal variation is a result of de novo
mutation.
Several review articles have been published that
compile the evidence for and the history of the phenomenon
of somaclonal variation; furthermore, speculation on the
basic nature of the mechanisms responsible for in vitro


97
1) To quantify the morphological variation that was
observed visually within groups and among indi
vidual plants;
2) To objectively evaluate and compare the extent of
variation observed among the different groups of
regenerated plants;
3) To determine whether observed variability for
character expression was related to differences in
tissue culture parameters or embryo source.
Results and Discussion
Cytological Evaluation
The first approach to plant characterization was to
determine whether the in vitro regimen resulted in plants
with chromosome number changes. The objectives of the
cytogentic characterizations were to determine if observed
morphological variation was related to changes at the whole
chromosome level (number, but not structure) and to deter
mine if media components or culture age had any effect on
mitotic fidelity. Aneuploids and polyploids have been found
in nucellar populations (4,16,45,82). Vardi (126) found
that one 'Shamouti' orange plant of 10 regenerated from
embryogenic callus that arose from isolated protoplasts was
tetraploid, but the other 9 were diploid. No data regarding
chromosome number of polyembryonic Citrus plants regenerated
from ovules or embryogenic callus have been published, but


Table 3.7. Origin and fresh weight increase of 'Hamlin' orange embryogenic callus lines.
Cultures were subcultured following initiation on MT medium supplemented with
-1 -1
0.01 mg 1 2,4-D/O.l mg 1 DZ. Fresh weight increase was determined after
28 days.
Line No.
Initiation medium
2,4-D/BA (mg l-1)
Initial explant
weight (g)
Fresh weight
g
increase
%
1
0.01/0.01
0.183
2.019
1103
2
0.01/0.01
0.194
1.908
984
3
0.01/0.01
0.352
2.384
677
4
0.01/0.01
0.171
0.643
376
5
0.01/0.01
0.143
1.080
755
6
0.01/0.01
0.248
1.095
441
7
0.01/0.1
0.401
2.218
553
8
0.01/0.1
0.278
2.774
998
9
0.01/0.1
0.417
2.248
539
10
1.0/0.1
0.017
0.941
5535
11
1.0/0.1
0.019
1.217
6405


CHAPTER
Page
Results 38
General Comments 38
Experiment 1: Production of Embryos and
Embryogenic Callus from Undeveloped
Ovules of Several Citrus Species ... 44
Experiment 2: Production of Embryos and
Embryogenic Callus by Undeveloped
Ovules from Immature Fruit of 'Hamlin'
Orange 50
Experiment 3: Growth and Habituation
of and Embryo Production by 'Hamlin'
Orange Embryogenic Callus 54
Discussion 58
Conclusion 62
IV EMBRYO DEVELOPMENT, GERMINATION, AND PLANT
ESTABLISHMENT 64
Introduction 64
Materials and Methods 65
Results 66
Discussion 72
Conclusions 79
V CHARACTERIZATION OF CITRUS PLANTS REGENERATED
FROM EMBRYOGENIC CULTURES 82
Introduction 82
Materials and Methods 84
General Remarks 84
Cytogenetic Characterization of
Regenerated Plants 85
Electrophoretic Characterization of
Regenerated Plants 87
Morphological Characterization of
Regenerated Plants 92
Results and Discussion 97
Cytological Evaluation 97
Electrophoretic Evaluation 99
Morphological Evaluation 103
Growth rate 110
Mean internode length 118
Petiole L/W ratio 121
Leaf blade L/W ratio 124
Conclusions 129
v


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1.Altman, A., and R. Goren. 1978. Development of citrus
bud explants in culture. J.Amer. Soc. Hort. Sci.,
103: 120-123.
2. Barlass, M., and K.G.M. Skene. 1982. In vitro plant-
let formation from Citrus species and hybrids.
Scientia Hort., 17: 333-341.
3. Barrett, H.C. 1978. Intergeneric hybridization of
citrus and other genera in citrus cultivar improvement.
Proc. Int. Soc. Citriculture, 2: 586-589.
4. Barrett, H.C., and D.J. Hutchison. 1978. Spontaneous
tetraploidy in apomictic seedlings of Citrus. Econ.
Bot., 32: 27-45.
5. Ben-Hayyim, G., and J. Kochba. 1983. Aspects of salt
tolerance in a NaCl-selected stable cell line of Citrus
sinensis. Plant Physiol., 72: 685-690.
6. Ben-Hayyim, G., A. Shani, and A. Vardi. 1982. Eval
uation of isozyme systems in Citrus to facilitate
identification of fusion products. Theor. Appl.
Genet., 64: 1-5.
7. Bono, R., L.F. de Cordova, and J. Soler. 1981.
'Arrufatina,1 'Esbal' and 'Guillermina,'
three Clementine mandarin mutations recently discovered
in Spain. Proc. Int. Soc. Citriculture, pp. 94-96.
8. Brettell, R.I.S., E. Thomas, and D.S. Ingram. 1980.
Reversion of Texas male-sterile cytoplasm maize in
culture to give fertile, T-toxin resistant plants.
Theor. Appl. Genet., 58: 55-58.
9. Browers, M.A., and T.J. Orton. 1982. Transmission of
gross chromosomal variability from suspension cultures
into regenerated celery plants. J. Hered.,
73: 159-162.
10.Burk, L.G., and J.F. Chaplin. 1980. Variation among
anther-derived haploids from a multiple disease-
resistant tobacco hybrid. Crop. Sci., 20: 334-338.
139


