Citation
Tissue culture of Trifolium polymorphum, T. carolinianum, Adesmia latifolia, A. bicolor and Lotononis bainesii

Material Information

Title:
Tissue culture of Trifolium polymorphum, T. carolinianum, Adesmia latifolia, A. bicolor and Lotononis bainesii
Creator:
Vidoz, Maria Laura ( Dissertant )
Quesenberry, Kenneth H. ( Thesis advisor )
Williams, Mary J. ( Thesis advisor )
Gallo, Maria ( Reviewer )
Wofford, David ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
2006
Language:
English

Subjects

Subjects / Keywords:
Bud culture ( jstor )
Cotyledons ( jstor )
Genotypes ( jstor )
In vitro fertilization ( jstor )
Index numbers ( jstor )
Organogenesis ( jstor )
Plant growth regulators ( jstor )
Plants ( jstor )
Protoplast culture ( jstor )
Species ( jstor )
Agronomy thesis, M.S
Dissertations, Academic -- UF -- Agronomy

Notes

Abstract:
Although they fix nitrogen, provide feed for livestock, improve soil properties and protect the soil from erosion, many forage legume species have been underutilized. Currently, breeding programs have an increased interest in several of the less-studied species that could improve pasture quality in subtropical regions of the world. Trifolium polymorphum Poir., T. carolinianum Michx., Adesmia latifolia (Spreng.) Vogel, A. bicolor (Poir.) DC. and Lotononis bainesii Baker are promising forages; however, they possess two major drawbacks: low seedling vigor and low dry matter production. The objective of this research was to develop in vitro plant regeneration protocols for these species that could assist breeding programs by potentially enabling the use of in vitro chromosome doubling techniques and genetic transformation. Experiments using three types of basal medium (MS, L2 and B5) were conducted with T. polymorphum and Adesmia spp. In the latter, the effect of different plant growth regulators (TDZ, BAP and IBA) alone or in combination was evaluated. Cotyledon culture on media supplemented with various plant growth regulators was assessed in T. carolinianum, Adesmia spp. and L. bainesii. Time required for shoot bud induction in A. latifolia was determined using immature leaflets as explants. Different leaf parts were evaluated for their morphogenetic potential in A. latifolia and L. bainesii. In T. polymorphum, only callus formation was achieved using primarily B5 basal medium. With T. carolinianum, cotyledon culture on MS + 10 micron TDZ for 30 days with transfer to MS + 1 micron TDZ gave superior shoot bud organogenesis (20% of genotypes). Plant regeneration of A. bicolor was achieved through immature leaflet culture on MS or L2 + 4.5 micron TDZ for 60 days followed by transfer to MS devoid of plant growth regulators for 45 days. The highest frequency of plant regeneration in A. latifolia was obtained using immature rachises cultured on MS + 10 micron TDZ for 20 days and then transferred to MS without plant growth regulators for bud elongation and rooting. L. bainesii plant regeneration (in 90% of genotypes) was achieved by culturing immature leaflets on MS + 4.5 micron TDZ for 30 days and then transferring cultures to MS + 0.044 micron BAP + 0.049 micron IBA for bud elongation and rooting. In conclusion, shoot organogenesis protocols were developed for four out of the five forage legume species evaluated, and plant regeneration was achieved with three of these species. Additional experiments to obtain rooting of T. carolinianum should be conducted. Studies to reduce the incidence of hyperhydricity in cultures of Adesmia spp. and to assess the influence of different plant growth regulators on shoot bud formation of L. bainesii are also needed.
Subject:
Adesmia, culture, legumes, Lotononis, organogenesis, plant, regeneration, tissue, Trifolium
General Note:
Title from title page of source document.
General Note:
Document formatted into pages; contains 108 pages.
General Note:
Includes vita.
Thesis:
Thesis (M.S.)--University of Florida, 2006.
Bibliography:
Includes bibliographical references.
General Note:
Text (Electronic thesis) in PDF format.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Vidoz, Maria Laura. 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.
Embargo Date:
3/1/2007
Resource Identifier:
649810216 ( OCLC )

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Full Text












TISSUE CULTURE OF Trifolium polymorphum, T. carolinianum, Adesmia latifolia, A.
bicolor AND Lotononis bainesii















By

MARIA LAURA VIDOZ


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Maria Laura Vidoz



































To my mother and to God, for their unconditional love.
















ACKNOWLEDGMENTS

I would like to thank God for revealing His presence throughout my life, for His

guidance during hard times and for His many blessings. I am thankful to my mother for

her unconditional support and encouragement, for devoting so much time to my

education, for giving me a deep love of nature, making it difficult to put into words how

much she means to me.

I would like to thank my supervisory committee chair (Dr. Kenneth Quesenberry)

for his advice and knowledge, and for introducing me to his wonderful family, all of

which helped me professionally and personally. I am also grateful to the other members

of my committee (Dr. Mimi Williams, Dr. Maria Gallo and Dr. David Wofford) for their

critics and suggestions.

I thank Judy Dampier for her technical support, friendship and advice, making my

stay in Gainesville a positive experience. I thank Loan Ngo for her technical assistance

and sincere friendship. I also appreciate the technical support of Lindsay and Kailey

Place.

I am thankful to Luis Mroginski and Dr. Hebe Rey, who introduced me to the tissue

culture world and constantly encouraged me to grow in my career.

I would like to thank Lorena, Carlos, Gaby, Raquel, Jorge and Sonali for their

company during these two years, and to all my friends in Argentina for their support.




















TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. .................... iv


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


LIST OF FIGURES .............. ...............x.....


AB STRAC T ................ .............. xii


CHAPTER


1 INTRODUCTION ................. ...............1.......... ......


2 LITERATURE REVIEW ................. ...............5................


Tissue Culture Concepts as Applied to the Fabaceae Family............... ................5
Applications of Tissue Culture for the Fabaceae Family .............. .....................
Production of Plants Free from Certain Specific Pathogens .............. ..... ...........10
Micropropagation ......___ .......___ ... ..._ .............1
Production of Dihaploid, Homozygous Plants .............. ......... .... ................12
Generation oflInterspecific Hybrids: Embryo Rescue and Protoplast Fusion.....12
Plant Regeneration after Transformation Protocols ............... ...... ..................1
Medium and Long-term Germplasm Conservation and Plant Material
Exchange ................. ...............15.................

3 MATERIALS AND METHODS .............. ...............17....


Plant M material ................. ...............17........ ......
Culture Conditions................ ... ............1
Evaluation and Experimental Design .............. ...............19....


4 TISSUE CULTURE OF Trifolium polymorphum AND T. carolinianum ...............21

Introducti on ................. ...............21._ ___.......
Materials and methods ............ ............ ...............22...
Trifolium polymorphum .....__ ................. ........__ .......... 2
Trifolium carolinianum .............. ...............23....
Results and Discussion .............. ...............23....
Trifolium polymorphum .....__ ................. ........__ .......... 2












Trifolium carolinianum .............. ...............25....
Conclusions............... ..............2


5 PLANT REGENERATION OF Adesmia latifolia AND A. bicolor ........._..._.............3 1


Introducti on ........._..._. ...._ ... ...............3_ 1....
Materials and Methods .............. ...............32....
Plant M material .............. ...............32...
Basal Media Experiment .............. ........... ..............3
Factorial Experiments with TDZ and BAP .............. ...............34....
Induction Time Experiments .............. ...............35....
Type of Explant Experiment ........._..__......_ .. ...............35..
Results and Discussion .................. ... ... ..............3
Effect of Basal Medium on Shoot Organogenesis .................... ...............3
Influence of TDZ on Organogenesis .............. ...............38....
Influence of BAP on Organogenesis ................. ...............40........... ...
Induction Time for Adventitious Bud Formation................ ..............4
Effect of Explant Type on Shoot Organogenesis ................. .......................44
Conclusions............... ..............4


6 PLANT REGENERATION OF Lotononis bainesii ........._. ....._.... ..............61


Introducti on ............. ..... .._ ...............61....
Materials and Methods .............. ...............62....

Cotyledon Culture .............. ...............62....
Leaflet Culture ....__. ................. .......__. ..........6

Type of Explants ....__. ................. ...............63. ....
Results and Discussion .............. ...............63....

Cotyledon Culture .............. ...............63....
Leaflet Culture ....__. ................. .......__. ..........6

Type of Explant ................. ...............67...............
Conclusions............... ..............6


7 SUMMARY AND CONCLUSIONS ................._.._.._ ......... ............7


APPENDIX


ADDITIONAL TABLES FOR TWO GENOTYPES OF Adesmia latifolia ........._.._........77


LIST OF REFERENCES ........._..... ...._... ...............86....


BIOGRAPHICAL SKETCH .............. ...............95....

















LIST OF TABLES


Table pg

3-1 Plant material used as a source of explants in the experiments. ............. ................20

4-1 Mean percentage of explants producing callus from petiole pieces of T.
polymorphum in three basal media after 30 days of culture. ............. ..................29

4-2 Number of buds per explant in the responsive genotypes of 7 carolinianum
cultured on medium supplemented with 10 CIM TDZ after 30 and 60 days of
culture. ........... ..... .. ...............29....

5-1 Effect of three basal media on percentage of adventitious bud formation, mean
number of buds and regeneration index in A. bicolor and A. latifolia after 60
days of culture. ............. ...............47.....

5-2 Effect of TDZ concentration on percentage of adventitious bud formation, mean
number of buds per explant, and regeneration index in A. latifolia after 30 days
of culture. ............. ...............48.....

5-3 Effect of TDZ concentration on percentage of adventitious bud formation, mean
number of buds per explant, regeneration index, number of shoots, shoot length
and percentage of ex vitro survival in A. latifolia. ........._.._. .....___ ........._.....48

5-4 Effect of BAP concentration on percentage of adventitious bud formation, mean
number of buds per explant and regeneration index in A. latifolia after 30 days
of culture. ............. ...............49.....

5-5 Effect of BAP concentration on percentage of adventitious bud formation, mean
number of buds per explant and regeneration index after 60 days of culture; and
shoot length after 90 days of culture in A. latifolia. ................ ...................4

5-6 Effect of different times of exposure to TDZ on bud formation percentage, mean
number of buds per explant and regeneration index in A. latifolia after 30 and 60
days of culture. ............. ...............50.....

5-7 Effect of short exposure to TDZ on bud formation percentage, mean number of
buds per explant and regeneration index in A. latifolia after 30 days of culture.....50

5-8 Effect of explant type on bud formation percentage, mean number of buds per
explant and regeneration index in A. latifolia after 30 days of culture. ..................5 1










6-1 ANOVA table showing the p-values corresponding to the effect of explant type
on bud formation percentage, mean number of buds per explant and regeneration
index in L. bainesii after 30 days of culture. .............. ...............70....

6-2 Effect of explant type on bud formation percentage, mean number of buds per
explant and regeneration index in L. bainesii after 30 days of culture. ..................70

A-1 Effect of different combinations of TDZ and IBA on percentage of adventitious
bud formation and mean number of buds per explant in A. latifolia Ul8.6 after
3 0 and 60 day s of cul ture. ............. ...............77.....

A-2 Effect of different combinations of TDZ and IBA on regeneration index, number
of shoots per explant and shoot length in A. latifolia Ul8.6 after 60 days of
culture. ...._.._................. ........_.._.........77

A-3 Effect of different combinations of TDZ and IBA on percentage of adventitious
bud formation and mean number of buds per explant in A. latifolia Ul8.8 after
30 and 60 days of culture. ............. ...............78.....

A-4 Effect of different combinations of TDZ and IBA on regeneration index, number
of shoots per explant and shoot length in A. latifolia Ul8.8 after 60 days of
culture. ...._.._................. ........_.._.........78

A-5 Effect of different combinations of TDZ and IBA on percentage of ex vitro
acclimatization in A. latifolia Ul8.6 and Ul8.8 after 20 days of transfer to ex
vitro conditions. ........._.._.. ...._... ...............79....

A-6 Effect of different combinations of BAP and IBA on percentage of adventitious
bud formation and mean number of buds per explant in A. latifolia Ul8.6 after
30 and 60 days of culture. ............. ...............79.....

A-7 Effect of different combinations of BAP and IBA on regeneration index and
shoot length in A. latifolia Ul8.6 after 60 days of culture. .................. ...............80

A-8 Effect of different combinations of BAP and IBA on percentage of adventitious
bud formation and mean number of buds per explant in A. latifolia Ul8.8 after
30 and 60 days of culture. ............. ...............80.....

A-9 Effect of different combinations of BAP and IBA on regeneration index and
shoot length in A. latifolia Ul8.8 after 60 days of culture. .................. ...............81

A-10 Effect of different times of exposure to TDZ on bud formation percentage, mean
number of buds per explant and regeneration index in A. latifolia Ul8.6 after 30
and 60 days of culture. ............. ...............81.....

A-11 Effect of different times of exposure to TDZ on bud formation percentage, mean
number of buds per explant and regeneration index in A. latifolia Ul8.8 after 30
and 60 days of culture. ............. ...............82.....










A-12 Effect of short exposure to TDZ on bud formation percentage, mean number of
buds per explant and regeneration index in A. latifolia Ul8.6 and Ul8.8 after 30
days of culture. ............. ...............82.....

A-13 Effect of explant type on bud formation percentage, mean number of buds per
explant and regeneration index in A. latifolia Ul8.6 and Ul8.8 after 30 days of
culture. ........... ..... .. ._ ...............83.....

















LIST OF FIGURES


Figure pg

4-1 Shoot bud organogenesis through cotyledon culture of 7 carolinianum ................30

5-1 Organogenesis in A. latifolia ............... ...............52....

5-2 Regression curve showing the effect of TDZ concentration on percentage of
adventitious bud formation in A. latifolia after 60 days of culture. .........................53

5-3 Regression curve showing the effect of TDZ concentration on regeneration
index in A. latifolia after 30 days of culture. ........._.._.. ...._.. ........_.._......5

5-4 Regression curve showing the effect of TDZ concentration on shoot number per
explant in A. latifolia after 90 days of culture. .............. ...............54....

5-5 Regression curve showing the effect of TDZ concentration on shoot length in A.
latifolia after 90 days of culture. ............. ...............54.....

5-6 Regression curve showing the effect of TDZ concentration on acclimatization
rate in A. latifolia after 30 days of transfer to ex vitro conditions. ........................55

5-7 Regression curve showing the effect of BAP concentration on percentage of
adventitious bud formation in A. latifolia after 30 days of culture. .........................55

5-8 Regression curve showing the effect of BAP concentration on regeneration
index in A. latifolia after 30 days of culture. ........._.._.. ...._.. ........_.._......5

5-9 Regression curve showing the effect of BAP concentration on percentage of
adventitious bud formation in A. latifolia after 60 days of culture. .........................56

5-10 Regression curve showing the effect of BAP concentration on mean number of
buds per explant in A. latifolia after 60 days of culture. ................... ...............5

5-11 Regression curve showing the effect of BAP concentration on regeneration
index in A. latifolia after 60 days of culture. ........._.._.. ...._.. ........_.._......5

5-12 Regression curve showing the effect of BAP concentration on shoot length in A.
latifolia after 90 days of culture. ............. ...............58.....

5-13 Regression curve sowing the effect of exposure to TDZ on bud formation
percentage in A. latifolia after 60 days of initiation of cultures. .............. ..............58










5-14 Regression curve sowing the effect of exposure to TDZ on mean number of
buds per explant in A. latifolia after 60 days of initiation of cultures. ..................59

5-15 Regression curve sowing the effect of exposure to TDZ on regeneration index in
A. latifolia after 60 days of initiation of cultures. ........._. ......__ ................59

5-16 Regression curve sowing the effect of short exposures to TDZ on percentage of
bud formation in A. latifolia after 30 days of initiation of cultures.............._.._.. ......60

5-17 Regression curve sowing the effect of short exposures to TDZ on regeneration
index in A. latifolia after 30 days of initiation of cultures. ................ ........_.._.....60

6-1 Organogenesi s in L. bainesii ................. ......... ...............71. ..

6-2 Number of buds produced per explant by 50 genotypes of L. bainesii after 30
days of culture from cotyledon and leaflets. ............. ...............72.....

6-3 Number of plants produced per explant after 90 days of culture by responsive
genotypes ofL. bainesii through cotyledon and leaflet culture. ............. ................72

6-4 Number of successfully acclimatized plants in the genotypes of L. bainesii that
were capable of plant regeneration through cotyledon and leaflet culture.. .............73

A-1 Regression curve sowing the effect of exposure to TDZ on bud formation
percentage in A. latifolia after 30 days of initiation of cultures..........._.._.. ............_.84

A-2 Regression curve sowing the effect of exposure to TDZ on mean number of
buds per explant in A. latifolia after 30 days of initiation of cultures. ................... ..84

A-3 Regression curve sowing the effect of exposure to TDZ on regeneration index in
A. latifolia after 30 days of initiation of cultures. .....___.............. ... ........._..._85
















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

TISSUE CULTURE OF Trifolium polymorphum, T. carolinianum, Adesmia latifolia, A.
bicolor AND Lotononis bainesii
By

Maria Laura Vidoz

December 2006

Chair: Kenneth H. Quesenberry
Cochair: Mary J. Williams
Major Department: Agronomy

Although they fix nitrogen, provide feed for livestock, improve soil properties and

protect the soil from erosion, many forage legume species have been underutilized.

Currently, breeding programs have an increased interest in several of the less-studied

species that could improve pasture quality in subtropical regions of the world. Trifolium

polymorphum Poir., T. carolinianum Michx., Adesmia latifolia (Spreng.) Vogel, A.

bicolor (Poir.) DC. and Lotononis bainesii Baker are promising forages; however, they

possess two maj or drawbacks: low seedling vigor and low dry matter production. The

obj ective of this research was to develop in vitro plant regeneration protocols for these

species that could assist breeding programs by potentially enabling the use of in vitro

chromosome doubling techniques and genetic transformation.

Experiments using three types of basal medium (MS, L2 and B5) were conducted

with 7: polymorphum and Adesmia spp. In the latter, the effect of different plant growth

regulators (TDZ, BAP and IBA) alone or in combination was evaluated. Cotyledon









culture on media supplemented with various plant growth regulators was assessed in T.

carolinianum, Adesmia spp. and L. bainesii. Time required for shoot bud induction in A.

latifolia was determined using immature leaflets as explants. Different leaf parts were

evaluated for their morphogenetic potential in A. latifolia and L. bainesii.

In T. polymorphum, only callus formation was achieved using primarily B5 basal

medium. With 7: carolinianum, cotyledon culture on MS + 10 CIM TDZ for 30 days with

transfer to MS + 1 C1M TDZ gave superior shoot bud organogenesis (20% of genotypes).

Plant regeneration ofA. bicolor was achieved through immature leaflet culture on MS or

L2 + 4.5 CIM TDZ for 60 days followed by transfer to MS devoid of plant growth

regulators for 45 days. The highest frequency of plant regeneration in A. latifolia was

obtained using immature rachises cultured on MS + 10 CIM TDZ for 20 days and then

transferred to MS without plant growth regulators for bud elongation and rooting. L.

bainesii plant regeneration (in 90% of genotypes) was achieved by culturing immature

leaflets on MS + 4.5 CIM TDZ for 30 days and then transferring cultures to MS + 0.044

CIM BAP + 0.049 CIM IBA for bud elongation and rooting.

In conclusion, shoot organogenesis protocols were developed for four out of the

five forage legume species evaluated, and plant regeneration was achieved with three of

these species. Additional experiments to obtain rooting of T. carolinianum should be

conducted. Studies to reduce the incidence of hyperhydricity in cultures of Adesmia spp.

and to assess the influence of different plant growth regulators on shoot bud formation of

L. bainesii are also needed.















CHAPTER 1
INTTRODUCTION

In addition to providing feed for livestock, forages offer multiple benefits such as

maintenance and improvement of soil characteristics, weed suppression, and protection of

soil from erosion. When used as cover crops, forages may also assist in the recovery of

degraded soils (Peters et al., 2003) by decreasing the loss of nutrients from leaching or

erosion (Tilman et al., 2002). Forages that exhibit two or more of these characteristics are

better accepted by farmers in agricultural systems (Peters et al., 2003).

The family Fabaceae comprises between 670 to 750 genera and 18,000 to 19,000

species, many of which constitute an important source of protein in human and animal

diets, balancing the amino acids provided by cereals (Graham & Vance, 2003).

According to Crews & Peoples (2004), legumes constitute a more environmentally

friendly source of nitrogen than synthetic fertilizers due to their ability to fix nitrogen,

reducing the risk of eutrophication and contamination of subterranean water.

However, numerous species of pasture legumes have thus far been underutilized

(Graham & Vance, 2003), although many of them could be used to increase the genetic

variability of their cultivated relatives or as a source of useful genes. For instance,

legumes bred for drought and salinity tolerance would be useful in water-stressed areas of

the world (Graham & Vance, 2003). In this scenario, forages that are naturally adapted to

those conditions will be particularly valuable.

With approximately 240 species, the genus Trifolium L. is one of the most

important genera in the Fabaceae family regarding the number of species it comprises









and their potential uses (Zohary & Heller, 1984). It is found in temperate to subtropical

regions of Europe, the Americas, Asia and Africa (Lange & Schifino-Wittmann, 2000),

but also occurs in the mountain and alpine zones in the tropics of West Africa and South

America (Zohary & Heller, 1984). At least 25 species are useful as forages.

Trifolium polymorphum is endemic to subtropical South America and 7

carolmianum is endemic to subtropical North America. Although common in native

grasslands, neither species has been the subject of crop improvement research through

plant breeding programs. T. polymorphum Poir. is a perennial subtropical species

described in the United States, Argentina, Brazil, Chile, Paraguay, Peru and Uruguay

(Quesenberry et al., 1997). It constitutes the only amphicarpic species in the genus and

has been used in some cytogenetic and reproductive studies (Lange & Schifino-

Wittmann, 2000). Agronomic evaluations conducted in New Zealand discovered one

accession with 154 g kg-l crude protein and 697 g kg-l digestibility (Dodd & Orr, 1995).

However, its marginal dry matter (DM) production and low seedling vigor is a maj or

limitation for its use as a forage legume.

Trifolium carolinianum Michx. is an annual species native to the southeastern US.

It produces seed and nodulates profusely, but like T. polymorphum, it has low seedling

vigor and low DM production. Genetic improvement through traditional breeding and

transformation can overcome these restrictions. Transformation, however, usually

requires an efficient protocol for plant regeneration to be in place. Although in vitro

regeneration protocols are available for several Trifolium species (Ding et al., 2003),

there are none for 7 polymorphum or T. carolinianum. Additionally, induction of

polyploidy can also be used to increase seedling vigor and overall plant size, as was









shown previously in the genus Trifolium (Taylor et al., 1976; Furuya et al., 2001). Once

again, the availability of an in vitro protocol would permit the use of additional

techniques to duplicate chromosomes in T. polymorphum as has been demonstrated in

bahiagrass (Paspalum notatum Fliigge) using colchicine, trifluralin and oryzalin

(Quesenberry et al., 2003). Moreover, the use of an in vitro protocol to induce

chromosome duplication could prevent the recovery of chimeric plants that often are

obtained when vegetative meristems are treated with chromosome doubling agents. In an

in vitro system single cells are doubled and, consequently, plant regeneration from these

single cells, as in the case of adventitious bud formation, renders a plant whose cells

uniformly contain duplicated chromosomes.

Adesmia DC. (Fabaceae) is the only genus of the South American tribe Adesmieae

(Benth.) Hutch and it comprises approximately 240 species of herbs and shrubs (Ulibarri

& Burkart, 2000). Many Adesmia species, such as A. bicolor (Poir.) DC., A. latifolia

(Spreng.) Vogel and A. punctat (Poir.) DC., constitute promising forage materials

because of their satisfactory winter growth, high crude protein values and good in vitro

organic matter digestibility. These species have been reported to be useful for soil cover

and erosion control (Coelho & Battistin, 1998; Tedesco et al., 2000; Tedesco et al.,

2001). In addition, A. bicolor showed tolerance to low phosphorous fertility and

possesses valuable morphological characteristics when compared to other legumes (Dodd

& Orr, 1995). In spite of these desirable characteristics, there are very few reports related

to the biology of the genus (Coelho & Battistin, 1998; Dodd & Orr, 1995; Tedesco et al.,

2000; Tedesco et al., 2001), and no reports regarding their use in tissue culture.










The genus Lotononis belongs to the tribe Crotalarieae and comprises approximately

150 species, from herbs to small shrubs (Jaftha et al., 2002). These species are distributed

from Southern Africa to the Mediterranean region and India, and are found under a range

of climates and geographical situations. L. bainesii Baker is a perennial herb whose

forage value has been demonstrated in Australia (Jaftha et al., 2002). Although it has

been previously reported to be a cleistogamous species, Real et al. (2004) have conducted

molecular studies that suggest that it requires pollinators to set seed and some genotypes

are self-incompatible. There is only one report on tissue culture ofL. bainessi, where a

low frequency of plant regeneration was obtained (Bovo et al., 1986).

Limited quantities of seeds may constitute a problem for release of new cultivars

(Frame, 2004) but tissue culture may offer a solution, shortening the period of time for

the availability of propagules. In addition, in cross pollinated species such as L. bainesii,

A. latifolia and A. bicolor, each seed is potentially a different genotype. Therefore, it is

not possible to propagate a plant exhibiting exceptional characteristics through seeds

since the progeny may segregate for the trait of interest. However, micropropagation

offers a solution for the mass propagation of an individual plant.















CHAPTER 2
LITERATURE REVIEW

All plant cells have the potential to be totitpotent, i.e., to be able to dedifferentiate,

divide and regenerate into whole plants (Loidl, 2004). This was the idea that Gottlieb

Haberlandt had in mind when he first attempted plant tissue culture in the early 20th

century (Caponetti et al., 2004). Although he failed in his venture to regenerate plants

from isolated tissues, his work attracted the attention of the scientific world and,

consequently, abundant research was developed on the topic.

Tissue Culture Concepts as Applied to the Fabaceae Family

Tissue culture is usually defined as a heterogeneous group of techniques in which

explants (protoplasts, cells, tissues or organs) are aseptically placed onto a culture

medium of defined chemical composition, and incubated under controlled conditions

(Mroginski et al., 2004b). There are three types of plant regeneration systems that are

used most frequently: micropropagation, organogenesis and somatic embryogenesis.

Micropropagation consists of the in vitro propagation of selected genotypes through

improved axillary shoot production from explants with pre-existing meristems (Kane,

2004). In contrast, the other two regeneration schemes are based on the use of non-

meristematic tissues as explants: organogenesis is the de novo formation of organs

(shoots, roots or flowers), and somatic embryogenesis is the production of embryos

without a previous fusion of gametes (Radice, 2004).

Members of the Fabaceae family have traditionally been regarded as recalcitrant to

in vitro regeneration, particularly in the case of cultivated grain legumes (Griga, 1999;









Veltcheva et al., 2005; Mundhara & Rashid, 2006). Veltcheva et al. (2005) suggest that

recalcitrance in grain legumes could be caused by the narrow genetic base of the

cultivated varieties that have undergone inbreeding and selection for long periods of time.

In addition, they suggest that in forage species the outbreeding and lower genotype

selection may account for easier identification of responsive genotypes.

Some of the factors that affect in vitro response of a given species are genotype,

explant, composition of the culture medium and conditions under which explants are

incubated (Radice, 2004). The genotype of the donor plant is one of the most critical

factors since it influences in vitro responses, from the establishment of the explant to the

regeneration of whole plants, as well as ex vitro, during the acclimatization of

regenerated plants. The importance of the genotype on in vitro plant regeneration of

cultivated peanut (Arachis hypogaea L.) has been demonstrated by Chengalrayan et al.

(1998), who assessed 16 genotypes for responsiveness in vitro using a protocol to induce

somatic embryogenesis. These authors found differences in the frequency of response at

each stage of the process and suggested that genotype could be the primary factor

influencing conversion of somatic embryos to plantlets. A similar experiment was carried

out in soybean (Glycine max (L.) Merrill), in which 17 breeding lines were evaluated for

their response and ability to regenerate plants through somatic embryogenesis (Tomlin et

al., 2002). Among these lines, a significant difference in the percentage of responsive

explants, number and quality of somatic embryos were observed. Other legume species in

which differences among genotypes were reported, particularly regarding somatic

embryogenesis are M~edicago sativa L. and T. pratense L. (Lakshmanan & Taji, 2000).










For T. pretense, Quesenberry and Smith (1993) increased genotype regeneration

frequency from less than 5% to almost 70%, after five cycles of recurrent selection.

The explant type used for culture establishment depends on the obj ectives that are

pursued since it determines responsiveness of the plant material in vitro (Lakshmanan &

Taji, 2000). The aspects that should be considered in explant selection are: explant tissue

(leaves, petals, anthers, roots, meristems, cotyledons, epicotyls, hypocotyls), explant size,

explanation time, topophysis and polyphenol oxidation (Kane, 2004). It is widely

accepted that immature zygotic embryos and young seedlings are the most responsive

explants to induce somatic embryogenesis in legume species. This is because areas where

cells show active division are more responsive to the embryogenic stimulus (Griga, 1999;

Mundhara & Rashid, 2006). However, a range of explants have been used with success to

induce somatic embryogenesis in Fabaceae family. These have included mature seeds,

shoot apices, seedlings, hypocotyls, cotyledons, leaves, petioles, internodes, roots,

endosperms, cell suspensions and protoplasts (Lakshmanan & Taji, 2000). For the

induction of organogenesis in legume species, a similar variety of explants has been used.

As an example, in Arachis, several explants have been capable of regenerating plants:

fully expanded leaves (Dunbar & Pittman, 1992), leaflets from young seedlings (Akasaka

et al., 2000), epicotyls, petioles (Cheng et al., 1992), cotyledons, embryo-axes, mature

whole seeds (Radhakrishnan et al., 2000), protoplasts (Li et al., 1993), mature zygotic

embryo-derived leaflets (Chengalrayan et al., 2001) and shoot apices (Radhakrishnan et

al., 1999).

Culture medium composition is determined by the type and concentration of

inorganic salts (macro- and micro-nutrients), organic compounds (sugar, vitamins,









activated charcoal, etc.), plant growth regulators (mainly auxins and cytokinins), gelling

agents or other support system and the gaseous atmosphere inside the culture vessel

(Radice, 2004). The most widely used basal medium for legume regeneration is MS

medium developed by Murashige and Skoog (1962) for callus cultures of tobacco

(Murashige & Skoog, 1962). This basal medium, which has a high salt concentration, has

been used to achieve plant regeneration in several legume genera, such as Arachis (Rey

& Mroginski, 2006), Astragalus (Luo et al., 1999), Cajanus (Singh et al., 2003), Cassia

(Agrawal & Sardar, 2006), Cicer (Chakraborti et al., Dalbergia (Singh & Chand, 2003),

Glycine (Tomlin et al., 2002), 2006), Lathyrus (Barik et al., 2005), Lotus (Akashi et at.,

2003), Pha~seolus (Delgado-Sanchez et al., 2006), Pisum (Loiseau et al., 1998), Trifolium

(Ding et al., 2003) and Vigna (Saini & Jaiwal, 2002). However, other basal media have

been specifically developed for certain legume species, such as G. max (Gamborg et al.,

1968) and T. pretense (Collins & Phillips, 1982). Several plant growth regulators have

been used with success in plant regeneration protocols for legume species, but the type of

response and effectiveness of the compounds are highly dependent on the species and

even on genotypes within a species. In general, auxins are used to induce somatic

embryogenesis, whereas cytokinins are used to induce organogenesis. Nevertheless, there

are some exceptions such as in T. repens L., M~edicago sativa and Pha~seolus spp., in

which it was possible to achieve somatic embryogenesis by using cytokinins instead of

auxins (Lakshmanan & Taji, 2000).

In addition to media composition factors, incubation conditions under which

explants are incubated must be controlled. These include temperature, light quality and

intensity, photoperiod, humidity and hygiene (Mroginski et al., 2004b). In general, the










temperature for incubation of cultures is between 23-29 oC, depending on optimal growth

requirement of the species (Radice, 2004). In some cases, when the process to be induced

is somatic embryogenesis, cultures are incubated in the dark, since light is not required

for this developmental pathway. In contrast, when organogenesis is to be induced,

cultures are usually kept under light conditions with a specific photoperiod. In addition,

light quality (spectral quality) and quantity (photon flux) are reported to have an

important role in morphogenetic processes in vitro and on the subsequent growth of the

regenerated structures (Lian et al., 2002).

Applications of Tissue Culture for the Fabaceae Family

Since its origin in the early 20th century, tissue culture procedures have been used

for a variety of purposes, such as basic studies of particular physiological processes

because the use of tissues instead of whole plants usually simplifies the study of the

phenomenon (Mroginski et al., 2004b). Another use of tissue culture is the production of

plants free from certain specific pathogens, generally viruses, through meristem or shoot

tip culture alone or combined with thermo/chemotherapy However, the most important

application of these techniques from an economic point of view is related to

micropropagation. This method is particularly important in horticulture, since it generally

maintains genetic stability (Kane, 2004), and allows propagation of periclinal chimeras.

