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Optimization of tissue culture medium for plant regeneration and inheritance studies of plant regeneration in red clover

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Optimization of tissue culture medium for plant regeneration and inheritance studies of plant regeneration in red clover
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Poerba, Yuyu Suryasari, 1961-
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English
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ix, 74 leaves : ; 29 cm.

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Callus ( jstor )
Clover ( jstor )
Cultural studies ( jstor )
Diameters ( jstor )
Embryos ( jstor )
Genetics ( jstor )
Genotypes ( jstor )
In vitro fertilization ( jstor )
Regeneration ( jstor )
Somatic embryogenesis ( jstor )
Agronomy thesis, Ph. D
Dissertations, Academic -- Agronomy -- UF
Plant tissue culture ( lcsh )
Red clover -- Genetics ( lcsh )
Regeneration (Botany) ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1996.
Bibliography:
Includes bibliographical references (leaves 67-72).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Yuyu Suryasari Poerba.

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OPTIMIZATION OF TISSUE CULTURE MEDIUM FOR PLANT REGENERATION AND INHERITANCE STUDIES OF PLANT REGENERATION IN RED CLOVER















By

YUYU SURYASARI POERBA



















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1996














ACKNOWLEDGMENTS


I wish to express my deepest gratitude to Dr. Kenneth H. Quesenberry, supervisory committee chairman, for his support, guidance, help, and encouragement throughout my Ph.D. program. I would like also to sincerely thank my supervisory members, Dr. Paul L. Pfahler, Dr. David S. Wofford, Dr. Michael E. Kane, and Dr. Jude W. Grosser, for their advice, suggestions, guidance, and support throughout my program. I am very grateful to all my committee members for showing such interest.

I would also like to extend my special thanks to Ms.

Loan Ngo and Mr. David Moon for their help in the laboratory and in the greenhouse; to my fellow students for their help, support, and encouragement; and to all the staff of the Agronomy Department for helping me throughout my study.

I certainly appreciates the opportunity for this

graduate study given by the Indonesian Institute of Sciences and the Agency for the Assessment and Application of Technology.

I would like to deeply thank my parents, brothers, sisters, and all my in laws, for their encouragement and support of my study. Finally, I would like to express my


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deepest gratitude and appreciation to my husband, Desendi Poerba, for being here throughout the study, help, love, support, encouragement, and making life so much easier and enjoyable; and to our sons, Yudson and Deyson, for making life so precious and joyful.












































111















TABLE OF CONTENTS



ACKNOWLEDGMENTS......................................... ii

LIST OF TABLES.......................................... vi

ABSTRACT................................................ viii

CHAPTERS

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

2 REVIEW OF LITERATURE .............................. 4

Red Clover Genetics .......................... 4
Red Clover Tissue Culture Medium Development. 5 Genetic Studies of Tissue Culture Response... 13
Quantitative Genetic Studies............. 14
Mendelian Genetic Studies ................ 15
Forage Legumes In vitro Selection Response... 17

3 OPTIMIZATION OF TISSUE CULTURE MEDIUM FOR PLANT
REGENERATION OF RED CLOVER ........................ 22

Materials and Methods ........................ 25
General Procedure ........................ 25
Experiment I.............................. 26
Experiment II............................. 28
Experiment III............................ 32
Experiment IV ............................ 32
Results and Discussion ....................... 33
Experiment I.............................. 33
Experiment II............................. 36
Experiment III............................ 41
Experiment IV ............................ 43

4 INHERITANCE STUDY OF PLANT REGENERATION IN RED
CLOVER............................................ 48
Materials and Methods ........................ 50
Mating Design ........................... 50
Hypocotyl Culture ....................... 51
Results and Discussion ....................... 52

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5 SUMMARY AND CONCLUSION............................ 63

REFERENCE LIST...................................................67

BIOGRAPHICAL SKETCH...................................... 73























































v














LIST OF TABLES


Table page

3.1 Media used in red clover regeneration as
proposed in the literature ........................ 27

3.2 Media systems used in regeneration study of red
clover ............................................ 27

3.3 Callus fresh weight gain on different media with
fixed genotypes....................................... 33

3.4 Number of embryos per explant on different media
with fixed genotypes .............................. 35

3.5 Number of developing embryos per explant on
different media with fixed genotypes............... 36

3.6 Callus fresh weight gain for 4 weeks of culture
with fixed growth regulator combinations........... 37

3.7 Mean squares of callus weight gain in different
auxin and cytokinin sources with fixed basal salts 38

3.8 Mean number of embryos per explant for 8 weeks of
culture with fixed callus induction................ 39

3.9 Mean number of embryos per explant for 12 weeks of
culture with fixed embryo induction media.......... 40

3.10 Mean number of embryos per explant on different
media at 8 weeks of culture........................ 42

3.11 Mean number of embryos per explant over genotypes
on different media at 12 weeks of culture 42

3.12 Mean number of embryos per explant at 8 weeks of
culture........................................... 44

3.13 Mean number of embryos per explant on different
genotypes at 8 weeks of culture.................... 44



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3.14 Mean number of embryos per explant on different
media sequences at 12 weeks of culture............. 45

3.15 Mean number of embryos per explant on different
genotypes at 12 weeks of culture................... 46

3.16 Mean number of embryos per explant on different
genotypes within each media system................. 46

4.1 Diallel cross of nine genotypes of red clover..... 50

4.2 Mean callus diameters of 36 crosses of red clover
genotypes 4 weeks after culture.................... 53

4.3 Analysis of variance of general combining ability
(GCA) and specific combining ability (SCA) effects
for callus formation .............................. 54

4.4 Estimates of general combining ability (GCA)
effects for callus diameter red clover............. 55

4.5 Estimates of specific combining ability (SCA)
effects for callus diameter for 4 weeks............ 56

4.6 Regeneration capacity index of 36 crosses of red
clover genotypes 12 weeks after culture............ 59

4.7 Analysis of variance of general combining ability
(GCA) and specific combining ability (SCA) effects
for regeneration capacity ......................... 60

4.8 Estimates of general combining ability (GCA)
effects for regeneration capacity of red clover... 60

4.9 Estimates of specific combining ability (SCA)
effects for regeneration capacity of red clover... 62
















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

OPTIMIZATION OF TISSUE CULTURE MEDIUM FOR PLANT REGENERATION AND INHERITANCE STUDIES OF PLANT REGENERATION IN RED CLOVER By

Yuyu Suryasari Poerba

May 1996


Chairman: Dr. Kenneth H. Quesenberry Major Department: Agronomy

Red clover (Trifolium pratense L.) is an important

forage species grown in temperate climates around the world. One of the problems encountered in red clover tissue culture is low plant regeneration frequency. A series of experiments were conducted to optimize tissue culture media for plant regeneration and to investigate the genetic basis of in vitro regeneration of red clover. An experiment to evaluate previously published plant regeneration protocols showed that the superior treatment was B5C (B5 basal medium plus 2.0 mg L-1 each of NAA , 2,4-D, and kinetin) for callus induction, LSP (L2 basal medium plus 0.002 mg L-' picloram and 0.2 mg L-1 BA) for shoot induction, and LSP for plant regeneration, each with a 4-week interval. An experimentally selected optimum media protocol for red clover regeneration was L2 basal viii








medium supplemented with 2.0 mg L-' NAA, 2,4-D, and kinetin for callus induction, with 0.002 mg L-' picloram + 0.2 mg L-' BA for embryo induction, and L2 basal medium supplemented with 0.002 mg L-' picloram + 0.1 mg L-1 BA for regeneration. With this protocol genotypes with an intermediate regeneration response on the B5 protocol showed increased regeneration. Nine genotypes of red clover, with a range in level of plant regeneration (low, intermediate, and high regeneration) on the B5 protocol, were used in the inheritance study of in vitro plant regeneration. Progenies of 36 crosses were tested for callus formation at four weeks of culture and plant regeneration at 12 weeks of culture using hypocotyl explants. Diallel analysis was performed on these data using Griffing's Method 4, Model I. Results obtained from the diallel experiment showed that crosses with C54TC, C34TC, or 7-1-5-34 as one parent were among the combinations showing the highest callus diameter and regeneration capacity. Those crosses with NEWRC1, NEWRC4, or NEWRC30 as one parent were among the lowest callus diameter and regeneration response. Both general combining ability and specific combining ability (SCA) were significant sources of variation for callus diameter and for plant regeneration. Although SCA effects were significant for a few crosses, the results generally showed that additive genetic effects were most important in controlling inheritance of these responses.