Ill
than any other group of plants. Additionally, comparisons
of subgroups within major groups (i.e. the second level of
analysis described in Materials and Methods) revealed no
significant differences for mean values for any of the
comparisons.
Figure 5.1 shows a plant type (thin pointed leaves,
short internodes, and low vigor) that was observed among
regenerated plants. Plants of this type rarely survived.
Examples of normal and abnormal vigor and leaf shapes are
shown in Figure 5.2 and 5.3. The 'Hamlin' orange plants
used for these photographs were the same age and originated
from the same initiation treatment. Examples of extreme
types such as this could be found in each of the groups of
regenerated plants and in the nucellar seedling population.
However, the growth rate extremes observed in each of the
groups of plants from embryogenic cultures usually exceeded
those observed in the nucellar population (except the
minimum value for plants from primary embryos and the
maximum values of plants from either secondary embryos or
embryos from 2,4-D media). Therefore, in terms of minimum
and maximum growth rates, the groups of plants produced from
embryogenic cultures were more variable because they con
tained more extreme types.
The F-tests performed to compare growth rate variances
(Table 5.6) indicated that the groups of plants from primary
embryos and embryogenic callus were equally variable, but
the secondary embryo group was less variable than the former


91
5) GPI = glucosephosphate isomerase (D-Glucose-6-phosphate
ketol isomerase, E.C.5.3.1.9) 100 mg Na2 fructose-6-
phosphate, 10 mg NADP, 20 mg MTT, 4 mg PMS, 100 mg
MgCl2, and 10 units of glucose-6-phosphate dehydro
genase in 100 ml 0.1 M Tris HC1, pH 8.0;
6) GOT = glutamate oxaloacetate transaminase (Aspartate
aminotransferase, L-aspartate:2-oxoglutarate amino
transferase, E.C.2.6.1.1) 500 mg L-aspartic acid,
70 mg a-keto glutaric acid, 50 mg pyridoxal 5' phos
phate, and 200 mg Fast Blue BB salt in 0.1 M Tris HC1,
pH 8.0;
7) IDH = isocitrate dehydrogenase (Threo-D-isocitrate:
NADP+ oxidoreductase E.C.1.1.1.42) 200 mg Na^ iso-
citric acid, 30 mg NADP, 30 mg NBT, 4 mg PMS, and
100 mg MgCl2 in 0.1 M Tris HC1, pH 8.0;
8) LAP = leucine amino peptidase (Aminoacy1-peptide
hydrolase, E.C.3.4.11.1) 40 mg L-leucine-g-naph-
thylamide HC1, 100 mg Black K salt, 50 ml Tris malate
(0.2 M Tris, 0.2 M maleic anhydride), 30 ml H20, and
0.2 N NaOH (modified from Scandalios (104);
9) MDH malate dehydrogenase (L-Malate:NAD+ oxidore
ductase, E.C.1.1.1.37) 20 mg NAD, 20 mg NBT, 4 mg
PMS, 20 ml 0.1 M malic acid (pHed to 7.0 with NaOH),
10 ml 1.0 M Tris HC1 (pH 8.0), and 70 ml H20;
10) ME = malic enzyme (L-Malate:NADP+ oxidoreductase-
(oxalacetate-decarboxylating), E.C.1.1.4.0) 30 mg
NADP, 30 mg NBT, 4 mg PMS, 100 mg MgCl2, 20 ml 0.1 M


137
vitro regime was less stringent and allowed more pre
existing variability to be expressed in regenerated plants.
Although media differences had no influence on the variation
observed, differences of culture age and embryogenic pathway
(direct or indirect) did affect the level of variability.
However, there was no consistent trend to these observa
tions. Plants from primary or callus-derived embryos were
equally variable for all traits, but plants from secondary
embryos were either more or less variable than the others,
depending on the trait studied. Regeneration via an inter
mediate callus stage did not increase variability among
Citrus regenerates. These findings differ from the reports
that document increased variability among regenerated plants
following increased time in culture.
'Hamlin' orange plants produced by direct or indirect
embryogenesis were evaluated in this study. Additional
studies should focus on the stability of 'Hamlin' plants
regenerated via protoplast and organogenic techniques to
expand the assessment of somaclonal variation of Citrus.
Similar studies should be undertaken with other cultivars
and species to determine whether the genetic background of
the Citrus material placed into culture has any relation to
the level of phenotypic stability observed. These suggested
studies may generate the information necessary to control
whether uniform or variant plants are produced from Citrus
tissue culture. The ability to control the plant types