This kind of chimera may be important in ornamental species and cannot be propagated

through organogenesis or somatic embryogenesis. Tissue culture may also be used for the

production of interspecific hybrids where zygotic embryos abort early in their

development and have to be rescued, or in the case of plants with a rudimentary embryo

(Mroginski et al., 2004b). The production of dihaploid, homozygous plants is also

possible through anther or ovule culture, which reduces the time required to achieve









homozygosis in breeding programs. Other applications of tissue culture include the

induction of somaclonal variation, production of secondary metabolites using cell culture,

generation of somatic hybrids through protoplast fusion, and plant regeneration after

transformation protocols. From the species preservation point of view, tissue culture

constitutes a valuable technique for medium and long-term germplasm conservation (in

vitro and cryoconservation), as well as plant material exchange since pathogen-free plants

are used for this purpose (Mroginski et al., 2004b).

Production of Plants Free from Certain Specific Pathogens

Since plants were first domesticated, diseases and pests have threatened crop

productivity. Some diseases caused by fungi and bacteria may be controlled if certain

practices are used during the cultivation of the crop. However, in the case of viruses, the

control is usually more difficult and in many cases the only indication of the presence of

a virus is a reduction in crop yields. Viral diseases are transmitted rapidly particularly

when the crop is vegetatively propagated (Kartha, 1984).

Meristem culture is one of the tools to eliminate viruses from plant material,

provided that the rate of virus multiplication and movement in the plant is lower than the

rate at which the meristematic region elongates. This is often the case since vascular

tissues do not reach the meristem. In the Fabaceae family, meristem culture has been

applied successfully to the rescue of interspecific hybrids between A. hypogaea and A.

stenosperma Krapov & W.C. Gregory, and A. hypogaea and A. otavioi, which showed

symptoms of peanut stripe virus (Radhakrishnan et al., 1999). Meristem culture with or

without thermo/chemotherapy- has also been used to eliminate peanut mottle virus, peanut

stripe virus and tomato spotted wilt virus from interspecific hybrids ofArachis that were

maintained vegetatively in the germplasm collection at the Southern Regional Plant









Introduction Station (Griffin, GA) (Dunbar et al., 1993a). In contrast, shoot tip culture

was not an effective procedure to regenerate plants free of the peanut mottle viruses.

Prasada Rao et al. (1995) excised seed axes from peanut stripe virus infected seed and

cultured them on a medium containing ribavirin to obtain peanut plants free of the virus.

Meristem culture has also been successfully applied to other legume genera, such as

Trifolium and Pha~seolus, for the production of plants free of common viruses (Phillips

and Collins, 1979; Veltcheva et al., 2005).

Micropropagation

Micropropagation, the true-to-type propagation of a genotype through tissue culture

techniques, is a useful tool in breeding programs. Among other advantages, it enables the

production of uniform plants from a selected genotype at a high multiplication rate

(Olmos et al., 2004). The stages for micropropagation from shoot explants are: a) donor

plant selection and preparation, b) axillary shoot proliferation, c) pretransplant or rooting,

and d) transfer to the natural environment (Kane, 2004).

Cultivated peanut has been reported to have limited reproductive efficiency, which

is a drawback when large populations are required for breeding purposes (Radhakrishnan

et al., 2000). Micropropagation may be used to overcome this situation, provided that an

efficient in vitro protocol is available. For this species, Radhakrishnan et al. (2000)

developed a high frequency micropropagation protocol from embryo axes and plant

regeneration from other juvenile explants. Successful micropropagation protocols have

also been developed for other species such as Vigna mungo (L.) Hepper, a grain legume

important in South Asia and Australia, where plants were regenerated from shoot tips,

embryo axes and cotyledonary nodes (Saini & Jaiwal, 2002).









Production of Dihaploid, Homozygous Plants

The production of haploid plants following anther, pollen or ovule culture is

desired in breeding programs because it would result in a reduction in the number of

cycles to achieve complete homozygosity after the duplication in the number of

chromosomes of the regenerated haploid plants. Several attempts have been made in the

Fabaceae family but with little success. For instance, in the genus Arachis, Bajaj et al.

(1980) reported androgenesis but no plant regeneration from pollen cultures ofA.

hypogaea and A. glabrata Benth. Bajaj et al. (1981) reported plant regeneration from

anther cultures of A. hypogaea and A. villosa Benth., with a chromosome number varying

from haploid to octaploid. In soybean, Rodrigues et al. (2004) studied the origin of

embryo-like structures from anther cultures using molecular techniques. They found both

homozygous and heterozygous structures, suggesting that embryogenesis and

androgenesis occurred simultaneously. Anther culture in Pha~seolus resulted in callus

formation with cell ploidy levels ranging from haploid to polyploidy (Veltcheva et al.,

2005).

Generation of Interspecific Hybrids: Embryo Rescue and Protoplast Fusion

In many cases, wild species offer traits of interest that could be useful in breeding

programs if incorporated into their cultivated relatives. Nevertheless, there may be

interspecific barriers that need to be overcome in order to be able to transfer the trait of

interest. One of the possibilities to circumvent this would be to use tissue culture

techniques to rescue the embryo before it aborts, followed by micropropagation of the

hybrid if it has low fertility.

Arachis villosulicarpa Hoehne, a wild relative of cultivated peanut (A. hypogaea),

is rich in oil and is resistant to Cercospora arachidicola and Cercosporichum personatum










(Mansur et al., 1993). However, hybrids between this species and A. hypogaea could not

be obtained due to cross compatibility barriers that caused abnormal endosperm

development (Pittman et al., 1984). Somatic hybridization may be used to overcome this

incompatibility, provided that an in vitro protocol is available. For this purpose, Mansur

et al. (1993) developed a protocol for plant regeneration from cotyledons, leaves and cell

suspensions of A. villosulicarpa.

In the genus Trifolium, a few interspecific hybrids have been produced with the aid

of tissue culture techniques, such as hybrids between red clover (T. pretense) and T.

sarosiense Hazsl. where aseptic embryo rescue was used before in situ abortion (Phillips

et al., 1982). Przywara et at. (1996) regenerated hybrid plants from crosses between T.

repens and 7 nigrescens Viv. using in vitro pollination followed by embryo rescue. If

hybrid embryos did not grow, they were transferred onto MS medium supplemented with

growth regulators and achieved plant regeneration through organogenesis.

To apply somatic hybridization for the recovery of interspecific hybrids, at least

one of the parents should be able to regenerate plants from protoplasts, but both should be

able to undergo protoplast culture. In addition, it should be possible to select the somatic

hybrids (Myers et al., 1989). There are reports on protoplast culture, which in most cases

resulted in plant regeneration, in several species of the genus Trifolium such as T.

fragiferum L. (Rybcznski, 1997), T. pretense (Myers et al., 1989; Radionenko et al.,

1994), T. repens (Webb et al., 1987), 7 resupinatum L. (Oelck et al., 1982) and T. rubens

L. (Grosser & Collins, 1984). Plant regeneration from protoplasts has also been obtained

in other legume genera such as Astragalus (Hou & Jia, 2004).









Other grain legume genera where embryo rescue techniques have been applied to

obtain interspecific hybrids are Cicer and Pha~seolus. In order to determine the phase

when embryo rescue should occur in wide hybrids, Clarke et al. (2006) used selfed

chickpea (Cicer arietinum L.) and selfed wild relatives to study the stage of embryo

development at which abortion occurs. Several protocols have been developed to

regenerate plants through embryo rescue after crosses between cultivated and wild

Pha~seolus species; however, no somatic hybridization has been reported for the genus

(Veltcheva et al., 2005).

Plant Regeneration after Transformation Protocols

The application of molecular approaches to plant breeding usually requires an

efficient in vitro system to regenerate plants from transformed single cells in order to

obtain nonchimeric plants (Gill & Ozias-Akins, 1999). In vitro protocols amenable to

molecular breeding have been developed for a wide range of legume genera: Arachis

(Ozias-Akins & Gill, 2001; Vidoz et al., 2006; Rey & Mroginski, 2006); Astraglus (Luo

et al., 1999), Cajanus (Singh et al., 2003), Cassia (Agrawal & Sardar, 2006), Cicer

(Chakraborti et al., 2006); Dalbergia (Singh & Chand, 2003); Glycine (Tomlin, 2002),

Lathyrus (Barik et al., 2005), Lotus (Lombari et al., 2003), Macroptilium (Ezura et al.,

2000), Pha~seolus (Delgado-Sanchez et al., 2006), Trifolium (Ding et al., 2003) and Vigna

(Saini & Jaiwal, 2002). Some examples of legume species in which tissue culture has

assisted transformation protocols are A. hypogaea (Ozias-Akins & Gill, 2001), Lotus

japonicus (Regel) K. Larsen (Lombari et al., 2003), G. max (Olhoft et al., 2003), 7

repens, 7 pratense and 7 subterraneum L. (Ding et al., 2003).










Medium and Long-term Germplasm Conservation and Plant Material Exchange

As civilization advances, the centers of diversity of many important plants for food

and forage are threatened. This situation implies the loss of valuable genes contained in

the wild relatives of the cultivated species that could be used in breeding programs. For

this reason, germplasm is kept in storage facilities, mainly as seeds, which require

considerable land and labor to be renewed. For many species belonging to the genus

Arachis, seed viability decreases abruptly after 2-3 years of storage. However, some

protocols have been developed to recover plants from seeds that would not germinate by

themselves through the in vitro culture of embryonic axes (Dunbar et al., 1993b; Morris,

et al., 1995).

Cryopreservation constitutes an alternative to the laborious and time consuming

storage of seeds. Not only does it allow for long-term storage, but it also ensures genetic

stability, requires little space and is low maintenance (Gagliardi et al., 2003). The ultra-

low temperatures of liquid nitrogen cause interruption of all biochemical reactions

protecting the plant material from physiological and genetic changes (Yamada et al.,

1991). In addition, plants are kept free from pathogens when propagated from plants that

have been indexed for the presence of specific microorganisms. Protocols for

cryopreservation have been developed for several species: A. burchellii Krapov. & W.C.

Greg., A. hypogaea, A. retusa Krapov. et al., (Gagliardi et al., 2003), A. macedoi Krapov.

& W.C. Greg., A. pietratrelliirtrt~t~r~rtrt Krapov. & W.C. Greg., A. prostrate Benth. A.

villosulicarpa (Gagliardi et al., 2002) and T. repens (Yamada et al., 1991) among others.

Medium-term conservation of germplasm can also be done by maintaining plants under

in vitro conditions, which has similar advantages as cryopreservation: little space, low

maintenance, and protection from pathogens. Moreover, plants kept in vitro are a ready









source of material in case the production of a large number of plants is required

(Bhojwani, 1981).

Although there are many reports on tissue culture of legume species, there are no in

vitro protocol s for 7: polymorphum, T. carolinianum, A. bicolor or A. latifolia, four

promising forage species. For L. bainesii there is only one report of plant regeneration

from cotyledons (Bovo et al., 1986). Therefore, the main obj ective of this research was to

develop protocols for plant regeneration of these species that may then be used to

improve their forage potential. Medium basal salts, plant growth regulators, explant type

and time of exposure were the main factors evaluated.















CHAPTER 3
MATERIALS AND METHODS

Procedures common to all experiments are described here. The modifications made

for specific experiments are described in corresponding chapters.

Plant Material

Experiments were carried out with four species representing three different genera

of the Leguminosae family: A. bicolor, A. latifolia, L. bainesii, T. carolinianum, and 7:

polymorphum (Table 3-1). Seeds were scarified using concentrated sulphuric acid (98%)

for 5 minutes (Adesmia spp., L. bainesii), 7 minutes (7: polymorphum) or 10 minutes (T.

carolinianum) and then were rinsed for 10 minutes in running tap water. Subsequently,

seeds were surface disinfected by immersion in a solution of sodium hypochlorite

containing 0.571 % W/V available chlorine for 5 minutes and rinsed with distilled sterile

water three times. Disinfected seeds were placed on half-strength Murashige and Skoog

(1962) (MS) basal medium, with 15 g L^' sucrose and 0.7 g L^1 agar (Sigmal A-1296)

in 100 mm diameter x 15 mm deep petri dishes.

Culture Conditions

Basal medium consisted of either MS, L2 (Collins & Phillips, 1982) or B5

(Gamborg et al., 1968). Media were prepared using MS basal salt mixture (Sigma

M/5524) and MS vitamins (Sigma~ M7150) mixed in proportions corresponding to

M/urashige and Skoog (1962), B5 basal salt mixture (Sigma" G5768) and B5 vitamins


1Use of brand name is for identification purposes only and does not imply exclusion of other similar brand
products.









(RPI~ G37010) were used in quantities specified in Gamborg et al. (19)68) or L2

concentrated stock solutions mixed in the adequate proportions according to Collins &

Phillips (1982). Concentrated stock solutions of plant growth regulators were prepared by

dissolving the correct quantity of the product { [TDZ= thidiazuo-;~n, 1-nphnyrl-3-(12,3

thiadiazol-5-yl)urea (Sigma~ P6186)], [BAP= benzylaminopurine (Sigma~ B3408)],

[KIN= kinetin, 6-furfurylaminopurine (Sigma~ KO7.53)], [PIC= picloram, 4-amino-3,5,6-

trichloropicolinic acid (Sigma~ P5575)], [2,4-D= 2,4-dichlorophenoxyacetic acid

(Sigma~ D6679)], [IBA= indolebutyric acid (Sigma~ 15386)] in distilled water and kept

frozen at -14oC.The pH of the media was adjusted to 5.8 with the addition of drops of 1 N

KOH or 1N HCI before the addition of agar (Sigma~ A-1296). Culture medium was

sterilized by autoclaving for 20 minutes at 0.103 Mpa.

For the first steps of all experiments, 100 mm diameter x 15 mm deep petri dishes

were used and explants were placed with the abaxial side down on 20 mL of culture

medium. After the induction, cultures were transferred onto MS medium with or without

the addition of 0.044 CIM BAP + 0.049 C1M IBA to achieve bud elongation and rooting of

shoots. To induce further growth of shoots, cultures were subsequently transferred to

magenta boxes (76.2 mm x 76.2 mm x 101.6 mm) (Magenta Corporation") containing 50

mL of MS devoid of plant growth regulators. Cultures were kept in a growth chamber at

26 & 2oC with a 16-hour photoperiod and 85 Clmol m-2 S-1 prOVided by cool fluorescent

lights.

Regenerated plants were removed from magenta boxes and rinsed under running

tap water to completely remove the culture medium and were then placed in plug trays

containing vermiculite and covered with a humidity dome. Trays were placed in a growth









chamber at 22 + 2oC with a 14-hour photoperiod. Plants were watered daily to keep a

high humidity level during the first two weeks and a solution containing 2 g/L of

Captan~ [4-cyclohexene-1,2-dicarboximide, N-(trichloromethyl) thio] was applied twice

during this period. During the third week, covers were removed gradually for longer

periods of time and plants were finally transferred to the greenhouse.

Evaluation and Experimental Design

Cultures were evaluated every 30 days for most experiments, in order to determine

the number of explants that died, remained irresponsive, produced callus or buds.

Whenever buds were regenerated, the number of buds per explant was also recorded.

Each treatment was applied to 10 explants and experiments were repeated 2-3 times.

Experiments were treated as completely random factorial experiments or completely

randomized designs.









Table 3-1. Plant material used as a source of explants in the experiments.
Species Accession No. Origin
A. bicolor U5 Lavalleja (Uruguay)
U6 Rocha (Uruguay)
U7 Paysandu (Uruguay)
U8 Tacuarembo (Uruguay)
U10 Paso de los Toros/Tacuarembo (Uruguay)
Ul l Durazno (Uruguay)
Ul2 Canelones (Uruguay)
Ul3 Rocha (Uruguay)
Ul4 Castillos/Rocha (Uruguay)
A. latifolia Ul7 Valle Eden/Tacuarembo (Uruguay)
Ul8 Mina de Corrales/Rivera (Uruguay)
Ul9 Velasquez/Rocha (Uruguay)
T. polymorphum Ul Estancia El Rinc6n/Florida (Uruguay)
U2 Uruguay
U3 Uruguay
CPI 87102
Pilar (Paraguay)
Rio Grande (Brazil)
Uruguay
T. carolinianum PI 516273 Gainesville, Florida, USA
L. bainesii Cv INIA Glencoe Uruguay
* U preceding a number is a locally assigned University of Florida number, CPI is
Commonwealth Plant Introduction number from Australia and PI is USDA/NPGS Plant
Introduction number















CHAPTER 4
TISSUE CULTURE OF Trifolium polymorphum AND 7 carolinianum

Introduction

The genus Trifolium originated in the Mediterranean region, but subsequently

spread to the Americas, Asia and Africa as well (Zohary & Heller, 1984). It consists of 8

sections and approximately 240 species, 25 of which are used as forage (Lange &

Schifino-Wittman, 2000). 7 polymorphum is a highly palatable forage legume native to

eastern Argentina, Uruguay, Paraguay, central Chile and southern Brazil (Speroni &

Izaguirre, 2003). It is an amphicarpic species that produces aerial and subterranean

flowers on the same individual. It has been reported that the latter produce seeds more

profusely, acting as a seed bank, whereas seed production from aerial flowers may be

affected by grazing and insect attacks. The above-ground flowers have a morphology that

seems to stimulate insect pollination; however, the species does not appear to exhibit self-

incompatibility and self-pollination can occur, even before anthesis (Speroni & Izaguirre,

2003). Other workers report that the above-ground flowers are almost exclusively cross-

pollinated (Daniel Real personal communication, 2006). I carolinianum is an annual

species and one of only three clovers native to the southeastern US. It is self-pollinated

and re-seeds abundantly, but due to small plant size, has only limited forage potential.

Plant regeneration has been achieved in several species of the genus Trifolium

including 7 repens, T7pratense, 7 subterraneum, 7 michelianum~~~~iiii~~~~iii Savi, 7 isthmocarpum

Brot. (Ding, et al., 2003), 7 nigrescens (Konieczny, 1995), and 7 rubens (Grosser &

Collins, 1984) via either organogenesis or somatic embryogenesis. Many of these










protocols have used explants consisting of immature zygotic embryos (Maheswaran &

Williams, 1984) or seedling derived explants such as hypocotyls and radicles (Heath et

al., 1993), cotyledons (Konieczny, 1999), or leaves (Radionenko et al., 1994). However,

these explants may not be appropriate for the propagation of selected genotypes in cross

pollinated species in which seeds coming from the same plant may exhibit different

genotypes. In this case, explants obtained from vegetative tissues in fully developed

plants are more suitable for the propagation of selected individuals. Trifolium plant

regeneration from non-meristematic tissues has been achieved using leaves (Rybcznski,

1997) and petioles (Quesenberry & Smith, 1993).

T. polymorphum and T. carolinianum exhibit low seedling vigor and low DM

production that limit their forage use. Therefore, in vitro chromosome duplication may be

used to increase plant vigor. In addition, chimeral plants could be avoided provided that

plant regeneration is obtained from single cells that were previously doubled by treatment

with chromosome doubling agents such as colchicine, trifluralin or oryzalin (Quesenberry

et al., 2003). Consequently, the obj ective of these experiments was to develop a plant

regeneration protocol that could be used for in vitro research to produce polyploid plants

of T. polymorphum and T. carolinianum with increased seedling vigor and DM

production.

Materials and methods

Trifolium polymorphum

Seeds from several accessions (Table 3-1) were germinated as described in chapter

3. Plants were maintained aseptically in magenta boxes containing MS medium devoid of

growth regulators and were transferred approximately monthly. Petioles from immature

fully expanded leaves were excised and cut on sterilized filter paper into pieces of 5 to 6-









mm length, which were used as explants. Five pieces were placed per Petri dish, onto one

of the three basal media prepared as indicated in chapter 3 with the addition of 4.5 C1M

TDZ. The three treatments were applied to all germinated genotypes and experiments

were repeated twice. Incubation conditions were the same as described in chapter 3 and

data was recorded after 30 days of culture. The experiment was statistically analyzed as a

factorial arrangement in a completely randomized design using PROC GLM from PC

SAS (SAS Institute, 2003). Tukey's HSD Multiple Range Test at p<;0.05 level was used

to compare the means of the basal media for each genotype.

Trifolium carolinianunt

Seeds were scarified and germinated as indicated in chapter 3. Cotyledons from 45

1-week old seedlings were excised and cut longitudinally along the midrib into two

pieces so that four cotyledon explants were obtained per genotype. Each explant was

placed onto MS alone or MS with 10 CIM TDZ, 10 CIM BAP or 10 CIM KINT. Five pieces

were placed per petri dish and the identity of the genotypes was maintained. Seedlings

without cotyledons were placed onto MS medium and kept in vitro for further

experiments. After 30 days of culture, regenerated buds were transferred onto L2 medium

supplemented with 1 CIM TDZ. Incubation conditions were the same as described in

chapter 3.

Results and Discussion

Trifoliant polymorphum

After 30 days of culture, the only response obtained was the formation of friable,

light brown callus that died after subculturing onto the fresh culture medium. The

statistical analyses revealed an effect of basal media, genotypes and basal medium x










genotype interaction (Table 4-1). Basal medium B5 was significantly better than the

others for the induction of callus formation in 9 of the 15 genotypes. In this experiment,

TDZ was used as the growth regulator since it has a high cytokinin activity (Huetteman

& Preece, 1993) and was reported to effectively induce organogenesis in a number of

species of the genus Trifolium and M~edicago (Ding et al., 2003). In addition, pieces of

petioles were chosen as explants, since this would allow for the propagation and

manipulation of selected genotypes. Petioles were successfully used as explants in T.

pratense (Quesenberry & Smith, 1993) and T. rubens (McGee et al., 1989).

The failure in the regeneration of shoot buds could be due to the plant growth

regulator used in the experiment, which may not be adequate to trigger the organogenic

process or might have been present in a concentration toxic for this species. It is also

possible that other auxins such as PIC or 2,4-D could be more effective. However, it

would be necessary to evaluate a larger number of genotypes since there were significant

differences among them.

Seedlings grew vigorously in vitro after slow germination, which started a week

after the scarification and continued for over a month. Nevertheless, after approximately

two months in culture, they started to decay producing few leaves even with frequent

transfers to fresh medium. This factor limited the number of replications and experiments

that could be carried out with this species. In order to determine if the presence of plant

growth regulators in the germination medium promoted multiple shoot formation, seeds

from different genotypes were scarified as in the first experiment and placed onto culture

media containing either 10 CIM TDZ or 1 CIM BAP. Germination was not enhanced but

multiple shoots were produced in some cases (data not shown). Nevertheless, the rate of










growth was very slow, and seedlings became chlorotic approximately a month after

germination started.

Trifolium carolinianum

After 30 days of culture, it was observed that all explants placed on the basal

medium devoid of plant growth regulators had died. Among the plant growth regulators

that were evaluated in the experiment, KIN was not effective for callus induction or bud

formation in any of the 45 genotypes and BAP only resulted in shoot bud formation in

one genotype and callus formation in one other. TDZ effectively induced shoot bud

organogenesis in 20% of the genotypes and callus formation in another 20% of the

explants. Moreover, the mean number of buds was considerably low (2. 1) suggesting that

the species may be recalcitrant to tissue culture (Table 4-2). In all cases, calli were small,

friable and light brown colored, but were already dead at the time the data was recorded.

Thirty days after transfer, the mean number of buds across genotypes increased

markedly from 2.1 to 18.7 and buds began to elongate although growth was slow (Figure

4-1). To achieve bud elongation, cultures were transferred to L2 medium because it has a

lower salt concentration than MS medium. In addition, the concentration of TDZ was

reduced from 10 to 1 CIM because it has been reported that TDZ may inhibit bud

elongation (Huetteman & Preece, 1993).

Almost all responses were with TDZ, indicating that this plant growth regulator

would be preferred for induction of organogenesis in T. carolinianum. In a previous

report, TDZ proved effective for shoot bud induction in several Trifolium species (Ding

et al., 2003). However, most of the reports on plant regeneration through organogenesis









in this genus mention the use of other plant growth regulators such as BAP (Heath et al.,

1993; Konieczny, 1999) and 2-isopentenyladenine (Konieczny, 2000).

Trifolium carolmiainum is an annual, self pollinated species, and therefore it has

high homogeneity and homozygosis is expected within populations and plants. For this

reason, in order to develop a tissue culture protocol, cotyledonary explants were used.

Considering that seeds from a single plant and even a population should exhibit similar

genotypes, this constitutes a suitable explant that is available in large quantities and can

be easily disinfected and handled. This is not the case in cross pollinated species because

the progeny of an individual plant may not reflect the trait of interest in the mother plant,

which generally limits the use of seed-derived explants.

Interestingly, there were unexpected differences in response among genotypes in

that explants from some genotypes were completely irresponsive while others produced

up to 35 buds. This might indicate that the population that was used in the study is not

highly homogeneous, probably due to the occurrence of some cross pollination. Another

possibility could be that the difference in the responsiveness of the genotypes was caused

by the treatment received by the plant material. For example, if some seeds had a thinner

coat, the scarification process with sulphuric acid could have affected the cotyledons and

their ability to regenerate in vitro. Another possible cause of variation could be size of the

cotyledons among seedlings, which may explain the difference in the number of buds in

the responsive genotypes.

Although it does not explain the variability observed in the experiment, it is

possible that the unresponsive genotypes were more sensitive to the high concentration of

salts in the culture medium. In this case, L2 basal medium should also be used for the









induction of organogenesis in this species. This would be in contrast to other work on

Trifolium spp which showed that the use of MS as the basal medium allowed the

development of an efficient protocol for 7 repens, E~pratense, 7 subterraneum, 7

michelianum and I isibinumpainl'"" (Ding et al., 2003).

Conclusions

In 7 polymorphum, a significant interaction between genotypes and basal media

was observed for callus production. There were also significant differences among

genotypes and culture media. However, the culture media tested to achieve plant

regeneration through organogenesis were not effective and yielded only callus. Among

the three basal media tested, a higher percentage of callus formation was observed with

B5. The lack of shoot organogenesis may have been caused by the type or level of plant

growth regulators, and therefore, other cytokinins or auxins should be tested. Moreover,

additional experiments are required in order to determine the best culture conditions to

maintain germplasm actively growing in vitro.

In contrast, 7 carolinian2um shoot bud formation was achieved in 20% of the

genotypes when the culture medium contained 10 C1M TDZ. This might suggest that in

this species a potent cytokinin is required in order to induce this morphogenetic process.

Callus formation was observed in another 20% of the genotypes, and the rest remained

non-responsive and died. This difference was not expected considering that it is a self

pollinated species, and populations are expected to be highly homogeneous. Shoot bud

elongation occurred in MS supplemented with 1 CIM TDZ but simultaneously, the

number of adventitious buds continued to increase. Additional experiments are being






28


conducted in order to achieve rooting of the regenerated shoots and an adequate growth

of the plantlets that would likely result in a higher acclimatization rate ex vitro.










Table 4-1. Mean percentage of explants producing callus from petiole pieces of T.
polymorphum in three basal media after 30 days of culture.
Basal medium
Genotype MS L2 B5
CPI. 1 0 a* 0 a 20 a
CPI.2 90 b 20 a 100 b
CPI.3 0 a 10 a 100 b
CPI.4 0 a 0 a 80 b
CPI.5 0 a 0 a 0
CPI.6 0 a 0 a 80 b
CPI.7 0 a 0 a 0
CPI.8 0 a 0 a 0
CPI.9 0 a 0 a 30 b
CPI.10 0 a 0 a 100 b
CPI.11 0 a 0 a 90 b
Urug. 1 0 a 0 a 90 b
Parag. 1 0 a 0 a 90 b
Parag.2 0 a 0 a 60 b
U 02.1 0 a 0 a 100 b
*Within rows, different letters indicate significant differences according to Tukey's HSD
Multiple Range test at p<;0.05 level.






Table 4-2. Number of buds per explant in the responsive genotypes of T. carolinianum
cultured on medium supplemented with 10 CIM TDZ after 30 and 60 days of
culture.


Genotype


30 days of culture


60 days of culture
35






























Figure 4-1. Shoot bud organogenesis through cotyledon culture of T. carolinianum, 15
days after transfer to L2 + 1 CIM TDZ (bar: 20 mm).















CHAPTER 5
PLANT REGENERATION OF Adesmia latipolia AND A. bicolor

Introduction

The tribe Adesmieae belongs to the Fabaceae family and consists of only one

genus, Adesmia DC., and approximately 240 species endemic to South America (Ulibarri

& Burkart, 2000). Most of these species of herbs and shrubs grow in the Andes

mountains and semi desert zones in the Patagonia. Some species, such as A. latifolia, A.

bicolor and A. punctat are considered promising forages because of their ability to grow

during the winter season, high crude protein value and high in vitro organic matter

digestibility (Tedesco et al., 2000). In addition, due to the stoloniferous morphology of

plants, they may be used for soil cover and erosion control (Coelho & Battistin, 1998).

Adesmia bicolor and A. latifolia are cross pollinated species although some self

pollination may occur (Tedesco et al., 2000). As a consequence, selected genotypes

cannot be propagated through seeds because their progeny could segregate for the trait of

interest. Even though these species could be multiplied vegetatively using stolons, tissue

culture could provide a more rapid and efficient method of propagation. In addition, an in

vitro protocol for plant regeneration from single cells, such as organogenesis and somatic

embryogenesis, could be used to double the chromosome number in an attempt to

increase plant productivity. Moreover, the development of a tissue culture system to

propagate and maintain plants in vitro would prove useful for germplasm exchange

(Mroginski et al., 2004b) since plants are protected from pathogens and do not require a

quarantine period. Another contribution of tissue culture techniques to plant breeding is










the in vitro selection for biotic and abiotic stresses, which has been applied in alfalfa for

resistance to specific fungal diseases (Dita et al., 2006).

It has been suggested that in forage legume populations there is greater

heterogeneity in comparison to grain legume populations, due to the lower selection

pressure of high seed producing genotypes (Veltcheva et al., 2005). This condition would

result in an easier identification of in vitro responsive genotypes. However, the

development of an efficient plant regeneration protocol implies the regulation of the

many factors that influence in vitro responses and not only the genotype. These other

factors include selection of an adequate explant, based on the obj ectives of the culture,

composition of the basal medium and plant growth regulators used to induce the

morphogenetic response, and conditions of incubation (Radice, 2004).

Tissue culture protocols have been developed for some forage legumes such as T.

pratense, 7: repens (Ding et al., 2003), A. pintoi Krapov. & W.C. Greg. (Rey &

Mroginski, 2006), A. glabrata (Vidoz et al., 2006), Lotus corniculatus L. (Akashi et al,

2003), Astragalus melilotoides Pall. (Hou & Jia, 2004) and Macroptilium atropurpureum

(DC.) Urb. (Ezura et al., 2000). Nevertheless, currently there are no reports on tissue

culture in the genus Adesmia. The obj ective of these experiments was to develop for the

first time, an in vitro plant regeneration system that could assist A. bicolor and A. latifolia

breeding programs.

Materials and Methods

Plant Material

Seeds ofA. bicolor and A. latifolia were scarified, sterilized and germinated as

described in chapter 3. Plants were maintained aseptically in magenta boxes on MS

medium without the addition of plant growth regulators. Unless indicated, explants









consisted of leaflets from immature leaves, at approximately 50% of expansion, that were

excised from those in vitro grown plants.

Basal Media Experiment

Seven genotypes of A. bicolor and 16 of A. latifolia were used as explant sources.

Leaflets were placed onto the three basal media indicated in chapter 3, with the addition

of 4.5 CIM TDZ. Each treatment was applied to 10 leaflets of each genotype and

experiments were repeated three times. After 60 days of culture, buds were transferred to

magenta boxes containing MS devoid of growth regulators for 45 days, where buds

elongated and rooted. Incubation and acclimatization of regenerated plants was

performed as described in chapter 3.

Response variables analyzed were: percent organogenesis (%= number of explants

that produced buds/total number of explants* 100), mean number of buds per explant (E

number of buds per explant/ number of explants that produced buds) and regeneration

index (index= % shoot bud formation mean number of buds per explant / 100). This

index was used in order to evaluate the influence of the basal medium on the percentage

of shoot bud formation and mean number of buds per explant simultaneously. A higher

index value indicates that a genotype is capable of producing a higher total number of

buds. These response variables were also used in subsequent experiments.

This experiment was statistically analyzed as a factorial arrangement in a

completely randomized design using PROC GLM from PC SAS (SAS Institute, 2003).

Tukey's HSD Multiple Range Test at p<;0.05 was used to compare the means of the basal

media for each genotype.