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CHAPTER 1
INTRODUCTION


Plant regeneration is a vital objective of tissue

culture, and is necessary for the application of molecular and somatic cell genetics (genetic transformation and somatic hybridization) to crop improvement. An understanding of the role of the genotype in the regeneration is important to allow selection of clones with superior regeneration capacity for specialized experiments. This understanding can be used to select clones that regenerate, and to enhance the efficiency of regeneration.

Selection of suitable genotypes as well as tissue

culture procedures, therefore, is essential in regeneration and genetic analysis of plants following in vitro selection methods. Desirable variation can be separated from undesirable by genetic analysis and breeding. Knowledge of the inheritance of regeneration capacity and of somaclonal variation should aid in finding and exploiting variation and in conducting transformation experiments.

Red clover is one of the few major crop species where most all phases of tissue culture, plant regeneration, and molecular genetic engineering can be performed. Although

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considerable research has been conducted on tissue culture of red clover, most of the media protocols for red clover are genotype-dependent; not all genotypes respond similarly to a particular media system. By developing tissue culture media on which a broad spectrum of genotypes will respond, in vitro plant regeneration would not be an obstacle in plant transformation studies.

Genetic studies of tissue culture response and

regeneration capacity have conducted by many researchers in crop species; however, these studies have lagged behind in red clover. Understanding the genetic mechanism of tissue culture response and regeneration capacity in red clover would assist in the selection and breeding of genotypes with high regeneration capacity. This would facilitate improvement via genetic transformation to be achieved effectively and efficiently.

Genotypic variation for in vitro plant regeneration in red clover was ascribed to the occurrence of additive genetic variance (Keyes et al., 1980). This trait was reported to be highly heritable and was responsive to recurrent selection (Quesenberry and Smith, 1993).

In this study, the optimization of tissue culture media and the role of genetics in plant regeneration of red clover were investigated using petiole and hypocotyl callus










cultures. A three step media protocol (i.e., callus induction media, embryo induction media, and embryo development media, each of four-week intervals) was used throughout these studies. The first part of the research was a series of tissue culture media sequence optimization experiments to improve plant regeneration of red clover. These studies were conducted using genotypes which had been selected for high regeneration capacity on the B5 media protocol (Quesenberry and Smith, 1993). The second part was a genetic study of plant regeneration from hypocotyl callus of nine red clover genotypes which had a range in levels of regeneration (high, medium, and low regeneration capacity) on B5 media protocol.












CHAPTER 2
LITERATURE REVIEW


Red Clover Genetics


Red clover is an important cross-pollinated forage

species grown in temperate climates around the world. In the natural form, it is a highly self-incompatible diploid (2n=2x=14). Red clover has very stable meiotic associations and is apparently sexually isolated from all other species of the genus Trifolium (Cope and Taylor, 1985). There are no naturally occurring polyploid forms, although chemically and sexually induced tetraploids (2n=4x=28) exist. The chemically induced tetraploids are grown predominantly in Europe, where they were first developed (Smith et al., 1985). They produce more forage and are superior in quality, persistence, and disease resistance compared with their diploid counterparts in Europe. Chemically induced red clover tetraploids are produced using either colchicine or N20 (Taylor et al., 1976). Sexually induced tetraploids have been produced by the union of gametes in which one gamete (unilateral, 2x-4x or 4x-2x) or both gametes (bilateral, 2x-2x) contribute the sporophytic chromosome number (2n) (Meglic and Smith, 1992).


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Red clover is a short-lived perennial and its

usefulness is limited by its lack of persistence (Phillips et al., 1982). A recent report by Phillips et al.(1992) indicated that interspecific hybridization between red clover and T. alpestre L. (diploid strongly perennial species) using in vitro embryo rescue techniques was successful, although the hybrids (2n=2n+1=15) were functionally sterile. The hybrid resembled T. alpestre more closely than the paternal parent, red clover. If the hybrid infertility barriers could be overcome through backcrossing, these wide hybrids potentially offer new genetic variability for the improvement of red clover (Phillips et al., 1992).

The perennial growth habit of red clover causes plants to be exposed to pathogens and other pests in the environment for several years. Several different fungal, bacterial, and viral diseases have been identified as factors limiting production and persistence. Incorporation of pest resistance genes by genetic transformation appears to be a valuable technique that can be used in red clover (Quesenberry et al., 1992).


Red Clover Tissue Culture Medium Development


One of the problems encountered in genetic

transformation experiments is a low frequency plant








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regeneration either from callus or cell suspensions. As early as 1979, research showed that whole plant regeneration could be achieved from tissue cultures of various Trifolium species (Collins et al., 1979; Collins and Phillips, 1980). Beach and Smith (1980) demonstrated whole plant regeneration from tissue cultures of T. pratense L. and T. incarnatum L. using Gamborg's B5 media (Gamborg et al., 1976) with growth regulator modifications. However, plant regeneration using most of these systems was at a low frequency and primarily from unadapted plant introduction material or wild Trifolium species (Quesenberry et al., 1992).

The capacity of gametophytic or sporophytic cells to form in vitro shoots or embryos which are competent to develop i.e. to regenerate, is a particular feature of plants. Plant regeneration via in vitro somatic embryogenesis and organogenesis has been used to regenerate a wide variety of plant species (Tisserat et al., 1979; Williams and Maheswaran, 1986). However, many agronomically-important crops still cannot be regenerated from culture, and others regenerate only at low frequency (McGee et al., 1989; Williams and Maheswaran, 1989).

The levels of shoot or embryo induction and plant

regeneration from in vitro tissue cultures are basically influenced by the genotype and the physiological status of










the donor plant, the source of explant tissues within the plant, the culture medium and culture conditions, and the interaction between all these factors (McGee et al., 1989; Tisserat et al., 1979; Williams and Maheswaran, 1986; Mathias and Simpson, 1986; Lazar et al., 1984a; Bregitzer, 1992).

In red clover, protocols have been developed for somatic embryogenesis and organogenesis that involve incubation on a series of different media for callus initiation, embryo induction, embryo development or shoot development, and plant regeneration (Beach and Smith, 1979, Grosser and Collins, 1984; Phillips and Collins, 1979, 1980, 1982; Collins and Phillips, 1982, Bhojwani et al., 1984; MacLean and Nowak, 1989); and for direct somatic embryogenesis from immature embryos (Maheswaran and Williams, 1984, 1986) or from petiole segments derived from established shoot cultures (McGee et al., 1989).

Beach and Smith (1979) developed a protocol specific for callus induction, and plant regeneration of red clover from seedling hypocotyls and excised pistils of two cultivars on B5 medium (Gamborg et al., 1968). The callus induction medium was B5 medium supplemented with 10 pM each of 2,4-dichloro phenoxy acetic acid (2,4-D), a-naphthaline acetic acid (NAA), and 6-furfuryl amino purine (kinetin). Hypocotyls produced more callus than pistils. Shoot buds








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formed on this medium in some cases. After four to five weeks, callus was transferred to B5 medium containing twice the normal concentration of thiamine, 10 pM NAA, and 15 pM adenine. Numerous shoots developed. Rooting of shoots was accomplished on B5 medium containing the higher concentration of thiamine and 1.1 pM NAA. The SH medium (Schenck and Hildebrandt, 1972) was a suitable substitute for B5 basal medium in callus induction. Most of the regenerated plants were normal, fertile diploids. A few abnormal, sterile plants were recovered.

Phillips and Collins (1979) investigated the growth of callus from seedling sections of five cultivars. Visual ratings of various combinations of NAA, indole-3-acetic acid (IAA), p-chlorophenoxy acetic acid (CPA), 2,4-D, 4-amino3,5,6-trichloro picolinic acid (picloram), kinetin, 6(y,ydimethylallylamino) purine (2iP), and 6-benzylamino purine (BAP) on the SH (Schenk and Hildebrandt, 1972), B5, and Murashige and Skoog (MS) (1962) basal media were carried out. The combination of 0.25 pM of picloram and 0.44 pM of BAP was optimal for callus initiation and cell proliferation. However the basal media were unsatisfactory.