3
Several alternative approaches to the problem of Citrus
cultivar improvement have been attempted or suggested. For
example, the use of monoembryonic or slightly polyembryonic
types as seed parents can maximize the number of zygotic
progeny produced (108). The search for nucellar or bud
mutant selections is a part of Citrus improvement programs
throughout the world (7,22,43,48,98,135). Use has been made
of mutagenizing radiation, as well, in attempts aimed at
producing improved forms (39,108,129). Other suggested
methods that may increase the number of genetic combinations
that could be evaluated take advantage of plant tissue
culture technology and include zygotic embryo rescue (93),
cell selection (5), and somatic hybridization
(126,127,130). Numerous reports have been published within
the last decade of variant phenotypes and heritable
mutations arising among plants of diverse genera that have
been produced by tissue culture techniques (30,67,80);
Scowcroft and Larkin coined the term "somaclonal variation"
to describe this phenomenon (67). Some of the variant
plants exhibited improvement of economically important
traits and have been, therefore, incorporated into breeding
programs and commercial enterprises (40,65,68,72). Thus
far, most studies of the phenotypic stability of tissue
culture-produced plants have focused on herbaceous, annual
species. However, aberrant plant phenotypes were found
among Citrus plants regenerated from cultured monoembryonic
nucelli (80).


Figure 5.3. Example of normal and abnormal 'Hamlin'
seedling leaf blade and petiole shape. Notice
broadened petiole, rounded apex, and serration
of margins of the abnormal phenotype.


Figure 5.2. Example of low and normal vigor among regen
erated 'Hamlin' plants of the same age and from
the same treatment.


CHAPTER V
CHARACTERIZATION OF CITRUS PLANTS
REGENERATED FROM EMBRYOGENIC CULTURES
Introduction
The difficulties encountered when standard plant
breeding methods are employed for Citrus cultivar improve
ment were discussed in Chapter I, along with the need for
alternative approaches to cultivar improvement. The reports
of variant plant types arising among several genera after in
vitro regeneration or propagation have been presented and
discussed in Chapter II. The potential of somaclonal
variation as an alternative method of Citrus germplasm
enhancement has also been discussed, as has the need for
information on stability of regenerated plants for genetic
transformation research. The results of a series of
studies, designed to characterize Citrus plants regenerated
from embryogenic cultures and to determine if evidence of
variability among regenerated plants exists, are recorded in
this chapter.
The results of the studies performed on the regenerated
plants were compared with those obtained from identical
studies of a population of nucellar seedlings to determine
the extent of variability among the products of in vivo and
82


Figure 5.1. Abnormal plant type (thin pointed leaves, short internodes, low vigor)
observed among regenerated Citrus plants. Plants of this type were produced
only from tissue culture. They rarely survived transfer to soil.


118
groups. However, each of the tissue culture plant groups
had greater variances than the nucellar seedling population
for growth rate. The differences observed between primary
and secondary group variances averaged out when the plants
from cultured undeveloped ovules were grouped according to
initiation medium. When grouped in this manner, all tissue
culture plant group variances were equal, and all were
greater than the variance of the nucellar population.
Therefore, growth substance amendments in the initiation
medium had no influence on the amount of variability for
growth rate observed among groups of regenerated 'Hamlin'
orange plants, but embryo source (i.e. primary, secondary,
or callus-derived) did have an effect on the amount of
growth rate variation observed. F-tests performed with
variances of subgroups within main groups revealed no
significant differences. The equality of subgroup variances
and means provided greater statistical validity to the
conclusions drawn from the comparisons of main group vari
ances .
Mean internode length
The summary conclusion regarding the relative order of
groups for MIL depended on the way that plants from ovule
cultures were grouped (Table 5.7). When plants were grouped
according to embryo source, no significant difference for
MIL between the group from primary embryos and the nucellar
seedlings was observed; likewise, MILs of the secondary


129
seedling traits that were evaluated are not known to be
correlated with any trait of horticultural interest, except
perhaps the dwarf growth habit. However, Navarro reported
that morphological variants from monoembryonic 'Clementine'
mandarin nucellar cultures persisted to maturity and
following budwood propagation (80) It certainly may be
possible that a number of other variations will be expressed
in these plants at maturity. For example, variants for
length of juvenile period, fruit type and quality, yield,
etc. could be of genetic or commercial horticultural value.
It is anticipated that at least some of the regenerated
'Hamlin' orange plants will be grown to maturity to evaluate
economically important characteristics and to identify
potentially useful variants.
Conclusions
1. All plants for which chromosome counts were made,
regardless of origin, were diploid, but not all diploid
plants were morphologically uniform. Therefore, these
specific variants were not related to chromosome number
changes. No evidence of tissue-culture induced cyto
genetic instability was found among the plants evalu
ated.
No electrophoretic variants were observed among any
plant group. Therefore, no visible mutations occurred
at these specific loci. The absence of variant
2.