Factorial Experiments with TDZ and BAP

Among the responsive genotypes in the basal media experiment, A. latifolia Ul8.6

and Ul8.8 were chosen as explant sources for subsequent experiments, because their

micropropagation rate (2.74 and 3.12 fold increase per month, respectively) provided

adequate amounts of tissue. Explants were placed onto MS supplemented with 0, 1, 10,

30 or 60 CIM TDZ alone or with the addition of either 0, 0. 1 or 1 CIM IBA, such that 15

treatments were used. These same concentrations and combinations were assayed

replacing TDZ by BAP. Each treatment was applied to 10 leaflets of each genotype and

experiments were repeated twice. After a month, cultures were transferred onto MS +

0.044 CIM BAP + 0.049 CIM IBA for 30 days to achieve bud elongation and then to

magenta boxes containing MS without growth regulators for the same period of time.

Incubation and acclimatization of regenerated plants was carried out as described in

chapter 3.

Since both genotypes responded similarly, data were analyzed and presented as a

combination of the two; however, results for each genotype are presented in the

Appendix (Tables A-1 to A-9). Data from subsequent experiments were also pooled for

genotypes. In addition to the variables mentioned above, shoot number, length of the

longest shoot and acclimatization rate (% survival= number of plants that survived after a

month / number of plants transferred to ex vitro conditions *100) were also determined.

Statistical analyses were performed as a factorial arrangement in a completely

randomized design using PROC GLM from PC SAS (SAS Institute, 2003). Tukey's HSD

Multiple Range Test at p<;0.05 was used to compare the means of treatments. Regression

analysis was performed using Microsoft Office Excel (Microsoft, 2003).









Induction Time Experiments

The same explants and sources from the previous experiment were used to evaluate

the influence of exposure time to TDZ. Leaflets were placed onto MS + 10 C1M TDZ for

0, 1, 4, 7, 14, 20 or 30 days and then transferred onto MS devoid of plant growth

regulators. Each treatment was applied to 10 leaflets of each genotype and experiments

were repeated twice. Forty-five days after the initiation of the experiment, all cultures

were transferred onto fresh medium for 30 days, and then into magenta boxes containing

the same culture medium. Incubation conditions were the same as described in chapter 3.

The experiment was analyzed as a completely randomized design using PROC

GLM from PC SAS (SAS Institute, 2003). Tukey's HSD Multiple Range Test at p<;0.05

was used to compare treatment means. Regression analysis was performed using

Microsoft Office Excel (Microsoft, 2003). Additional data for each genotype are

presented in Appendix (Tables A-10 to A-12).

Type of Explant Experiment

The same two genotypes ofA. latifolia were used as explant sources. Leaflets were

excised and placed onto sterile filter paper where they were divided into petiole, rachis

and leaflets. Petioles and rachises were subsequently divided into 5-6 mm pieces. All

petiole and rachises pieces obtained from each leaf and eight leaflets were placed in each

petri dish containing MS + 10 C1M TDZ. This procedure was repeated 10 times for

mature, fully expanded and immature, actively expanding leaves from both genotypes.

Thirty days after the initiation of the experiment, explants were transferred onto MS

medium without growth regulators to achieve bud elongation. The conditions under

which cultures were incubated were as those indicated in chapter 3.









Statistical analysis was performed as a factorial arrangement in a completely

randomized design using PROC GLM from PC SAS (SAS Institute, 2003). Tukey's HSD

Multiple Range Test at p<;0.05 was used to compare the means of treatments. Additional

data for each genotype separately is presented in Appendix (Table A-13).

Results and Discussion

Effect of Basal Medium on Shoot Organogenesis

After 60 days of culture, shoot bud formation was observed in 18 of the 23

genotypes evaluated (Table 5-1). Four of the non-responsive genotypes (U5.3, U5.5,

Ul2.5 and Ul3.1) corresponded to A. bicolor and one (Ul9.3) to A. latifolia. Both

genotype and basal medium had highly significant effects on the percentage of shoot bud

formation. The interaction between these factors was also highly significant.

The basal medium MS resulted in a higher number of responsive genotypes and

was superior to L2 in two of them. The average percentage of shoot bud formation across

all genotypes was not significantly different between MS and L2 (22 and 16.6%,

respectively); however, both were superior to B5 (5.5%). In general, higher percentages

were obtained in those genotypes derived from accession Ul8 of A. latifolia, whereas

those corresponding to A. bicolor were the least responsive.

For the variable mean number of buds per explant, only genotype was a significant

source of variation. This was also evident when the average number of buds per explant

across genotypes is considered. This may indicate that the basal medium may have had a

maj or influence during the period in which explants dedifferentiated or acquired

competence to respond to the plant growth regulator stimulus, but once induction had

occurred, it had a low influence on the number of buds arising per explant. In all cases,









the mean number of shoot buds arising per explant was rather low and never higher than

five, although up to 20 buds per individual explant were formed in some cases.

The regeneration index proved to be useful in this study involving numerous

genotypes since in some cases a high number of buds per explant were produced, but a

low percentage of explants was responsive. Even though the identification of highly

responsive genotypes was not the main purpose of this experiment, this index proved

useful for the selection of genotypes that could be used in further experiments to adjust

the in vitro plant regeneration protocol. Averaging regeneration index data over

genotypes, the means for MS and L2 were significantly higher than for B5. However, the

index masked the effect of genotype x medium interaction observed on percentage, since

the statistical analysis revealed only genotype and basal medium effects.

The results of this experiment suggested that MS medium produced higher

proliferation of shoot buds in A. latifolia and A. bicolor than the other basal media.

Although MS was originally developed for callus culture of tobacco (Murashige and

Skoog, 1962) and B5 and L2 were developed for legume species (Gamborg et al., 1968;

Collins & Phillips, 1982), MS basal medium has been successfully used for tissue culture

of several legume genera including forages like Trifolium and M~edicago (Ding et al.,

2003), grain legumes like Cicer (Chakraborti et al., 2006) and Vigna (Saini & Jaiwal,

2002) and trees such as Dalbergia and Cassia (Singh & Chand, 2003; Agrawal & Sardar,

2006). Moreover, shoot bud induction was possible using TDZ as the plant growth

regulator, which is in agreement with reports on other legume species as A. hypogagea

(Akasaka et al, 2000), A. correntina (Burkart) Krapov. & W. C. Greg. (Mroginski et al.,









2004a), Cajanus cajan (L.) Millsp. (Singh et al., 2003) and Vigna radiata (L.) R. Wilczek

(Mundhara & Rashid, 2006).

Regarding the acclimatization ex vitro of regenerated plants, the overall

performance of plants obtained in MS treatment was markedly higher (72.4% survival ex

vitro) than those of plants obtained in L2 (38.7%) or B5 (45.6%) (Figure 5-1 E). This

higher survival rate of plants regenerated on MS may be due to reduced problems

associated with hyperhydricity. One of the factors that lead to this condition characterized

by morphological and physiological abnormalities is water availability (Hazarika, 2006).

The higher salt concentration of MS basal medium may result in lower water availability

for plant material, so that the incidence of hyperhydricity is lower in MS than in L2 or B5

medium. Low survival rates could also be due to other disorders common to plants

exhibiting heterotrophic growth in vitro, such as a low photosynthetic rate, abnormalities

in structure and functioning of stomata, and poor mesophyll and vascular system

development (Hazarika, 2006).

Influence of TDZ on Organogenesis

After 15 days of culture, shoot buds were observed in all plant growth regulator

combinations in both genotype 18.6 and 18.8 of A. latifolia. Similarly, TDZ has been

successfully used to induce shoot formation in other legume genera; however, in some

species such as Vigna radiata (Mundhara & Rashid, 2006), A. hypogaea (Gill & Ozias-

Akins, 1999), Trifolium spp (Ding et al., 2003) and C. cajan (Singh et al., 2003), explants

consisted of seedling-derived parts which are usually much more responsive than non-

juvenile tissues.

In most of these Adesmia explants, callus formation was rare and, in some cases,

buds seemed to arise almost directly from the surface of the explant. This is a desirable









characteristic since prolonged maintenance in tissue culture may result in somaclonal

variation (Ozias-Akins & Gill, 2001). For the percentage of explants exhibiting bud

organogenesis and mean number of buds per explant, there was a highly significant effect

for TDZ but not for IBA concentration or its interaction with TDZ concentration. Since

IBA effect was not significant, the average across IBA concentrations is presented. Only

the treatment without TDZ was significantly differed from the rest for the percentage of

shoot bud formation. A separate analysis was performed by excluding the 0 TDZ

treatment in order to detect differences among TDZ concentrations and acquire a more

accurate understanding of the response (Tables 5-2 and 5-3). Similarly a regression

analysis was carried out without considering the 0 TDZ treatment and the simplest model

that best explained culture responses was chosen in each case. When the 0 TDZ

treatment was dropped, numerically the percentage adventitious bud formation increased

with increasing TDZ concentration at 30 days of culture, but this increase was not

significant (Table 5-2). By 60 days of culture, there was a significant quadratic effect of

TDZ concentration (R2=0.96) (Table 5-3 and Figure 5-2). Regardless of TDZ

concentration the percentage of responsive explants increased from 30 to 60 days of

culture which indicates that some explants required a longer period of time to become

competent and differentiate buds (Appendix Tables A-1 and A-3).

The same statistical procedures and considerations described above were applied to

the other response variables. Even when the 0 TDZ treatment was dropped from the

analysis, TDZ concentration did not affect mean number of buds per explant at either 30

or 60 days (Tables 5-2 and 5-3). In some cases, the number of buds per explant of A.

latifolia Ul8.8 actually decreased at 60 days of culture at higher concentrations of TDZ









(Appendix Table A-3) due to the death of some buds. These bud deaths were probably

due to some phytotoxic effect of this potent plant growth regulator, as reported in peanut

(Kanyand et al., 1994).

The same regeneration index as in the basal media experiment was applied here

(Tables 5-2 and 5-3). This index showed a quadratic response with increasing TDZ

concentrations at 30 days of culture (R2= 1) (Figure 5-3.), but by 60 days of culture the

effect of TDZ concentration had disappeared (Table 5-3). Shoot number (Table 5-3 and

Figure 5-4) and shoot length (Table 5-3 and Figure 5-5) showed a linear decrease with

increasing concentrations of TDZ (R2= 0.87 and 0.79, respectively) (Figures 5-1 A-D).

The reduction in shoot elongation is a common response reported in several species when

TDZ is added to the culture medium (Huetteman & Preece, 1993). Shoot length is an

important factor for the success in ex vitro acclimatization since longer shoots may have

more reserves to produce roots which promote higher survival rates (Table 5-3). This was

evident in both A. latifolia genotypes (Appendix Tables A-2, A-4 and A-5) where ex vitro

survival decreased linearly with increasing concentrations of TDZ (R2=0.88) (Figure 5-

6). In addition, there was a higher incidence of hyperhydricity in plants regenerated in

medium containing 30 and 60 CIM TDZ, probably caused by a hormonal imbalance in the

culture medium (Hazarika, 2006).

Influence of BAP on Organogenesis

For most variables considered in this study, the effect of BAP concentration was

significant whereas the effect of IBA and the interaction between these factors were not.

Consequently, the mean across IBA concentrations was obtained and values were treated

as in the previous experiment regarding Tukey's test and regression analysis. After 30

days of culture, there was a quadratic effect of BAP concentration (R2=0.24) (Figure 5-7)









although the percentage of explants that had produced buds was low and some explants

remained non-responsive (Table 5-4). By 60 days of culture, the response to BAP

concentration remained quadratic (R2=0.57), but the percentage of adventitious bud

formation increased in at all concentrations (Table 5-5 and Figure 5-9). This response is

similar to what has been reported for A. hypogaea, where the addition of BAP to the

culture medium required a longer period to induce shoot organogenesis than when was

TDZ was added to the medium (Kanyand et al., 1994). Bud formation occurred in

explants of most treatments at 60 days after initiation of cultures (Table 5-5 and

Appendix Tables A-6 and A-8). Additionally, an increase in the percentage of bud

formation occurred in those that had responded earlier. This pattern was also observed in

the number of buds per explant. Similar to percentage of bud organogenesis, regeneration

index response to increasing BAP concentration after 30 days of culture was quadratic

(R2=0.25) (Figure 5-8) being highest for explants placed onto 10 CIM BAP. After 60 days

of culture, the regeneration index response was still quadratic (R2=0.48) (Figure 5-11i),

but the index values were significantly lower for explants cultured onto 1 CIM BAP than

for those placed onto 10-60 CIM BAP (Tables 5-4 and 5-5).

For mean number of buds per explant, there were no differences among treatments

after 30 days of culture, but after 60 days, the response to BAP concentrations was

quadratic (R2=0.56) (Tables 5-4 and 5-5, Figure 5-10). At 90 days of culture, shoot length

also exhibited a quadratic response to BAP concentration (R2=0.50) (Figure 5-12),

reaching a maximum at 10-30 CIM BAP and then decreased (Table 5-5, Appendix Tables

A-7 and A-9). This reduction in length at high levels of the plant growth regulator may









have resulted from the induction of numerous shoot primordia that competed among each

other and/or to the higher number of hyperhydric shoots.

The addition of BAP to basal medium has proved useful to induce organogenesis in

some legume genera such as Desnzodium (Rey & Mroginski, 1997), Aeschynonsene (Rey

& Mroginski, 1996) and Trifolium (Heath et al., 1993). Interestingly, BAP was less

effective than TDZ for shoot bud induction in A. latifolia. This is in agreement with a

report in A. hypogaea, in which several cytokinins were tested but TDZ proved to be the

most efficient (Akasaka et al., 2000).

Induction Time for Adventitious Bud Formation

In order to determine the minimum time of exposure to TDZ required for shoot bud

induction in A. latifolia, explants were maintained in a culture medium supplemented

with this plant growth regulator for increasing periods of time. It was observed that one

and four days of culture were enough to induce organogenesis in A. latifolia Ul8.8 and

Ul8.6, although with a very low percentage of bud formation (Appendix Tables A-10

and A-11).

In general, the percentage of bud organogenesis increased as explants remained for

longer periods of time in contact with TDZ. There were no significant differences

between the 1- and 4-day treatments or among 7- to 30-day treatments (Table 5-6). An

increase in the percentage of responsive explants was also observed from 30 to 60 days

after the initiation of cultures. This suggests that, as in the case of four to seven days of

exposure to TDZ, a period of over 30 days may be required for buds to arise even though

cells have been induced to follow this developmental pathway. In most cases the mean

number of buds per explant increased from 30 to 60 days after the initiation of cultures.

Exceptions to this were the case when some buds became stunted and died. However,









there were no significant differences in mean bud number when explants were placed

between on TDZ 4 and 30 days (Table 5-6). Regarding the regeneration index, there were

significant differences between 1 or 4 days on TDZ and 7 to 30 days on the medium with

this plant growth regulator after 30 days of culture. Thirty days later, the highest index

value was observed for the longest treatment, but it was not significantly different from 7

to 20 days of culture on TDZ. Several studies showed that in order to regenerate normal

shoots in A. hypogaea, explants should not remain in a medium supplemented with TDZ

for more than 7 days at 45.4 CIM TDZ or 21 days at 4.54 CIM TDZ (Akasaka et al., 2000).

In A. latifolia there were no significant differences in shoot length after culture of

explants on TDZ for 1 to 30 days (data not shown). Regression analysis for all response

variables showed a quadratic increase for length of time on TDZ supplemented medium

(R2 fTOm 0.80 to 0.95) (Figures 5-13 to 5-15 and Appendix Figures A-1 to A-3).

Another experiment was conducted with shorter intervals between treatments to

determine more precisely the number of days required for buds to arise. The percentage

of shoot bud formation was highest for 10 days of culture on TDZ containing medium,

however, it was not significantly different from percentages obtained after 5 to 9 days of

culture on TDZ (Table 5-7). There were no differences among treatments regarding the

mean number of buds per explant, and the regeneration index showed a significant

difference only between 10 days of culture on TDZ and 1 or 2 days of exposure to this

plant growth regulator but not with longer exposures (Table 5-7). In addition, the

percentages of response were lower than in the first experiment (Appendix Table A-12).

Regression analysis showed a linear increase of percentage of shoot bud formation and

regeneration index with increasing exposure to TDZ (R2= 0.92 and 0.93, respectively)










(Figures 5-16 and 5-17), but this is probably because treatments corresponded to the first

phase of the quadratic model observed in the previous experiment.

Effect of Explant Type on Shoot Organogenesis

Results from an exploratory experiment with several genotypes ofA. latifolia

revealed that leaflets were more responsive than petioles, suggesting that the part of the

leaf used as explant had a maj or influence on the in vitro responsiveness. The percentage

of bud formation was significantly higher when rachises were used as explants (Table 5-

8). Leaflet explants produced a significantly higher percentage of organogenesis than

petioles, which in general remained non-responsive. In the best case, only 16%

organogenesis was achieved in contrast to the 90% responsiveness that was obtained

using rachises from immature leaflets (Appendix Table A-13). These response patterns

were observed in both immature and mature leaves. Mean number of buds per explant

and bud formation index also showed the higher responsiveness of rachises over the other

two types of explants. For these response variables, there were no significant differences

between leaflet and petiole explants (Table 5-8).

The higher frequency of shoot bud formation from rachises may be due to the

presence of intercalary meristems in the rachises because they are the last segment of the

leaf to undergo the maturation process. Nevertheless, this does not explain that this

explant, when excised from mature leaves, gave almost as high responses as when

harvested from actively expanding ones. The only disadvantage of using rachises would

be the lower amount of plant material in cases in which few or small plants are available,

since each leaf provides between 10 to 20 leaflets, but each rachis from immature leaves

gives no more than two explants.










Regarding explant age, there were no significant differences between immature and

mature leaves although explants from expanding leaves were more responsive than those

from fully expanded, mature ones. This may be due to the meristematic activity of young

leaves with less differentiated cells that could readily respond to an external stimulus, i.e.

TDZ. In A. hypogaea, leaf developmental stage is a primary factor in somatic embryo

induction, as unfolded leaflets usually lose embryogenic potential (Baker & Wetzstein,

1998). Nevertheless, shoot bud formation was possible in a number of wild Arachis

species (Dunbar & Pittman, 1992). In contrast to what might be expected, cotyledons of

A. bicolor and A. latifolia were completely irresponsive in vitro when cultured onto PIC,

2,4-D or TDZ (data not shown).

Conclusions

It was possible to achieve plant regeneration from A. latifolia and A. bicolor

through immature leaflet culture. Shoot bud organogenesis was successfully induced in

several genotypes of both species using MS, L2 or B5 as the basal medium. Even though

there were no marked differences in bud formation frequency between MS and L2, the

former one promoted a higher ex vitro survival of regenerated plants and therefore was

used in subsequent experiments. Interestingly, A. bicolor proved to be much less

responsive than A. latifolia and all regenerated plants failed to acclimatize ex vitro.

The plant growth regulator TDZ was more efficient than BAP for shoot bud

induction, not only in the frequency of bud formation, but also in the time required for

buds to arise from explants. In general, the percentage of organogenesis increased with

higher levels of TDZ, but this was associated with a reduction in bud elongation. Shoot

length was a critical factor in ex vitro acclimatization of regenerated plants and as a

consequence, plants originated in TDZ concentrations of 1 and 10 CIM showed higher









survival rates. Despite the lower organogenesis frequency of explants cultured on BAP,

this plant growth regulator did not affect shoot length as drastically as TDZ, and in

general the most effective concentrations were 10 and 30 CIM BAP.

Shoot bud organogenesis was observed after exposures to TDZ as short as one day,

although the percentage of bud formation increased with prolonged culture in a medium

containing this plant growth regulator up to 10 days. Exposure for 30 days to 10 CIM TDZ

allowed a higher frequency of organogenesis and did not show a negative effect in shoot

elongation, which was observed in the previous experiment at higher levels of the plant

growth regulator.

A maj or influence of explant type on shoot bud formation of A. latifolia was

revealed in a follow-up experiment. A higher frequency of shoot bud formation and mean

number of buds per explant was obtained when rachises were used as explants. The age

of the source leaf was not as important as the part of the leaf placed into culture.

Nevertheless, an increase in bud formation was achieved when using explants from

immature, expanding leaves instead of mature, fully expanded ones.

A suggested protocol for A. latifolia plant regeneration is as follows:

1. Use immature rachises as explant source (immature leaflets may be used with

some reduction in efficiency if large amounts of explant tissue are desired)

2. Culture on MS + 10 CIM TDZ for 20 days

3. Transfer to MS with no plant growth regulators for bud elongation and rooting












Table 5-1. Effect of three basal media on percentage of adventitious bud formation (%),
mean number of buds (No.) and regeneration index (Indext) in A. bicolor
(U5.2-Ul3.3) and A. latifolia (Ul7. 1-Ul9.6) after 60 days of culture.
Genotype Basal Medium
MS L2 B5
% No. Indext % No. Index % No. Index
U5.2 3.3 a* 1.7 0.2 a 3.0 a 1.0 0.1 a 0" a 0 a
U5.3 Oa 0 Oa Oa 0 Oa Oa 0 Oa
U5.5 Oa 0 Oa Oa 0 Oa Oa 0 Oa
Ul2.2 0" a 0 a 3.3 a 0.7 0.1 a 0" a 0 a
Ul2.5 Oa 0 Oa Oa 0 Oa Oa 0 Oa
Ul3.1 Oa 0 Oa Oa 0 Oa Oa 0 Oa
Ul3.3 6.7 a 0.7 0.1 a 3.3 a 0.3 0 a 3.3 a 0.3 0 a
Ul7.1 50.0 b 1.9 1.1 a 6.7 a 1.7 0.2 a 3.3 a 0.3 0 a
Ul7.2 53.3 b 3.7 1.9 a 26.7 ab 2.8 1.1 a 16.7 a 1.1 0.3 a
Ul7.3 3.3 a 0.3 0 a 0" a 0 a 0" a 0 a
Ul8.1 63.8 ab 3.6 1.8ab 80.0 b 3.4 2.9 b 36.7 a 2.9 1.0 a
Ul8.2 78.6 b 2.3 1.9b 37.2 a 3.7 1.5ab 13.3 a 0.8 0.2 a
Ul8.3 17.4 a 2.2 0.4 a 24.4 a 1.9 0.5 a 3.3 a 1 0.1 a
Ul8.4 23.3 a 0.7 0.5 a 16.7 a 1.1 0.6 a 0" a 0 a
Ul8.6 56.7 b 1.6 0.9 a 46.7 1.2 0.8 a 6.7 a 0.3 0.1 a
Ul8.8 46.7 b 2.6 1.1ab 80.0 b 3.9 2.6 b 13.3 a 4.1 0.8 a
Ul8.9 53.3 b 2.1 1.3 a 23.3 ab 1.6 0.4 a 16.4 a 0.7 0.2 a
Ul9.1 3.3 a 0.3 0 a 0" a 0 a 0" a 0 a
Ul9.2 3.3 a 0.3 0 a 3.3 a 1.7 0.2 a 0" a 0 a
Ul9.3 Oa 0 Oa Oa 0 Oa Oa 0 Oa
Ul9.4 5.6 a 0.5 0.1 a 4.2 a 0.3 0 a 0" a 0 a
Ul9.5 13.3 a 0.6 0.2 a 0" a 0 a 3.3 a 0.3 0 a
Ul9.6 23.3 a 3.9 2.7 b 23.3 a 1.8 1.2ab 9.1 a 1.6 0.4 a
Mean 22.0 B 1.3A 0.6B 16.6B 1.2A 0.5B 5.5A 0.6A 0.1A
* Within rows, means for a given variable followed by different lower case letters
indicate significant differences according to Tukey's HSD Multiple Range Test at p<;0.05
level. Means over genotypes followed by different upper case letters indicate significant
differences according to Tukey's HSD Multiple Range Test at p<;0.05 level. "f Index= %
shoot bud formation x mean number of buds / 100












Table 5-2. Effect of TDZ concentration on percentage of adventitious bud formation
(ABF), mean number of buds per explant, and regeneration index (Indext) in
A. latifolia after 30 days of culture.
TDZ (CIM) ABF Buds Indextf
---%--- ---No.---
0 0 0 0
1 52.5a* 1.7a 0.9a
10 57.5a 1.9a 1.1a
30 67.5a 2.0a .a
60 71.7a 2.3a 1.6b
Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05. "flndex= % shoot bud formation x mean number of
buds / 100




Table 5-3. Effect of TDZ concentration on percentage of adventitious bud formation
(ABF), mean number of buds per explant, and regeneration index (Indext)
after 60 days of culture; number of shoots and shoot length after 90 days of
culture; and percentage of ex vitro survival in A. latifolia.
TDZ (CIM) ABF Buds Indextf Shoots Length Survival
--%-- ---No.--- ---No.--- ---mm--- -----%-----
0 0 0 0 0 0 0
1 65.8a* 2.7a 1.7 a 10.4" 31.3c 76.5b
10 79.2 a 2.8a 2.3 a 7.8bc 18.2b 60.3b
30 87.5 b 2.5a 2.2 a 4.5ab 5.3ab 15.0a
60 85.8 b 2.7a 2.4 a 3.3a 2.6a 4.2a
Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05. "f Index= % shoot bud formation x mean number of
buds / 100










Table 5-4. Effect of BAP concentration on percentage of adventitious bud formation
(ABF), mean number of buds per explant and regeneration index (Indext) in
A. latifolia after 30 days of culture.
BAP (C1M) ABF Buds Indext
---%--- ---No.---
0 0 0 0
1 2.5a* 0.3 a 0 a
10 18.3b 0.8 a 0.2 b
30 1.7a 0.2 a 0 a
60 0.8a 0.1 a 0 a
Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05. "f Index= % shoot bud formation x mean number of
buds / 100


Table 5-5. Effect of BAP concentration on percentage of adventitious bud formation
(ABF), mean number of buds per explant and regeneration index (Indext)
after 60 days of culture; and shoot length after 90 days of culture in A.


latifolia.
BAP (CIM)


Indext


Buds
---No.---


Length
---mm---
0


ABF
--%--


1 13.3a* 1.4 a 0.2 a 15. 0
10 65.8 b 3.2 b 2.2 b 41.7 b
30 57.5 b 2.9 b 1.7 b 30.0 a
60 50.0 b 2.9 b 1.6 b 17.5 a
* Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05. "f Index= % shoot bud formation x mean number of
buds / 100













30 days of culture 60 days of culture
Days in TDZ ABF Buds Indext ABF Buds Index
---%--- ---No.--- ---%--- ---No.---
0 Oa Oa Oa Oa Oa Oa
1 2.5 a 0.3 a 0 a 2.5 a 0.3 ab 0 a
4 12.5 a 0.8 ab 0.2 a 30.0 a 2.1 bc 0.6 a
7 57.5 b 2.0 bc 1.2 b 77.5 b 2.0 bc 1.5 abc
14 70.0 b 1.7 bc 1.2 b 77.5 b 2.5 c 2.0 bc
20 72.5 b 2.6 c 1.9 b 87.5 b 2.3 c 2.0 bc
30 80.0 b 1.8 bc 1.4 b 90.0 b 2.8 c 2.5 c
* Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05. "f Index= % shoot bud formation x mean number of
buds / 100


Table 5-7. Effect of short exposure to TDZ on bud formation percentage (ABF), mean
number of buds per explant and regeneration index (Indext) in A. latifolia
after 30 days of culture.
Days in TDZ ABF Buds Indext
---%--- ---No.---
0 Oa Oa Oa
1 Oa Oa Oa
2 2.5 a 0.3 a 0 a
3 5.0 a 0.5 a 0.1 a
4 5.0 a 1.0" a .1ab
5 7.5 a 0.8" a .1ab
6 12.5 a 0.8 a 0.2 ab
7 17.5 ab a.1 0.2 ab
8 20.0 a 1.4 a 0.3 ab
9 27.5 a 1.4 a 0.4 ab
10 35.0 b 0.9 a 0.4 b
Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05. "f Index= % shoot bud formation x mean number of
buds / 100


I


Table 5-6. Effect of different times of exposure to TDZ on bud formation percentage
(ABF), mean number of buds per explant and regeneration index (Indext) in
A. latifolia after 30 and 60 days of culture, respectively.










Table 5-8. Effect of explant type on bud formation percentage (ABF), mean number of
buds per explant and regeneration index (Indext) in A. latifolia after 30 days
of culture.
Immature Mature
Type of explants leaves leaves Mean
Petioles 9.1 8.0 8.6 a
ABF ---%--- Rachis 80.0 65.0 72.5 0
Leaflets 38.1 20.6 29.4 b
Petioles 0.8 0.4 0.6 a
Buds --No.-- Rachi s 2.3 2.7 2.5 b
Leaflets 1.2 1.0 1.1 a
Petioles 0.2 0.1 0.15 a
Indext Rachis 2.3 2.7 2.5 b
Leaflets 0.6 0.3 0.5 a
* Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05. "f Index= % shoot bud formation x mean number of
buds / 100






52







ri, J' ~u~IA














Figure53 5-.Ogngnsi nA aio ia.AB hotbdfrain 0dy fe
initatio of ult resadC, D)soteogto90dyafrinitonf
cutrs;AC)i S+ MTD n ),D n S+6 p D (a:2
mm.E)Scessfu a clmtzio of reeeae pat,4 dy fe

trnse to exvtr odiin(a:50m )














y = -0.0144x2 + 1.1876x + 66.162
R2 = 0.96


100
00
80
70
60
% 50
40
30
20
10
0


0 10 20 30 40 50 60 70
TDZ (micromolar)

Figure 5-2. Regression curve showing the effect of TDZ concentration on percentage of
adventitious bud formation (%) in A. latifolia after 60 days of culture.


y = -1E-04x2 + 0.0182x + 0.8968
R2 .. I


0 10 20 30 40 50 60 70
TDZ (micromolar)


Figure 5-3. Regression curve showing the effect of TDZ concentration on regeneration
index (Index) in A. latifolia after 30 days of culture.















y = -0.1159x + 9.4273
12
R2= 0.87
10

s

6

S4




0 10 20 30 40 50 60 70

TDZ (micromolar)

Figure 5-4. Regression curve showing the effect of TDZ concentration on shoot number
per explant (No. shoots) in A. latifolia after 90 days of culture.


y = -0.4487x + 25.642
R2 = 0.79













S 10 20 30 40 50 60 7


TDZ (micromolar)


Figure 5-5. Regression curve showing the effect of TDZ concentration on shoot length
(Length) in A. latifolia after 90 days of culture.










y = -1.2482x + 70.534
00
R2 = 0.88
80
70
60
S50
S40
30
20
10

-0

TDZ (micromolar)



Figure 5-6. Regression curve showing the effect of TDZ concentration on acclimatization
rate (% survival) in A. latifolia after 30 days of transfer to ex vitro conditions.





y = -0.0031x2 + 0.0434x + 8.2682
20
R2 = 0.24
18
16
14
12
% 10







0 10 20 30 40 50 60 70
BAP (micromolar)

Figure 5-7. Regression curve showing the effect of BAP concentration on percentage of
adventitious bud formation (%) in A. latifolia after 30 days of culture.












y = -3E-05x2 + 9E-05x + 0.0986
0.3
R2 = 0.25

0.2


0.2



S0.1


0.1

0 10 20 30 40 50 60 70

BAP (micromolar)


Figure 5-8. Regression curve showing the effect of BAP concentration on regeneration
index (Index) in A. latifolia after 30 days of culture.


y = -0.0364x2 + 2.6051x + 22.813
R2 = 0.57


80

70

60

5 0

% 40
30

20

10

0


0 10 20 30 40 50 60 70

BAP (micromolar)


Figure 5-9. Regression curve showing the effect of BAP concentration on percentage of
adventitious bud formation (%) in A. latifolia after 60 days of culture.














y = -0.0011x2 + 0.086x + 1.7276
4.0
R2 = 0.56
3.5

3.0

en 2.5

co 2.0

2 1.5

1.0

0.5

0.0
0 10 20 30 40 50 60 70

BAP (micromolar)



Figure 5-10. Regression curve showing the effect of BAP concentration on mean number
of buds per explant (No. Buds) in A. latifolia after 60 days of culture.


y = -0.0011x2 + 0.0825x + 0.6343
R2 = 0.48














0 10 20 30 40 50 60 71


BAP (micromolar)



Figure 5-11i. Regression curve showing the effect of BAP concentration on regeneration
index (Index) in A. latifolia after 60 days of culture.











y = -0.0195x2 + 1.0944x + 20.841
R2 = 0.50













0 10 20 30 40 50 60 71


BAP (micromolar)


Figure 5-12. Regression curve showing the effect of BAP concentration
(Length) in A. latifolia after 90 days of culture.


on shoot length


y = -0.1948x2 + 8.6392x + 1.817
R2 = 0.91


120

100

80

% 60

40

20


0 5 10 15 20
Days in TDZ


25 30


Figure 5-13. Regression curve sowing the effect of exposure to TDZ on bud formation
percentage (%) in A. latifolia after 60 days of initiation of cultures.














y = -0.0052x2 + 0.2274x + 0.3826
3.5
R2 = 0.80


2.5



S1.5



0.5


0 5 10 15 20 25 30 35

Days in TDZ

Figure 5-14. Regression curve sowing the effect of exposure to TDZ on mean number of
buds per explant (No. Buds) in A. latifolia after 60 days of initiation of
cultures.