An improved basal medium (L2) was experimentallydeveloped (Phillips and Collins, 1979). The L2 medium contained a lower concentration of NH4 and higher concentrations of PO3-, K , Mg2+, and Ca2 than that of MS








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medium. The minor salts formulation was similar to that of SH medium, with adjustment in the concentration of several salts. The organic formulation (vitamins and sucrose) was similar to that of Linsmaier and Skoog (1965), with higher concentrations of thiamine and myo-inositol. Fresh weight increases were evaluated comparing the L2, MS, SH, and Miller inorganic formulation using the growth regulators and organic formulation of the L2 medium. No statistical differences were detected between the L2 and MS inorganic formulation; however, the L2 formulation was superior to the others tested. Vigorous calli from both mature and immature vegetative and reproductive explant sources were obtained on L2 medium.

Plant regeneration from callus culture was studied by Phillips and Collins (1979). Meristem-derived callus regenerated from 30 to 80% of the genotypes while nonmeristem-derived callus regenerated from only 1%. Plants regenerated from calli cultured on various combinations of 2,4-D, NAA, CPA, picloram, BAP, and kinetin. Combinations including 2,4-D yielded more plants than a combination of picloram and BAP. Plants were recovered efficiently when callus grown on medium containing 2,4-D was transferred to a medium containing a low concentration of picloram and a high concentration of BAP. Plants were rooted on a half-strength L2 medium supplemented with nicotinic acid and a vitamin-








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like analog, 3-amino pyridine. All regenerated plants were diploid and normal in appearance.

Phillips and Collins (1980) evaluated various

combinations of IAA, NAA, picloram, 2,4-D, BAP, and adenine in solidified L2 basal medium to regenerate plants from cell suspension cultures and to satisfy the conditions for the application of genetic selection procedures at the cellular level. Concentrations of 2,4-D at 23pM to 0.14pM were statistically superior to other concentrations. L2 medium containing 45DM 2,4-D and 15pM adenine was optimal for initiating plant regeneration. Plants regenerated via somatic embryogenesis, as evidenced by the developmental pattern of regeneration. Some somatic embryos developed complete shoots and roots on the callus induction medium. Shoot development of somatic embryos was most efficient on a L2 medium containing a low concentration of picloram and a moderate concentration of BAP. Most regenerated plants were normal, fertile diploids.

Phillips and Collins (1982) summarized the in vitro

culture protocols and the composition of media used for red clover. Explant preparation (including seedling germination), callus induction and proliferation, induction of somatic embryogenesis and plant regeneration, rooting of shoots, and establishment of plants in soil were described.










Bhojwani et al. (1984) studied intra-varietal variation for in vitro plant regeneration in Trifolium. Plant regeneration of red clover was successfully accomplished when calli derived from anthers (somatic tissues of the anther) was transferred to embryo development medium. The callus induction medium was MS basal medium supplemented with 0.1 mg L-1 2,4-D and 0.5 mg L-1 BAP or 1 mg L-' 2,4-D and 2 mg L- 2iP with or without casein hydrolysate. The embryo development medium was MS basal medium containing 0.05 mg L- 2,4-D and 0.5 mg L-' 2iP.

MacLean and Nowak (1989) studied plant regeneration of red clover using media sequences described by Beach and Smith (1979) and Collins and Phillips (1982), and a combination of media from both sequences. They observed that plant regeneration was best accomplished when callus tissues derived from B5 callus initiation medium (Beach and Smith, 1979) were incubated on SPL medium (Phillips and Collins, 1982) using regenerative genotypes.

Bhojwani and White (1982) developed a method for the culture of mesophyll protoplast of T. repens that gave a high frequency of cell division. The protoplast-derived calli were transferred to B5 solid medium or to MS medium supplemented with various growth regulators to obtain plant regeneration. Callus initiation medium contained B5 basal








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salt supplemented with 2 mg L- each of 2,4-D, NAA, and kinetin. Plant regeneration medium was MS basal medium supplemented with 0.5 mg L- 2iP, and 0.1 mg L- IAA.

Maheswaran and Williams (1984, 1986) developed direct somatic embryogenesis of red clover using immature embryos as explants. Embryoids were induced directly from the hypocotyl region of these embryos on EC6 basal medium (Maheswaran and Williams, 1984) containing 0.05 mg L-' BAP and 1.0 mg L-' yeast extract. Lowering of the yeast extract level to one half or zero at a BAP level of 0.05 mg L-1 gave precocious germination of the immature embryo explant or abnormal growth of the explanted zygotic embryo without initiation of embryoids. Increasing the BAP level above

0.05 mg L-' gave a shift from direct somatic embryogenesis to the formation of a nodular morphogenic callus.

McGee et al. (1989) developed a more rapid, one-step

regeneration system for Trifolium petiole segment of shoots cultured in vitro. They observed that petioles from in vitro Trifolium shoot cultures excised and placed on regeneration medium (L2 basal medium supplemented with 0.015 mg L-' picloram and 0.06 mg L-1 BAP), provided an improved system for studying the cellular and molecular events of somatic embryogenesis as compared with callus cultures. This system has been used to select isogenic cell lines of








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T. rubens showing high- or low-frequency somatic embryogenesis, and to examine polypeptide translational profiles during culture of high-frequency, low-frequency or non-embryonic cell lines of T. rubens and T. pratense (McGee et al., 1989).

Comparing the methods above, it is important to

consider types and concentrations of growth regulators, and to determine the timing of changes in growth regulator ratios in order to have an efficient system of plant regeneration of red clover.


Genetic Studies of Tissue Culture Response


Plant development from callus tissue culture has been shown to be under genetic control in various species and numerous experiments have shown that nuclear genes control the in vitro response (Frankenberger et al., 1981; Komatsuda et al., 1989; Chowdhury et al., 1991; Quimio and Zapata, 1990; Zhou and Konzak, 1992; Rackocszy-Trojanowska and Malepszy, 1993). Plant tissue culture and regeneration capacity has been studied using both quantitative and qualitative genetic approaches.








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Ouantitative Genetic Studies


Diallel analysis of shoot formation from cauliflower tissue culture was performed as early as 1974 (Buiatti et al., 1974), indicating that gene action was additive, but with a low heritability. Additive gene action also accounted for a large part of the shoot-forming variation between tomato F1s in a diallel cross (Frankenberger et al., 1981). For maize somatic embryogenesis, additive variance was high compared to non-additive or dominance variation (Nesticky et al., 1983).

Additive genetic variance was also the most significant source of variation for somatic embryogenesis in rice (Peng and Hodges, 1989) and wheat (Chevrier et al., 1990). Most of these results suggested that parents producing highly responsive hybrids can be identified. In rice anther culture, reciprocal effects on percent of explants producing callus and percent green-plant regeneration were not significant, indicating that both characters were under strict nuclear control and were not influenced by chloroplast and mitochondrial genomes. Further analysis showed gene action for both characters to be partially dominant with the high response being highly recessive and controlled by a few genes. There were no indications of inter-allelic interaction (Quimio and Zapata, 1990).








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Genetic variation for maize callus growth and shoot regeneration ability were observed, although a large proportion of the variation was additive type, positive heterosis increased the tissue culture response in maize somatic embryogenesis (Tomes and Smith, 1985). Although many differences between genotypes for in vitro tissue culture responses were reported to be additive and heritable (Keyes et al., 1980; Tomes and Smith, 1985), some genetic studies indicated that dominance, additive x additive and dominance x additive genetic effects were important (Chu and Croughan, 1990).