Table 3.2. Effect of cultivar and culture conditions on the number of primary embryos
produced by undeveloped ovules from Citrus fruit harvested 8 months after
anthesis. Embryos were counted 56 days after culture initiation.
Cultivar
Treatment
/ i 11 a
(mg 1 )
500.0 ME
Light
500.0 ME
No light
0.01 2,4-D
Light
0.01 2,4-D/
0.1 BA
Light
0.01 2,4-D/
0.1 DZ
Light
Total
Hamlin orange
145
93
97
35
132
502
Pell Navel orange
251
133
172
155
258
969
Pineapple orange
168
98
118
100
148
632
Orlando tngelo
55
69
23
18
29
194
Marsh grapefruit
27
54
115
80
19
295
Owari satsuma
0
0
0
0
0
0
Key lime
52
113
28
15
53
261
Bearss lemon
0
17
0
0
0
17
Total
698
577
553
403
639
2870
£
Each cultivar x treatment combination utilized 120 ovules except Orlando
(2,4-D = 100 ovules).
4^


53
Table 3.6. Effect of media amendments on embryo produc
tion by undeveloped ovules from 'Hamlin' orange
fruit harvested 12 weeks after anthesis.
Cultures were held under a 16 h photoperiod and
were evaluated 28 days after initiation.
Treatment (mg 1 *)
Percent3
responsive
Ovules
Number
embryos
produced
Number
embryos/
ovule
cultured
Number
embryo/
responsive
ovule
500.
0 ME
7.9
(960)b
187
0.20
2.46
0.01
2.4-D/0.1 BA
7.3
(960)
130
0.14
1.86
0.01
2.4-D/0.1 DZ
6.8
(900)
113
0.13
1.85
MT
5.0
(100)
6
0.06
1.20
aResponsive ovules are those from which embryos were
produced.
^Number in parentheses is the number of ovules evaluated.


67
Table 4.1. Germination and development of roots and shoots
from primary embryos of several Citrus cultivars
cultured on MT medium supplemented with 1.0 mg
l'1 GA3-
Cultivar
Embryos
: number
(percentage)
Cultured
Producing
roots
Producing
shoots
Producing
both
Hamlin orange
174
149
(86)
101
(58)
84
(48)
Pell Navel orange
230
210
(91)
142
(62)
131
(57)
Pineapple orange
96
86
(90)
62
(65)
57
(59)
Marsh grapefruit
100
97
(97)
80
(80)
78
(78)
Orlando tngelo
79
63
(80)
54
(68)
45
(57)
Key lime
45
31
(69)
21
(47)
20
(44)
Bearss lemon
9
9(100)
4
(44)
4
(44)
Total
733
645
(88)
464
(63)
419
(57)


75
(balanced germination) eliminated 43% of the embryos that
had survived the first step.
The ability of germinated embryos to survive transfer
from the tissue culture environment to soil and the external
environment was the third requirement for successful
regeneration. Although many embryos germinated, the balance
between root and shoot growth was not always favorable for
plant survival. Transplants with normal roots but minimal
shoot growth usually degenerated in soil before commencement
of shoot elongation, presumably because of the inability of
the shoot to support the energy demand for continued growth
and development. Likewise, transplants with normal or
excessive shoot development but minimal root growth rarely
survived, possibly because the roots could not supply
nutrients and water that were directly available to the
shoots previously from the culture medium. In general, only
those embryos that had balanced root and shoot growth
survived the transfer to soil. Once plants were actively
growing in soil, successful acclimatization to lower
humidity and greater light intensity was accomplished by
gradual exposure to both. Greater plant mortality resulted
from the transfer of germinated embryos to soil than from
acclimatization. When plant death did occur in this phase
of the regeneration process, it usually resulted from
degeneration in the absence of growth after germination and
transplanting or from soil-borne pathogens that attacked


CHAPTER IV
EMBRYO DEVELOPMENT, GERMINATION, AND
PLANT ESTABLISHMENT
Introduction
Although in vitro somatic embryogenesis has been
observed in numerous plant genera, information on the
survival of plants regenerated from embryogenic cultures has
rarely been published. Citrus is unique among economically
important woody perennial plants because, as discussed in
the Literature Review (Chapter II) and demonstrated in
Chapter III, many species of the genus are capable of in
vivo and in vitro somatic embryogenesis. Numerous schemes
have been proposed to utilize the embryogenic capability of
Citrus in plant improvement programs. Additionally, the
potential of mass in vitro propagation of Citrus has come
under scrutiny in Florida because of a replant shortage
following severe freezes and an outbreak of Citrus canker
(Xanthomonas campestri pv. citri). Consequently, there has
been interest in the efficiency of the regeneration process,
in the degree of plant survival, and in the phenotypic
stability of regenerated plants. The objectives of the
research detailed in this chapter were to regenerate plants
from the Citrus embryos produced in the studies described in
64


119
embryo and embryogenic callus groups were not significantly
different. However, the former pair had greater MILs than
the latter. There were no differences between primary and
secondary group means when plants were grouped on the basis
of initiation treatment, as was the case with growth rate
differences. The nucellar population had a significantly
larger MIL than the ME or 2,4-D groups, which did not differ
from each other. All groups had significantly larger MILs
than the embryogenic callus group. Growth substance compo
nents in the culture initiation media did not affect group
MILs, but significant differences between group means were
associated with embryo source.
Although 3 of the 6 plants from tissue culture iden
tified as having growth rates that differed significantly
from group means also had correspondingly different MILs,
not all individual plants that were of extreme relative
stature (i.e. growth rate) were correspondingly extreme for
MIL. However, a non-statistical perusal of the data compar
ing individual plant MILs and growth rates indicated that
shorter plants (i.e. those with lower growth rates) gen
erally had smaller MILs, and taller plants (i.e. those with
higher growth rates) had larger MILs. Several exceptions to
this generalization were noted in all groups of plants.
These differences of growth rate and internode length
resulted in visually obvious differences in terms of rela
tive plant size and/or foliage density among certain indi
vidual plants. Individual plants with MIL values less than