3 y = -0.0036x2 + 0.1875x + 0.0024
R2 = 0.95
2.5








0.5


0 5 10 15 20 25 30 35

Days in TDZ


Figure 5-15. Regression curve sowing the effect of exposure to TDZ on regeneration
index (Index) in A. latifolia after 60 days of initiation of cultures.













y = 3.3636x 4.7727
R2= 0.92


y = 0.0418x 0.05
RM 0.93














0 24 6 810 1:


40

35

30

25

% 20
15

10

5

O


Days in TDZ


Figure 5-16. Regression curve sowing the effect of short exposures to TDZ on percentage
of bud formation (%) in A. latifolia after 30 days of initiation of cultures.


0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0


Days in TDZ

Figure 5-17. Regression curve sowing the effect of short exposures to TDZ on
regeneration index (Index) in A. latifolia after 30 days of initiation of cultures.















CHAPTER 6
PLANT REGENERATION OF Lotononis bainesii

Introduction

The genus Lotononis consists of approximately 150 species, from herbs to small shrubs,

belonging to the Fabaceae family, tribe Crotalarieae (Jaftha et al., 2002). Their broad distribution

from Southern Africa to the Mediterranean region and India indicates that these species grow

under dissimilar environments from the climatological and geographical point of view. Among

Lotononis species, L. divaricata (Eckl. & Zeyh.) Benth., L. tenella (E. Mey.) Eckl. & Zeyh. and

L. laxa Eckl. & Zeyh. have forage potential for arid areas, and L. bainesii Baker, a perennial

herb, is a valuable forage in Australia (Jaftha et al., 2002). The increasing interest in this species

has motivated some recent molecular studies to determine its mode of reproduction to assist

breeding programs (Real et al., 2004). These studies have reported that, although it has been

previously considered a cleistogamous species, it should be treated as an allogamous species in

improvement programs. Some seed may be produced by self pollination, but self incompatibility

may also be found in some genotypes.

Due to the allogamous nature of L. bainesii, seeds from a certain plant may correspond to

different genotypes. Therefore, specific genotypes cannot be propagated through seeds and other

means of multiplication would be useful. This could be overcome if a plant regeneration protocol

is developed that allows the propagation of selected plants. One such protocol has been

developed for L. bainessi by Bovo et al. (1986), who obtained a low frequency of plant

regeneration from cotyledons and leaflets. Moreover, a tissue culture protocol may be used to

solve another maj or constraint in this species, low seedling vigor, through the duplication of









chromosomes using chromosome doubling agents such as colchicine, oryzalin and trifluralin

(Quesenberry et al., 2003). Cells with duplicated chromosomes would be induced to follow

either organogenesis or somatic embryogenesis, producing solid plants instead of chimeras that

would likely result if other techniques were used. In addition, a tissue culture protocol that

results in plant regeneration from single cells would open the possibility of genetic

transformation of this species (Ozias-Akins & Gill, 2001). The objective of these experiments

was to develop an efficient tissue culture protocol for L. bainesii, which could then be used in

genetic improvement programs of the species.

Materials and Methods

Cotyledon Culture

Seeds ofL. bainesii cy. INTIA Glencoe were scarified and germinated as described in

chapter 3. Fifty 1-week-old seedlings were randomly selected and their cotyledons were excised

and cut longitudinally along the midrib into two pieces so that four cotyledonary explants were

obtained per genotype. Each explant was placed with the abaxial side down onto MS alone or

MS with 4.5 CIM TDZ, 4.14 CIM PIC or 4.52 CIM 2,4-D. Five explants were placed per petri dish

and the identity of genotypes was maintained. Seedlings without cotyledons were placed onto

MS medium and kept in vitro for subsequent experiments. After 30 days of culture, regenerated

buds were subcultured onto MS medium supplemented with 0.044 CIM BAP + 0.049 C1M IBA for

a month. Developing plants were then transferred to magenta boxes that contained MS lacking

growth regulators for the same period of time before acclimatization ex vitro. Incubation and

acclimatization were the same as those described in chapter 3.

Leaflet Culture

Genotypes used in this experiment were the same as in the cotyledon experiment. Explants

consisted of pieces of leaflets (ca. 4 mm2) including the midvein, harvested from immature










expanding leaves of each genotype growing under aseptic conditions. Treatments, incubation and

acclimatization were the same as in the previous experiment.

Type of Explants

One of the genotypes that had performed well in the cotyledon experiment was used as the

explant source. Leaflets were excised and placed onto sterile filter paper where they were

divided into petiole, petiole tip (in which the three leaflets are inserted) and leaflets. Petiole

pieces (divided into 5- to 6-mm portions), the petiole tip and three leaflets corresponding to each

trifoliate leaf were placed in a petri dish containing MS supplemented with 10 CIM TDZ. This

procedure was repeated 10 times for both mature and immature leaves. Thirty days after the

initiation of the experiment, explants were subcultured to MS without growth regulators.

Incubation conditions were the same as those described in chapter 3. This experiment was

statistically analyzed as a factorial arrangement in a completely randomized design (3 parts of

the leaf x 2 stages of development) using PROC GLM from PC SAS (SAS Institute, 2003).

Tukey's HSD Multiple Range Test at p<;0.05 was used to compare the means of treatments.

Results and Discussion

Cotyledon Culture

In the absence of growth regulators, cotyledon explants on MS basal medium remained

non-responsive and gradually turned brown. Conversely, after 30 days of culture on medium

supplemented with PIC, all genotypes produced light brown or light green friable callus of less

than 1 cm in diameter. When this callus was transferred onto MS + 0.044 CIM BAP + 0.049 CIM

IBA, these calli did not show further growth and died. Similar results were observed when 2,4-D

was used as a growth regulator in the culture medium (Figure 6-1 A,B). However, in six










genotypes, a few short roots were produced from callus on 2,4-D induction medium, but they did

not continue growing upon transfer to MS + 0.044 CIM BAP + 0.049 CIM IBA.

The addition of TDZ to the bud induction culture medium resulted in shoot bud

organogenesis in 27 out of 50 genotypes tested (Figure 6-2 A). Callus and bud formation started

approximately 7 and 15 days after the initiation of cultures, respectively (Figure 6-1 C,D). Callus

was in general dark green with dark brown areas. Considering only responsive genotypes, the

mean number of buds per cotyledonary explant was 13.6. Sixty days after culture, 48% of the

total number of buds was capable of regenerating plants (177 plants/ 367 buds). In some

genotypes, the number of plants after 90 days of culture was superior to the number of buds after

30 days, indicating that during that period new buds were produced and regenerated whole plants

(Figure 6-3 A). In other genotypes, not all the buds present after 30 days resulted in plant

regeneration. Moreover, nine genotypes produced buds but they did not elongate and finally

died.

The lack of elongation and death of buds probably resulted from the hyperhydricity of

tissues, which might have been caused by a hormonal imbalance in the culture medium. It was

observed that only 39% of plants transferred to ex vitro conditions were capable of successful

acclimatization 30 days after the transfer (Figures 6-1 E and 6-4 A). This low survival rate is

likely associated with the high incidence of hyperhydricity in regenerated plants. Besides the

influence of plant growth regulators in the culture medium, this abnormality may also result from

high water availability in the culture vessel and low light levels (Hazarika, 2006).

The growth regulator, TDZ, has been used for organogenesis and plant regeneration from

embryo- or seedling-derived explants in several species of legumes including A. hypogaea (Gill

& Ozias-Akins, 1999), K. radiata (Mundhara & Rashid, 2006), Trifolium spp., M~edicago sp.p










(Ding et al., 2003) and C. cajan (Singh et al., 2003). Nevertheless, in the latter, somatic

embryogenesis was obtained at higher concentrations of TDZ than those that had induced

organogenesis. Similarly, there are reports of somatic embryo induction using TDZ in A.

hypogaea (Murthy et al., 1995). Interestingly, PIC and 2,4-D were not effective for non zygotic

embryo induction in L. bainesii, but both plant growth regulators were reported to be effective in

A. hypogaea (Griga, 1999).

Leaflet Culture

Since L. bainesii is an allogamous species and not all progeny will necessarily reflect the

superior performance of an individual plant, cotyledons are not the most suitable explants when

the purpose is propagation of outstanding genotypes. Therefore, the previous experiment was

repeated using leaflets as explants, since they are available throughout the year and offer the

possibility of large scale propagation of selected genotypes as well as development of an in vitro

chromosome doubling protocol.

Thirty days after the initiation of cultures it was observed that, when growth regulators

were absent, explants remained irresponsive except in 22 genotypes in which roots up to 10-cm

long were produced from the cut surface of the midvein. This suggests that the endogenous level

of auxins in the leaflets may be enough to induce rhizogenesis in the absence of an exogenous

supply of plant growth regulators. Probably, the high levels of auxins are responsible for the

formation of vigorous roots in plants maintained in vitro not only from basal nodes but also from

those not in contact with the culture medium. Some of these genotypes corresponded to those

that had produced roots from callus in the previous experiment. When the culture medium

contained PIC or 2,4-D, responses were similar to those observed in the cotyledon experiment.

Although some calli were larger (up to 1.5 cm in diameter), they did not exhibit further growth









when transferred onto MS + 0.044 CIM BAP + 0.049 CIM IBA. The addition of 2,4-D resulted in

root formation in seven genotypes as well.

The growth regulator, TDZ, was effective in inducing shoot bud organogenesis in 45 out of

50 genotypes tested, four of which were also non-responsive in the cotyledon experiment.

However, the mean number of buds (3.8) was considerably lower than that obtained when

cotyledon pieces were used (13.6) (Figures 6-1 F and 6-2 B). After 90 days of culture, a 22%

increase in the number of regenerated plants compared to the number of buds at 30 days was

observed (211 plants at 90 days vs. 173 buds at 30 days) (Figure 6-3 B and 6-1 G). This might be

due to buds continuing to be formed on MS + 0.044 CIM BAP + 0.049 C1M IBA. But since this

medium had such a low concentration of plant growth regulators, more probably these buds were

already induced before the subculture. Even though the total number of plants obtained from

leaflet culture was higher than that for cotyledons, only 21% were successfully acclimatized

when transferred ex vitro (Figure 6-4 B). This high plant mortality was likely due to

hyperhydricity, of which numerous plants showed symptoms; however, manipulation of culture

conditions might have resulted in higher survival rates. It is possible that the vigorous growth of

in vitro regenerated plants resulted in high ethylene accumulation, which has been reported to

favor hyperhydricity (Hazarika, 2006).

It is interesting that more genotypes were responsive when explants consisted of leaflets

rather than cotyledons. In general, juvenile explants are preferred since they are more likely to

undergo organogenesis or somatic embryogenesis. For example, in A. hypogaea, seedlings more

than 21-days old failed to undergo somatic embryogenesis using TDZ compared to up to 97% of

6-day-old seedlings (Murthy et al., 1995). The lower cotyledon response in L. bainesii may have

been caused by the scarification/surface disinfection procedures seed received, although this is









unlikely given that much stronger pretreatments. have been reported with no negative effects

(Bovo et al, 1986). These authors obtained better responses with cotyledons as explants (66% of

bud formation in the best culture medium vs. 54% when using leaflets). In the present study,

however, a higher frequency of organogenesis was obtained following leaflet culture (90% of

explants producing buds vs. 54% of cotyledon pieces producing buds). Additionally, results in

this experiment differ from those reported by Bovo et al. (1986) in that shoots readily rooted in

spite of being in a culture medium with the potent cytokinin TDZ so rooting was not a critical

factor in whole plant regeneration. Veltcheva et al. (2005) suggest that forage legume

populations are markedly heterogeneous, resulting in an easier identification of in vitro

responsive genotypes. Organogenic genotypes of L. bainesii cy INIA Glencoe were easily

identified, which may be due to a shorter breeding history than those in grain legumes in which

the narrow genetic base limits the discovery of regenerating genotypes.

Type of Explant

Since the previous experiment showed that leaflets were capable of producing shoot buds

in the presence of TDZ in most of the genotypes tested, another experiment was performed to

asses the influence of the part and age of the leaf in shoot organogenesis. Callus formation

started within a week of initiation of cultures and shoot buds began to arise after 15 days of

culture in the six types of explants used.

For percentage of shoot bud formation, leaf part had a significant effect, whereas leaf age

and interaction between leaf part and leaf age were not significant (Table 6-1). Bud

organogenesis from leaflets was similar (85.8% from insertion explants and 83.3% from leaflet

explants) and higher than petioles (48.3%). The number of buds per explant ranged from 1.1 to

3.4 (Table 6-2) but, similar to bud organogenesis percentage, there was no leaf age by leaf part

interaction for number of buds per explant (Table 6-1). In contrast to percentage of bud










organogenesis, the mean number of buds per explant did not differ among parts of the leaf, but

did differ due to leaf age. Explants from immature leaves gave a higher number of buds per

explant (2.9) than those from mature ones (1.5) (Table 6-2).

Considering the regeneration index, there was a significant effect of the part and age of the

leaf but not a significant interaction (Table 6-1). The mean regeneration index for petiole

explants (1.1) was significantly lower than the index for leaflet or leaflet insertion area (2.4 in

both cases). However, the mean regeneration index for explants from immature leaves (2.4) was

significantly higher than the index for explants excised from mature leaves (1.5). Leaflet

insertion area from immature leaves had the highest index (3.4) over all combinations.

The advantage of using immature leaves has been reported in A. hypogaea, in which the

frequency of somatic embryo formation decreased considerably as leaflets unfolded (Baker &

Wetzstein, 1998). In A. villosulicarpa, the best organogenic frequency and mean number of buds

per explant were obtained when mature fully expanded leaves were used as explants (Dunbar &

Pittman, 1992). In L. bainesii, Bovo et al. (1986) reported up to 54% bud formation when pieces

of fully expanded leaflets from greenhouse grown plants were placed onto the best culture

medium. Although these authors used larger explants (6 mm2), Organogenesis frequency was

lower than in the present experiment. This could be due to the conditions under which mother

plants were grown, a factor that greatly affects in vitro responsiveness (Radice, 2004).

Conclusions

It was possible to regenerate plants from over 50% of L. bainesii cy INIA Glencoe

genotypes that were evaluated through cotyledon culture and 90% of genotypes through leaflet

culture in a medium composed of MS +4.5 CIM TDZ. Bud elongation and rooting was obtained

upon transfer onto MS + 0.044 C1M BAP + 0.049 CIM IBA. Although immature leaflet culture










resulted in a higher number of responsive genotypes, plants regenerated from cotyledons

exhibited a higher survival rate when transferred to ex vitro conditions. Nevertheless, in both

cases survival rates were low and this situation was related with the incidence of hyperhydricity

in cultures. When the culture medium was supplemented with either PIC or 2,4-D, friable light

green or light brown callus formation was obtained, but this callus did not show further growth

when subcultured.

Regarding the type of explants, the experiment carried out with one genotype of L. bainesii

revealed that leaflet insertion areas from expanding leaves and pieces of leaflets were more

efficient for shoot bud induction. For both mature and immature leaves, the lowest frequencies of

shoot bud formation were obtained when pieces of petioles were used as explants. Mean number

of buds per explant and regeneration index were higher for explants collected from immature

leaves.

A suggested protocol for L. bainesii plant regeneration is as follows:

1. Use immature leaflets as explants, which are as efficient as leaflet insertion areas but a

larger amount of tissue is obtained per leaf

2. Culture on MS + 4.5 CIM TDZ for 30 days

3. Transfer to MS + 0.044 CIM BAP + 0.049 CIM IBA for bud elongation and rooting

Although these experiments have improved the regeneration frequency reported for this

species, it is likely that the optimization of other factors besides the type of explant would result

in a more efficient protocol. Additional experiments regarding the regulation of plant growth

regulator concentrations and combinations, time required for organogenesis induction and

control of factors that influence hyperhydricity will be conducted to increase the regeneration

frequency and ex vitro establishment rate.





























Table 6-2. Effect of explant type on bud formation percentage, mean number of buds per explant
and regeneration index in L. bainesii after 30 days of culture.
Leaf part Immature Mature Mean
Petioles 38.3 58.3 48.3b
Leaflet insertion 100.0 71.5 85.8a
Bud formation %a
Leaflets 90.0 76.7 83.4"
Mean 76.1A 68.8A
Petioles 2.2 1.6 1.9 a
Leaflet insertion 3.4 1.1 2.3 a
Number of buds
Leaflets 3.0 1.8 2.4"
Mean 2.9A 1.5B
Petioles 1.0 1.2 1.1b
Regeneration Leaflet insertion 3.4 1.4 2.4a
indext Leaflets 2.8 1.9 2.4a
Mean 2.4A 1.5B
* For each parameter, means within columns (a,b,c) or rows (A,B) with different letters indicate
significant differences according to Tukey's HSD Multiple Range Test at p<;0.05. "f Index= (%
shoot bud formation x mean number of buds) / 100


Table 6-1. ANOVA table showing the p-values corresponding to the effect of explant type on
bud formation percentage (%), mean number of buds per explant (No.) and
regeneration index (Indext) in L. bainesii after 30 days of culture.
Source df p-value
% No. Indextf
Leaf parts 2 0.004 0.606 0.032
Leaf age 1 0.454 0.003 0.048
LP x LA 2 0.120 0.294 0.119
Error 54
Jf Index=( % shoot bud formation x mean number of buds) / 100













je /4b
L~
C


G


ii

~rS,
il
CL

r Ir 9
L
r


Figure 6-1. Organogenesis in L. bainesi. A) Cotyledon cultures 20 days after initiation of
experiments in MS basal media supplemented with 2,4-D, B) PIC and C) TDZ (bar:
20 mm). D) Shoot bud formation from cotyledons 45 days after culture (bar: 20 mm).
E) Plants successfully acclimatized 45 days after transfer to ex vitro conditions (Bar:
50 mm). F), G) Shoot bud proliferation from leaflet explants originated in MS + 10
CIM TDZ 60 and 90 days after initiation of cultures, respectively (bar: 20 mm).


A






F


-
s~





k:


F~J~ ~1~ "m~i~Pr~s~i~ rh















40 A

35

o
25

20



10

5


0 1-9 10-19 20-29 30-39 40-49 50-59 60-69
Number of buds


3A

25


40 B

35



25

20



10

5-


0 1-9 10-19 20-29 30-39 40-49 50-59 60-69
Number of buds


Figure 6-2. Number of buds produced per explant by 50 genotypes of L. bainesii after 30 days of
culture from A) cotyledon and B) leaflets.


30B

25


20


15


E 10

z5


0 1-9 10-19 20-29 30-39 0 1-9 10-19 20-29 30-39
Number of plants Number of plants

Figure 6-3. Number of plants produced per explant after 90 days of culture by responsive
genotypes ofL. bainesii through A) cotyledon and B) leaflet culture.















v01 20









5 5

0 1-4 5-9 10-14 15-19 0 1-4 5-9 10-14 15-19
Number of plants Number of plants

Figure 6-4. Number of successfully acclimatized plants in the genotypes of L. bainesii that were
capable of plant regeneration through A) cotyledon and B) leaflet culture.















CHAPTER 7
SUMMARY AND CONCLUSIONS

Grain legumes are generally regarded as recalcitrant to in vitro plant regeneration

due to a narrow genetic base that results in low genetic variability and a more difficult

identification of responsive genotypes. It is thought to be easier to identify in vitro

responsive genotypes of forage legumes, because they have usually undergone fewer

selection cycles and populations are more heterogeneous (Veltcheva et al., 2005). Results

reported here concur with this concept since shoot bud organogenesis was achieved in 20

to 90% of genotypes in four out of Hyve forage legume species evaluated.

Petiole culture of T. polymorphum on three different basal media supplemented

with TDZ was not effective to induce bud organogenesis, at least for those genotypes

tested. Possibly, this species is more recalcitrant to tissue culture than the others. Further

studies will be conducted to determine the most suitable conditions for in vitro plant

growth (using these aseptically grown plants as an explant source) as well as to determine

the culture conditions required for shoot bud induction, i.e., basal medium composition,

and plant growth regulator type, concentration and combinations. In T. carolinianum, an

annual native clover species, shoot bud formation was achieved for the first time through

cotyledon culture. Additional experiments are being conducted to achieve further

elongation of shoots and rooting, before regenerated plants are transferred to ex vitro

conditions to evaluate the establishment rate.

In A. bicolor, A. latifolia and Lotononis bainesii, plant regeneration was achieved

via organogenesis in 54 to 90% of the genotypes tested. Nevertheless, additional










experiments should be conducted to improve the frequency of bud formation and to

reduce the incidence of hyperhydricity in culture, which should result in higher ex vitro

survival rates.

Overall, MS was a suitable basal medium for shoot bud induction in all species

tested, except for 7 polymorphum where the only response was callus formation on B5

basal medium. In A. latifolia, L2 was as effective as MS for shoot bud organogenesis, but

plants regenerated in L2 showed a lower establishment rate when transferred to ex vitro

conditions. Among plant growth regulators tested, TDZ was efficient in all species except

T. polymorphum, where it only induced callus formation. In A. latifolia, shoot bud

formation was observed in TDZ concentrations ranging from 1 to 60 CIM. The induction

time experiment in A. latifolia revealed that 20 days of culture on medium containing

TDZ was sufficient to obtain shoot organogenesis. In agreement with other literature,

immature leaf tissues were in general more responsive in L. bainesii and Adesmia spp.

Immature rachises proved to be superior explants for organogenesis induction in A.

latifolia, and leaflet insertion areas or leaflets from immature leaves were more

responsive than pieces of petioles in L. bainesii. Although cotyledon culture resulted in

shoot bud formation in 7 carolinianum and L. bainesii, in Adesmia spp. cotyledons

remained unresponsive.

Suggested protocols for the studied species are:

T. carolinianum

1. Cotyledon culture on MS + 10 C1M TDZ for 30 days

2. Transfer of organogenic cultures on MS + 1 CIM TDZ, where new buds continue

to arise and short shoots are produced from those buds already differentiated









A. bicolor

1. Immature leaflet culture on MS or L2 + 4.5 C1M TDZ for 60 days

2. Transfer to MS devoid of plant growth regulators for 45 days, where buds

elongated and rooted.

A. latifolia

1. Immature rachis culture on MS + 10 CIM TDZ for 20 days

2. Transfer to MS with no plant growth regulators for bud elongation and rooting

L. bainesii

1. Culture immature leaflets on MS + 4.5 CIM TDZ for 30 days

2. Transfer to MS + 0.044 CIM BAP + 0.049 CIM IBA for bud elongation and

rootmng

In conclusion, in vitro plant regeneration protocols were developed for four

promising legume species. These protocols could be used to assist in breeding programs

to improve seedling vigor and DM production. This would be important for cattle

production in those countries where these species are native. Additionally, this may

allow the development of a legume alternative to perennial peanut in the state of Florida,

since to-date it is the only introduced forage legume species that has shown long term

persistence. The main drawbacks of perennial peanut are its vegetative propagation,

which increases the establishment cost, and low production in the year after planting. In

contrast, Adesmia spp. and L. bainesii cultivars could be propagated through seeds,

reducing the cost for farmers. Currently, field studies are being conducted to evaluate

field performance ofAdesmia spp. and L. bainesii to the Florida environment.



















Table A-1. Effect of different combinations of TDZ and IBA on percentage of
adventitious bud formation (ABF) and mean number of buds per explant in A.
latipolia Ul8.6 after 30 and 60 days of culture.
30 days of culture 60 days of culture
IBA (C1M) IBA (C1M)
0 0.1 1 0 0.1 1
ABF Buds ABF Buds ABF Buds ABF Buds ABF Buds ABF Buds
TDZ % No. % No. % No. % No. % No. % No.


APPENDIX
ADDITIONAL TABLES FOR TWO GENOTYPES OF Adesmia latifolia


Oa
45ab
60b
70b
85b


Oa
1.3b
2.8cd
1.9bc
3.1d


Oa
55bc
40ab
75bc
1000


Oa
1.7b
2b
2.3b
1.9b


0 a
75b
55b
75b
50b


Oa
1.7b
2b
2.1b
2.2b


Oa
3.1bc
3.6bc
2.1b
3.70


Oa
60bc
55b
80bc
95e


Oa
2.2b
2.6b
2.8b
2.4b


Oa
2.6b
2.4b
2.7b
2.7b


* Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05


Table A-2. Effect of different combinations of TDZ and IBA on regeneration index
(Indext), number of shoots per explant and shoot length in A. latifolia Ul8.6
after 60 days of culture.


IBA (CIM)
0.1


Shoots
-No.-


Index Shoots
-No.-


Index



Oa
1.3 ab
1.5 ab
2.2 b
2.3 b


Shoots
-No.-


Index


Length
-mm-

Oa
17.5 c
8 bc
8 bc
7.5 ab


TDZ
(I-M)


Oa "
2.2 bc
3.4 c
1.8ab
3.3 c


Oa
10 b
13ab
6.5a
9a


Length
-mm-

Oa

25 b
13 b
5 ab
4 b


Ib
10 b
9 a
5.5a


Length
-mm-



25 b
23 b
6.5 b
4ab


Oa
2.3 b
2 b
2.3 b
2.3 b


Oa
13b
8ab
8ab
5 ab


* Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05. "f Index= % shoot bud formation x mean number of
buds / 100












Table A-3. Effect of different combinations of TDZ and IBA on percentage of
adventitious bud formation (ABF) and mean number of buds per explant in A.
latifolia Ul8.8 after 30 and 60 days of culture.
30 days of culture 60 days of culture
IBA (CIM) IBA (CIM)
0 0.1 1 0 0.1 1
ABF Buds ABF Buds ABF Buds ABF Buds ABF Buds ABF Buds
TDZ % No. % No. % No. % No. % No. % No.


Table A-4. Effect of different combinations of TDZ and IBA on regeneration index
(Indext), number of shoots per explant and shoot length in A. latifolia Ul8.8
after 60 days of culture.
IBA (CIM)
0 0.1 1
Indext Shoots Length Index Shoots Length Index Shoots Length
TDZ -No.- -mm- -No.- -mm- -No.- -mm-

0 Oa* Oa Oa Oa Oa Oa Oa Oa
1 1.7" b b 53 b 1.4 ab 7.5a 35 a 1.7 b 10 b 33 a
10 2.5 b 6 ab 40 ab 2.2 b 5 a 16 a 2 b 5.5ab 10 a
30 2.8b 2a 8.5a 2.1b 1.5a 3.5a 2b Oa Oa
60 2.9 b 0 a 0 1.5ab 0 a 0 2.1 b 0 a 0
* Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05. "f Index= % shoot bud formation x mean number of
buds / 100


Oa*
60b
80b
80b
80b


Oa
2 b
1.5 b
1.6b
1.8b


Oa
1.4b
1.3ab
1.8 b
2.3 b


Oa Oa
35ab 2.3b
65 b 1.7b
55 b 2.2b
65 b 2.5b


Oa
70 b
95 b
100b
100b


Oa
2.5b
2.6b
2.8b
2.9b


Oa Oa
45ab 3.4b
75 b 2.9b
80 b 2.6b
70 b 2.1b


Oa
65b
70b
95b
85b


Oa
2.7b
2.9b
2.1b
2.4b


* Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05







79




Table A-5. Effect of different combinations of TDZ and IBA on percentage of ex vitro
acclimatization (%) in A. latifolia Ul8.6 and Ul8.8 after 20 days of transfer to
ex vitro conditions.
U 18.6 U 18.8
IBA (CIM) IBA (C1M)
TDZ (M) 0 0.1 1 0 0.1 1


Table A-6. Effect of different combinations of BAP and IBA on percentage of
adventitious bud formation (ABF) and mean number of buds per explant in A.
latifolia Ul8.6 after 30 and 60 days of culture.
30 days of culture 60 days of culture
IBA (C1M) IBA (CIM)
0 0.1 1 0 0.1 1
ABF Buds ABF Buds ABF Buds ABF Buds ABF Buds ABF Buds
BAP % No. % No. % No. % No. % No. % No.


Oa
86 b
86 b
13ab
Oa


Oa
49 ab
100 b
28a"b
Oa


Oa
96 b
65 ab
50ab
25a"b


Oa
83 b
25 ab
Oa
Oa


Oa Oa
63 a 83 b
50 a 36 a
Oa Oa
Oa Oa
according to Tukey's


* Within columns, different letters indicate significant differences
HSD Multiple Range Test at p<;0.05


Oa
5 a
40b
45 b
10ab


Oa
1.5ab
3.9b
3.8 b
3ab


Oa
10 a
85b
25 a
15 a


Oa
4 b
3.5b
2ab
1.5ab


Oa
10ab
45b
40 b
45 b


Oa
0.5 a
2.7a
2.9 a
1.8 a


* Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05


































Table A-8. Effect of different combinations of BAP and IBA on percentage of
adventitious bud formation (ABF) and mean number of buds per explant in A.
latifolia Ul8.8 after 30 and 60 days of culture.
30 days of culture 60 days of culture
IBA (CIM) IBA (CIM)
0 0.1 1 0 0.1 1
ABF Buds ABF Buds ABF Buds ABF Buds ABF Buds ABF Buds
BAP
(CIM) % No. % No. % No. % No. % No. % No.
O Oa* 0 0 Oa 0 Oa 0 Oa Oa Oa Oa Oa
1 Sa 0.5 0"Oa 0 Oa 20" a l 35ab 1.3" a" Oa
10 10 a 0.5 45b 1.3 20 a 1.2 60b 2.8 b 90 c 3.9 b 75b 2.5 b
30 5 a 0.5 0" a 0" a 80b 2.9 b 65bc 3.5 b 90b 2.3 b
60 5 a 0.5 0" a 0" a 75b 4.8 c 85bc 3.3 b 70b 3 b
* Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05


Table A-7. Effect of different combinations of BAP and IBA on regeneration index
(Indext) and shoot length in A. latifolia Ul8.6 after 60 days of culture.
IBA (CIM)


0.1
Index Length
|--mm--


Indext

|
0.2 ab
1.5 bc
1.6 c
0.3a"b


Index
| de
0 a
0.1 a
1.4 a
1.2 a
0.9a


Length
--mm--


Length
--mm--


BAP (CIM)


0 a
0.4 a
3 b
0.5 a
0.3a


0
40
42.5
5
0


* Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05. "f Index= % shoot bud formation x mean number of
buds / 100












Table A-9. Effect of different combinations of BAP and IBA on regeneration index
(Indext) and shoot length in A. latifolia Ul8.8 after 60 days of culture.
IBA (CIM)
0 0.1 1
Indext Length Index Length Index Length
BAP (uM) | --mm-- |--mm-- |--mm--
0 Oa* 0 Oa 0 Oa 0
1 0.4 ab 30 0.4" a 0" a
10 1.7 bc 40 3.5 b 58 1.9 b 50
30 2.4c"d 40 2.3b 33 2.1b 33
60 3.6d 30 2.8 b 20 2.1 b 20
Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05. "f Index= % shoot bud formation x mean number of
buds / 100







Table A-10. Effect of different times of exposure to TDZ on bud formation percentage
(ABF), mean number of buds per explant and regeneration index (Indext) in
A. latifolia Ul8.6 after 30 and 60 days of culture, respectively.
30 days of culture 60 days of culture
ABF Buds Indext ABF Buds Index
Days in TDZ --%-- --No.-- --%-- --No.--


0 Oa Oa Oa Oa 0 0
1 Oa Oa Oa Oa 0 0
4 25 ab 1.7 b 0.4 ab 35 ab 2.8 0.9
7 80 bc 2.3 b 1.8 c 85 bc 2.3 1.9
14 75 bc 1.6 b 1.2 bc 75 bC 2.2 1.7
20 70 bc 2.9 b 2 c 85 bc 2.6 2.2
30 90 1.9 b 1.8 c 95 c 3 3
* Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05. "f Index= % shoot bud formation x mean number of
buds / 100












Table A-1 1. Effect of different times of exposure to TDZ on bud formation percentage
(ABF), mean number of buds per explant and regeneration index (Indext) in
A. laifolia Ul8.8 after 30 and 60 days of culture, respetively
30 days of culture 60 days of culture
ABF Buds Indext ABF Buds Index
Days in TDZ --%-- --No.-- --%-- --No.--
0 Oa* Oa Oa Oa Oa 0
1 5 a 0.5" ab .1ab 5 a 0.5 a 0.1
4 0 a 0 a 0 25 a 1.3 a 0.4
7 35 b 1.8 ab 0.6 abc 70 b 1.6 ab 1.2
14 65 b 1.8 ab 1.2 bc 80 b 2.9 b 2.3
20 75 b 2.2 b 1.7 c 90 b 2 ab 1.9
30 70 b 1.6 ab a. bc 85 b 2.5 b 2.1
* Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05. "f Index= % shoot bud formation x mean number of
buds / 100


Table A-12. Effect of short exposure to TDZ on bud formation percentage (ABF), mean
number of buds per explant and regeneration index (Indext) in A. latifolia
Ul8.6 and Ul8.8 after 30 das of culture.
Ul8.6 Ul8.8
ABF Buds Indext ABF Buds Index
Days in TDZ --%-- --No.-- --%-- --No.--


0
0
0.5
1
0.5
1
1.2
1
1.9
1.8
0.7


0




1.5
0.5
0.5
1.2


1.1


0
0
5
10
5
5
20
15
25
25
25


Oai

0 a
Oa
5 ab
10ab
5 ab
20 abc
15 a
30 bc
45 c


0
1
2
3
4
5
6
7
8
9
10


* Different letters indicate significant differences according to Tukey's HSD Multiple
Range Test at p<:0.05. "f Index= % shoot bud formation x mean number of buds / 100













Table A-13. Effect of explant type on bud formation percentage (ABF), mean number of
buds per explant and regeneration index (Indext) in A. latifolia Ul8.6 and
Ul8.8 after 30 days of culture.