Studies designed to analyze the inheritance of anther

culture ability in maize demonstrated that general combining abilities were highly significant (Petolino and Thompson, 1988), with a high heritability. A predominantly additive gene action exists for microspore-derived embryo induction (Koba et al., 1993) and green plant regeneration (Zhou and Konzak, 1992; Shimada et al., 1993). Estimated heritability was high for anther culture ability (Lazar et al., 1984b). Mendelian Genetic Studies


Genotypes which produce callus, somatic, or androgenic embryos in vitro have been tested for their ability to sexually transmit these traits, and sometimes reciprocal








16

crosses have been performed. All the analyses of segregating populations demonstrated that there was a genetic component for tissue-culture traits. In wheat anther culture, the green plant regeneration trait segregated among backcross populations (Zhou and Konzak, 1992), and this trait was attributed to nuclear genes. Similar evidence was obtained from somatic embryogenesis experiments, and it has been demonstrated that callus induction rate and regeneration ability are controlled by independent genetic systems in wheat (Chowdhury et al., 1991) and barley (Komatsuda et al., 1989).

Most studies conducted to determine the number of genes controlling the various components of tissue culture responses have indicated that one, two, or a few nuclear genes were involved. Recessive nuclear genes were responsible for regeneration capacity either from leaf disc in tomato (Frankenberger et al., 1981), or from immature inflorescence callus in rye (Rakocszy-Trojanowska and Malepszy, 1993). On the contrary for alfalfa (Reisch and Bingham, 1980; Wan et al., 1988; Ray and Bingham, 1989; Hernadez and Christie, 1990; Kielly and Bowley, 1992; Yu and Pauls, 1993), dominant genes seem to control somatic embryogenesis or anther culture ability (Taylor and Veilleux, 1992). A few reports suggest that complementary genes are necessary for the whole tissue culture process








17

(Wan et al., 1988; Hernandez and Christie, 1990; Yu and Pauls, 1993).


Forage Legumes In vitro Selection Response


Genetic variation for in vitro response has been reported for a number of forage legumes, especially in alfalfa (Bingham et al., 1975), red clover (Keyes et al., 1980) and Desmodium (Wofford et al., 1992). In red clover, genotypic variation was ascribed to the occurrence of additive genetic variance (Keyes et al. 1980) whereas in diploid alfalfa, the capacity for somatic embryogenesis was controlled by two dominant genes (Reisch and Bingham, 1980).

In alfalfa, regeneration was genotype-specific and only a few genotypes in certain cultivars have been isolated with the ability to regenerate plants from explant derived calli (Arcioni et al., 1990a). Two selected genotypes from the cultivar 'Adriana' were shown to regenerate through somatic embryogenesis (Arcioni et al., 1990b). Studies of the genetic control of somatic embryogenesis have been carried out in diploid (Reisch and Bingham, 1980) and tetraploid alfalfa (Wan et al., 1988; Hernandez-Fernandez and Christie, 1989) and all the studies showed that this trait was under the control of at least two independent loci although with different interactions. The high heritability of








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embryogenesis allowed an increase in the regeneration capacity of a cultivar from 12 to 67% (Bingham et al., 1975).

Plant regeneration from callus was found to be highly heritable in alfalfa (Bingham et al., 1975; Wan et al., 1988) and red clover (Quesenberry and Smith, 1993). Regeneration was increased from 12% in a standard alfalfa cultivar to 67% after three generations (Bingham et al., 1975). Genetic analysis of regeneration in diploid alfalfa confirmed that regeneration was relatively simply inherited. Segregation for regeneration in F1, F2, and backcross generations involving crosses between the high regenerator and genotypes with very low regeneration indicated that regeneration was dominant and fitted a two-locus model (Reisch and Bingham, 1980). A dominant allele at each locus appeared necessary to explain high regeneration in which >75% of callus colonies produced buds. However, it is not known how many different modes of genetic control may exist for regeneration in alfalfa.

Recently, Crea et al. (1995) studied the genetic

control of somatic embryogenesis in the alfalfa cultivar Adriana. The results indicated that the somatic embryogenesis observed in the cv. Adriana was under genetic control. The segregation ratio of the progenies from intercrossing or selfing embryogenic genotypes gave a








19

population which segregated following the two dominant loci model proposed by Wan et al.(1988). The possibility of increasing the frequency of embryogenic plants has been demonstrated. From the 3% frequency of regenerable genotypes observed in the cv. Adriana (Arcioni et al., 1990b), they were able to obtain by selfing and intercrossing a seed population in which 50% of plants were embryogenic.

Keyes et al (1980) performed genetic studies evaluating variation in callus cultures of red clover. The callus induction medium used was KB2 medium, consisting of the L2 basal medium supplemented with Beach and Smith (1979) callus growth regulators i.e 2 mg L-' each of 2,4-D, NAA, and kinetin. Plant regeneration media used were LSE (Phillips and Collins, 1980) and the plant regeneration medium of Beach and Smith (1979). Additive genetic variance was a significant source of variability for most in vitro traits evaluated, including rapid callus growth, colony vascularization, root initiation, chlorophyll production, and somatic embryogenesis. These traits were highly heritable. Dominance genetic variance was significant for only a few in vitro characters such as callus morphology, percentage of colonies initiating roots, and total number of roots. Maternal and cytoplasmic factors were significant








20

primarily in early subcultures. No significant differences for embryogenesis were attributable to differences in the regeneration media used. Furthermore, no interaction of additive genetic effects with regeneration media were observed. These results indicated that improvement in the frequency of plantlet regeneration from callus of red clover could effectively be achieved by breeding and selection for embryonic types (Keyes et al., 1980).

Recently, Quesenberry and Smith (1993) reported

successful recurrent selection for plant regeneration of red clover from callus tissue culture. After five cycles of recurrent phenotypic selection for increased plant regeneration via somatic embryogenesis from callus tissue culture, the percentage of regenerating plants increased from 4 to 72%. Number of regenerating plants per petiole explant was > 200 for selected individuals compared with <10 in base population individuals. They reported that plant regeneration from callus tissue of red clover was a highly heritable trait. Narrow sense heritability estimates ranged from 40 to 50%.

Although much research has been conducted on tissue

culture of red clover, most of the media developed for red clover are genotype-dependent. Not all genotypes responded similarly in a particular media system. By developing tissue culture media which support regeneration from a broad








21

spectrum of genotypes, in vitro plant regeneration would not be an obstacle in transformation experiments. Genetic studies of this trait have been conducted extensively in many species; however, in red clover, this area of research has received limited attention. Understanding the genetic mechanism of the tissue culture and regeneration response of red clover would be helpful in the selection and breeding for these traits so that the improvement could be achieved efficiently and effectively.














CHAPTER 3
OPTIMIZATION OF TISSUE CULTURE MEDIUM FOR
PLANT REGENERATION OF RED CLOVER


One of the problems encountered in genetic

transformation experiments is a low frequency of plant regeneration either from callus or cell suspensions. Much research has been conducted on tissue culture of red clover; however, the frequency of plant regeneration using most of these systems was at low (McGee et al., 1989) and primarily from unadapted plant introduction materials or wild Trifolium species (Quesenberry et al., 1992).

Red clover protocols for somatic embryogenesis and organogenesis that involve incubation on a series of different media for callus initiation, embryo induction and embryo development or shoot development, and plant regeneration have been presented (Beach and Smith, 1979, Phillips and Collins, 1979, 1982; Collins and Phillips, 1982, Bhojwani et al., 1984; MacLean and Nowak, 1989; Myers et al., 1989). Other research has demonstrated direct somatic embryogenesis from immature embryos (Maheswaran and Williams, 1984, 1986) or from petiole segments derived from established shoot cultures (McGee et al., 1989).


22








23

A protocol specific for callus induction, and plant regeneration of red clover from seedling hypocotyls and excised pistils using B5 basal salt (Gamborg et al., 1968) has been reported. The callus induction medium was B5 medium supplemented with 10 pM each of 2,4-D, NAA, and kinetin. Calli were then transferred at 4 to 5 weeks to B5 medium containing twice the normal concentration of thiamine, 10 pM NAA, and 15 pM adenine. Rooting of developed shoots was accomplished on B5 medium containing the higher concentration of thiamine and 1.1 pM NAA.