36
The number of ovules producing embryos directly without
an intermediate callus stage, the number of embryos pro
duced, and the number of ovules with proliferating callus
were recorded 56 days after culture initiation. Only
embryos that had reached cotyledonary or heart-shaped stages
of development were counted; these were designated primary
embryos. Further observation of cultures for embryo produc
tion and callus development were made. Embryos that devel
oped after primary embryos were removed from cultures for
germination (see Chapter IV) were designated secondary
embryos. It was not possible in all cases to determine
whether the secondary embryos originated via direct embryo-
genesis from the cultured ovules or from the small clumps of
embryogenic callus that began to proliferate.
Experiment 2; Production of Embryos and Embryogenic Callus
by Undeveloped Ovules from Immature Fruit of 'Hamlin'
Orange
Undeveloped ovules were isolated from immature 'Hamlin'
orange fruit harvested in April and May, 1984, 8 and
12 weeks after anthesis, respectively. In April, 120 ovules
(6 plates with 20 ovules each) were cultured on each of the
following amended media: 1) 500.0 mg 1 1 ME (16 h light),
2) 500.0 mg 1_1 ME (No light), 3) 0.01 mg l"1 2,4-D,
4) 2,4-D/0.1 mg 1_1 BA, and 5) 2,4-D/0.1 mg 1_1 DZ. The
number of responsive ovules (those producing embryos) and
the number of embryos produced were recorded 56 days after
culture initiation. In May, 1000 ovules (50 plates with


144
56. Kobayashi, S., H. Uchimiya. and I. Ikeda. 1983. Plant
regeneration from 'Trovita' orange protoplasts. Japan.
J. Breed, 33: 119-122.
57. Kochba, J., and J. Button. 1974. The stimulation of
embryogenesis and embryoid development in habituated
ovular callus from the 'Shamonti' orange (Citrus
sinensis) as affected by tissue age and sucrose concen
tration. Z. Pflanzenphysiol., 73: 415-421.
58. Kochba, J., J. Button, P. Spiegel-Roy, C.H. Bornman,
and M. Kochba. 1974. Stimulation of rooting of Citrus
embryoids by gibberellic acid and adenine sulfate.
Ann. Bot., 38: 795-802.
59. Kochba, J., and P. Spiegel-Roy. 1973. Effect of
culture media on embryoid formation from ovular callus
of 'Shamonti' orange (Citrus sinensis). Z.
Pflanzenzuchtg., 69: 156-162.
60. Kochba, J., and P. Spiegel-Roy. 1977a. Cell and
tissue culture for breeding and developmental studies
of citrus. HortScience, 12: 110-114.
61. Kochba, J., and P. Spiegel-Roy. 1977b. The effects of
auxins, cytokinins and inhibitors on embryogenesis in
habituated ovular callus of the 'Shamonti' orange
(Citrus sinensis). Z. Phlanzenphysiol., 81: 283-288.
62. Kochba, J., P. Spiegel-Roy, H. Neumann, and S. Saad.
1978. Stimulation of embryogenesis in citrus ovular
callus by ABA, Ethephon, CCC and Alar and its sup
pression by GA^. Z. Phlanzenphysiol. 89: 427-432.
63. Kochba, J., P. Spiegel-Roy, H. Neumann, and S. Saad.
1982. Effect of carbohydrates on somatic embryogenesis
in subcultured nucellar callus of Citrus cultivars. Z.
Pflanzenphysiol., 105: 359-368.
64. Kochba, J., P. Spiegel-Roy, and H. Safran. 1972.
Adventive plants from ovules and nucelli in citrus.
Planta, 106: 237-245.
65.Krishnamurthi, M., and J. Tlaskal. 1974. Fiji disease
resistant Saccharum officinarum var Pindar subclones
from tissue cultures. Proc. Int. Soc. Sugar Cane
Technol. lj>: 130-137.
Larkin, P.J., S.A. Ryan, R.I.S. Brettell, and W.R.
Scowcroft. 1984. Heritable somaclonal variation in
wheat. Theor. Appl Genet., 67: 443-455.
66.


71
Table 4.3. Germination of embryos and survival of plants
from embryogenic cultures of several Citrus
cultivars by initial treatment. Embryos and
plants from all cultivars were included in each
initial treatment group.
Embryos
placed Germinated Established Regeneration
Initial
treatment
on
No.
GA_
3a
embryos.
No. %D
plants
No.
%c
survival
ME (light)
174
25
94
54
49
52
28
ME (dark)
146
25
87
60
62
71
43
2,4-D
143
26
84
59
37
44
26
2,4-D/BA
130
32
68
52
27
40
21
2,4-D/DZ
140
22
86
61
64
74
46
g
(Number of embryos placed on GA. medium/total number of
primary embryos) x 100.
(Number of germinated embryos/number of embryos placed on
GA^ medium) x 100.
0
(Number of established plants/number of germinated embryos)
x 100.
(Number of established plants/number of embryos placed on
GA^ medium) x 100.