Indextl ABF
--%--


Ul8.6
ABF Buds
--%-- --No.--


Ul8.8
Buds
--No.--


Index


Types of
explants
Petioles
Rachi s
Leaflets
Petioles
Rachis
Leaflets


10.3a
90
48.8bc
16 a
80 ed
37.5 ab


1.5 ab
3.5 b
1.3 ab
0.8 a
4.3 c
a 1


0.3 a
3.5
0.8 a
0.2 a
4.3 b
0.6 a


7.9 a
70"
27.5ab
0 a
50 bc
3.8 a


Immature
leaves

Mature
leaves


* Within columns, different letters indicate significant differences according to Tukey's
HSD Multiple Range Test at p<;0.05. "f Index= % shoot bud formation x mean number of
buds / 100










































y = -0.0061x2 + 0.238x + 0.0791
R2 = 0.88


=-0.1565x2 + 7.3789x 3.5616


90
80
70
60
50
40
30
20
10
0


0 5 10 15 20 25 30 35
Days in TDZ

Figure A-1. Regression curve sowing the effect of exposure to TDZ on bud formation
percentage (%) in A. latifolia after 30 days of initiation of cultures.


0 5 10 15 20
Days in TDZ


25 30


Figure A-2. Regression curve sowing the effect of exposure to TDZ on mean number of
buds per explant (No. Buds) in A. latifolia after 30 days of initiation of
cultures.












2 r
1.8 R2 = 0.90

1.6
1.4
1.2*



0.6
0.4
0.2*


0 5 10 15 20 25 30 35

Days in TDZ


Figure A-3. Regression curve sowing the effect of exposure to TDZ on regeneration
index (Index) in A. latifolia after 30 days of initiation of cultures.
















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Full Text

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TISSUE CULTURE OF Trifolium polymorphum, T. carolin ianum, Adesmia latifolia, A. bicolor AND Lotononis bainesii By MARIA LAURA VIDOZ A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Maria Laura Vidoz

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To my mother and to God, for their unconditional love.

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iv ACKNOWLEDGMENTS I would like to thank God fo r revealing His presence throughout my life, for His guidance during hard times and for His many bl essings. I am thankful to my mother for her unconditional support and encouragemen t, for devoting so much time to my education, for giving me a deep love of natu re, making it difficult to put into words how much she means to me. I would like to thank my supervisory co mmittee chair (Dr. Kenneth Quesenberry) for his advice and knowledge, and for introduc ing me to his wonderf ul family, all of which helped me professionally and personally. I am also grateful to the other members of my committee (Dr. Mimi Williams, Dr. Ma ria Gallo and Dr. David Wofford) for their critics and suggestions. I thank Judy Dampier for her technical support, friendship and advice, making my stay in Gainesville a positive experience. I th ank Loan Ngo for her technical assistance and sincere friendship. I also appreciate th e technical support of Lindsay and Kailey Place. I am thankful to Luis Mroginski and Dr. Hebe Rey, who introduced me to the tissue culture world and constantly encour aged me to grow in my career. I would like to thank Lorena, Carlos, Gaby, Raquel, Jorge and Sonali for their company during these two years, and to all my friends in Argentina for their support.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.............................................................................................................x ABSTRACT......................................................................................................................xii CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................5 Tissue Culture Concepts as A pplied to the Fabaceae Family.......................................5 Applications of Tissue Culture for the Fabaceae Family.............................................9 Production of Plants Free from Certain Specific Pathogens...............................10 Micropropagation................................................................................................11 Production of Dihaploid, Homozygous Plants....................................................12 Generation of Interspecific Hybrids: Embryo Rescue and Protoplast Fusion.....12 Plant Regeneration after Tr ansformation Protocols............................................14 Medium and Long-term Germplasm C onservation and Plant Material Exchange..........................................................................................................15 3 MATERIALS AND METHODS...............................................................................17 Plant Material..............................................................................................................17 Culture Conditions......................................................................................................17 Evaluation and Experimental Design.........................................................................19 4 TISSUE CULTURE OF Trifolium polymorphum AND T. carolinianum .................21 Introduction.................................................................................................................21 Materials and methods................................................................................................22 Trifolium polymorphum .......................................................................................22 Trifolium carolinianum .......................................................................................23 Results and Discussion...............................................................................................23 Trifolium polymorphum .......................................................................................23

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vi Trifolium carolinianum .......................................................................................25 Conclusions.................................................................................................................27 5 PLANT REGENERATION OF Adesmia latifolia AND A. bicolor ..........................31 Introduction.................................................................................................................31 Materials and Methods...............................................................................................32 Plant Material......................................................................................................32 Basal Media Experiment.....................................................................................33 Factorial Experiments with TDZ and BAP.........................................................34 Induction Time Experiments...............................................................................35 Type of Explant Experiment...............................................................................35 Results and Discussion...............................................................................................36 Effect of Basal Medium on Shoot Organogenesis..............................................36 Influence of TDZ on Organogenesis...................................................................38 Influence of BAP on Organogenesis...................................................................40 Induction Time for Adventitious Bud Formation................................................42 Effect of Explant Type on Shoot Organogenesis................................................44 Conclusions.................................................................................................................45 6 PLANT REGENERATION OF Lotononis bainesii ...................................................61 Introduction.................................................................................................................61 Materials and Methods...............................................................................................62 Cotyledon Culture...............................................................................................62 Leaflet Culture.....................................................................................................62 Type of Explants..................................................................................................63 Results and Discussion...............................................................................................63 Cotyledon Culture...............................................................................................63 Leaflet Culture.....................................................................................................65 Type of Explant...................................................................................................67 Conclusions.................................................................................................................68 7 SUMMARY AND CONCLUSIONS.........................................................................74 APPENDIX ADDITIONAL TABLES FOR TWO GENOTYPES OF Adesmia latifolia .....................77 LIST OF REFERENCES...................................................................................................86 BIOGRAPHICAL SKETCH.............................................................................................95

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vii LIST OF TABLES Table page 3-1 Plant material used as a source of explants in the experiments...............................20 4-1 Mean percentage of explants producing callus from petiole pieces of T. polymorphum in three basal media after 30 days of culture....................................29 4-2 Number of buds per explan t in the responsive genotypes of T. carolinianum cultured on medium supplemented with 10 M TDZ after 30 and 60 days of culture.......................................................................................................................29 5-1 Effect of three basal media on percenta ge of adventitious bud formation, mean number of buds and regeneration index in A. bicolor and A. latifolia after 60 days of culture..........................................................................................................47 5-2 Effect of TDZ concentration on percenta ge of adventitious bud formation, mean number of buds per explant, and regeneration index in A. latifolia after 30 days of culture..................................................................................................................48 5-3 Effect of TDZ concentration on percenta ge of adventitious bud formation, mean number of buds per explant, regenerati on index, number of shoots, shoot length and percentage of ex vitro survival in A. latifolia ....................................................48 5-4 Effect of BAP concentration on percen tage of adventitious bud formation, mean number of buds per explant and regeneration index in A. latifolia after 30 days of culture..................................................................................................................49 5-5 Effect of BAP concentration on percen tage of adventitious bud formation, mean number of buds per explant and regenera tion index after 60 days of culture; and shoot length after 90 days of culture in A. latifolia ..................................................49 5-6 Effect of different times of exposur e to TDZ on bud formation percentage, mean number of buds per explant and regeneration index in A. latifolia after 30 and 60 days of culture..........................................................................................................50 5-7 Effect of short exposure to TDZ on bud formation percentage, mean number of buds per explant and regeneration index in A. latifolia after 30 days of culture.....50 5-8 Effect of explant type on bud forma tion percentage, mean number of buds per explant and regeneration index in A. latifolia after 30 days of culture...................51

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viii 6-1 ANOVA table showing the p-values corres ponding to the effect of explant type on bud formation percentage, mean number of buds per explant and regeneration index in L. bainesii after 30 days of culture............................................................70 6-2 Effect of explant type on bud forma tion percentage, mean number of buds per explant and regeneration index in L. bainesii after 30 days of culture...................70 A-1 Effect of different combinations of TDZ and IBA on percentage of adventitious bud formation and mean number of buds per explant in A. latifolia U18.6 after 30 and 60 days of culture.........................................................................................77 A-2 Effect of different combinations of TDZ and IBA on regeneration index, number of shoots per explant and shoot length in A. latifolia U18.6 after 60 days of culture.......................................................................................................................77 A-3 Effect of different combinations of TDZ and IBA on percentage of adventitious bud formation and mean number of buds per explant in A. latifolia U18.8 after 30 and 60 days of culture.........................................................................................78 A-4 Effect of different combinations of TDZ and IBA on regeneration index, number of shoots per explant and shoot length in A. latifolia U18.8 after 60 days of culture.......................................................................................................................78 A-5 Effect of different combinations of TDZ and IBA on percentage of ex vitro acclimatization in A. latifolia U18.6 and U18.8 after 20 days of transfer to ex vitro conditions.........................................................................................................79 A-6 Effect of different combinations of BAP and IBA on percentage of adventitious bud formation and mean number of buds per explant in A. latifolia U18.6 after 30 and 60 days of culture.........................................................................................79 A-7 Effect of different combinations of BAP and IBA on regeneration index and shoot length in A. latifolia U18.6 after 60 days of culture.......................................80 A-8 Effect of different combinations of BAP and IBA on percentage of adventitious bud formation and mean number of buds per explant in A. latifolia U18.8 after 30 and 60 days of culture.........................................................................................80 A-9 Effect of different combinations of BAP and IBA on regeneration index and shoot length in A. latifolia U18.8 after 60 days of culture.......................................81 A-10 Effect of different times of exposur e to TDZ on bud formation percentage, mean number of buds per explant and regeneration index in A. latifolia U18.6 after 30 and 60 days of culture..............................................................................................81 A-11 Effect of different times of exposur e to TDZ on bud formation percentage, mean number of buds per explant and regeneration index in A. latifolia U18.8 after 30 and 60 days of culture..............................................................................................82

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ix A-12 Effect of short exposure to TDZ on bud formation percentage, mean number of buds per explant and regeneration index in A. latifolia U18.6 and U18.8 after 30 days of culture..........................................................................................................82 A-13 Effect of explant type on bud forma tion percentage, mean number of buds per explant and regeneration index in A. latifolia U18.6 and U18.8 after 30 days of culture.......................................................................................................................83

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x LIST OF FIGURES Figure page 4-1 Shoot bud organogenesis th rough cotyledon culture of T. carolinianum ................30 5-1 Organogenesis in A. latifolia ....................................................................................52 5-2 Regression curve showing the effect of TDZ concentration on percentage of adventitious bud formation in A. latifolia after 60 days of culture..........................53 5-3 Regression curve showing the effect of TDZ concentration on regeneration index in A. latifolia after 30 days of culture.............................................................53 5-4 Regression curve showing the effect of TDZ concentration on shoot number per explant in A. latifolia after 90 days of culture..........................................................54 5-5 Regression curve showing the effect of TDZ concentration on shoot length in A. latifolia after 90 days of culture...............................................................................54 5-6 Regression curve showing the effect of TDZ concentration on acclimatization rate in A. latifolia after 30 days of transfer to ex vitro conditions............................55 5-7 Regression curve showing the effect of BAP concentration on percentage of adventitious bud formation in A. latifolia after 30 days of culture..........................55 5-8 Regression curve showing the effect of BAP concentration on regeneration index in A. latifolia after 30 days of culture.............................................................56 5-9 Regression curve showing the effect of BAP concentration on percentage of adventitious bud formation in A. latifolia after 60 days of culture..........................56 5-10 Regression curve showing the effect of BAP concentration on mean number of buds per explant in A. latifolia after 60 days of culture...........................................57 5-11 Regression curve showing the effect of BAP concentration on regeneration index in A. latifolia after 60 days of culture.............................................................57 5-12 Regression curve showing the effect of BAP concentration on shoot length in A. latifolia after 90 days of culture...............................................................................58 5-13 Regression curve sowing the effect of exposure to TDZ on bud formation percentage in A. latifolia after 60 days of initiation of cultures...............................58

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xi 5-14 Regression curve sowing the effect of exposure to TDZ on mean number of buds per explant in A. latifolia after 60 days of initiation of cultures......................59 5-15 Regression curve sowing the effect of exposure to TDZ on regeneration index in A. latifolia after 60 days of in itiation of cultures.....................................................59 5-16 Regression curve sowing the effect of short exposures to TDZ on percentage of bud formation in A. latifolia after 30 days of in itiation of cultures..........................60 5-17 Regression curve sowing the effect of short exposures to TDZ on regeneration index in A. latifolia after 30 days of initiation of cultures.......................................60 6-1 Organogenesis in L. bainesii ....................................................................................71 6-2 Number of buds produced per explant by 50 genotypes of L. bainesii after 30 days of culture from cotyledon and leaflets.............................................................72 6-3 Number of plants produced per explan t after 90 days of culture by responsive genotypes of L. bainesii through cotyledon and leaflet culture...............................72 6-4 Number of successfully acclima tized plants in the genotypes of L. bainesii that were capable of plant regeneration through cotyledon and leaflet culture...............73 A-1 Regression curve sowing the effect of exposure to TDZ on bud formation percentage in A. latifolia after 30 days of initiation of cultures...............................84 A-2 Regression curve sowing the effect of exposure to TDZ on mean number of buds per explant in A. latifolia after 30 days of initiation of cultures......................84 A-3 Regression curve sowing the effect of exposure to TDZ on regeneration index in A. latifolia after 30 days of in itiation of cultures.....................................................85

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xii Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science TISSUE CULTURE OF Trifolium polymorphum, T. carolin ianum, Adesmia latifolia, A. bicolor AND Lotononis bainesii By Maria Laura Vidoz December 2006 Chair: Kenneth H. Quesenberry Cochair: Mary J. Williams Major Department: Agronomy Although they fix nitrogen, provide feed for livestock, improve soil properties and protect the soil from erosion, many forage legume species have been underutilized. Currently, breeding programs have an increase d interest in several of the less-studied species that could improve pasture quality in subtropi cal regions of the world. Trifolium polymorphum Poir. T. carolinianum Michx., Adesmia latifolia (Spreng.) Vogel, A. bicolor (Poir.) DC. and Lotononis bainesii Baker are promising forages; however, they possess two major drawbacks: low seedling vigor and low dry matter production. The objective of this research was to develop in vitro plant regeneration protocols for these species that could assist breeding prog rams by potentially enabling the use of in vitro chromosome doubling techniques and genetic transformation. Experiments using three types of basal medium (MS, L2 and B5) were conducted with T. polymorphum and Adesmia spp. In the latter, the effect of different plant growth regulators (TDZ, BAP and IBA) alone or in combination was evaluated. Cotyledon

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xiii culture on media supplemented with various plant growth regulators was assessed in T. carolinianum Adesmia spp and L. bainesii Time required for shoot bud induction in A. latifolia was determined using immature leaflets as explants. Different leaf parts were evaluated for their morphogenetic potential in A. latifolia and L. bainesii In T. polymorphum, only callus formation was achieved using primarily B5 basal medium. With T. carolinianum cotyledon culture on MS + 10 M TDZ for 30 days with transfer to MS + 1 M TDZ gave superior shoot bud or ganogenesis (20% of genotypes). Plant regeneration of A. bicolor was achieved through immature leaflet culture on MS or L2 + 4.5 M TDZ for 60 days followed by transf er to MS devoid of plant growth regulators for 45 days. The highest fr equency of plant regeneration in A. latifolia was obtained using immature rachises cultured on MS + 10 M TDZ for 20 days and then transferred to MS without plant growth regulators for bud elongation and rooting. L. bainesii plant regeneration (in 90% of genotype s) was achieved by culturing immature leaflets on MS + 4.5 M TDZ for 30 days a nd then transferring cu ltures to MS + 0.044 M BAP + 0.049 M IBA for bud elongation and rooting. In conclusion, shoot organoge nesis protocols were devel oped for four out of the five forage legume species evaluated, and pl ant regeneration was ach ieved with three of these species. Additional experiments to obtain rooting of T. carolinianum should be conducted. Studies to reduce the inciden ce of hyperhydricity in cultures of Adesmia spp. and to assess the influence of different pl ant growth regulators on shoot bud formation of L. bainesii are also needed.

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1 CHAPTER 1 INTRODUCTION In addition to providing feed for livestock, forages offer multiple benefits such as maintenance and improvement of soil charact eristics, weed suppre ssion, and protection of soil from erosion. When used as cover crops, fo rages may also assist in the recovery of degraded soils (Peters et al., 2003) by decrea sing the loss of nutrients from leaching or erosion (Tilman et al., 2002). Fora ges that exhibit two or more of these characteristics are better accepted by farmers in agricu ltural systems (Peters et al., 2003). The family Fabaceae comprises between 670 to 750 genera and 18,000 to 19,000 species, many of which constitute an important source of protein in human and animal diets, balancing the amino acids provid ed by cereals (Graham & Vance, 2003). According to Crews & Peoples (2004), legum es constitute a more environmentally friendly source of nitrogen than synthetic fert ilizers due to their ability to fix nitrogen, reducing the risk of eutrophication and c ontamination of subterranean water. However, numerous species of pasture le gumes have thus far been underutilized (Graham & Vance, 2003), although many of them could be used to increase the genetic variability of their cultivated relatives or as a source of useful genes. For instance, legumes bred for drought and salinity tolerance would be useful in water-stressed areas of the world (Graham & Vance, 2003). In this scen ario, forages that are naturally adapted to those conditions will be particularly valuable. With approximately 240 species, the genus Trifolium L. is one of the most important genera in the Fabaceae family rega rding the number of species it comprises

PAGE 15

2 and their potential uses (Zohary & Heller, 1984). It is found in temperate to subtropical regions of Europe, the Americas, Asia and Africa (Lange & Schifino-Wittmann, 2000), but also occurs in the mountai n and alpine zones in the tropi cs of West Africa and South America (Zohary & Heller, 1984). At leas t 25 species are useful as forages. Trifolium polymorphum is endemic to subtropical South America and T. carolinianum is endemic to subtropical North America. Although common in native grasslands, neither species has been the s ubject of crop improvement research through plant breeding programs. T. polymorphum Poir. is a perennial subtropical species described in the United States, Argentina, Brazil, Chile, Paraguay, Peru and Uruguay (Quesenberry et al., 1997). It constitutes th e only amphicarpic species in the genus and has been used in some cytogenetic an d reproductive studies (Lange & SchifinoWittmann, 2000). Agronomic evaluations c onducted in New Zealand discovered one accession with 154 g kg-1 crude protein and 697 g kg-1 digestibility (D odd & Orr, 1995). However, its marginal dry matter (DM) production and low seedling vigor is a major limitation for its use as a forage legume. Trifolium carolinianum Michx. is an annual species na tive to the southeastern US. It produces seed and nodul ates profusely, but like T. polymorphum it has low seedling vigor and low DM production. Genetic impr ovement through traditional breeding and transformation can overcome these restri ctions. Transformation, however, usually requires an efficient protocol for plant regeneration to be in place. Although in vitro regeneration protocols are available for several Trifolium species (Ding et al., 2003), there are none for T. polymorphum or T. carolinianum Additionally, induction of polyploidy can also be used to increase s eedling vigor and overal l plant size, as was

PAGE 16

3 shown previously in the genus Trifolium (Taylor et al., 1976; Fur uya et al., 2001). Once again, the availability of an in vitro protocol would permit the use of additional techniques to duplicate chromosomes in T. polymorphum as has been demonstrated in bahiagrass ( Paspalum notatum Flgge) using colchicine, trifluralin and oryzalin (Quesenberry et al., 2003). Moreover, the use of an in vitro protocol to induce chromosome duplication could pr event the recovery of chimeric plants that often are obtained when vegetative merist ems are treated with chromosome doubling agents. In an in vitro system single cells are doubled and, consequently, plant regeneration from these single cells, as in th e case of adventitious bud formation, renders a plant whose cells uniformly contain duplicated chromosomes. Adesmia DC. (Fabaceae) is the only genus of the South American tribe Adesmieae (Benth.) Hutch and it comprises approximately 240 species of herbs and shrubs (Ulibarri & Burkart, 2000). Many Adesmia species, such as A. bicolor (Poir.) DC., A. latifolia (Spreng.) Vogel and A. punctata (Poir.) DC., constitute promising forage materials because of their satisfactory winter gr owth, high crude protein values and good in vitro organic matter digestibility. These species have been reported to be useful for soil cover and erosion control (Coelho & Battistin, 1998; Tedesco et al., 2000; Tedesco et al., 2001). In addition, A. bicolor showed tolerance to low phosphorous fertility and possesses valuable morphological characterist ics when compared to other legumes (Dodd & Orr, 1995). In spite of these desirable char acteristics, there are ve ry few reports related to the biology of the genus (Coelho & Batt istin, 1998; Dodd & Orr, 1995; Tedesco et al., 2000; Tedesco et al., 2001), and no reports regarding their use in tissue culture.

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4 The genus Lotononis belongs to the tribe Crotalar ieae and comprises approximately 150 species, from herbs to small shrubs (Jafth a et al., 2002). These species are distributed from Southern Africa to the Mediterranean region and Indi a, and are found under a range of climates and geographical situations. L. bainesii Baker is a perennial herb whose forage value has been demonstrated in Au stralia (Jaftha et al., 2002). Although it has been previously reported to be a cleistogam ous species, Real et al (2004) have conducted molecular studies that suggest that it requires pollinators to set seed and some genotypes are self-incompatible. There is on ly one report on tissue culture of L. bainessi where a low frequency of plant regenera tion was obtained (Bovo et al., 1986). Limited quantities of seeds may constitute a problem for release of new cultivars (Frame, 2004) but tissue culture may offer a solution, shortening the period of time for the availability of propagul es. In addition, in cross pol linated species such as L. bainesii A. latifolia and A. bicolor each seed is potentially a differe nt genotype. Therefore, it is not possible to propagate a plant exhibiting exceptional ch aracteristics through seeds since the progeny may segregate for the tr ait of interest. However, micropropagation offers a solution for the mass propa gation of an individual plant.

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5 CHAPTER 2 LITERATURE REVIEW All plant cells have the potentia l to be totitpotent, i.e., to be able to dedifferentiate, divide and regenerate into whole plants (L oidl, 2004). This was the idea that Gottlieb Haberlandt had in mind when he first atte mpted plant tissue culture in the early 20th century (Caponetti et al., 2004) Although he failed in his ve nture to regenerate plants from isolated tissues, his work attracted the attention of the scientific world and, consequently, abundant research was developed on the topic. Tissue Culture Concepts as A pplied to the Fabaceae Family Tissue culture is usually defined as a he terogeneous group of techniques in which explants (protoplasts, cells, tissues or or gans) are aseptically placed onto a culture medium of defined chemical composition, and incubated under c ontrolled conditions (Mroginski et al., 2004b). Ther e are three types of plant re generation systems that are used most frequently: micropropagation, or ganogenesis and somatic embryogenesis. Micropropagation consists of the in vitro propagation of selected genotypes through improved axillary shoot production from explan ts with pre-existing meristems (Kane, 2004). In contrast, the other two regene ration schemes are based on the use of nonmeristematic tissues as explants: organogenesis is the de novo formation of organs (shoots, roots or flowers), and somatic embryogenesis is the production of embryos without a previous fusion of gametes (Radice, 2004). Members of the Fabaceae family have tradit ionally been regarded as recalcitrant to in vitro regeneration, particularly in the case of cultivated grain legumes (Griga, 1999;

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6 Veltcheva et al., 2005; Mundhara & Rashid, 2006). Veltcheva et al. (2005) suggest that recalcitrance in grain legume s could be caused by the narrow genetic base of the cultivated varieties that have undergone inbreeding and selecti on for long periods of time. In addition, they suggest that in forage species the outbreeding and lower genotype selection may account for easier iden tification of responsive genotypes. Some of the factors that affect in vitro response of a given species are genotype, explant, composition of the culture medium and conditions under which explants are incubated (Radice, 2004). The genotype of th e donor plant is one of the most critical factors since it influences in vitro responses, from the establis hment of the explant to the regeneration of whole plants, as well as ex vitro, during the acclimatization of regenerated plants. The importance of the genotype on in vitro plant regeneration of cultivated peanut ( Arachis hypogaea L.) has been demonstrated by Chengalrayan et al. (1998), who assessed 16 genotypes for responsiveness in vitro using a protocol to induce somatic embryogenesis. These authors found diff erences in the freque ncy of response at each stage of the process and suggested that genotype could be the primary factor influencing conversion of soma tic embryos to plantlets. A similar experiment was carried out in soybean ( Glycine max (L.) Merrill), in which 17 br eeding lines were evaluated for their response and ability to regenerate pl ants through somatic embryogenesis (Tomlin et al., 2002). Among these lines, a significant difference in the percentage of responsive explants, number and quality of somatic em bryos were observed. Other legume species in which differences among genotypes were re ported, particularly regarding somatic embryogenesis are Medicago sativa L. and T. pratense L. (Lakshmanan & Taji, 2000).

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7 For T. pratense Quesenberry and Smith (1993) increased genotype regeneration frequency from less than 5% to almost 70%, after five cycles of recurrent selection. The explant type used for culture estab lishment depends on the objectives that are pursued since it determines respons iveness of the plant material in vitro (Lakshmanan & Taji, 2000). The aspects that should be consider ed in explant selecti on are: explant tissue (leaves, petals, anthers, roots, meristems, cotyledons, epicotyls, hypocotyls), explant size, explantation time, topophysis and polyphenol oxidation (Kane, 2004). It is widely accepted that immature zygotic embryos and young seedlings are the most responsive explants to induce somatic embryogenesis in le gume species. This is because areas where cells show active division are more responsiv e to the embryogenic s timulus (Griga, 1999; Mundhara & Rashid, 2006). However, a range of explants have been used with success to induce somatic embryogenesis in Fabaceae family. These have included mature seeds, shoot apices, seedlings, hypocotyls, cotyledon s, leaves, petioles, internodes, roots, endosperms, cell suspensions and protoplasts (Lakshmanan & Taji, 2000). For the induction of organogenesis in legume species, a si milar variety of explants has been used. As an example, in Arachis several explants have been capable of regenerating plants: fully expanded leaves (Dunbar & Pittman, 1992) leaflets from young seedlings (Akasaka et al., 2000), epicotyls, petio les (Cheng et al., 1992), cotyle dons, embryo-axes, mature whole seeds (Radhakrishnan et al., 2000), pr otoplasts (Li et al., 1993), mature zygotic embryo-derived leaflets (Chenga lrayan et al., 2001) and shoo t apices (Radhakrishnan et al., 1999). Culture medium composition is determin ed by the type and concentration of inorganic salts (macroand micro-nutrient s), organic compounds (sugar, vitamins,

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8 activated charcoal, etc.), plant growth regulat ors (mainly auxins and cytokinins), gelling agents or other support system and the gase ous atmosphere inside the culture vessel (Radice, 2004). The most widely used basa l medium for legume regeneration is MS medium developed by Murashige and Skoog (1962) for callus cultures of tobacco (Murashige & Skoog, 1962). This basal medium, which has a high salt concentration, has been used to achieve plant regeneration in several legume genera, such as Arachis (Rey & Mroginski, 2006), Astragalus (Luo et al., 1999), Cajanus (Singh et al., 2003), Cassia (Agrawal & Sardar, 2006), Cicer (Chakraborti et al., Dalbergia (Singh & Chand, 2003), Glycine (Tomlin et al., 2002), 2006), Lathyrus (Barik et al., 2005), Lotus (Akashi et at., 2003), Phaseolus (Delgado-Sanchez et al., 2006), Pisum (Loiseau et al., 1998), Trifolium (Ding et al., 2003) and Vigna (Saini & Jaiwal, 2002). Howe ver, other basal media have been specifically developed for certain legume species, such as G. max (Gamborg et al., 1968) and T. pratense (Collins & Phillips, 1982). Several plant growth regulators have been used with success in pl ant regeneration protocols for le gume species, but the type of response and effectiveness of the compounds are highly de pendent on the species and even on genotypes within a species. In gene ral, auxins are used to induce somatic embryogenesis, whereas cytokinins are used to induce organogenesis. Nevertheless, there are some exceptions such as in T. repens L., Medicago sativa and Phaseolus spp., in which it was possible to achieve somatic embr yogenesis by using cytokinins instead of auxins (Lakshmanan & Taji, 2000). In addition to media composition fact ors, incubation conditions under which explants are incubated must be controlled. Th ese include temperature, light quality and intensity, photoperiod, humidity and hygiene (M roginski et al., 2004b). In general, the

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9 temperature for incubation of cultures is be tween 23-29 C, depending on optimal growth requirement of the species (Radice, 2004). In some cases, when the process to be induced is somatic embryogenesis, cultures are incubate d in the dark, since li ght is not required for this developmental pathway. In contra st, when organogenesis is to be induced, cultures are usually kept under light conditions with a specific photoperiod. In addition, light quality (spectra l quality) and quantity (photon flux) are reported to have an important role in morphogenetic processes in vitro and on the subsequent growth of the regenerated structures (Lian et al., 2002). Applications of Tissue Cult ure for the Fabaceae Family Since its origin in the early 20th century, tissue culture procedures have been used for a variety of purposes, such as basic stud ies of particular ph ysiological processes because the use of tissues instead of whol e plants usually simplifies the study of the phenomenon (Mroginski et al., 2004b). Another use of tissue culture is the production of plants free from certain specific pathogens, generally viruses, through meristem or shoot tip culture alone or combined with thermo/chemotherapy. Ho wever, the most important application of these techniques from an economic point of view is related to micropropagation. This method is particularly im portant in horticulture, since it generally maintains genetic stability (Kane, 2004), and allows propagation of periclinal chimeras. This kind of chimera may be important in ornamental species a nd cannot be propagated through organogenesis or somatic embryogenesis. Tissue culture may also be used for the production of interspecific hybrids where zygotic embryos abort early in their development and have to be rescued, or in the case of plants w ith a rudimentary embryo (Mroginski et al., 2004b). The production of dihaploid, homozygous plants is also possible through anther or ovule culture, wh ich reduces the time required to achieve

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10 homozygosis in breeding programs. Other appl ications of tissue culture include the induction of somaclonal variation, production of secondary metabolites using cell culture, generation of somatic hybrids through prot oplast fusion, and plant regeneration after transformation protocols. From the species preservation point of view, tissue culture constitutes a valuable technique for medi um and long-term germplasm conservation ( in vitro and cryoconservation), as well as plant ma terial exchange since pathogen-free plants are used for this purpose (Mroginski et al., 2004b). Production of Plants Free fr om Certain Specific Pathogens Since plants were first domesticated, di seases and pests have threatened crop productivity. Some diseases caused by fungi a nd bacteria may be controlled if certain practices are used during the cultivation of the crop. However, in the case of viruses, the control is usually more difficult and in many cases the only indication of the presence of a virus is a reduction in crop yields. Viral di seases are transmitted rapidly particularly when the crop is vegetatively propagated (Kartha, 1984). Meristem culture is one of the tools to eliminate viruses from plant material, provided that the rate of virus multiplication a nd movement in the plant is lower than the rate at which the meristematic region elonga tes. This is often the case since vascular tissues do not reach the meristem. In the Fa baceae family, meristem culture has been applied successfully to the rescue of interspecific hybrids between A. hypogaea and A. stenosperma Krapov & W.C. Gregory, and A. hypogaea and A. otavioi, which showed symptoms of peanut stripe virus (Radhakris hnan et al., 1999). Meristem culture with or without thermo/chemotherapy has also been used to eliminate peanut mottle virus, peanut stripe virus and tomato spotted wilt virus from interspecific hybrids of Arachis that were maintained vegetatively in the germplasm collection at the Southern Regional Plant

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11 Introduction Station (Griffin, GA ) (Dunbar et al., 1993a). In c ontrast, shoot tip culture was not an effective procedure to regenerate plants free of the peanut mottle viruses. Prasada Rao et al. (1995) excised seed axes fr om peanut stripe viru s infected seed and cultured them on a medium containing ribavirin to obtain peanut plants free of the virus. Meristem culture has also been successfully applied to other legume genera, such as Trifolium and Phaseolus for the production of plants free of common viruses (Phillips and Collins, 1979; Veltcheva et al., 2005). Micropropagation Micropropagation, the true-totype propagation of a genotype through tissue culture techniques, is a useful tool in breeding programs. Among ot her advantages, it enables the production of uniform plants from a select ed genotype at a high multiplication rate (Olmos et al., 2004). The stages for micropropa gation from shoot expl ants are: a) donor plant selection and preparation, b) axillary shoo t proliferation, c) pretransplant or rooting, and d) transfer to the natural environment (Kane, 2004). Cultivated peanut has been reported to have limited reproductive efficiency, which is a drawback when large populations are re quired for breeding purposes (Radhakrishnan et al., 2000). Micropropagation ma y be used to overcome this situation, provided that an efficient in vitro protocol is available. For this species, Radhakrishnan et al. (2000) developed a high frequency micropropagation protocol from embryo axes and plant regeneration from other juvenile explants Successful micropropagation protocols have also been developed for other species such as Vigna mungo (L.) Hepper, a grain legume important in South Asia and Australia, where plants were regenerated from shoot tips, embryo axes and cotyledonary nodes (Saini & Jaiwal, 2002).