Other research led to development of a new basal medium called L2 (Phillips and Collins, 1979). This medium contained a lower concentration of NH4 and higher concentrations of PO3-, K', Mg2', and Ca2{ than MS medium. The minor salts formulation was similar to SH medium, with adjustment in the concentration of several salts. The organic formulation (vitamins and sucrose) was similar to that of Linsmaier and Skoog (1965), with higher concentrations of thiamine and myo-inositol. Phillips and Collins (1982) described explant preparation (including seedling germination), callus induction and proliferation, induction of somatic embryogenesis and plant regeneration, rooting of shoots, and establishment of plants in soil.

MacLean and Nowak (1989) studied plant regeneration of red clover using media sequences described by Beach and








24

Smith (1979) and Collins and Phillips (1982), and a combination of media from both sequences. They concluded that optimum plant regeneration was obtained when callus was initiated on B5 callus medium (Beach and Smith, 1979) but then transferred after four weeks to LSP medium (Phillips and Collins, 1982).

Working with white clover (T. repens L.), Bhojwani and White (1982) developed a method for the culture of mesophyll protoplasts that gave a high frequency of cell division. Protoplast-derived calli were transferred to B5 solid medium or to MS medium supplemented with various growth regulators to obtain plant regeneration. The callus initiation medium contained B5 basal salt supplemented with 2 mg L- each of 2,4-D, NAA, and kinetin. Plant regeneration medium was MS basal medium supplemented with 0.5 mg L- 2iP, and 0.1 mg L-' IAA.

Comparing the methods above, it is important to

consider types and concentrations of growth regulators, and to determine timing of changes in growth regulators ratios in order to have an efficient system of plant regeneration of red clover.








25

Materials and Methods


General Procedure


Three-step media protocols (callus induction - embryo induction - plant regeneration) were used throughout these studies. For callus induction, petiole sections (3 to 5 mm long) were excised from sterile plant cultures maintained in Magenta boxes on B5 medium without growth regulators and transferred to the callus induction media. All explants were incubated for 4 weeks to induce callus formation and growth in a growth chamber. Cultures were maintained at 26 � 20C C with a 16-h day, 85 pEm-2s-1. Irradiation was supplied by cool-white fluorescent light. Factorial experiments in Randomized Complete Block (RCB) were used as an experimental design throughout these studies. Six replicates of 4 to 6 explants per replicate were initially established. After 4 weeks, two replicates of each medium treatment were sacrificed for fresh callus weight gain data. The remaining 4 replicates were transferred onto embryo media under the same environmental conditions.

After 4 additional weeks, two replicates of each medium treatment were sacrificed for determination on number of embryos per explant. The remaining two replicates were transferred into embryo germination media for a 4-week period. The number of developing embryos for each genotype








26

cultured on the various media systems were recorded at the end of 8- and 12-week culture periods. Analyses of variance were performed as a factorial experiment in a randomized block design for callus fresh weight gain (4 weeks of culture), number of embryoids (8 weeks of culture), and number of developing embryos per explant (12 weeks of culture), respectively. Before analysis, data for both number of embryos per explant and number of developing embryos per explant were transformed using square root of (x + 0.5). Differences among genotype means and among media system means were compared by Duncan's Multiple Range Test (DMRT), and were declared different at P < 0.05.


Experiment I


Six genotypes (7-1-5-36, 7-1-5-43, C34TC, 8-2-16-5, 82-16-39, FLMR6-23-3) of red clover that previously had been determined to be good regenerators on the protocol of Beach and Smith (1979) were tested for plant regeneration capacity using six media sequences proposed in the literature. Three callus induction media (L2, B5C, and KB2) were used in this study. L2 medium contained L2 basal salt plus 0.06 mg Lpicloram and 0.1 mg L-1 BAP (Phillips and Collins, 1979); B5C medium contained B5 basal salt supplemented with 2.0 mg L-' each of NAA, 2,4-D mg L-1, and kinetin (Beach & Smith,








27

1979). KB2 contained L2 basal medium supplemented with 2.0

mg L- each of NAA, 2,4-D, and kinetin (Keyes et al., 1980).

The various media to which calli were transferred are listed

in Table 3.1, and the six media sequences are listed in

Table 3.2.


Table 3.1. Media used in red clover regeneration as proposed in the literature

Growth regulators
Medium Salts & Proposed use
vitamins Auxin Cytokinin

------(mg L-)-----L2 L2 PIC, 0.06 BAP, 0.1 Callus induction B5C B5 NAA, 2.0 KIN, 2.0 Callus induction 2,4-D, 2.0 and regeneration KB2 L2 NAA, 2.0 KIN 2.0 Callus induction 2,4-D, 2.0 and regeneration LSE L2 2,4-D, 0.01 Adenine, Somatic embryo
2.0 induction
B5E B5 NAA, 2.0 Adenine, Embryo germination
2.0
MSR MS IAA, 0.1 2iP, 0.5 Embryo germination LSP L2 PIC, 0.002 BAP, 2.0 Shoot development B5R B5 NAA, 0.2 - Root development




Table 3.2. Media systems used in regeneration study of red clover

Media sequences
No. Reference
Callus Embryo Shoot

1 L2 ---- LSE ---- LSP Phillips and Collins (1980)
2 B5C ---- B5E ---- B5R Beach and Smith (1979)
3 KB2 ---- LSE ---- LSP Keyes et al. (1980) 4 KB2 ---- B5E ---- B5R Keyes et al. (1980)
5 B5C ---- LSP ---- LSP MacLean and Nowak (1989) 6 B5C ---- MSR ---- MSR Bhojwani and White (1982)








28

Data were analyzed as a 3 x 6, 6 x 6, and 6 x 6

factorial in a RCB design for: callus fresh weight gain (4 weeks of culture), number of embryos or organized structures per explant (8 weeks of culture), and number of developing embryos per explant (12 weeks of culture), respectively. Experiment II


Six genotypes (7-1-5-36, FLMR6-23-3, C25TC, C34TC,

C51TC, and C54TC) of red clover, which had exhibited a high level of plant regeneration capacity on B5 protocol (Beach and Smith, 1979), were used to study the effects of various combinations and concentrations of auxin and cytokinin on different basal media for callus formation and plant regeneration response. Four basal media (L2, B5, SH, and MS) and three combinations of auxin and cytokinin (0.06 mg L-1 picloram + 0.10 mg L-1 BAP; 2.0 mg L- NAA + 2.0 mg L2,4-D + 2.0 mg L-1 kinetin; and 2.0 mg L- NAA + 2.0 mg L2,4-D + 0.10 mg L- BAP) were used for callus initiation. The twelve treatments were:

1. L2 basal salt plus 0.06 mg L- picloram + 0.10 mg L- BAP 2. L2 basal salt plus 2.0 mg L-' NAA + 2.0 mg L- 2,4-D + 2.0
mg L- kinetin
3. L2 basal salt plus 2.0 mg L- NAA + 2.0 mg L- 2,4-D +
0.10 mg L-1 BAP
4. B5 basal salt plus 0.06 mg L- picloram + 0.10 mg L- BAP 5. B5 basal salt plus 2.0 mg L-1 NAA + 2.0 mg L-1 2,4-D + 2.0
mg L- kinetin
6. B5 basal salt plus 2.0 mg L-' NAA + 2.0 mg L- 2,4-D +
0.10 mg L- BAP
7. SH basal salt plus 0.06 mg L-1 picloram + 0.10 mg L- BAP








29

8. SH basal salt plus 2.0 mg L-1 NAA + 2.0 mg L-' 2,4-D + 2.0
mg L- kinetin
9. SH basal salt plus 2.0 mg L-1 NAA + 2.0 mg L-Y 2,4-D +
0.10 mg L- BAP
10. MS basal salt plus 0.06 mg L- picloram + 0.10 mg L-Y BAP 11. MS basal salt plus 2.0 mg L- NAA + 2.0 mg L- 2,4-D +
2.0 mg L-1 kinetin
12. MS basal salt plus 2.0 mg L- NAA + 2.0 mg L-1 2,4-D +
0.10 mg L-1 BAP

Data for callus weight gain were analyzed as 6 x 4 x 3

factorial experiment in Randomized Complete Block Design

with two replications. The four media producing the best

quantity and quality of callus were chosen (i.e. number 2,

5, 6, and 8) from the 12 callus induction media. These four

media were then used for somatic embryo induction in

combination with six different auxin-cytokinin

concentrations. The six combination of auxin and cytokinin

were:

1. none
2. 0.01 mg L-1 2,4-D + 2.0 mg L-' adenine
3. 0.01 mg L-1 2,4-D + 0.1 mg L-1 BAP 4. 2.0 mg L- NAA + 2.0 mg L- adenine 5. 2.0 mg L- NAA + 2.0 mg L- kinetin
6. 0.002 mg L-1 picloram + 0.2 mg L- BAP.