103
supports the ideas that the undeveloped ovules used to
initiate cultures were unfertilized and that the embryos
produced were of maternal origin. Likewise, the somatic
origin of the nucellar seedling population was supported.
'Hamlin' orange is heterozygous for alleles at at least 4 of
the enzymes used for plant evaluation (GPI, IDH, LAP, and
SDH) (Table 5.4). Assuming independent segregation at those
loci, about 94% of any plants resulting from self-pollina
tion could have been detected because their banding patterns
would have differed from typical 'Hamlin' patterns at at
least one of the loci. Likewise, most hybrid plants
resulting from cross pollination could have been identified
if they existed in these populations because other Citrus
species and cultivars differ from 'Hamlin' in allelic
composition at these loci (123). Therefore, the observed
zymogram uniformly excluded the possibility that some of
either the nucellar seedlings or the plants from undeveloped
ovules (from primary or secondary embryos) were of zygotic
origin.
Morphological Evaluation
The third approach taken to characterize plants was the
evaluation of morphological variability. In contrast to the
stability exhibited by all plants from all groups for
chromosome number and electrophoretic profile, differences
among individual plants from all groups were observed for


89
All zymograms were visualized on starch gels (approxi
mately 10%). Electrode and gel buffers used are listed in
Table 5.3. Fully expanded young leaves were harvested for
sample extraction. Sample wicks of No. 1 Whatman filter
paper were placed on the under side of leaves lying on a
glass plate. Pressure applied to the sample wick with a
pestle resulted in release of leaf cell contents and absorp
tion by the wick. The sample wicks were inserted into the
gel prior to electrophoresis for approximately 15-18 hours
at 180-225 V.
The following enzyme activity stains, as described by
Vallejos (124) were utilized to evaluate all plants:
1) G6PD = glucose-6-phospate dehydrogenase (D-Glucose-
6-phospate:NADP+ 1-oxidoreductase, E.C.1.1.1.49)
(except 20 mg of NADP were used rather than 15 mg);
2) SDH = shikimic acid dehydrogenase (Shikimate:NADP+
oxidoreductase, E.C.1.1.1.25);
3) PGD = 6-phosphogluconate dehydrogenase (6-Phospho-D-
gluconate:NADP+ 2-oxidoreductase, E.C.1.1.1.44).
Other enzyme activity stains utilized in this research
include:
4) ACP = acid phosphatase (Orthophosphoryl-monoester
phosphohydrolase, acid optimum E.C.3.1.3.2) 100 mg
a-naphthyl acid phosphate monosodium, 50 mg Black K
salt, 5 ml 1.0 M sodium acetate pHed to 4.7 with
glacial acetic acid, and 95 ml H20;


LIST OF TABLES
Table Page
3.1. Effects of cultivar and culture conditions on
the percentage of undeveloped Citrus ovules
that produced embryos 45
3.2. Effect of cultivar and culture conditions on
the number of primary embryos produced by
undeveloped ovules from Citrus fruit harvested
8 months after anthesis 46
3.3. Effect of cultivar and culture conditions on
the efficiency of embryo production by
undeveloped ovules from Citrus fruit harvested
8 months after anthesis 48
3.4. Effect of cultivar and culture conditions on
secondary proliferation from undeveloped Citrus
ovules 49
3.5. Effect of culture conditions on embryo produc
tion by undeveloped ovules from 'Hamlin' orange
fruit harvested 8 weeks after anthesis 51
3.6. Effect of media amendments on embryo production
by undeveloped ovules from 'Hamlin' orange
fruit harvested 12 weeks after anthesis .... 53
3.7. Origin and fresh weight increase of 'Hamlin'
orange embryogenic callus lines 55
3.8. Fresh weight increase and cotyledonary embryo
production by 'Hamlin' orange embryogenic callus
sublines 56
3.9. Results of subjective visual examination of
callus proliferation and embryo production from
'Hamlin' orange embryogenic callus 57
4.1. Germination and development of roots and shoots
from primary embryos of several Citrus
cultivars 67
Vll


39
within 7-14 days after culture initiation. The integuments
of the ovules then split and one to several developing
primary embryos emerged without prior callus production.
Frequently, other less-developed globular structures were
produced from within the ovule or from embryos already
present (Fig. 3.1). Some of these structures continued
normal development, became cotyledonary embryos, and were
designated secondary embryos. A creamy-white friable callus
arose on occasion from the proliferating embryos. Although
no histological studies were done, this callus appeared to
be composed of proliferating proembryos and was distin
guished from the proliferating secondary embryos only by the
relative proportion of developed and undeveloped structures
(see Fig. 3.2).
The secondary proliferation of embryos and callus
described above was fairly common, but long-term prolifera
tion was more rare. Much of the initial proliferation
ceased after several weeks. Alternatively, in a number of
instances embryo differentiation halted, but embryo growth
continued to produce "pseudobulbils" (large green or white
spherical structures). Pseudobulbil proliferation resulted
in callus line termination and prevented plant regeneration
from occurring, despite the fact that the pseudobulbils
could be induced to form roots (data not presented). On
occasion, embryogenic callus proliferations arose from the
surface of cultured pseudobulbils. In general, though,
pseudobulbil production represented an undesirable,