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12 Production of Dihaploid, Homozygous Plants The production of haploid plants followi ng anther, pollen or ovule culture is desired in breeding programs because it woul d result in a reduction in the number of cycles to achieve complete homozygosity after the duplication in the number of chromosomes of the regenerated haploid plants Several attempts have been made in the Fabaceae family but with little succe ss. For instance, in the genus Arachis Bajaj et al. (1980) reported androgenesis but no plan t regeneration from pollen cultures of A. hypogaea and A. glabrata Benth. Bajaj et al. (1981) re ported plant regeneration from anther cultures of A. hypogaea and A. villosa Benth., with a chromosome number varying from haploid to octaploid. In soybean, Rodr igues et al. (2004) st udied the origin of embryo-like structures from anther cultures using molecular techniques. They found both homozygous and heterozygous structures suggesting that embryogenesis and androgenesis occurred simultaneously. Anther culture in Phaseolus resulted in callus formation with cell ploidy levels ranging fr om haploid to polyploi dy (Veltcheva et al., 2005). Generation of Interspecific Hybrids: Embryo Rescue and Protoplast Fusion In many cases, wild species offer traits of interest that could be useful in breeding programs if incorporated into their cultivat ed relatives. Nevertheless, there may be interspecific barriers that need to be overcome in order to be able to transfer the trait of interest. One of the possibilities to circum vent this would be to use tissue culture techniques to rescue the em bryo before it aborts, follo wed by micropropagation of the hybrid if it has low fertility. Arachis villosulicarpa Hoehne, a wild relative of cultivated peanut ( A. hypogaea ), is rich in oil and is resistant to Cercospora arachidicola and Cercosporidium personatum

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13 (Mansur et al., 1993). However, hybrids between this species and A. hypogaea could not be obtained due to cross compatibility barriers that caused abnormal endosperm development (Pittman et al., 1984). Somatic hybridization may be used to overcome this incompatibility, provided that an in vitro protocol is available. For this purpose, Mansur et al. (1993) developed a protoc ol for plant regeneration from cotyledons, leaves and cell suspensions of A. villosulicarpa In the genus Trifolium a few interspecific hybrids have been produced with the aid of tissue culture techni ques, such as hybrids between red clover ( T. pratense ) and T. sarosiense Hazsl. where aseptic embryo rescue was used before in situ abortion (Phillips et al., 1982). Przywara et at (1996) regenerated hybrid pl ants from crosses between T. repens and T. nigrescens Viv. using in vitro pollination followed by embryo rescue. If hybrid embryos did not grow, they were tran sferred onto MS medium supplemented with growth regulators and ac hieved plant regeneration through organogenesis. To apply somatic hybridization for the rec overy of interspecifi c hybrids, at least one of the parents should be able to regenerate plants from protoplasts, but both should be able to undergo protoplast cult ure. In addition, it should be po ssible to select the somatic hybrids (Myers et al., 1989). There are report s on protoplast culture, which in most cases resulted in plant regeneration, in several species of the genus Trifolium such as T. fragiferum L. (Rybcznski, 1997), T. pratense (Myers et al., 1989; Radionenko et al., 1994), T. repens (Webb et al., 1987), T. resupinatum L. (Oelck et al., 1982) and T. rubens L. (Grosser & Collins, 1984). Plant regenerati on from protoplasts has also been obtained in other legume genera such as Astragalus (Hou & Jia, 2004).

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14 Other grain legume genera where embryo resc ue techniques have been applied to obtain interspecific hybrids are Cicer and Phaseolus In order to determine the phase when embryo rescue should occur in wide hybrids, Clarke et al (2006) used selfed chickpea ( Cicer arietinum L.) and selfed wild relatives to study the stage of embryo development at which abortion occurs. Seve ral protocols have been developed to regenerate plants through embryo rescue af ter crosses between cultivated and wild Phaseolus species; however, no somatic hybridization has been reported for the genus (Veltcheva et al., 2005). Plant Regeneration after Transformation Protocols The application of molecular approaches to plant breeding usually requires an efficient in vitro system to regenerate plants from transformed single cells in order to obtain nonchimeric plants (G ill & Ozias-Akins, 1999). In vitro protocols amenable to molecular breeding have been develope d for a wide range of legume genera: Arachis (Ozias-Akins & Gill, 2001; Vidoz et al., 2006; Rey & Mroginski, 2006); Astragalus (Luo et al., 1999), Cajanus (Singh et al., 2003), Cassia (Agrawal & Sardar, 2006), Cicer (Chakraborti et al., 2006); Dalbergia (Singh & Chand, 2003); Glycine (Tomlin, 2002), Lathyrus (Barik et al., 2005), Lotus (Lombari et al., 2003), Macroptilium (Ezura et al., 2000), Phaseolus (Delgado-Sanchez et al., 2006), Trifolium (Ding et al., 2003) and Vigna (Saini & Jaiwal, 2002). Some ex amples of legume species in which tissue culture has assisted transformation protocols are A. hypogaea (Ozias-Akins & Gill, 2001), Lotus japonicus (Regel) K. Larsen (Lombari et al., 2003), G. max (Olhoft et al., 2003), T. repens T. pratense and T. subterraneum L. (Ding et al., 2003).

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15 Medium and Long-term Germplasm Conservation and Plant Material Exchange As civilization advances, the centers of diversity of ma ny important plants for food and forage are threatened. This situation imp lies the loss of valuable genes contained in the wild relatives of the cult ivated species that could be used in breeding programs. For this reason, germplasm is kept in storage f acilities, mainly as seeds, which require considerable land and labor to be renewe d. For many species belonging to the genus Arachis seed viability decreases abruptly after 2-3 years of storage. However, some protocols have been developed to recover pl ants from seeds that would not germinate by themselves through the in vitro culture of embryonic axes (Dunbar et al., 1993b; Morris, et al., 1995). Cryopreservation constitutes an alternativ e to the laborious and time consuming storage of seeds. Not only does it allow for l ong-term storage, but it also ensures genetic stability, requires little space and is low main tenance (Gagliardi et al., 2003). The ultralow temperatures of liquid nitrogen cause interruption of all bi ochemical reactions protecting the plant material from physiol ogical and genetic changes (Yamada et al., 1991). In addition, plants are kept free from pa thogens when propagated from plants that have been indexed for the presence of specific microorganisms. Protocols for cryopreservation have been deve loped for several species: A. burchellii Krapov. & W.C. Greg., A. hypogaea A. retusa Krapov. et al., (Gag liardi et al., 2003), A. macedoi Krapov. & W.C. Greg., A. pietrarellii Krapov. & W.C. Greg., A. prostrata Benth. A. villosulicarpa (Gagliardi et al., 2002) and T. repens (Yamada et al., 1991) among others. Medium-term conservation of germplasm can al so be done by mainta ining plants under in vitro conditions, which has similar advantages as cryopreservation: little space, low maintenance, and protection from pathogens. Moreover, plants kept in vitro are a ready

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16 source of material in case the production of a large num ber of plants is required (Bhojwani, 1981). Although there are many reports on tissue culture of legume species, there are no in vitro protocols for T. polymorphum T. carolinianum A. bicolor or A. latifolia four promising forage species. For L. bainesii there is only one report of plant regeneration from cotyledons (Bovo et al., 1986). Therefore, the main objective of this research was to develop protocols for plant regeneration of these species that may then be used to improve their forage potential. Medium basal sa lts, plant growth regul ators, explant type and time of exposure were the main factors evaluated.

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17 CHAPTER 3 MATERIALS AND METHODS Procedures common to all experiments are described here. The modifications made for specific experiments are desc ribed in corresponding chapters. Plant Material Experiments were carried out with four sp ecies representing three different genera of the Leguminosae family: A. bicolor A. latifolia L. bainesii T. carolinianum and T. polymorphum (Table 3-1). Seeds were scarified us ing concentrated sulphuric acid (98%) for 5 minutes ( Adesmia spp ., L. bainesii ), 7 minutes ( T. polymorphum ) or 10 minutes ( T. carolinianum ) and then were rinsed for 10 minutes in running tap wate r. Subsequently, seeds were surface disinfected by immersi on in a solution of sodium hypochlorite containing 0.571 % W/V available chlorine for 5 minutes and rinsed wi th distilled sterile water three times. Disinfected seeds were placed on half-strengt h Murashige and Skoog (1962) (MS) basal medium, with 15 g L-1 sucrose and 0.7 g L-1 agar (Sigma1 A-1296) in 100 mm diameter x 15 mm deep petri dishes. Culture Conditions Basal medium consisted of either MS L2 (Collins & Phillips, 1982) or B5 (Gamborg et al., 1968). Media were prepared using MS basal salt mixture (Sigma M5524) and MS vitamins (Sigma M7150) mixed in proportions corresponding to Murashige and Skoog (1962), B5 basal salt mixture (Sigma G5768) and B5 vitamins 1Use of brand name is for identification purposes only and does not imply exclusion of other similar brand products.

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18 (RPI G37010) were used in quantities speci fied in Gamborg et al. (1968) or L2 concentrated stock solutions mixed in th e adequate proportions according to Collins & Phillips (1982). Concentrated stock solutions of plant growth regulators were prepared by dissolving the correct quantity of the product {[TDZ= thidiazuron, 1-phenyl-3-(1,2,3thiadiazol-5-yl)urea (Sigma P6186)], [BAP= benzylaminopurine (Sigma B3408)], [KIN= kinetin, 6-furfurylaminopurine (Sigma K0753)], [PIC= picloram, 4-amino-3,5,6trichloropicolinic acid (Sigma P5575)], [2,4-D= 2,4dichlorophenoxyacetic acid (Sigma D6679)], [IBA= indoleb utyric acid (Sigma I5386)] in distilled water and kept frozen at -14C.The pH of the media was adju sted to 5.8 with the addition of drops of 1 N KOH or 1N HCl before the addition of agar (Sigma A-1296). Culture medium was sterilized by autoclaving for 20 minutes at 0.103 Mpa. For the first steps of all experiments, 100 mm diameter x 15 mm deep petri dishes were used and explants were placed with the abaxial side down on 20 mL of culture medium. After the induction, cultures were tran sferred onto MS medi um with or without the addition of 0.044 M BAP + 0.049 M IBA to achieve bud el ongation and rooting of shoots. To induce further growth of shoots, cultures were subsequently transferred to magenta boxes (76.2 mm x 76.2 mm x 101.6 mm) (Magenta Corporation) containing 50 mL of MS devoid of plant growth regulators. Cultures were kept in a growth chamber at 26 2C with a 16-hour photoperiod and 85 mol m-2 s-1 provided by cool fluorescent lights. Regenerated plants were removed from magenta boxes and rinsed under running tap water to completely remove the culture medium and were then placed in plug trays containing vermiculite and covered with a hum idity dome. Trays were placed in a growth

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19 chamber at 22 2C with a 14-hour photoperiod. Plants were watered daily to keep a high humidity level during the first two week s and a solution containing 2 g/L of Captan [4-cyclohexene-1,2-dicarboximide, N-(t richloromethyl) thio] was applied twice during this period. During the third week, covers were removed gradually for longer periods of time and plants were fi nally transferred to the greenhouse. Evaluation and Experimental Design Cultures were evaluated every 30 days for mo st experiments, in order to determine the number of explants that died, remain ed irresponsive, produced callus or buds. Whenever buds were regenerated, the number of buds per explant was also recorded. Each treatment was applied to 10 explants and experiments were repeated 2-3 times. Experiments were treated as completely ra ndom factorial experiments or completely randomized designs.

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20 Table 3-1. Plant material used as a so urce of explants in the experiments. Species Accession No. Origin A. bicolor U5 Lavalleja (Uruguay) U6 Rocha (Uruguay) U7 Paysandu (Uruguay) U8 Tacuarembo (Uruguay) U10 Paso de los Toros/Tacuarembo (Uruguay) U11 Durazno (Uruguay) U12 Canelones (Uruguay) U13 Rocha (Uruguay) U14 Castillos/Rocha (Uruguay) A. latifolia U17 Valle Eden/Tacuarembo (Uruguay) U18 Mina de Corrales/Rivera (Uruguay) U19 Velsquez/Rocha (Uruguay) T. polymorphum U1 Estancia El Rinc n/Florida (Uruguay) U2 Uruguay U3 Uruguay CPI 87102 Pilar (Paraguay) Rio Grande (Brazil) Uruguay T. carolinianum PI 516273 Gainesville, Florida, USA L. bainesii Cv INIA Glencoe Uruguay U preceding a number is a locally assigned University of Florida number, CPI is Commonwealth Plant Introduction number from Australia and PI is USDA/NPGS Plant Introduction number

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21 CHAPTER 4 TISSUE CULTURE OF Trifolium polymorphum AND T. carolinianum Introduction The genus Trifolium originated in the Mediterr anean region, but subsequently spread to the Americas, Asia and Africa as we ll (Zohary & Heller, 1984). It consists of 8 sections and approximately 240 species, 25 of which are used as forage (Lange & Schifino-Wittman, 2000). T. polymorphum is a highly palatable forage legume native to eastern Argentina, Uruguay, Paraguay, central Chile and s outhern Brazil (Speroni & Izaguirre, 2003). It is an amphicarpic species that produ ces aerial and subterranean flowers on the same individual. It has been reported that the latter produce seeds more profusely, acting as a seed bank, whereas s eed production from aerial flowers may be affected by grazing and insect attacks. Th e above-ground flowers have a morphology that seems to stimulate insect pollination; however, the species does not appear to exhibit selfincompatibility and self-pollination can occur, even before anthesis (Speroni & Izaguirre, 2003). Other workers report that the above-gr ound flowers are almost exclusively crosspollinated (Daniel Real personal communication, 2006). T. carolinianum is an annual species and one of only three cl overs native to the southeastern US. It is self-pollinated and re-seeds abundantly, but due to small pl ant size, has only limited forage potential. Plant regeneration has been achieved in several species of the genus Trifolium including T. repens T.pratense T. subterraneum T. michelianum Savi, T. isthmocarpum Brot. (Ding, et al., 2003), T. nigrescens (Konieczny, 1995), and T. rubens (Grosser & Collins, 1984) via either organogenesis or somatic embryogenesis. Many of these

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22 protocols have used explants consisting of immature zygotic embryos (Maheswaran & Williams, 1984) or seedling derived explants such as hypocotyls and radicles (Heath et al., 1993), cotyledons (Konieczny, 1999), or leaves (Radionenko et al., 1994). However, these explants may not be appropriate for the propagation of selected genotypes in cross pollinated species in which seeds coming from the same plant may exhibit different genotypes. In this case, explants obtained from vegetative tissues in fully developed plants are more suitable for the pr opagation of selected individuals. Trifolium plant regeneration from non-meristematic tissues has been achieved usi ng leaves (Rybcznski, 1997) and petioles (Quesenberry & Smith, 1993). T. polymorphum and T. carolinianum exhibit low seedling vigor and low DM production that limit their forage use. Therefore, in vitro chromosome duplication may be used to increase plant vigor. In addition, ch imeral plants could be avoided provided that plant regeneration is obtained from single ce lls that were previous ly doubled by treatment with chromosome doubling agents such as colchi cine, trifluralin or oryzalin (Quesenberry et al., 2003). Consequently, th e objective of these experime nts was to develop a plant regeneration protocol th at could be used for in vitro research to produce polyploid plants of T. polymorphum and T. carolinianum with increased seedling vigor and DM production. Materials and methods Trifolium polymorphum Seeds from several accessions (Table 3-1) were germinat ed as described in chapter 3. Plants were maintained aseptically in magenta boxes containing MS medium devoid of growth regulators and were transferred a pproximately monthly. Petioles from immature fully expanded leaves were excised and cut on st erilized filter paper into pieces of 5 to 6-

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23 mm length, which were used as explants. Five pieces were placed per Petri dish, onto one of the three basal media prepared as indi cated in chapter 3 with the addition of 4.5 M TDZ. The three treatments were applied to all germinated genotypes and experiments were repeated twice. Incubation conditions we re the same as described in chapter 3 and data was recorded after 30 days of culture. Th e experiment was statistically analyzed as a factorial arrangement in a completely ra ndomized design using PROC GLM from PC SAS (SAS Institute, 2003). Tukeys HSD Multiple Range Test at p 0.05 level was used to compare the means of the basal media for each genotype. Trifolium carolinianum Seeds were scarified and germinated as i ndicated in chapter 3. Cotyledons from 45 1-week old seedlings were excised and cut longitudinally along the midrib into two pieces so that four cotyledon explants were obtained per genotype. Each explant was placed onto MS alone or MS with 10 M TDZ, 10 M BAP or 10 M KIN. Five pieces were placed per petri dish and the identity of the genotypes was maintained. Seedlings without cotyledons were placed onto MS medium and kept in vitro for further experiments. After 30 days of culture, regene rated buds were transferred onto L2 medium supplemented with 1 M TDZ. Incubation conditions we re the same as described in chapter 3. Results and Discussion Trifolium polymorphum After 30 days of culture, the only response obtained was the formation of friable, light brown callus that died after subcu lturing onto the fresh culture medium. The statistical analyses revealed an effect of basal media, genotypes and basal medium x

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24 genotype interaction (Table 4-1). Basal me dium B5 was significantly better than the others for the induction of callus formation in 9 of the 15 genotypes. In this experiment, TDZ was used as the growth regulator sin ce it has a high cytokini n activity (Huetteman & Preece, 1993) and was repor ted to effectively induce or ganogenesis in a number of species of the genus Trifolium and Medicago (Ding et al., 2003). In addition, pieces of petioles were chosen as explants, since this would allow for the propagation and manipulation of selected genotypes. Petioles were successfully used as explants in T. pratense (Quesenberry & Smith, 1993) and T. rubens (McGee et al., 1989). The failure in the regeneration of shoot buds could be due to the plant growth regulator used in the experiment, which may not be adequate to trigger the organogenic process or might have been present in a concen tration toxic for this species. It is also possible that other auxins such as PIC or 2,4-D could be more effective. However, it would be necessary to evaluate a larger num ber of genotypes since there were significant differences among them. Seedlings grew vigorously in vitro after slow germination, which started a week after the scarification and c ontinued for over a month. Nevertheless, after approximately two months in culture, they started to decay producing few leaves even with frequent transfers to fresh medium. This factor limite d the number of replications and experiments that could be carried out with this species. In order to determine if the presence of plant growth regulators in the germination medi um promoted multiple shoot formation, seeds from different genotypes were scarified as in the first experiment and placed onto culture media containing either 10 M TDZ or 1 M BAP. Germination was not enhanced but multiple shoots were produced in some cases (data not shown). Nevertheless, the rate of

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25 growth was very slow, and seedlings becam e chlorotic approximately a month after germination started. Trifolium carolinianum After 30 days of culture, it was observed that all explants placed on the basal medium devoid of plant grow th regulators had died. Among the plant growth regulators that were evaluated in the experiment, KIN was not effective for callus induction or bud formation in any of the 45 genotypes and BA P only resulted in shoot bud formation in one genotype and callus formation in one other. TDZ effectively induced shoot bud organogenesis in 20% of the genotypes and callus formation in another 20% of the explants. Moreover, the mean number of buds was considerab ly low (2.1) suggesting that the species may be recalcitrant to tissue culture (Table 4-2). In all cases, calli were small, friable and light brown colored, but were already dead at the time the data was recorded. Thirty days after transfer, the mean nu mber of buds across genotypes increased markedly from 2.1 to 18.7 and buds began to el ongate although growth was slow (Figure 4-1). To achieve bud elongation, cultures were transferred to L2 me dium because it has a lower salt concentration than MS medium. In addition, the concentration of TDZ was reduced from 10 to 1 M because it has been repor ted that TDZ may inhibit bud elongation (Huetteman & Preece, 1993). Almost all responses were with TDZ, indi cating that this plan t growth regulator would be preferred for i nduction of organogenesis in T. carolinianum. In a previous report, TDZ proved effective fo r shoot bud induction in several Trifolium species (Ding et al., 2003). However, most of the reports on plant regeneration through organogenesis

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26 in this genus mention the use of other plant growth regulators such as BAP (Heath et al., 1993; Konieczny, 1999) and 2-isope ntenyladenine (Konieczny, 2000). Trifolium carolinianum is an annual, self pollinate d species, and therefore it has high homogeneity and homozygosis is expected within popula tions and plants. For this reason, in order to develop a tissue culture pr otocol, cotyledonary explants were used. Considering that seeds from a single plant and even a population should exhibit similar genotypes, this constitutes a suitable explant that is available in large quantities and can be easily disinfected and handled. This is not the case in cross pollinated species because the progeny of an individual plant may not reflec t the trait of interest in the mother plant, which generally limits the use of seed-derived explants. Interestingly, there were unexpected di fferences in response among genotypes in that explants from some genotypes were comp letely irresponsive while others produced up to 35 buds. This might indicate that the popu lation that was used in the study is not highly homogeneous, probably due to the o ccurrence of some cro ss pollination. Another possibility could be that the difference in the responsiveness of the genotypes was caused by the treatment received by the plant material. For example, if some seeds had a thinner coat, the scarification process with sulphuric acid could have affected the cotyledons and their ability to regenerate in vitro Another possible cause of va riation could be size of the cotyledons among seedlings, which may explain the difference in the number of buds in the responsive genotypes. Although it does not explain the variability observed in the experiment, it is possible that the unresponsive ge notypes were more sensitive to the high concentration of salts in the culture medium. In this case, L2 basal medium should also be used for the

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27 induction of organogenesis in this species. Th is would be in contrast to other work on Trifolium spp which showed that the use of MS as the basal medium allowed the development of an efficient protocol for T. repens T.pratense T. subterraneum T. michelianum and T. isthmocarpum (Ding et al., 2003). Conclusions In T. polymorphum a significant interaction betw een genotypes and basal media was observed for callus production. There we re also significant differences among genotypes and culture media. However, the culture media tested to achieve plant regeneration through organogenesis were not effective and yielded only callus. Among the three basal media tested, a higher percentage of callus formation was observed with B5. The lack of shoot organogenesis may have been caused by the type or level of plant growth regulators, and therefore, other cytoki nins or auxins should be tested. Moreover, additional experiments are required in order to determine the best culture conditions to maintain germplasm actively growing in vitro In contrast, T. carolinianum shoot bud formation was achieved in 20% of the genotypes when the culture medium contained 10 M TDZ. This might suggest that in this species a potent cytokinin is required in order to induce this morphogenetic process. Callus formation was observed in another 20% of the genotypes, and the rest remained non-responsive and died. This difference was not expected considering that it is a self pollinated species, and populations are expect ed to be highly homogeneous. Shoot bud elongation occurred in MS supplemented with 1 M TDZ but simultaneously, the number of adventitious buds continued to increase. Additional experiments are being

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28 conducted in order to achieve ro oting of the regenerated shoo ts and an adequate growth of the plantlets that would likely re sult in a higher acclimatization rate ex vitro

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29 Table 4-1. Mean percentage of explants producing callus from petiole pieces of T. polymorphum in three basal media after 30 days of culture. Basal medium Genotype MS L2 B5 CPI.1 0 a* 0 a 20 a CPI.2 90 b 20 a 100 b CPI.3 0 a 10 a 100 b CPI.4 0 a 0 a 80 b CPI.5 0 a 0 a 0 a CPI.6 0 a 0 a 80 b CPI.7 0 a 0 a 0 a CPI.8 0 a 0 a 0 a CPI.9 0 a 0 a 30 b CPI.10 0 a 0 a 100 b CPI.11 0 a 0 a 90 b Urug.1 0 a 0 a 90 b Parag.1 0 a 0 a 90 b Parag.2 0 a 0 a 60 b U 02.1 0 a 0 a 100 b *Within rows, different letters indicate significant differences according to Tukeys HSD Multiple Range test at p 0.05 level. Table 4-2. Number of buds per expl ant in the responsive genotypes of T. carolinianum cultured on medium supplemented with 10 M TDZ after 30 and 60 days of culture. Genotype 30 days of culture 60 days of culture 8 4 35 9 1 8 10 3 21 11 2 14 23 2 22 24 3 28 32 1 5 41 1 13 43 2 22

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30 Figure 4-1. Shoot bud organogenesi s through cotyledon culture of T. carolinianum 15 days after transfer to L2 + 1 M TDZ (bar: 20 mm).

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31 CHAPTER 5 PLANT REGENERATION OF Adesmia latifolia AND A. bicolor Introduction The tribe Adesmieae belongs to the Fab aceae family and consists of only one genus, Adesmia DC., and approximately 240 species endemic to South America (Ulibarri & Burkart, 2000). Most of these species of herbs and shrubs grow in the Andes mountains and semi desert zones in the Patagonia. Some species, such as A. latifolia A. bicolor and A. punctata are considered promising forages because of their ability to grow during the winter season, high crude protein value and high in vitro organic matter digestibility (Tedesco et al., 2000). In addi tion, due to the stoloniferous morphology of plants, they may be used for soil cover a nd erosion control (Coelho & Battistin, 1998). Adesmia bicolor and A. latifolia are cross pollinated species although some self pollination may occur (Tedesco et al., 2000) As a consequence, selected genotypes cannot be propagated through seeds because thei r progeny could segregate for the trait of interest. Even though these species could be multiplied vegetatively using stolons, tissue culture could provide a more rapid and effi cient method of propagation. In addition, an in vitro protocol for plant regeneration from singl e cells, such as organogenesis and somatic embryogenesis, could be used to double th e chromosome number in an attempt to increase plant productivity. Moreover, the de velopment of a tissue culture system to propagate and maintain plants in vitro would prove useful for germplasm exchange (Mroginski et al., 2004b) since plants are protected from pa thogens and do not require a quarantine period. Another contribution of tissu e culture techniques to plant breeding is

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32 the in vitro selection for biotic and ab iotic stresses, which has b een applied in alfalfa for resistance to specific fungal diseases (Dita et al., 2006). It has been suggested that in forage legume populations there is greater heterogeneity in comparison to grain legum e populations, due to the lower selection pressure of high seed producing genotypes (V eltcheva et al., 2005). This condition would result in an easier identification of in vitro responsive genotypes. However, the development of an efficient plant regenera tion protocol implies the regulation of the many factors that influence in vitro responses and not only the genotype. These other factors include selection of an adequate e xplant, based on the objectives of the culture, composition of the basal medium and plan t growth regulators used to induce the morphogenetic response, and conditi ons of incubation (Radice, 2004). Tissue culture protocols have been devel oped for some forage legumes such as T. pratense T. repens (Ding et al., 2003), A. pintoi Krapov. & W.C. Greg. (Rey & Mroginski, 2006), A. glabrata (Vidoz et al., 2006), Lotus corniculatus L. (Akashi et al, 2003), Astragalus melilotoides Pall. (Hou & Jia, 2004) and Macroptilium atropurpureum (DC.) Urb. (Ezura et al., 2000). Nevertheless, currently there are no reports on tissue culture in the genus Adesmia The objective of these experiments was to develop for the first time, an in vitro plant regeneration system that could assist A. bicolor and A. latifolia breeding programs. Materials and Methods Plant Material Seeds of A. bicolor and A. latifolia were scarified, sterilized and germinated as described in chapter 3. Plants were main tained aseptically in magenta boxes on MS medium without the addition of plant grow th regulators. Unless indicated, explants

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33 consisted of leaflets from immature leaves, at approximately 50% of expansion, that were excised from those in vitro grown plants. Basal Media Experiment Seven genotypes of A. bicolor and 16 of A. latifolia were used as explant sources. Leaflets were placed onto the three basal me dia indicated in chapter 3, with the addition of 4.5 M TDZ. Each treatment was applied to 10 leaflets of each genotype and experiments were repeated three times. After 60 days of culture, buds were transferred to magenta boxes containing MS devoid of gr owth regulators for 45 days, where buds elongated and rooted. Incubation and acclim atization of regenerated plants was performed as described in chapter 3. Response variables analyzed were: percent organogenesis (%= number of explants that produced buds/total number of explants *100), mean number of buds per explant ( number of buds per explant/ number of expl ants that produced buds) and regeneration index (index= % shoot bud formation mean number of buds per explant / 100). This index was used in order to evaluate the infl uence of the basal medium on the percentage of shoot bud formation and mean number of buds per explant simultaneously. A higher index value indicates that a genotype is capable of produc ing a higher total number of buds. These response variables were also used in subsequent experiments. This experiment was statistically anal yzed as a factorial arrangement in a completely randomized design using PROC GLM from PC SAS (SAS Institute, 2003). Tukeys HSD Multiple Range Test at p 0.05 was used to compare the means of the basal media for each genotype.

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34 Factorial Experiments with TDZ and BAP Among the responsive genotypes in the basal media experiment, A. latifolia U18.6 and U18.8 were chosen as explant sources fo r subsequent experiments, because their micropropagation rate (2.74 and 3.12 fold in crease per month, respectively) provided adequate amounts of tissue. Explants were placed onto MS supplemented with 0, 1, 10, 30 or 60 M TDZ alone or with the addition of either 0, 0.1 or 1 M IBA, such that 15 treatments were used. These same concen trations and combinations were assayed replacing TDZ by BAP. Each treatment was a pplied to 10 leaflets of each genotype and experiments were repeated twice. After a month, cultures were transferred onto MS + 0.044 M BAP + 0.049 M IBA for 30 days to achieve bud elongation and then to magenta boxes containing MS without growth regulators for the same period of time. Incubation and acclimatization of regenerated plants was carried out as described in chapter 3. Since both genotypes responded similarly, data were analyzed and presented as a combination of the two; however, results for each genotype are presented in the Appendix (Tables A-1 to A-9). Data from s ubsequent experiments were also pooled for genotypes. In addition to the variables men tioned above, shoot number, length of the longest shoot and acclimatization rate (% survival= number of pl ants that survived after a month / number of plan ts transferred to ex vitro conditions *100) were also determined. Statistical analyses were performed as a factorial arrangement in a completely randomized design using PROC GLM from PC SAS (SAS Institute, 2003). Tukeys HSD Multiple Range Test at p 0.05 was used to compare the means of treatments. Regression analysis was performed using Micr osoft Office Excel (Microsoft, 2003).

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35 Induction Time Experiments The same explants and sources from the prev ious experiment were used to evaluate the influence of exposure time to TDZ. Leaflets were placed onto MS + 10 M TDZ for 0, 1, 4, 7, 14, 20 or 30 days and then transfe rred onto MS devoid of plant growth regulators. Each treatment was applied to 10 leaflets of each genotype and experiments were repeated twice. Forty-five days after the initiation of the e xperiment, all cultures were transferred onto fresh medium for 30 da ys, and then into magenta boxes containing the same culture medium. Incubation conditions were the same as described in chapter 3. The experiment was analyzed as a comp letely randomized design using PROC GLM from PC SAS (SAS Institute, 2003). Tukeys HSD Multiple Range Test at p 0.05 was used to compare treatment means. Regression analysis was performed using Microsoft Office Excel (Microsoft, 2003). Additional data for each genotype are presented in Appendix (Tables A-10 to A-12). Type of Explant Experiment The same two genotypes of A. latifolia were used as explant sources. Leaflets were excised and placed onto sterile filter paper where they were divided into petiole, rachis and leaflets. Petioles and rachises were s ubsequently divided into 5-6 mm pieces. All petiole and rachises pieces obtained from each leaf and eight leaflets were placed in each petri dish containing MS + 10 M TDZ. This procedure was repeated 10 times for mature, fully expanded and immature, activel y expanding leaves from both genotypes. Thirty days after the initiation of the expe riment, explants were transferred onto MS medium without growth regulators to achieve bud elongation. The conditions under which cultures were incubated were as those indicated in chapter 3.