The 24 media combinations were:

1. B5C(5)--> B5 basal salt without growth regulators
2. B5C(5)--> B5 basal salt + 0.01 mg L-1 2,4-D + 2.0 mg L1
adenine
3. B5C(5)--> B5 basal salt + 0.01 mg L- 2,4-D + 0.1 mg LBAP
4. B5C(5)--> B5 basal salt + 2.0 mg L- NAA + 2.0 mg Ladenine
5. B5C(5)--> B5 basal salt + 2.0 mg L- NAA + 2.0 mg Lkinetin
6. B5C(5)--> B5 basal salt + 0.002 mg L-1 picloram + 0.2 mg
mg L- BAP
7. B5C(6)--> B5 basal salt without growth regulators








30

8. B5C(6)--> B5 basal salt + 0.01 mg L-1 2,4-D + 2.0 mg L-1
adenine
9. B5C(6)--> B5 basal salt + 0.01 mg L-1 2,4-D + 0.1 mg L-'
BAP
10. B5C(6)--> B5 basal salt + 2.0 mg L-1 NAA + 2.0 mg L-1
adenine
11. B5C(6)--> B5 basal salt + 2.0 mg L-1 NAA + 2.0 mg L-1
kinetin
12. B5C(6)--> B5 basal salt + 0.002 mg L-' picloram + 0.2 mg
L-1 BAP
13. L2C(2)--> L2 basal salt without growth regulators 14. L2C(2)--> L2 basal salt + 0.01 mg L-' 2,4-D + 2.0 mg Ladenine
15. L2C(2)--> L2 basal salt + 0.01 mg L- 2,4-D + 0.1 mg L-1
BAP
16. L2C(2) --> L2 basal salt + 2.0 mg L- NAA + 2.0 mg L-1
adenine
17. L2C(2)--> L2 basal salt + 2.0 mg L-1 NAA + 2.0 mg L-1
kinetin
18. L2C(2)--> L2 basal salt + 0.002 mg L-' picloram + 0.2 mg
L-1 BAP
19. SHC(8)--> SH basal salt without growth regulators 20. SHC(8)--> SH basal salt + 0.01 mg L-' 2,4-D + 2.0 mg L-1
adenine
21. SHC(8)--> SH basal salt + 0.01 mg L-1 2,4-D + 0.1 mg L-1
BAP
22. SHC(8) --> SH basal salt + 2.0 mg L-1 NAA + 2.0 mg L-1
adenine
23. SHC(8)--> SH basal salt + 2.0 mg L-1 NAA + 2.0 mg L-1
kinetin
24. SHC(8)--> SH basal salt + 0.002 mg L-' picloram + 0.2 mg
L-1 BAP

The number embryoids or organized structures per

explant were analyzed as 6 x 4 x 6 factorial in a randomized

block design with two replications. Five best media based

on number and quality of embryos were chosen from these 24

somatic embryo induction media (i.e. number 1, 4, 8, 12, and

18). These were combined with the four different

concentrations of auxin and cytokinin below for embryo

development i.e.:








31

1. none
2. 0.2 mg L-1 NAA
3. 0.002 mg L- picloram + 0.2 mg L- BAP 4. 0.002 mg L-Y picloram + 0.1 mg L- BAP

The 20 media combinations were:

1. B5C(5)--> B5--> B5 basal salt without growth regulators
2. B5C(5)--> B5--> B5 basal salt + 0.2 mg L- NAA
3. B5C(5)--> B5--> B5 basal salt + 0.002 mg L-' picloram +
0.2 mg L- BAP
4. B5C(5)--> B5--> B5 basal salt + 0.002 mg L- picloram +
0.1 mg L- BAP
5. B5C(5)--> BSE(4)--> B5 basal salt (no growth regulators)
6. B5C(5)--> BSE(4)--> B5 basal salt + 0.2 mg L- NAA
7. B5C(5)--> BSE(4)--> B5 basal salt + 0.002 mg L- picloram
+ 0.2 mg L- BAP
8. B5C(5)--> BSE(4)--> B5 basal salt + 0.002 mg L- picloram
+ 0.1 mg L- BAP
9. B5C(6)--> BSE(2)--> B5 basal salt (no growth regulators)
10. B5C(6)--> BSE(2)--> B5 basal salt + 0.2 mg L- NAA 11. B5C(6)--> BSE(2)--> B5 basal salt + 0.002 mg L-'
picloram + 0.2 mg L- BAP
12. B5C(6)--> BSE(2)--> B5 basal salt + 0.002 mg L-Y
picloram + 0.1 mg L- BAP
13. B5C(6)--> BSE(6)--> B5 basal salt (no growth regulators) 14. B5C(6)--> BSE(6)--> B5 basal salt + 0.2 mg L- NAA 15. B5C(6)--> BSE(6)--> B5 basal salt + 0.002 mg L- picloram
+ 0.2 mg L- BAP
16. B5C(6)--> BSE(6)--> B5 basal salt + 0.002 mg L-' picloram
+ 0.1 mg L-1 BAP
17. L2C(2)--> LSE(6)--> L2 basal salt (no growth regulators) 18. L2C(2)--> LSE(6)--> L2 basal salt + 0.2 mg L- NAA 19. L2C(2)--> LSE(6)--> L2 basal salt + 0.002 mg L- picloram
+ 0.2 mg L- BAP
20. L2C(2)--> LSE(6)--> L2 basal salt + 0.002 mg L- picloram
+ 0.1 mg L- BAP

The number of developing embryos at 12 weeks was

analyzed as 6 x 5 x 4 factorial in an RCB design with two

replications.








32

Experiment III


Six genotypes (NEWRC 12, NEWRC 25, NEWRC 32, NEWRC 37, NEWRC 56, and NEWRC 98) of red clover which had a high plant regeneration response on the B5 protocol (Beach and Smith, 1979) were used. Three media systems, i.e the best media system from Exp. I and II, and the standard media system of Beach and Smith (1979), were compared for their callus formation and plant regeneration response. The three media systems were:

1. B5C - B5E - B5R (standard)

2. B5C - LSP - LSP (Exp. I)

3. L2C(2) - LSE(6) - L2(20) (Exp. II)

All data were analyzed as 3 x 6 factorial in a

randomized complete block design with two replicates. Experiment IV


Ten genotypes (NEWRC1, NEWRC4, NEWRC30, NEWRC 64,

NEWRC74, FLMR6-23-3, C34TC, C54TC, 7-1-5-36) of red clover which had a range in levels of plant regeneration capacity on B5 protocol (Beach and Smith, 1979) were used. The same three media systems used in Exp. III were evaluated. Response variables measured were callus formation and plant regeneration capacity. All data were analyzed as 3 x 10 factorial in an RCBD with two replications.








33

Results and Discussions


Experiment 1


The criteria used to select the optimal red clover

culture media for tissue culture responses included growth in culture and regeneration capacity because cell division should allow for the rapid proliferation of callus from petiole tissues. Growth was determined by measuring fresh weight gain (g) of callus over 4 weeks.

The analysis of variance showed highly significant effects of red clover genotypes for callus fresh weight gain. The genotype by media interaction was also significant for this trait. There was no effect of media for this variable. Because of the interaction between genotypes and media, further analysis was conducted for each genotype to test the differences in callus fresh weight gain among different media (Table 3.3). Table 3.3. Callus fresh weight gain on different media with fixed genotypes

Callus Genotype induction Mean media 1 2 3 4 5 6

------------------------ g ---------------------L2 0.21 a 0.10 a 0.08 b 0.09 a 0.10 a 0.09 b 0.11 ab KB2 0.16 ab0.11 a 0.09 b 0.09 a 0.07 a 0.11 ab 0.10 b B5C 0.15 b 0.11 a 0.15 a 0.13 a 0.07 a 0.13 a 0.12 a Means followed by the same letter within a column are not different (P = 0.05).