76
inactive or weakly-growing plants, and not from adverse
reactions to increased light and decreased humidity levels.
The data listed in Table 4.3 suggested that the ini
tiation treatment used to produce embryos may have had some
influence on plant survival. Specifically, embryos produced
from the ME/dark or 2,4-D/DZ treatments had greater regen
eration survival percentages than embryos from the other
treatments. As pointed out in the Results (Chapter IV), the
influence of initiation treatment on regenerate survival was
not manifested through differences in embryo development and
germination; rather, the greatest differences were observed
at the transfer and acclimatization step. The mechanism by
which initial treatment influences transplant survival
percentage is unclear although it may be related to embryo
production capacity because the treatments most effective
for embryo production were generally the treatments with the
highest rate of regenerate survival. The effect of initia
tion treatment on regeneration survival percentage was not
cultivar specific; ME/dark- or 2,4-D/DZ-produced embryos had
the greatest regeneration survival percentage for 5 of
6 cultivars.
Cultivar differences were observed for total embryo
production (Chapter III), the percentage of embryos develop
ing normally, the percentage of embryos that underwent
balanced germination, and the percentage of plants that
survived transfer to soil and acclimatization. However,
overall there were no great differences among cultivars for


127
of this question, it was significant that such variant types
were found among the tissue culture plants for a trait as
conservatively expressed as leaf blade shape. The statis
tical evidence suggesting greater variability of tissue
culture groups, and the visual evidence of more plants with
variant leaf types in the tissue culture groups supported
the idea that tissue culture plants were more variable for
leaf blade shape than the nucellar population.
Morphological evaluations of 'Hamlin' orange plants
were made to document the existence of individual plants
with at least one distinct form of phenotypic variation.
Such variants may have been of genetic origin (mutations of
individual genes controlling qualitative traits, of one or
several genes with additive effects, or both), or epigentic
in nature (e.g., residual growth regulator effects on plant
growth, abnormal initial shoot or leaf growth followed by
normal development, or environmental influences on character
expression). Several variant plant types were found with
greater frequency among all tissue culture groups, and these
groups were more morphologically variable than nucellar
seedlings. To minimize the influence of all nongenetic
factors on vegetative character expression, morphological
evaluations of all plants were delayed until several common
growth flushes had taken place. Therefore, the plants with
extreme vegetative seedling characteristics, from all


22
from fruit of 3 polyembryonic and 4 monoembryonic Citrus
types. They coined the phrase "primordium cell of nucellar
embryo" (PCNE) to describe certain unique, densely-
cytoplasmic cells characterized by a large nucleus with a
conspicuous nucleolus that were found within the nucellus of
the polyembryonic types, as early as anthesis or shortly
afterwards (depending on cultivar). These cells initiated
division shortly after the first division of the fertilized
egg (approximately 50 days after pollination), and subse
quently, developed into nucellar embryos. Such embryogenic
cells were found also in unfertilized ovules from mature
fruit, but most of the nucellar tissue was degenerated
(112). No PCNEs were found in monoembryonic Citrus ovules,
regardless of the stage of fruit development (54). Tisserat
and Murashige (120,121) demonstrated that the chalazal half
of monoembryonic C. medica L. ovules suppressed embryo-
genesis in Daucus carota L. cv. Queen Anne's Lace and C.
reticulata cv. Ponkan embryogenic callus. High levels of
ethanol production and concentrations of IAA, ABA, and GA
several times higher in C. medica than C. reticulata ovules
were noted and suggested as the source of embryogenic-
suppressive activity.
Although no PCNEs have been found in monoembryonic
Citrus nucelli, some monoembryonic Citrus ovules synthesize
embryogenic-suppressive compounds, and some attempts to
produce embryos from monoembryonic ovules have not been
successful (73,74), adventive embryogenesis ill vitro by


KEY TO ABBREVIATIONS
ABA:
abscisic acid.
ATP:
adenosine 5' triphosphate disodium salt.
BA:
N-(phenylmethyl)-1 H-purin-6-amine,
(benzyladenine).
CH:
casein hydrolysate.
DZ:
butanedioic acid mono-(2,2-dimethylhyrazide),
(daminozide).
GA3:
gibberellic acid.
H:
histidine buffer.
IAA:
indole acetic acid.
Kn:
kinetin.
LBTC:
lithium borate/Tris-citrate buffer.
L/W:
leaf blade or petiole length/width ratio.
ME:
malt extract.
MIL:
mean internode length.
MS:
Murashige and Skoog basal medium.
MT:
Murashige and Tucker medium for Citrus.
MTT:
3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-
tetrazolium bromide.
NAA:
napthyl acetic acid.
NAD:
3-nicotinamide adenine dinucleotide phosphate
disodium salt.
Na2EDTA:
disodium ethylenediaminetetraacetic acid.
NBT:
nitro blue tetrazolium.
x