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36 Statistical analysis was performed as a factorial arrangement in a completely randomized design using PROC GLM from PC SAS (SAS Institute, 2003). Tukeys HSD Multiple Range Test at p 0.05 was used to compare the means of treatments. Additional data for each genotype separately is presented in Appendix (Table A-13). Results and Discussion Effect of Basal Medium on Shoot Organogenesis After 60 days of culture, shoot bud fo rmation was observed in 18 of the 23 genotypes evaluated (Table 5-1). Four of the non-responsive genotypes (U5.3, U5.5, U12.5 and U13.1) corresponded to A. bicolor and one (U19.3) to A. latifolia Both genotype and basal medium had highly signifi cant effects on the percentage of shoot bud formation. The interaction be tween these factors was al so highly significant. The basal medium MS resulted in a hi gher number of responsive genotypes and was superior to L2 in two of them. The av erage percentage of shoot bud formation across all genotypes was not significantly diffe rent between MS and L2 (22 and 16.6%, respectively); however, both were superior to B5 (5.5%). In general, higher percentages were obtained in those genotypes derived from accession U18 of A. latifolia whereas those corresponding to A. bicolor were the least responsive. For the variable mean number of buds per explant, only genotype was a significant source of variation. This was al so evident when the average number of buds per explant across genotypes is considered. This may indi cate that the basal medium may have had a major influence during the period in whic h explants dedifferentiated or acquired competence to respond to the plant growth regulator stimulus, but once induction had occurred, it had a low influence on the number of buds arising per expl ant. In all cases,

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37 the mean number of shoot buds arising per e xplant was rather low and never higher than five, although up to 20 buds per individual explant were formed in some cases. The regeneration index proved to be us eful in this study involving numerous genotypes since in some cases a high number of buds per explant were produced, but a low percentage of explants was responsive. Even though the identification of highly responsive genotypes was not the main purpose of this experiment, this index proved useful for the selection of genotypes that coul d be used in further experiments to adjust the in vitro plant regeneration protocol. Aver aging regeneration index data over genotypes, the means for MS and L2 were si gnificantly higher than for B5. However, the index masked the effect of genotype x medium interaction observed on percentage, since the statistical analysis revealed only genotype an d basal medium effects. The results of this experiment suggest ed that MS medium produced higher proliferation of shoot buds in A. latifolia and A. bicolor than the other basal media. Although MS was originally developed for callus culture of tobacco (Murashige and Skoog, 1962) and B5 and L2 were developed for legume species (Gamborg et al., 1968; Collins & Phillips, 1982), MS basal medium ha s been successfully used for tissue culture of several legume genera including forages like Trifolium and Medicago (Ding et al., 2003), grain legumes like Cicer (Chakraborti et al., 2006) and Vigna (Saini & Jaiwal, 2002) and trees such as Dalbergia and Cassia (Singh & Chand, 2003; Agrawal & Sardar, 2006). Moreover, shoot bud induction was po ssible using TDZ as the plant growth regulator, which is in agreement with reports on other legume species as A. hypogagea (Akasaka et al, 2000), A. correntina (Burkart) Krapov. & W. C. Greg. (Mroginski et al.,

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38 2004a), Cajanus cajan (L.) Millsp. (Si ngh et al., 2003) and Vigna radiata (L.) R. Wilczek (Mundhara & Rashid, 2006). Regarding the acclimatization ex vitro of regenerated plants, the overall performance of plants obtained in MS trea tment was markedly hi gher (72.4% survival ex vitro ) than those of plants obtained in L2 ( 38.7%) or B5 (45.6%) (Figure 5-1 E). This higher survival rate of plants regenera ted on MS may be due to reduced problems associated with hyperhydricity. One of the fact ors that lead to this condition characterized by morphological and physiological abnormalities is water availability (Hazarika, 2006). The higher salt concentration of MS basal me dium may result in lower water availability for plant material, so that the incidence of hype rhydricity is lower in MS than in L2 or B5 medium. Low survival rates could also be due to other disorders common to plants exhibiting heterotrophic growth in vitro such as a low photosynthetic rate, abnormalities in structure and functioning of stomata, and poor mesophyll and vascular system development (Hazarika, 2006). Influence of TDZ on Organogenesis After 15 days of culture, shoot buds were observed in all plant growth regulator combinations in both genotype 18.6 and 18.8 of A. latifolia. Similarly, TDZ has been successfully used to induce shoot formation in other legume genera; however, in some species such as Vigna radiata (Mundhara & Rashid, 2006), A. hypogaea (Gill & OziasAkins, 1999), Trifolium spp (Ding et al., 2003) and C. cajan (Singh et al., 2003), explants consisted of seedling-derived parts which ar e usually much more responsive than nonjuvenile tissues. In most of these Adesmia explants, callus formation was rare and, in some cases, buds seemed to arise almost directly from th e surface of the explant. This is a desirable

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39 characteristic since prolonged maintenance in tissue culture may result in somaclonal variation (Ozias-Akins & Gill, 2001). For the percentage of explants exhibiting bud organogenesis and mean number of buds per expl ant, there was a highly significant effect for TDZ but not for IBA concentration or its interaction with TD Z concentration. Since IBA effect was not significant, the average ac ross IBA concentrations is presented. Only the treatment without TDZ was significantly diffe red from the rest for the percentage of shoot bud formation. A separate analysis was performed by excluding the 0 TDZ treatment in order to detect differences among TDZ concentrations and acquire a more accurate understanding of the response (Tab les 5-2 and 5-3). Similarly a regression analysis was carried out wit hout considering the 0 TDZ treatment and the simplest model that best explained culture responses wa s chosen in each case. When the 0 TDZ treatment was dropped, numerical ly the percentage adventitious bud formation increased with increasing TDZ concentration at 30 da ys of culture, but this increase was not significant (Table 5-2). By 60 days of culture there was a significant quadratic effect of TDZ concentration (R2=0.96) (Table 5-3 and Figure 5-2). Regardless of TDZ concentration the percentage of responsive explants increased from 30 to 60 days of culture which indicates that some explants required a longer period of time to become competent and differentiate buds (Appendix Tables A-1 and A-3). The same statistical procedur es and considerations descri bed above were applied to the other response vari ables. Even when the 0 TDZ treatment was dropped from the analysis, TDZ concentration did not affect mean number of bu ds per explant at either 30 or 60 days (Tables 5-2 and 5-3). In some cases, the number of buds per explant of A. latifolia U18.8 actually decreased at 60 days of cu lture at higher concentrations of TDZ

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40 (Appendix Table A-3) due to the death of some buds. These bud deaths were probably due to some phytotoxic effect of this potent plan t growth regulator, as reported in peanut (Kanyand et al., 1994). The same regeneration index as in the ba sal media experiment was applied here (Tables 5-2 and 5-3). This index showed a quadratic response with increasing TDZ concentrations at 30 days of culture (R2= 1) (Figure 5-3.), but by 60 days of culture the effect of TDZ concentration had disappeared (Table 5-3). Shoot number (Table 5-3 and Figure 5-4) and shoot length (T able 5-3 and Figure 5-5) s howed a linear d ecrease with increasing concentrations of TDZ (R2= 0.87 and 0.79, respectively) (Figures 5-1 A-D). The reduction in shoot elongation is a common response reported in several species when TDZ is added to the cultur e medium (Huetteman & Preece, 1993). Shoot length is an important factor for the success in ex vitro acclimatization since longer shoots may have more reserves to produce roots which promote higher survival rates (T able 5-3). This was evident in both A. latifolia genotypes (Appendix Tables A-2, A-4 and A-5) where ex vitro survival decreased linearly with in creasing concentrations of TDZ (R2=0.88) (Figure 56). In addition, there was a hi gher incidence of hyperhydricity in plants regenerated in medium containing 30 and 60 M TDZ, proba bly caused by a hormonal imbalance in the culture medium (Hazarika, 2006). Influence of BAP on Organogenesis For most variables considered in this study, the effect of BAP concentration was significant whereas the effect of IBA and the interaction between these factors were not. Consequently, the mean across IBA concentrat ions was obtained and values were treated as in the previous experiment regarding T ukeys test and regression analysis. After 30 days of culture, there was a quadrat ic effect of BAP concentration (R2=0.24) (Figure 5-7)

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41 although the percentage of expl ants that had produced buds was low and some explants remained non-responsive (Table 5-4). By 60 days of culture, the response to BAP concentration remained quadratic (R2=0.57), but the percentage of adventitious bud formation increased in at all concentrations (Table 5-5 and Figure 5-9). This response is similar to what has been reported for A. hypogaea, where the addition of BAP to the culture medium required a longer period to induce shoot organogenesis than when was TDZ was added to the medium (Kanyand et al., 1994). Bud formation occurred in explants of most treatments at 60 days after initiation of cu ltures (Table 5-5 and Appendix Tables A-6 and A-8). Additionally, an increase in the percentage of bud formation occurred in those that had responded earlier. This pattern was also observed in the number of buds per explant. Similar to percentage of bud organogenesis, regeneration index response to increasing BAP concentra tion after 30 days of culture was quadratic (R2=0.25) (Figure 5-8) being highe st for explants placed onto 10 M BAP. After 60 days of culture, the regeneration inde x response was still quadratic (R2=0.48) (Figure 5-11), but the index values were significantly lowe r for explants cultured onto 1 M BAP than for those placed onto 10-60 M BAP (Tables 5-4 and 5-5). For mean number of buds per explant, there were no differences among treatments after 30 days of culture, but after 60 days the response to BAP concentrations was quadratic (R2=0.56) (Tables 5-4 and 5-5, Figure 5-10). At 90 days of culture, shoot length also exhibited a quadratic re sponse to BAP concentration (R2=0.50) (Figure 5-12), reaching a maximum at 10-30 M BAP and then decreased (Table 5-5, Appendix Tables A-7 and A-9). This reduction in length at hi gh levels of the plant growth regulator may

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42 have resulted from the induction of numerous shoot primordia that competed among each other and/or to the higher nu mber of hyperhydric shoots. The addition of BAP to basal medium has proved useful to induce organogenesis in some legume genera such as Desmodium (Rey & Mroginski, 1997), Aeschynomene (Rey & Mroginski, 1996) and Trifolium (Heath et al., 1993). Interestingly, BAP was less effective than TDZ for shoot bud induction in A. latifolia This is in agreement with a report in A. hypogaea in which several cytokinins were tested but TDZ proved to be the most efficient (Akasaka et al., 2000). Induction Time for Adventitious Bud Formation In order to determine the minimum time of exposure to TDZ required for shoot bud induction in A. latifolia explants were maintained in a culture medium supplemented with this plant growth regulator for increasi ng periods of time. It was observed that one and four days of culture were enough to induce organogenesis in A. latifolia U18.8 and U18.6, although with a very low percentage of bud formation (Appendix Tables A-10 and A-11). In general, the percentage of bud organogene sis increased as explants remained for longer periods of time in contact with TD Z. There were no significant differences between the 1and 4-day treatments or am ong 7to 30-day treatments (Table 5-6). An increase in the percentage of responsive e xplants was also observed from 30 to 60 days after the initiation of cultures. This suggests that, as in the cas e of four to seven days of exposure to TDZ, a period of over 30 days ma y be required for buds to arise even though cells have been induced to follow this deve lopmental pathway. In most cases the mean number of buds per explant increased from 30 to 60 days after the in itiation of cultures. Exceptions to this were the case when some buds became stunted and died. However,

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43 there were no significant differences in mean bud number when e xplants were placed between on TDZ 4 and 30 days (Table 5-6). Re garding the regeneration index, there were significant differences between 1 or 4 days on TDZ and 7 to 30 days on the medium with this plant growth regulator after 30 days of culture. Thirty days la ter, the highest index value was observed for the longest treatment, but it was not significan tly different from 7 to 20 days of culture on TDZ. Several studies showed that in order to regenerate normal shoots in A. hypogaea explants should not remain in a medium supplemented with TDZ for more than 7 days at 45.4 M TDZ or 21 days at 4.54 M TDZ (A kasaka et al., 2000). In A. latifolia there were no significant differences in shoot length after culture of explants on TDZ for 1 to 30 days (data not s hown). Regression analysis for all response variables showed a quadratic increase for length of time on TDZ supplemented medium (R2 from 0.80 to 0.95) (Figures 5-13 to 5-15 and Appendix Figures A-1 to A-3). Another experiment was conducted with s horter intervals betw een treatments to determine more precisely the number of days re quired for buds to arise. The percentage of shoot bud formation was highest for 10 da ys of culture on TDZ containing medium; however, it was not significantly different from percentages obta ined after 5 to 9 days of culture on TDZ (Table 5-7). There were no differences among treatments regarding the mean number of buds per explant, and th e regeneration index showed a significant difference only between 10 days of culture on TDZ and 1 or 2 days of exposure to this plant growth regulator but not with longer exposures (T able 5-7). In addition, the percentages of response were lower than in the first experiment (Appendix Table A-12). Regression analysis showed a linear increase of percentage of shoot bud formation and regeneration index with in creasing exposure to TDZ (R2= 0.92 and 0.93, respectively)

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44 (Figures 5-16 and 5-17), but th is is probably because treatmen ts corresponded to the first phase of the quadratic model obser ved in the previous experiment. Effect of Explant Type on Shoot Organogenesis Results from an exploratory experi ment with several genotypes of A. latifolia revealed that leaflets were more responsive th an petioles, suggesting that the part of the leaf used as explant had a major influence on the in vitro responsiveness. The percentage of bud formation was significantly higher when r achises were used as explants (Table 58). Leaflet explants produced a significantly higher per centage of organogenesis than petioles, which in general remained non-re sponsive. In the best case, only 16% organogenesis was achieved in contrast to the 90% responsiveness that was obtained using rachises from immature leaflets (Appendix Table A-13 ). These response patterns were observed in both immature and mature l eaves. Mean number of buds per explant and bud formation index also showed the higher responsiveness of rachises over the other two types of explants. For these response vari ables, there were no significant differences between leaflet and petiole explants (Table 5-8). The higher frequency of shoot bud formati on from rachises may be due to the presence of intercalary meristems in the rachis es because they are the last segment of the leaf to undergo the maturation process. Neve rtheless, this does not explain that this explant, when excised from mature leaves, gave almost as high responses as when harvested from actively expanding ones. The onl y disadvantage of using rachises would be the lower amount of plant material in cases in which few or small plants are available, since each leaf provides between 10 to 20 leaf lets, but each rachis from immature leaves gives no more than two explants.

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45 Regarding explant age, there were no si gnificant differences between immature and mature leaves although explants from expandi ng leaves were more responsive than those from fully expanded, mature ones. This may be due to the meristematic activity of young leaves with less differentiated cells that could readily respond to an external stimulus, i.e. TDZ. In A. hypogaea leaf developmental stage is a primary factor in somatic embryo induction, as unfolded leaflets usually lose embryogenic pot ential (Baker & Wetzstein, 1998). Nevertheless, shoot bud formation was possible in a number of wild Arachis species (Dunbar & Pittman, 1992). In contrast to what might be expected, cotyledons of A. bicolor and A. latifolia were completely irresponsive in vitro when cultured onto PIC, 2,4-D or TDZ (data not shown). Conclusions It was possible to achiev e plant regeneration from A. latifolia and A. bicolor through immature leaflet culture. Shoot bud organogenesis was successfully induced in several genotypes of both species using MS, L2 or B5 as the basal medium. Even though there were no marked differences in bud formation frequency between MS and L2, the former one promoted a higher ex vitro survival of regenerated plants and therefore was used in subsequent experiments. Interestingly, A. bicolor proved to be much less responsive than A. latifolia and all regenerated plan ts failed to acclimatize ex vitro. The plant growth regulator TDZ was more efficient than BAP for shoot bud induction, not only in the fre quency of bud formation, but al so in the time required for buds to arise from explants. In general, th e percentage of organoge nesis increased with higher levels of TDZ, but this was associat ed with a reduction in bud elongation. Shoot length was a critical factor in ex vitro acclimatization of regenerated plants and as a consequence, plants originated in TDZ concentrations of 1 and 10 M showed higher

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46 survival rates. Despite the lower organogenesis frequency of explants cultured on BAP, this plant growth regulator did not affect shoot length as drastically as TDZ, and in general the most effective concentrations were 10 and 30 M BAP. Shoot bud organogenesis was observed after ex posures to TDZ as short as one day, although the percentage of bud formation incr eased with prolonged culture in a medium containing this plant growth regulator up to 10 days. Exposure for 30 days to 10 M TDZ allowed a higher frequency of organogenesis a nd did not show a negative effect in shoot elongation, which was observed in the previous experiment at higher levels of the plant growth regulator. A major influence of explant type on shoot bud formation of A. latifolia was revealed in a follow-up experiment. A higher frequency of shoot bud formation and mean number of buds per explant was obtained when rachises were used as explants. The age of the source leaf was not as important as the part of the leaf placed into culture. Nevertheless, an increase in bud formation was achieved when using explants from immature, expanding leaves instead of mature, fully expanded ones. A suggested protocol for A. latifolia plant regeneration is as follows: 1. Use immature rachises as explant sour ce (immature leaflets may be used with some reduction in efficiency if large amounts of explant tis sue are desired) 2. Culture on MS + 10 M TDZ for 20 days 3. Transfer to MS with no plant growth regulators for bud elongation and rooting

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47 Table 5-1. Effect of three basal media on per centage of adventitious bud formation (%), mean number of buds (No.) and regeneration index (Index) in A. bicolor (U5.2-U13.3) and A. latifolia (U17.1-U19.6) after 60 days of culture. Genotype Basal Medium MS L2 B5 % No. Index% No. Index % No. Index U5.2 3.3 a* 1.7 0.2 a 3.0 a 1.0 0.1 a 0 a 0 0 a U5.3 0 a 0 0 a 0 a 0 0 a 0 a 0 0 a U5.5 0 a 0 0 a 0 a 0 0 a 0 a 0 0 a U12.2 0 a 0 0 a 3.3 a 0.7 0.1 a 0 a 0 0 a U12.5 0 a 0 0 a 0 a 0 0 a 0 a 0 0 a U13.1 0 a 0 0 a 0 a 0 0 a 0 a 0 0 a U13.3 6.7 a 0.7 0.1 a 3.3 a 0.3 0 a 3.3 a 0.3 0 a U17.1 50.0 b 1.9 1.1 a 6.7 a 1.7 0.2 a 3.3 a 0.3 0 a U17.2 53.3 b 3.7 1.9 a 26.7 ab 2.8 1.1 a 16.7 a 1.1 0.3 a U17.3 3.3 a 0.3 0 a 0 a 0 0 a 0 a 0 0 a U18.1 63.8 ab 3.6 1.8ab 80.0 b 3.4 2.9 b 36.7 a 2.9 1.0 a U18.2 78.6 b 2.3 1.9 b 37.2 a 3.7 1.5ab 13.3 a 0.8 0.2 a U18.3 17.4 a 2.2 0.4 a 24.4 a 1.9 0.5 a 3.3 a 1 0.1 a U18.4 23.3 a 0.7 0.5 a 16.7 a 1.1 0.6 a 0 a 0 0 a U18.6 56.7 b 1.6 0.9 a 46.7 1.2 0.8 a 6.7 a 0.3 0.1 a U18.8 46.7 b 2.6 1.1ab 80.0 b 3.9 2.6 b 13.3 a 4.1 0.8 a U18.9 53.3 b 2.1 1.3 a 23.3 ab 1.6 0.4 a 16.4 a 0.7 0.2 a U19.1 3.3 a 0.3 0 a 0 a 0 0 a 0 a 0 0 a U19.2 3.3 a 0.3 0 a 3.3 a 1.7 0.2 a 0 a 0 0 a U19.3 0 a 0 0 a 0 a 0 0 a 0 a 0 0 a U19.4 5.6 a 0.5 0.1 a 4.2 a 0.3 0 a 0 a 0 0 a U19.5 13.3 a 0.6 0.2 a 0 a 0 0 a 3.3 a 0.3 0 a U19.6 23.3 a 3.9 2.7 b 23.3 a 1.8 1.2ab 9.1 a 1.6 0.4 a Mean 22.0 B 1.3A 0.6B 16.6B 1.2A 0.5B 5.5A 0.6A 0.1A Within rows, means for a given variable followed by different lower case letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05 level. Means over genotypes followed by differe nt upper case letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05 level. Index= % shoot bud formation x mean number of buds / 100

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48 Table 5-2. Effect of TDZ concentration on pe rcentage of adventitious bud formation (ABF), mean number of buds per ex plant, and regeneration index (Index) in A. latifolia after 30 days of culture. TDZ (M) ABF Buds Index ---%-----No.--0 0 0 0 1 52.5a* 1.7a 0.9a 10 57.5a 1.9a 1.1a 30 67.5a 2.0a 1.4ab 60 71.7a 2.3a 1.6b Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= % shoot bud formation x mean number of buds / 100 Table 5-3. Effect of TDZ concentration on pe rcentage of adventitious bud formation (ABF), mean number of buds per ex plant, and regeneration index (Index) after 60 days of culture; number of s hoots and shoot length after 90 days of culture; and percentage of ex vitro survival in A. latifolia TDZ (M) ABF Buds Index Shoots Length Survival --%----No.-----No.----mm-------%----0 0 0 0 0 0 0 1 65.8a* 2.7a 1.7 a 10.4c 31.3c 76.5b 10 79.2 ab 2.8a 2.3 a 7.8bc 18.2b 60.3b 30 87.5 b 2.5a 2.2 a 4.5ab 5.3ab 15.0a 60 85.8 b 2.7a 2.4 a 3.3a 2.6a 4.2a Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= % shoot bud form ation x mean number of buds / 100

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49 Table 5-4. Effect of BAP concentration on pe rcentage of adventitious bud formation (ABF), mean number of buds per ex plant and regeneration index (Index) in A. latifolia after 30 days of culture. BAP ( M) ABF Buds Index ---%-----No.--0 0 0 0 1 2.5a* 0.3 a 0 a 10 18.3b 0.8 a 0.2 b 30 1.7a 0.2 a 0 a 60 0.8a 0.1 a 0 a Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= % shoot bud form ation x mean number of buds / 100 Table 5-5. Effect of BAP concentration on pe rcentage of adventitious bud formation (ABF), mean number of buds per ex plant and regeneration index (Index) after 60 days of culture; and shoot le ngth after 90 days of culture in A. latifolia BAP ( M) ABF Buds Index Length --%----No.-----mm--0 0 0 0 0 1 13.3a* 1.4 a 0.2 a 15.0 a 10 65.8 b 3.2 b 2.2 b 41.7 b 30 57.5 b 2.9 b 1.7 b 30.0 ab 60 50.0 b 2.9 b 1.6 b 17.5 a Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= % shoot bud form ation x mean number of buds / 100

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50 Table 5-6. Effect of different times of e xposure to TDZ on bud formation percentage (ABF), mean number of buds per ex plant and regeneration index (Index) in A. latifolia after 30 and 60 days of culture, respectively. 30 days of culture 60 days of culture Days in TDZ ABF Buds Index ABF Buds Index ---%-----No.-----%-----No.--0 0 a 0 a 0 a 0 a 0 a 0 a 1 2.5 a 0.3 a 0 a 2.5 a 0.3 ab 0 a 4 12.5 a 0.8 ab 0.2 a 30.0 a 2.1 bc 0.6 ab 7 57.5 b 2.0 bc 1.2 b 77.5 b 2.0 bc 1.5 abc 14 70.0 b 1.7 bc 1.2 b 77.5 b 2.5 c 2.0 bc 20 72.5 b 2.6 c 1.9 b 87.5 b 2.3 c 2.0 bc 30 80.0 b 1.8 bc 1.4 b 90.0 b 2.8 c 2.5 c Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= % shoot bud form ation x mean number of buds / 100 Table 5-7. Effect of short exposure to TDZ on bud formation percentage (ABF), mean number of buds per explant and regeneration index (Index) in A. latifolia after 30 days of culture. Days in TDZ ABF Buds Index ---%-----No.--0 0 a 0 a 0 a 1 0 a 0 a 0 a 2 2.5 a 0.3 a 0 a 3 5.0 a 0.5 a 0.1 ab 4 5.0 a 1.0 a 0.1 ab 5 7.5 ab 0.8 a 0.1 ab 6 12.5 ab 0.8 a 0.2 ab 7 17.5 ab 1.1 a 0.2 ab 8 20.0 ab 1.4 a 0.3 ab 9 27.5 ab 1.4 a 0.4 ab 10 35.0 b 0.9 a 0.4 b Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= % shoot bud form ation x mean number of buds / 100

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51 Table 5-8. Effect of explant type on bud form ation percentage (ABF ), mean number of buds per explant and regeneration index (Index) in A. latifolia after 30 days of culture. Type of explants Immature leaves Mature leaves Mean Petioles 9.1 8.0 8.6 a Rachis 80.0 65.0 72.5 c ABF ---%--Leaflets 38.1 20.6 29.4 b Petioles 0.8 0.4 0.6 a Rachis 2.3 2.7 2.5 b Buds --No.-Leaflets 1.2 1.0 1.1 a Petioles 0.2 0.1 0.15 a Rachis 2.3 2.7 2.5 b Index Leaflets 0.6 0.3 0.5 a Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= % shoot bud fo rmation x mean number of buds / 100

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52 A B C D E Figure 5-1. Organogenesis in A. latifolia A), B) Shoot bud formation 60 days after initiation of cultures and C), D) shoot elongation 90 days af ter initiation of cultures; A), C) in MS + 1 M TDZ and B), D) in MS + 60 M TDZ (bar: 20 mm). E) Successful acclimatization of regenerated plants, 45 days after transfer to ex vitro conditions (Bar: 50 mm).

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53 Figure 5-2. Regression curve showing the effect of TDZ concentration on percentage of adventitious bud formation (%) in A. latifolia after 60 days of culture. Figure 5-3. Regression curve showing the eff ect of TDZ concentration on regeneration index (Index) in A. latifolia after 30 days of culture. y = -0.0144x2 + 1.1876x + 66.162 R2 = 0.96 0 10 20 30 40 50 60 70 80 90 100 010203040506070 TDZ (micromolar) % y = -1E-04x2 + 0.0182x + 0.8968 R2 = 1 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 010203040506070 TDZ (micromolar)Index

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54 Figure 5-4. Regression curve showing the eff ect of TDZ concentration on shoot number per explant (No. shoots) in A. latifolia after 90 days of culture. Figure 5-5. Regression curve showing the effect of TDZ concentration on shoot length (Length) in A. latifolia after 90 days of culture. y = -0.4487x + 25.642 R2 = 0.79 -5 0 5 10 15 20 25 30 35 010203040506070 TDZ (micromolar)Length (mm) y = -0.1159x + 9.4273 R2 = 0.87 0 2 4 6 8 10 12 010203040506070 TDZ (micromolar)No. Shoots

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55 Figure 5-6. Regression curve showing the effect of TDZ concentration on acclimatization rate (% survival) in A. latifolia after 30 days of transfer to ex vitro conditions. Figure 5-7. Regression curve showing the effect of BAP concentration on percentage of adventitious bud formation (%) in A. latifolia after 30 days of culture. y = -1.2482x + 70.534 R2 = 0.88 -10 0 10 20 30 40 50 60 70 80 90 010203040506070 TDZ (micromolar)% survival y = -0.0031x2 + 0.0434x + 8.2682 R2 = 0.24 0 2 4 6 8 10 12 14 16 18 20 010203040506070 BAP (micromolar) %

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56 Figure 5-8. Regression curve showing the eff ect of BAP concentration on regeneration index (Index) in A. latifolia after 30 days of culture. Figure 5-9. Regression curve showing the effect of BAP concentration on percentage of adventitious bud formation (%) in A. latifolia after 60 days of culture. y = -0.0364x2 + 2.6051x + 22.813 R2 = 0.57 0 10 20 30 40 50 60 70 80 010203040506070 BAP (micromolar) % y = -3E-05x2 + 9E-05x + 0.0986 R2 = 0.25 0.0 0.1 0.1 0.2 0.2 0.3 010203040506070 BAP (micromolar)Index

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57 Figure 5-10. Regression curve showing the eff ect of BAP concentration on mean number of buds per explant (No. Buds) in A. latifolia after 60 days of culture. Figure 5-11. Regression curve showing the eff ect of BAP concentration on regeneration index (Index) in A. latifolia after 60 days of culture. y = -0.0011x2 + 0.086x + 1.7276 R2 = 0.56 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 010203040506070 BAP (micromolar)No. Buds y = -0.0011x2 + 0.0825x + 0.6343 R2 = 0.48 0.0 0.5 1.0 1.5 2.0 2.5 010203040506070 BAP (micromolar)Index

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58 Figure 5-12. Regression curve showing the eff ect of BAP concentration on shoot length (Length) in A. latifolia after 90 days of culture. Figure 5-13. Regression curve sowing the e ffect of exposure to TDZ on bud formation percentage (%) in A. latifolia after 60 days of initiation of cultures. y = -0.0195x2 + 1.0944x + 20.841 R2 = 0.50 0 5 10 15 20 25 30 35 40 45 010203040506070 BAP (micromolar)Length (mm) y = -0.1948x2 + 8.6392x + 1.817 R2 = 0.91 0 20 40 60 80 100 120 05101520253035 Days in TDZ %

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59 Figure 5-14. Regression curve sowing the eff ect of exposure to TD Z on mean number of buds per explant (No. Buds) in A. latifolia after 60 days of initiation of cultures. Figure 5-15. Regression curve sowing the e ffect of exposure to TDZ on regeneration index (Index) in A. latifolia after 60 days of initiation of cultures. y = -0.0052x2 + 0.2274x + 0.3826 R2 = 0.80 0 0.5 1 1.5 2 2.5 3 3.5 05101520253035 Days in TDZNo. Buds y = -0.0036x2 + 0.1875x + 0.0024 R2 = 0.95 0 0.5 1 1.5 2 2.5 3 05101520253035 Days in TDZIndex

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60 Figure 5-16. Regression curve sowing the effect of short exposures to TDZ on percentage of bud formation (%) in A. latifolia after 30 days of initiation of cultures. Figure 5-17. Regression curve sowing the effect of short exposures to TDZ on regeneration index (Index) in A. latifolia after 30 days of initiation of cultures. y = 3.3636x 4.7727 R2 = 0.92 0 5 10 15 20 25 30 35 40 024681012 Days in TDZ % y = 0.0418x 0.05 R2 = 0.93 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 024681012 Days in TDZIndex

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61 CHAPTER 6 PLANT REGENERATION OF Lotononis bainesii Introduction The genus Lotononis consists of approximately 150 species, from herbs to small shrubs, belonging to the Fabaceae family, tribe Crotalarieae (Jaftha et al ., 2002). Their broad distribution from Southern Africa to the Med iterranean region and India indi cates that these species grow under dissimilar environments from the climat ological and geographical point of view. Among Lotononis species, L. divaricata (Eckl. & Zeyh.) Benth., L. tenella (E. Mey.) Eckl. & Zeyh. and L. laxa Eckl. & Zeyh. have forage potential for arid areas, and L. bainesii Baker a perennial herb, is a valuable forage in Australia (Jaftha et al., 2002). The increasing in terest in this species has motivated some recent molecular studies to determine its mode of reproduction to assist breeding programs (Real et al., 2004). These st udies have reported that, although it has been previously considered a cleistoga mous species, it should be treated as an allogamous species in improvement programs. Some seed may be produ ced by self pollination, but self incompatibility may also be found in some genotypes. Due to the allogamous nature of L. bainesii seeds from a certain plant may correspond to different genotypes. Therefore, specific genotypes cannot be pr opagated through seeds and other means of multiplication would be useful. This coul d be overcome if a plant regeneration protocol is developed that allows the propagation of selected plants. One such protocol has been developed for L. bainessi by Bovo et al. (1986), who obtaine d a low frequency of plant regeneration from cotyledons and leaflets. Moreov er, a tissue culture protocol may be used to solve another major constraint in this species low seedling vigor, through the duplication of

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62 chromosomes using chromosome d oubling agents such as colchici ne, oryzalin and trifluralin (Quesenberry et al., 2003). Cells with duplicat ed chromosomes would be induced to follow either organogenesis or somatic embryogenesis, producing solid plan ts instead of chimeras that would likely result if other techniques were us ed. In addition, a tissue culture protocol that results in plant regeneration from single ce lls would open the possibility of genetic transformation of this species (Ozias-Akins & Gill, 2001). The objective of these experiments was to develop an efficient tissue culture protocol for L. bainesii which could then be used in genetic improvement programs of the species. Materials and Methods Cotyledon Culture Seeds of L. bainesii cv. INIA Glencoe were scarified and germinated as described in chapter 3. Fifty 1-week-old seedlings were rando mly selected and their cotyledons were excised and cut longitudinally along the midrib into two pi eces so that four cotyledonary explants were obtained per genotype. Each explant was placed w ith the abaxial side down onto MS alone or MS with 4.5 M TDZ, 4.14 M PIC or 4.52 M 2,4-D. Five explants were placed per petri dish and the identity of genotypes was maintained. Seedlings without cotyledons were placed onto MS medium and kept in vitro for subsequent experiments. Afte r 30 days of culture, regenerated buds were subcultured onto MS medium supplemented with 0.044 M BAP + 0.049 M IBA for a month. Developing plants were then transferre d to magenta boxes that contained MS lacking growth regulators for the same pe riod of time before acclimatization ex vitro Incubation and acclimatization were the same as those described in chapter 3. Leaflet Culture Genotypes used in this experiment were the sa me as in the cotyledon experiment. Explants consisted of pieces of leaflets (ca. 4 mm2) including the midvein, harvested from immature