34

With genotypes 2(7-1-5-34), 4(8-2-16-5), and 5(8-2-1639) no significant differences were observed in callus fresh weight gain on the three different callus induction media (CIM). However with genotypes 1(7-1-5-36), 3(C34TC), and 6(FLMR6-23-3) significant differences among three media were observed. L2 medium gave the highest callus fresh weight gain for genotype 1, whereas B5C medium showed the highest callus weight gain for genotype 3 and 6. Over all genotypes, callus weight gain was highest on B5C and lowest on KB2.

Highly significant differences were observed among genotypes, media, and their interactions for number of embryos at eight weeks. For this reason, further analyses were conducted on each genotype to determine the differences in response on different media of a given genotype (Table 3.4). In all genotypes (except 6) significant differences were observed in number of embryoids per explant on the six media tested. In all genotypes, media system 1 (L2-LSE) resulted in lowest number of embryos per explant. Averaged over all genotypes, media system 5 (B5C-LSP) gave the highest number of embryos per explant.








35

Table 3.4. Number of embryos per explant on different media with fixed genotypes

Genotype
Media Mean
it 2 3 4 5 6

1 0Od 0c 0c 0c 0d 0a 0c 2 38 b 25 a 62 ab 60 b 53 c 7 a 37 a 3 16 c 0 a 85 a 53 b 99 a 1 a 29 b 4 34 b 7 b 45 b 95 a 67 b 4 a 35 ab
5 45 ab 20 ab 59 ab 63 b 73 b 3 a 39 a 6 57 a 17 ab 61 ab 54 b 66 b 0 a 36 a Means followed by the same letter within a column are not different (P = 0.05).
t 1 = 7-1-5-36, 2 = 7-1-534, 3 = C34TC, 4 = 8-2-16-5, 5 = 82-16-39, 6 = FLMR6-23-3.
t 1 = L2-LSE-LSP, 2 = B5C-B5E-B5R, 3 = KB2-LSE-LSP, 4 = B5CB5E-B5R, 5 = B5C-LSP-LSP, 6 = B5C-MSR-MSR.


Highly significant differences in number of developing embryos at 12 weeks were observed for genotypes, media, and for the interaction between genotype by media. Further analyses were conducted to evaluate differences in number of developing embryos among different media for each genotypes (Table 3.5).

Over all genotypes, significant differences were observed in number of developing embryos on the six different media systems tested (Table 3.5). Averaged over all genotypes, media system 1 (L2-LSE-LSP) had lowest number of developing embryos or shoots per explant, whereas the five other media systems were not different for mean number of developing embryos or shoots per explant with media system 5 (B5C-LSP-LSP) having the numerically highest number








36

of developing embryos per explant. Genotype 6 generally had the lowest number of developing embryos per explant of any media systems tested, except in media 5(B5C-LSP-LSP). Genotype 3 generally had the highest mean number of developing embryos per explant.


Table 3.5. Number of developing embryos per explant on different media with fixed genotypes Genotype
Media Mean
it 2 3 4 5 6

1it 0 c 0 c 13 b 1 b 0 d 0 c 2 b 2 65 b 60 a 137 a 131 a 82 a 10 bc 73 a 3 159 a 5 bc 131 a 93 a 135 a 3 bc 69 a 4 82 b 17 b 87 a 109 a 109 b 15 b 63 a 5 96 ab 26 ab 110 a 92 a 96 bc 46 a 74 a 6 99 ab 27 ab 105 a 102 a 86 cd 8 bc 63 a Means followed by the same letter within a column are not different (P = 0.05)
t 1 = 7-1-5-36, 2 = 7-1-534, 3 = C34TC, 4 = 8-2-16-5, 5 = 82-16-39, 6 = FLMR6-23-3.
t 1 = L2-LSE-LSP, 2 = B5C-B5E-B5R, 3 = KB2-LSE-LSP, 4 = B5CB5E-B5R, 5 = B5C-LSP-LSP, 6 = B5C-MSR-MSR.


Experiment II


Callus fresh gain weight (g) after 4 weeks in culture showed that genotypes were significantly different. However, since the main focus of this work was to develop media satisfactory for many genotypes, data were analyzed over genotypes.

The analysis of variance showed highly significant

effects for basal salts, growth regulators and basal salt by








37

growth regulator interaction. These results revealed that both basal salt and growth regulator, as well as their interactions were important for callus fresh weight gain. Thus, further analysis was necessary to understand the differences in response related to basal salt and/or growth regulator combinations (Table 3.6).


Table 3.6. Callus fresh weight gain for 4 weeks of culture with fixed growth regulator combinations Growth regulator combination Basal salt
it 2 3

-------------------g- ----------------------L2 0.087 a 0.072 b 0.067 ab B5 0.072 bc 0.096 a 0.079 a SH 0.076 ab 0.079 ab 0.063 b MS 0.061 c 0.071 c 0.067 c Mean 0.074 A 0.080 B 0.067 C Means within a column followed by the same lower case letter are not different (P = 0.05). Means within a row followed by the same upper case letter are not different (P = 0.05). t 1 = 0.06 mg L-1 picloram + 0.10 mg L- BAP
2 = 2.0 mg L-1 NAA + 2.0 mg L-1 2,4-D + 2.0 mg L- kinetin
3 = 2.0 mg L-1 NAA + 2.0 mg L-1 2,4-D + 0.10 mg L-1 BAP.


Within growth regulator combination 1 (0.06 mg Lpicloram + 0.10 mg L-1 BAP) L2 basal salts had the best callus growth. However, within growth regulator 2 (2.0 mg L- each of 2,4-D, NAA, and kinetin) the B5 basal salts had the highest callus production. With growth regulator combination 3 (2.0 mg L-1 2,4-D, 2.0 mg L-' NAA and 0.1 mg L-








38

BAP) the best callus fresh weight gain was obtained with B5 basal salt although this treatment was lower than treatment 2 with B5. In general, callus fresh weight gain was lowest using MS basal salt with any growth regulator combination. Over all basal salts, the growth regulator combination 3 had the lowest mean of callus fresh weight gain.

A contrast procedure was used to investigate

differences between auxin and cytokinin sources in callus production (Table 3.7). Auxin source significantly influenced callus production in the L2 basal salt with 0.06 mg L-1 picloram being superior to 2.0 mg L-1 NAA and 2,4-D. Table 3.7. Mean squares of callus weight gain in different auxin and cytokinin sources with fixed basal salts Basal salt
Growth regulator
L2 B5 SH MS Auxin (1 vs 3) 0.074* 0.010 0.043 0.005 Cytokinin (2 vs 3) 0.001 0.070* 0.065* 0.049*

* Significant at P = 0.05 level of probability Cytokinin source was significantly different for callus production in B5, SH, and MS basal salts but not L2. The

2.0 mg L-' kinetin treatment was superior to 0.10 mg L-1 BAP.

Analysis of variance showed highly significant effects of callus induction media (CIM), embryo induction media (EIM), and CIM by EIM interaction. These results revealed that both CIM and EIM as well as the CIM by EIM interaction








39

were important in the number of embryos per explant.

Because there was an interaction between CIM and EIM,

further analysis was necessary to determine the differences

in response related to CIM and EIM combination (Table 3.8).


Table 3.8. Mean number of embryos per explant for 8 weeks of culture with fixed callus induction media

Embryo Callus induction media (CIM) induction Mean media (EIM) B5C(5) B5C(6) L2C(2) SHC(8)

It 36 a 43 a 28 a 19 a 32 a 2 22 ab 41 a 43 a 18 a 31 ab 3 30 a 47 a 21 a 23 a 30 ab
4 21 ab 23 a 33 a 18 a 24 c 5 23 ab 36 a 25 a 10 a 23 c 6 3 b 33 a 45 a 21 a 26 bc

Mean 23 B 37 A 32 A 18 B

Means within a column followed by the same lower case letter are not different (P = 0.05). Means within a row followed by the same upper case letter are not different (P = 0.05). t 1 = basal salt without growth regulators
2 = basal salt with 0.01 mg L-1 2,4-D + 2.0 mg L-' adenine
3 = basal salt with 0.01 mg L-' 2,4-D + 0.1 mg L-1 BAP 4 = basal salt with 2.0 mg L-1 NAA + 2.0 mg L-1 adenine 5 = basal salt with 2.0 mg L-1 NAA + 2.0 mg L-1 kinetin
6 = basal salt with 0.002 mg L-1 picloram + 0.2 mg L-1 BAP.