15
The genetic background of the initial plant material
placed into culture has been shown to result in differences
in the degree of variation observed among regenerated
plants. More electrophoretic and morphologic variants were
found among sugar cane somaclones derived from a chromo-
somally unstable parent than those derived from a stable
line (42). McCoy et al. (71) used 2 oat cultivars to
regenerate plants and found that one produced more plants
with various cytogenetic abnormalities than the other. Sun
et al. (113) used 18 rice cultivars (9 from each of 2 eco-
geographic races). They reported that from one race no
polyploids were produced, and mutations were found in only 1
of 9 cultivar somaclone families. The other race produced
varying numbers of polyploids, and mutations were noted
among families from 5 of 9 cultivars. Therefore, the amount
of variation observed was related to the ecogeographic
origin of the cultivar cultured. Prat (89) used 2 different
lines in the work with N. sylvestris; an original line
(selfed for 7 generations) and a diploid androgenetic line
derived from the original line (5 cycles of pollen culture
and 2 selfed generations of one doubled haploid plant).
More morphological variation was obtained from the original
than from the androgenetic line. Prat speculated that this
resulted from differences in mutation rates between the
lines. Navarro et al. (80) reported that although aberrant
Citrus plants were obtained from cultured nucelli of mono-
embryonic cultivars, the polyembryonic types they studied


120
the minimum of the nucellar population were found in all
groups, but no plants were found with an MIL greater than
the maximum calculated for the nucellar population. There
fore, no concrete evidence of greater variability among
tissue culture groups was provided by these comparisons of
maxima and minima.
When plants were grouped according to initiation media,
all tissue culture groups were more variable than the
nucellar seedling population for MIL, but no differences
among the tissue culture groups were revealed in F-tests.
However, when plants were grouped on the basis of embryo
source, it was found that the primary and secondary groups
were not significantly different from each other or from
callus-derived plants or nucellar seedlings. Only the
callus group was more variable than the nucellar seedlings.
(This was possible because variances were compared in a
pairwise manner.) The greater variability of ME and 2,4-D
averaged out when plants were grouped according to embryo
source, and no differences could be detected then among
groups, except for the slightly more variable callus-derived
group of plants. The statistical evidence presented above
suggests (perhaps less emphatically than the evidence for
growth rate) that tissue culture derived plants were
slightly more variable than the nucellar population for MIL.
As with the analysis of growth rate variation, F-tests
performed with variances of subgroups within main groups


Table 5.6. Summary of F-tests and t-tests comparing growth rates of the following groups
of 'Hamlin' orange plants: Plants from primary embryos (Io); plants from
secondary embryos (2); plants from embryogenic callus (C) ; plants from ME
initiation treatments (ME); plants from 2,4-D initiation treatments (2,4-D);
and nucellar seedlings (N).
Comparison
A vs. B
F
df
P>Fa
A:Bb
t
df
P> t C 2
L:Bb
1
2
1.73
66+59
0.0331
+
5.55
121.9
0.0001
+
1
C
1.01
66 + 65
0.9643
=
3.97
131
0.0001
+
1
N
2.92
66+99
0.0001
+
3.03
96.4
0.0032
+
2
C
1.71
65+59
0.0373
-
-1.16
120.5
0.2494
=
2
N
1.68
59 + 99
0.0220
+
-4.02
100.9
0.0001
-
C
N
2.89
65+99
0.0001
+
-2.04
94.9
0.0437
-
ME
2,4-D
1.49
65+60
0.1180
=
0.65
125
0.5148
=
ME
C
1.19
65 + 65
0.4901
=
1.84
130
0.0687
=
ME
N
3.43
65+99
0.0001
+
0.38
90.3
0.7071
=
Q
l
(N
C
1.26
65 + 60
0.3713
=
1.29
125
0.1983
=
2,4-D
N
2.30
60 + 99
0.0002
+
-0.47
92.2
0.6373
=
Probability of
a greater
F value.
bIf
A>B, then +;
if A then -,
if A=B, then
0
Probability of a greater absolute value of t.
106


Table 5.7. Summary of F-tests and t-tests comparing mean internode length of groups
of 'Hamlin' orange plants. Abbreviations used for groups are as listed in the
title of Table 5.6.
Comparison
A vs. B
F
df
P>Fa
A:Bb
t
df
P> t C A
LiBb
1
2
1.04
66 + 59
0.8913
=
11.07
125
0.0001
+
1
C
1.34
65 + 66
0.2382
-
8.74
131
0.0001
+
1
N
1.15
66+99
0.5721
=
-1.90
165
0.0588
=
2
C
1.39
65+59
0.2014
=
-1.62
124
0.1078
=
2
N
1.11
59 + 99
0.6446
=
-14.41
158
0.0001
-
C
N
1.54
65+99
0.0522
+ / =
-11.32
118.4
0.0001
-
ME
2,4-D
1.27
65+60
0.3471
=
1.02
125
0.3107
=
ME
C
1.61
65 + 65
0.0584
=
3.65
130
0.0004
+
ME
N
2.47
65+99
0.0001
+
-5.36
99.7
0.0001
-
2,4-D
C
1.26
60+65
0.3571
=
2.69
125
0.0081
+
2,4-D
N
1.94
60 + 99
0.0034
+
-7.02
97.7
0.0001
-
Probability of
a greater
F value.
bIf
A>B, then +;
if A then -, if A=B, then

Q
Probability of a greater absolute value of t.
107