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63 expanding leaves of each genotype growing under aseptic conditions. Treatments, incubation and acclimatization were the same as in the previous experiment. Type of Explants One of the genotypes that had performed well in the cotyledon experiment was used as the explant source. Leaflets were excised and pl aced onto sterile filter paper where they were divided into petiole, petiole tip (in which the th ree leaflets are inserted) and leaflets. Petiole pieces (divided into 5to 6-mm portions), the petio le tip and three leaflets corresponding to each trifoliate leaf were placed in a petri dish containing MS supplemented with 10 M TDZ. This procedure was repeated 10 times for both mature and immature leaves. Thirty days after the initiation of the experiment, explants were s ubcultured to MS without growth regulators. Incubation conditions were the same as those described in chapter 3. This experiment was statistically analyzed as a fact orial arrangement in a completely randomized design (3 parts of the leaf x 2 stages of development) using PROC GLM from PC SA S (SAS Institute, 2003). Tukeys HSD Multiple Range Test at p 0.05 was used to compare the means of treatments. Results and Discussion Cotyledon Culture In the absence of growth regulators, coty ledon explants on MS basal medium remained non-responsive and gradually turn ed brown. Conversely, after 30 days of culture on medium supplemented with PIC, all genotypes produced li ght brown or light green friable callus of less than 1 cm in diameter. When this callus was transferred onto MS + 0.044 M BAP + 0.049 M IBA, these calli did not show further growth a nd died. Similar results were observed when 2,4-D was used as a growth regulator in the culture medium (Figure 6-1 A,B). However, in six

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64 genotypes, a few short roots were produced from callus on 2,4-D induction medium, but they did not continue growing upon transfer to MS + 0.044 M BAP + 0.049 M IBA. The addition of TDZ to the bud inducti on culture medium re sulted in shoot bud organogenesis in 27 out of 50 genotypes tested (F igure 6-2 A). Callus and bud formation started approximately 7 and 15 days after the initiation of cultures, resp ectively (Figure 6-1 C,D). Callus was in general dark green with dark brown ar eas. Considering only responsive genotypes, the mean number of buds per cotyledonary explant was 13.6. Sixty days after culture, 48% of the total number of buds was capable of regenera ting plants (177 plants/ 367 buds). In some genotypes, the number of plants after 90 days of culture was superior to the number of buds after 30 days, indicating that during th at period new buds were produced and regenerated whole plants (Figure 6-3 A). In other genotypes, not all the buds present after 30 da ys resulted in plant regeneration. Moreover, nine genotypes produced buds but they did not elongate and finally died. The lack of elongation and death of buds probably resulted from the hyperhydricity of tissues, which might have been caused by a horm onal imbalance in the culture medium. It was observed that only 39% of plants transferred to ex vitro conditions were capable of successful acclimatization 30 days after the transfer (Figures 6-1 E and 6-4 A). This low survival rate is likely associated with the high incidence of hype rhydricity in regenerated plants. Besides the influence of plant growth regulators in the cult ure medium, this abnormality may also result from high water availability in the culture ve ssel and low light levels (Hazarika, 2006). The growth regulator, TDZ, has been used for organogenesis and plant regeneration from embryoor seedling-derived explants in several species of legumes including A. hypogaea (Gill & Ozias-Akins, 1999), V. radiata (Mundhara & Rashid, 2006), Trifolium spp. Medicago sp.p

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65 (Ding et al., 2003) and C. cajan (Singh et al., 2003). Neverthe less, in the latter, somatic embryogenesis was obtained at higher concentrat ions of TDZ than t hose that had induced organogenesis. Similarly, there are reports of somatic embryo induction using TDZ in A. hypogaea (Murthy et al., 1995). Interestingly, PIC a nd 2,4-D were not effective for non zygotic embryo induction in L. bainesii but both plant growth regulators we re reported to be effective in A. hypogaea (Griga, 1999). Leaflet Culture Since L. bainesii is an allogamous species and not al l progeny will necessarily reflect the superior performance of an individual plant, coty ledons are not the most suitable explants when the purpose is propagation of out standing genotypes. Therefore, the previous experiment was repeated using leaflets as explants, since they are available throughout the year and offer the possibility of large scale propagation of sel ected genotypes as well as development of an in vitro chromosome doubling protocol. Thirty days after the initiation of cultures it was observed that, when growth regulators were absent, explants remained irresponsive exce pt in 22 genotypes in which roots up to 10-cm long were produced from the cut surface of the mi dvein. This suggests that the endogenous level of auxins in the leaflets may be enough to i nduce rhizogenesis in the absence of an exogenous supply of plant growth regulators. Probably, the high levels of auxins are responsible for the formation of vigorous roots in plants maintained in vitro not only from basal nodes but also from those not in contact with the culture medium. Some of thes e genotypes corresponded to those that had produced roots from callus in the pr evious experiment. When the culture medium contained PIC or 2,4-D, responses were similar to those observed in the cotyledon experiment. Although some calli were larger (up to 1.5 cm in di ameter), they did not exhibit further growth

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66 when transferred onto MS + 0.044 M BAP + 0.049 M IBA. The addition of 2,4-D resulted in root formation in seven genotypes as well. The growth regulator, TDZ, was effective in inducing shoot bud organog enesis in 45 out of 50 genotypes tested, four of which were also non-responsive in the cotyledon experiment. However, the mean number of buds (3.8) was considerably lower than that obtained when cotyledon pieces were used (13.6) (Figures 6-1 F and 6-2 B). After 90 days of culture, a 22% increase in the number of regenerated plants co mpared to the number of buds at 30 days was observed (211 plants at 90 days vs. 173 buds at 30 days) (Figure 63 B and 6-1 G). This might be due to buds continuing to be formed on MS + 0.044 M BAP + 0.049 M IBA. But since this medium had such a low concentrat ion of plant growth regulators, more probably these buds were already induced before the subculture. Even thou gh the total number of plants obtained from leaflet culture was higher than that for coty ledons, only 21% were su ccessfully acclimatized when transferred ex vitro (Figure 6-4 B). This high plant mortality was likely due to hyperhydricity, of which numerous plants showed symptoms; however, manipulation of culture conditions might have resulted in higher survival ra tes. It is possible that the vigorous growth of in vitro regenerated plants resulted in high ethyle ne accumulation, which has been reported to favor hyperhydricity (Hazarika, 2006). It is interesting that more genotypes were res ponsive when explants consisted of leaflets rather than cotyledons. In general, juvenile expl ants are preferred since they are more likely to undergo organogenesis or somatic em bryogenesis. For example, in A. hypogaea seedlings more than 21-days old failed to undergo somatic embryoge nesis using TDZ compared to up to 97% of 6-day-old seedlings (Murt hy et al., 1995). The lowe r cotyledon response in L. bainesii may have been caused by the scarification/surface disinfecti on procedures seed received, although this is

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67 unlikely given that much stronger pretreatment s have been reported with no negative effects (Bovo et al, 1986). These authors obta ined better responses with coty ledons as explants (66% of bud formation in the best culture medium vs. 54 % when using leaflets). In the present study, however, a higher frequency of organogenesis wa s obtained following leaflet culture (90% of explants producing buds vs. 54% of cotyledon pi eces producing buds). A dditionally, results in this experiment differ from those reported by Bovo et al. (1986) in that shoots readily rooted in spite of being in a culture medi um with the potent cytokinin TDZ so rooting was not a critical factor in whole plant regeneration. Veltcheva et al. (2005) suggest that forage legume populations are markedly heterogeneous, re sulting in an easie r identification of in vitro responsive genotypes. Organogenic genotypes of L. bainesii cv INIA Glencoe were easily identified, which may be due to a shorter breedin g history than those in grain legumes in which the narrow genetic base limits the discovery of regenerating genotypes. Type of Explant Since the previous experiment showed that l eaflets were capable of producing shoot buds in the presence of TDZ in most of the genotype s tested, another experiment was performed to asses the influence of the part and age of the leaf in shoot organogenesis. Callus formation started within a week of initia tion of cultures and shoot buds be gan to arise after 15 days of culture in the six type s of explants used. For percentage of shoot bud formation, leaf part had a significant effect, whereas leaf age and interaction between leaf part and leaf age were not significant (Table 6-1). Bud organogenesis from leaflets was similar (85.8% fr om insertion explants and 83.3% from leaflet explants) and higher than petiole s (48.3%). The number of buds per explant ranged from 1.1 to 3.4 (Table 6-2) but, similar to bud organogenesis pe rcentage, there was no leaf age by leaf part interaction for number of buds per explant (Table 6-1). In contrast to percentage of bud

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68 organogenesis, the mean number of buds per e xplant did not differ among parts of the leaf, but did differ due to leaf age. Explants from immatu re leaves gave a higher number of buds per explant (2.9) than those from ma ture ones (1.5) (Table 6-2). Considering the regeneration inde x, there was a significant effect of the part and age of the leaf but not a significant inte raction (Table 6-1). The mean regeneration index for petiole explants (1.1) was significantly lower than the inde x for leaflet or leaflet insertion area (2.4 in both cases). However, the mean regeneration index for explants from immature leaves (2.4) was significantly higher than the inde x for explants excised from mature leaves (1.5). Leaflet insertion area from immature leaves had th e highest index (3.4) over all combinations. The advantage of using immature leaves has been reported in A. hypogaea in which the frequency of somatic embryo formation decreased considerably as leafle ts unfolded (Baker & Wetzstein, 1998). In A. villosulicarpa the best organogenic frequenc y and mean number of buds per explant were obtained when mature fully ex panded leaves were used as explants (Dunbar & Pittman, 1992). In L. bainesii Bovo et al. (1986) reported up to 54% bud formation when pieces of fully expanded leaflets from greenhouse grow n plants were placed onto the best culture medium. Although these authors us ed larger explants (6 mm2), organogenesis frequency was lower than in the present experiment. This c ould be due to the condi tions under which mother plants were grown, a fact or that greatly affects in vitro responsiveness (Radice, 2004). Conclusions It was possible to regenerate plants from over 50% of L. bainesii cv INIA Glencoe genotypes that were evaluated through cotyledon cultu re and 90% of genot ypes through leaflet culture in a medium composed of MS +4.5 M TDZ. Bud elonga tion and rooting was obtained upon transfer onto MS + 0.044 M BAP + 0.049 M IBA. Although immature leaflet culture

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69 resulted in a higher number of responsive ge notypes, plants regenerated from cotyledons exhibited a higher survival rate when transferred to ex vitro conditions. Nevertheless, in both cases survival rates were low a nd this situation was related with the incidence of hyperhydricity in cultures. When the culture medium was supplem ented with either PIC or 2,4-D, friable light green or light brown callus form ation was obtained, but this callu s did not show further growth when subcultured. Regarding the type of explants, the expe riment carried out with one genotype of L. bainesii revealed that leaflet insertion areas from expa nding leaves and pieces of leaflets were more efficient for shoot bud induction. For both mature a nd immature leaves, the lowest frequencies of shoot bud formation were obtained when pieces of petioles were used as explants. Mean number of buds per explant and regenera tion index were higher for expl ants collected from immature leaves. A suggested protocol for L. bainesii plant regeneration is as follows: 1. Use immature leaflets as explants, which are as efficient as leafle t insertion areas but a larger amount of tissue is obtained per leaf 2. Culture on MS + 4.5 M TDZ for 30 days 3. Transfer to MS + 0.044 M BAP + 0.049 M IBA for bud elongation and rooting Although these experiments have improved the regeneration frequency reported for this species, it is likely that the optim ization of other factors besides the type of explant would result in a more efficient protocol. Additional experi ments regarding the regu lation of plant growth regulator concentrations and combinations time required for organogenesis induction and control of factors that influe nce hyperhydricity will be conducted to increase the regeneration frequency and ex vitro establishment rate.

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70 Table 6-1. ANOVA table showing th e p-values corresponding to th e effect of explant type on bud formation percentage (%), mean number of buds per explant (No.) and regeneration index (Index) in L. bainesii after 30 days of culture. Source df p-value % No. Index Leaf parts 2 0.004 0.606 0.032 Leaf age 1 0.454 0.003 0.048 LP x LA 2 0.120 0.294 0.119 Error 54 Index=( % shoot bud formation x mean number of buds) / 100 Table 6-2. Effect of explant type on bud formati on percentage, mean number of buds per explant and regeneration index in L. bainesii after 30 days of culture. Leaf part Immature Mature Mean Petioles 38.3 58.3 48.3b* Leaflet insertion 100.0 71.5 85.8a Leaflets 90.0 76.7 83.4a Bud formation % Mean 76.1A 68.8 A Petioles 2.2 1.6 1.9 a Leaflet insertion 3.4 1.1 2.3 a Leaflets 3.0 1.8 2.4 a Number of buds Mean 2.9A 1.5B Petioles 1.0 1.2 1.1b Leaflet insertion 3.4 1.4 2.4a Leaflets 2.8 1.9 2.4a Regeneration index Mean 2.4A 1.5B For each parameter, means within columns (a,b,c) or rows (A,B) with diffe rent letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= (% shoot bud formation x mean number of buds) / 100

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71 Figure 6-1. Organogenesis in L. bainesi. A) Cotyledon cultures 20 days after initiation of experiments in MS basal media supplemente d with 2,4-D, B) PIC and C) TDZ (bar: 20 mm). D) Shoot bud formation from cotyle dons 45 days after culture (bar: 20 mm). E) Plants successfully acclimatized 45 days after transfer to ex vitro conditions (Bar: 50 mm). F), G) Shoot bud proliferation from leaflet explants orig inated in MS + 10 M TDZ 60 and 90 days after initiation of cultures, respec tively (bar: 20 mm). B E F G A C D

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72 Figure 6-2. Number of buds produced per explant by 50 genotypes of L. bainesii after 30 days of culture from A) cotyledon and B) leaflets. Figure 6-3. Number of plants produced per ex plant after 90 days of culture by responsive genotypes of L. bainesii through A) cotyledon an d B) leaflet culture. 0 5 10 15 20 25 30 35 40 4501-910-1920-2930-3940-4950-5960-69Number of budsNumber of g enot yp es 0 5 10 15 20 25 30 35 40 4501-910-1920-2930-3940-4950-5960-69Number of budsNumber of g enot yp esA B 0 5 10 15 20 25 3001-910-1920-2930-39Number of plantsNumber of g enot yp es 0 5 10 15 20 25 3001-910-1920-2930-39Number of plantsNumber of g enot yp esA B

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73 Figure 6-4. Number of successfully acclim atized plants in the genotypes of L. bainesii that were capable of plant regeneration through A) cotyledon and B) leaflet culture. 0 5 10 15 20 2501-45-910-1415-19Number of plantsNumber of g enot yp es 0 5 10 15 20 2501-45-910-1415-19Number of plantsNumber of g enot yp esA B

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74 CHAPTER 7 SUMMARY AND CONCLUSIONS Grain legumes are generally regarded as recalcitrant to in vitro plant regeneration due to a narrow genetic base that results in low genetic variability and a more difficult identification of responsive genotypes. It is thought to be easier to identify in vitro responsive genotypes of forage legumes, because they have usually undergone fewer selection cycles and populations are more heterogeneous (Ve ltcheva et al., 2005). Results reported here concur with this concept si nce shoot bud organogenesis was achieved in 20 to 90% of genotypes in four out of fi ve forage legume species evaluated. Petiole culture of T. polymorphum on three different basal media supplemented with TDZ was not effective to induce bud or ganogenesis, at least for those genotypes tested. Possibly, this species is more recalcitra nt to tissue culture than the others. Further studies will be conducted to determin e the most suitable conditions for in vitro plant growth (using these aseptically grown plants as an explant source) as well as to determine the culture conditions required for shoot bud induction, i.e., basal medium composition, and plant growth regulator type, co ncentration and combinations. In T. carolinianum, an annual native clover species, shoot bud formation was achie ved for the first time through cotyledon culture. Additional experiments are being conducted to achieve further elongation of shoots and rooting, before re generated plants are transferred to ex vitro conditions to evaluate the establishment rate. In A. bicolor, A. latifolia and Lotononis bainesii plant regeneration was achieved via organogenesis in 54 to 90% of the genotypes tested. Nevertheless, additional

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75 experiments should be conducted to improve the frequency of bud formation and to reduce the incidence of hyperhydricity in culture, which should result in higher ex vitro survival rates. Overall, MS was a suitable basal medium for shoot bud induction in all species tested, except for T. polymorphum where the only response was callus formation on B5 basal medium. In A. latifolia L2 was as effective as MS for shoot bud organogenesis, but plants regenerated in L2 showed a lower establishment rate when transferred to ex vitro conditions. Among plant growth regulators teste d, TDZ was efficient in all species except T. polymorphum where it only induced callus formation. In A. latifolia shoot bud formation was observed in TDZ concentrati ons ranging from 1 to 60 M. The induction time experiment in A. latifolia revealed that 20 days of culture on medium containing TDZ was sufficient to obtain shoot organogene sis. In agreement with other literature, immature leaf tissues were in general more responsive in L. bainesii and Adesmia spp Immature rachises proved to be superior explants for organogenesis induction in A. latifolia, and leaflet insertion areas or leafle ts from immature leaves were more responsive than pieces of petioles in L. bainesii Although cotyledon cult ure resulted in shoot bud formation in T. carolinianum and L. bainesii in Adesmia spp. cotyledons remained unresponsive. Suggested protocols for th e studied species are: T. carolinianum 1. Cotyledon culture on MS + 10 M TDZ for 30 days 2. Transfer of organogeni c cultures on MS + 1 M TDZ, where new buds continue to arise and short shoots are produced from those buds already differentiated

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76 A. bicolor 1. Immature leaflet culture on MS or L2 + 4.5 M TDZ for 60 days 2. Transfer to MS devoid of plant grow th regulators for 45 days, where buds elongated and rooted. A. latifolia 1. Immature rachis culture on MS + 10 M TDZ for 20 days 2. Transfer to MS with no plant growth regulators for bud elongation and rooting L. bainesii 1. Culture immature leaflets on MS + 4.5 M TDZ for 30 days 2. Transfer to MS + 0.044 M BAP + 0.049 M IBA for bud elongation and rooting In conclusion, in vitro plant regeneration protocol s were developed for four promising legume species. Thes e protocols could be used to assist in breeding programs to improve seedling vigor and DM producti on. This would be important for cattle production in those countries where these sp ecies are native. A dditionally, this may allow the development of a legume alternative to perennial peanut in the state of Florida, since to-date it is the only introduced forage legume speci es that has shown long term persistence. The main drawbacks of pere nnial peanut are its vegetative propagation, which increases the establishment cost, and low production in the year after planting. In contrast, Adesmia spp and L. bainesii cultivars could be propagated through seeds, reducing the cost for farmers. Currently, fi eld studies are being conducted to evaluate field performance of Adesmia spp. and L. bainesii to the Florida environment.

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77 APPENDIX ADDITIONAL TABLES FOR TWO GENOTYPES OF Adesmia latifolia Table A-1. Effect of different combina tions of TDZ and IBA on percentage of adventitious bud formation (ABF) and m ean number of buds per explant in A. latifolia U18.6 after 30 and 60 days of culture. 30 days of culture 60 days of culture IBA ( M) IBA ( M) 0 0.1 1 0 0.1 1 ABF Buds ABF Buds ABF Buds ABF Buds ABF Buds ABF Buds TDZ ( M) % No. % No. % No. % No. % No. % No. 0 0a* 0a 0a 0a 0 a 0a 0a 0a 0a 0a 0a 0a 1 45ab 1.3b 55bc 1.7b 75b 1.7b 70b 3.1bc 60bc 2.2b 85b 2.6b 10 60b 2.8cd 40ab 2b 55b 2b 95b 3.6bc 55b 2.6b 85b 2.4b 30 70b 1.9bc 75bc 2.3b 75b 2.1b 85b 2.1b 80bc 2.8b 85b 2.7b 60 85b 3.1d 100c 1.9b 50b 2.2b 90b 3.7c 95c 2.4b 75b 2.7b Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05 Table A-2. Effect of different combinati ons of TDZ and IBA on regeneration index (Index), number of shoots per explant and shoot length in A. latifolia U18.6 after 60 days of culture. IBA ( M) 0 0.1 1 Index Shoots LengthIndex Shoots LengthIndex Shoots Length TDZ ( M) -No.-mm-No.-mm-No.-mm0 0 a* 0 a 0 a 0 a 0 a 0 a 0 a 0 a 0 a 1 2.2 bc 10 b 25 b 1.3 ab 11 b 25 b 2.3 b 13b 17.5 c 10 3.4 c 13ab 13 b 1.5 ab 10 b 23 b 2 b 8 ab 8 bc 30 1.8 ab 6.5a 5 ab 2.2 b 9 a 6.5 b 2.3 b 8 ab 8 bc 60 3.3 c 9a 4 b 2.3 b 5.5a 4 ab 2.3 b 5 ab 7.5 ab Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= % shoot bud form ation x mean number of buds / 100

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78 Table A-3. Effect of different combina tions of TDZ and IBA on percentage of adventitious bud formation (ABF) and m ean number of buds per explant in A. latifolia U18.8 after 30 and 60 days of culture. 30 days of culture 60 days of culture IBA ( M) IBA ( M) 0 0.1 1 0 0.1 1 ABF Buds ABF Buds ABF Buds ABF Buds ABF Buds ABF Buds TDZ ( M) % No. % No. % No. % No. % No. % No. 0 0a* 0 a 0 a 0 a 0 a 0 a 0 a 0 a 0 a 0 a 0 a 0 a 1 60b 2 b 45b 1.4b 35ab2.3b 70 b 2.5b 45ab 3.4b 65b 2.7b 10 80b 1.5 b 45b 1.3ab 65 b 1.7b 95 b 2.6b 75 b 2.9b 70b 2.9b 30 80b 1.6b 50b 1.8 b 55 b 2.2b 100b 2.8b 80 b 2.6b 95b 2.1b 60 80b 1.8b 50b 2.3 b 65 b 2.5b 100b 2.9b 70 b 2.1b 85b 2.4b Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05 Table A-4. Effect of different combinati ons of TDZ and IBA on regeneration index (Index), number of shoots per explant and shoot length in A. latifolia U18.8 after 60 days of culture. IBA ( M) 0 0.1 1 Index Shoots LengthIndex Shoots LengthIndex Shoots Length TDZ ( M) -No.-mm-No.-mm-No.-mm0 0a* 0a 0 a 0 a 0 a 0 a 0 a 0 a 0 a 1 1.7 b 11b 53 b 1.4 ab 7.5a 35 a 1.7 b 10 b 33 a 10 2.5 b 6 ab 40 ab 2.2 b 5 a 16 a 2 b 5.5ab 10 a 30 2.8 b 2 a 8.5 a 2.1 b 1.5a 3.5 a 2 b 0 a 0 a 60 2.9 b 0 a 0 a 1.5 ab 0 a 0 a 2.1 b 0 a 0 a Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= % shoot bud form ation x mean number of buds / 100

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79 Table A-5. Effect of different combina tions of TDZ and IBA on percentage of ex vitro acclimatization (%) in A. latifolia U18.6 and U18.8 after 20 days of transfer to ex vitro conditions. U 18.6 U 18.8 IBA ( M) IBA ( M) TDZ ( M) 0 0.1 1 0 0.1 1 0 0a* 0 a 0 a 0 a 0 a 0 a 1 86 b 49 ab 96 b 83 b 63 a 83 b 10 86 b 100 b 65 ab 25 ab 50 a 36 ab 30 13 ab 28 ab 50 ab 0 a 0 a 0 a 60 0 a 0 a 25 ab 0 a 0 a 0 a Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05 Table A-6. Effect of different combina tions of BAP and IBA on percentage of adventitious bud formation (ABF) and m ean number of buds per explant in A. latifolia U18.6 after 30 and 60 days of culture. 30 days of culture 60 days of culture IBA ( M) IBA ( M) 0 0.1 1 0 0.1 1 ABF Buds ABF Buds ABF BudsAB F Buds ABF Buds ABF Buds BAP ( M) % No. % No. % No. % No. % No. % No. 0 0a* 0 0 a 0 0 a 0 0 a 0 a 0 a 0 a 0 a 0 a 1 0 a 0 5 a 1 5 a 0.5 5 a 1.5ab10 a 4 b 10ab0.5 a 10 10 a 0.5 25 b 1 0 a 0 40 b 3.9 b 85 b 3.5 b 45 b 2.7 a 30 0 a 0 5 a 0.5 0 a 0 45 b 3.8 b 25 a 2 ab 40 b 2.9 a 60 0 a 0 0 a 0 0 a 0 10ab 3 ab 15 a 1.5ab 45 b 1.8 a Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05

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80 Table A-7. Effect of different combinati ons of BAP and IBA on regeneration index (Index) and shoot length in A. latifolia U18.6 after 60 days of culture. IBA ( M) 0 0.1 1 Index Length Index Length Index Length BAP ( M) --mm---mm---mm-0 0a* 0 0 a 0 0 a 0 1 0.2 ab 20 0.4 a 40 0.1 a 0 10 1.5 bc 30 3 b 42.5 1.4 a 30 30 1.6 c 20 0.5 a 5 1.2 a 50 60 0.3 ab 20 0.3 a 0 0.9 a 15 Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= % shoot bud form ation x mean number of buds / 100 Table A-8. Effect of different combina tions of BAP and IBA on percentage of adventitious bud formation (ABF) and m ean number of buds per explant in A. latifolia U18.8 after 30 and 60 days of culture. 30 days of culture 60 days of culture IBA ( M) IBA ( M) 0 0.1 1 0 0.1 1 ABF Buds ABF Buds ABF Buds ABF Buds ABF Buds ABF Buds BAP ( M) % No. % No. % No. % No. % No. % No. 0 0a* 0 0 a 0 0 a 0 0a 0 a 0 a 0 a 0 a 0 a 1 5 a 0.5 0 a 0 0 a 0 20 a 1 a 35ab 1.3 a 0 a 0 a 10 10 a 0.5 45b 1.3 20 a 1.2 60b 2.8 b 90 c 3.9 b 75b 2.5 b 30 5 a 0.5 0 a 0 0 a 0 80b 2.9 b 65bc 3.5 b 90b 2.3 b 60 5 a 0.5 0 a 0 0 a 0 75b 4.8 c 85bc 3.3 b 70b 3 b Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05

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81 Table A-9. Effect of different combinati ons of BAP and IBA on regeneration index (Index) and shoot length in A. latifolia U18.8 after 60 days of culture. IBA ( M) 0 0.1 1 Index Length Index Length Index Length BAP ( M) --mm---mm---mm-0 0 a* 0 0 a 0 0 a 0 1 0.4 ab 30 0.4 a 0 0 a 0 10 1.7 bc 40 3.5 b 58 1.9 b 50 30 2.4 cd 40 2.3 b 33 2.1 b 33 60 3.6d 30 2.8 b 20 2.1 b 20 Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= % shoot bud form ation x mean number of buds / 100 Table A-10. Effect of different times of exposure to TDZ on bud formation percentage (ABF), mean number of buds per ex plant and regeneration index (Index) in A. latifolia U18.6 after 30 and 60 days of culture, respectively. 30 days of culture 60 days of culture ABF Buds Index ABF Buds Index Days in TDZ --%---No.---%---No.-0 0a* 0 a 0 a 0 a 0 0 1 0 a 0 a 0 a 0 a 0 0 4 25 ab 1.7 b 0.4 ab 35 ab 2.8 0.9 7 80 bc 2.3 b 1.8 c 85 bc 2.3 1.9 14 75 bc 1.6 b 1.2 bc 75 bc 2.2 1.7 20 70 bc 2.9 b 2 c 85 bc 2.6 2.2 30 90 c 1.9 b 1.8 c 95 c 3 3 Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= % shoot bud form ation x mean number of buds / 100

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82 Table A-11. Effect of different times of exposure to TDZ on bud formation percentage (ABF), mean number of buds per ex plant and regeneration index (Index) in A. latifolia U18.8 after 30 and 60 days of culture, respectively. 30 days of culture 60 days of culture ABF Buds Index ABF Buds Index Days in TDZ --%---No.---%---No.-0 0a* 0 a 0 a 0 a 0 a 0 1 5 a 0.5 ab 0.1 ab 5 a 0.5 a 0.1 4 0 a 0 a 0 a 25 a 1.3 a 0.4 7 35 b 1.8 ab 0.6 abc 70 b 1.6 ab 1.2 14 65 b 1.8 ab 1.2 bc 80 b 2.9 b 2.3 20 75 b 2.2 b 1.7 c 90 b 2 ab 1.9 30 70 b 1.6 ab 1.1 abc 85 b 2.5 b 2.1 Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= % shoot bud form ation x mean number of buds / 100 Table A-12. Effect of short exposure to TDZ on bud formation percentage (ABF), mean number of buds per explant and regeneration index (Index) in A. latifolia U18.6 and U18.8 after 30 days of culture. U18.6 U18.8 ABF Buds Index ABF Buds Index Days in TDZ --%---No.---%---No.-0 0 0 0 0a* 0 0 1 0 0 0 0 a 0 0 2 5 0.5 0.1 0 a 0 0 3 10 1 0.2 0 a 0 0 4 5 0.5 0.1 5 ab 1.5 0.2 5 5 1 0.1 10 ab 0.5 0.1 6 20 1.2 0.3 5 ab 0.5 0.1 7 15 1 0.2 20 abc 1.2 0.3 8 25 1.9 0.5 15 ab 1 0.2 9 25 1.8 0.4 30 bc 1 0.3 10 25 0.7 0.4 45 c 1.1 0.5 Different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= % shoot bud formation x mean number of buds / 100

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83 Table A-13. Effect of explant type on bud formation percentage (ABF), mean number of buds per explant and regeneration index (Index) in A. latifolia U18.6 and U18.8 after 30 days of culture. U18.6 U18.8 ABF Buds Index ABF Buds Index Types of explants --%---No.---%---No.-Petioles 10.3a* 1.5 ab 0.3 a 7.9 a 0.1 0 Rachis 90 d 3.5 bc 3.5 b 70 c 1 1 Immature leaves Leaflets 48.8bc 1.3 ab 0.8 a 27.5ab 1.1 0.4 Petioles 16 a 0.8 a 0.2 a 0 a 0 0 Rachis 80 cd 4.3 c 4.3 b 50 bc 1 1 Mature leaves Leaflets 37.5 ab 1.1 a 0.6 a 3.8 a 0.9 0.1 Within columns, different letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 0.05. Index= % shoot bud form ation x mean number of buds / 100

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84 Figure A-1. Regression curve sowing the e ffect of exposure to TDZ on bud formation percentage (%) in A. latifolia after 30 days of initiation of cultures. Figure A-2. Regression curve sowing the effect of exposure to TDZ on mean number of buds per explant (No. Buds) in A. latifolia after 30 days of initiation of cultures. y = -0.1565x2 + 7.3789x 3.5616 R2 = 0.91 0 10 20 30 40 50 60 70 80 90 05101520253035 Days in TDZ % y = -0.0061x2 + 0.238x + 0.0791 R2 = 0.88 0 0.5 1 1.5 2 2.5 3 05101520253035 Days in TDZNo. Buds

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85 Figure A-3. Regression curve sowing the e ffect of exposure to TDZ on regeneration index (Index) in A. latifolia after 30 days of initiation of cultures. y = -0.0037x2 + 0.1617x 0.1002 R2 = 0.90 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 05101520253035 Days in TDZIndex

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95 BIOGRAPHICAL SKETCH Maria Laura Vidoz was born in Reconquista, Santa Fe, Argentina, on 3 November, 1975. At the age of two, her family moved to Corrientes, Argentina. She attended the Colegio San Jose from 1980 to 1993. In 1994 sh e began her undergraduate studies at the Facultad de Ciencias Agrarias, Universidad Nacional del Nordeste. In 2002, she received her Agronomy Engineer degree. She worked from 1995 to 1997 at the Instituto de Botanica del Nordeste and then from 1997 to 2004 at the Vegetal Tissue Culture Laboratory, at the same univers ity. She began her graduate ca reer at the University of Florida in January 2005, under the direction of Dr. Kenneth Quesenberry. In December 2006 she was awarded a Master of Science de gree in Agronomy with an emphasis in plant breeding and genetics. After graduati on, Laura plans to pursue her Ph.D. before returning to Argentina to work in the field plant physiology.