When EIM were evaluated within each CIM, the number of

embryos was significantly different only within B5C(5).

Over all CIM, EIM l(without growth regulators) had the

highest number of embryos per explant, although it was not

significantly different from EIM 2(0.01 mg L-1 2,4-D and 2.0

mg L-1 adenine) and EIM 3(0.01 mg L-1 and 0.1 mg L-1).








40

The analysis of variance for number of embryos per

explant after 12 weeks in culture showed significant effects

(P = 0.01) EIM and EIM by embryo development media (EDM)

interaction. No significant main effects for EDM were

observed. Because there was interaction between EIM and

EDM, further analysis was conducted to examine differences

within each EIM (Table 3.9).


Table 3.9. Mean number of embryos per explant for 12 weeks of culture with fixed embryo induction media

Embryo Embryo induction media (EIM) development Mean media (EDM) it 2 3 4 5

1* 55 a 36 a 39 a 41 a 62 a 47 a 2 17 b 28 a 39 a 39 a 73 a 39 a 3 26 b 39 a 51 a 34 a 69 a 44 a 4 30 b 44 a 33 a 50 a 69 a 45 a

Mean 32 B 37 B 40 B 41 B 68 A

Means within a column followed by the same lower case letter are not different (P = 0.05). Means within a row followed by the same upper case letter are not different (P = 0.05). t 1 = B5C(5)-B5 basal salt without growth regulators
2 = B5C(5)-B5 basal salt + 2.0 mg L-1 each of NAA and
adenin
3 = B5C(6)-B5 basal salt + 0.01 mg L-1 2,4-D + 2.0 mg L-1
adenin
4 = B5C(6)-B5 basal salt + 0.002 mg L-1 picloram + 0.2 mg
L- BAP
5 = L2C(2)-L2 basal salt + 0.002 mg L-' picloram + 0.2 mg
L- BAP
t 1 = basal salt without growth regulators
2 = basal salt + 0.2 mg L- NAA
3 = basal salt + 0.002 mg L-' picloram + 0.2 mg L-1 BAP 4 = basal salt + 0.002 mg L- picloram + 0.1 mg L- BAP








41

When EDM were evaluated within each EIM, number of embryos were significantly different only within EIM 1 [ B5C(5)-B5 without growth regulators]. Over all EIM, EDM 1 (without growth regulators) had the highest number of embryos per explant, although it was not significantly different from three others. Over all EDM, EIM 5 i.e. L2C(2)-LSE(6) had the highest number of developing embryos per explant. The protocol L2C(2)-LSE(6)-L2(20) was selected from this experiment for further testing. Experiment III


For the response variable callus fresh gain weight (g) after 4 weeks in culture, the analysis of variance showed significant genotypes effects. No significant effects were observed for media or for the interaction of media by genotype. However, since the focus of this research was medium optimization, these differences are only of marginal interest. It is important that the genotype by media component was not significant.

The analysis of variance for number of embryos at eight weeks showed highly significant differences among media. After 4 weeks, there were no genotype by media interaction effects. There were also no significant differences among genotypes. Media sequence L2C(2)-LSE(6) yielded the highest number of embryos per explant (Table 3.10).








42

Table 3.10. Mean number of embryos per explant on different media at 8 weeks of culture Media Number of embryo per explant B5C-LSP 78 b L2C(2)-LSE(6) 120 a B5C-B5E 84 b Means followed by the same letter are not different (P =
0.05).


For the variable number of developing embryos at twelve weeks of culture, differences were observed among genotypes, and among media system. The medium combination L2C(2)LSE(6)-L2(20) gave the highest number of developing embryos per explant, while B5C-B5E-B5R was the poorest in number of developing embryos per explant (Table 3.11).


Table 3.11. Mean number of embryos per explant over genotypes on different media at 12 weeks of culture Media Number of embryo per explant B5C-LSP-LSP 126 b L2C(2)-LSE(6)-L2(20) 175 a B5C-B5E-B5R 88 b Means followed by the same letter are not different (P =
0.05)


The finding of no genotype by media interaction again suggests that this group of genotypes responds similarly on the three media. Mean number developing embryos per explant varied among genotypes from 112 to 163 (data not shown).








43

Experiment IV


No significant differences among media or genotypes for callus fresh weight gain at four weeks of culture were observed. This finding is of interest since the genotypes were selected for variable regeneration response. This finding suggests that callus mass may not be related to regeneration response in this group of genotypes.

This result is similar to Myers et al. (1989), who

found that cell growth and protoplast division frequency in vitro were not associated with regeneration potential in the red clover genotypes which they tested. A high rate of callus growth in culture could be ideal for regeneration to take place, but not entirely necessary. Some of their genotypes which produced the most callus had a low frequency of regeneration, and their most highly regenerable genotype showed only average division frequencies in protoplast culture.

After eight weeks of culture, highly significant

differences in number of embryos per explant were observed among genotypes and among media, but there were no genotype by media interactions. Media system L2C(2)-LSE(6) yielded the highest number of embryos per explant although it was not significantly different to media system 1 (B5C-LSP) (Table 3.12).








44

Table 3.12. Mean number of embryos per explant at 8 weeks of culture

Media Number of embryo per explant

B5C-LSP-LSP 45 ab L2C(2)-LSE(6)-L2(20) 53 a B5C-B5E-B5R 37 b

Means followed by the same letter are not different (P =
0.05).


The fact that the standard B5 medium on which this germplasm

was developed yielded the lowest number of embryos shows

that either of the newly selected media may be superior to

the B5 medium. Genotype C54TC resulted in the highest

number of embryos per explant (Table 3.13), while NEWRC 4

was the lowest in the number of embryos per explant.


Table 3.13. Mean number of embryos per explant on different genotypes at 8 weeks of culture

Genotype Number of embryo per explant

7-1-5-36 66 b Selected as high regeneration 7-1-5-34 61 b C34TC 65 b C54TC 89 a
-----------------------------------------------------------FLMR6-23-3 22 c Selected as intermediate C64TC 71 ab regeneration C74TC 69 b
----------------------------------------------------------NEWRC 1 4 cd Selected as low regeneration NEWRC 4 2 d NEWRC 30 4 cd

Means followed by the same letter are not different (P =
0.05).








45

The analysis of variance at twelve weeks of culture for number of developing embryos per explant showed highly significant differences among genotypes and among the media systems tested. No significant differences were observed for genotype by media interaction. Media L2C(2)-LSE(6)L2(20) again yielded the highest number of embryos per explant, although it was not significantly different from media system B5C-LSP-LSP (Table 3.14).


Table 3.14. Mean number of embryos per explant on different media sequences at 12 weeks of culture Media Number of embryos per explant B5C-LSP-LSP 62 a L2C(2)-LSE(6)-L2(20) 66 a B5C-B5E-B5R 51 b Means followed by the same letter are not different (P =
0.05).


Genotype 7-15-36 had the highest number of developing embryos per explant, while genotype NEWRC 30 was the lowest in the number of developing embryos per explant (Table

3.15). When genotypes were evaluated within each medium, the number of embryos per explant was significantly different within each medium (Table 3.16). Furthermore, genotype C64TC and C74TC, which previously was rated as intermediate regeneration on media III (B5 protocol) showed increased regeneration on either media I (B5C-LSP-LSP) or media II [L2C(2)-LSP-L2(20)].








46

Table 3.15. Mean number of embryos per explant on different genotypes at 12 weeks of culture

Genotype Number of embryos per explant

7-1-5-36 107 a Selected as high regeneration 7-1-5-34 74 c C34TC 93 ab C54TC 109 a

FLMR6-23-3 26 d Selected as intermediate C64TC 87 bc regeneration C74TC 88 bc

NEWRC 1 5 e Selected as low regeneration NEWRC 4 4 e NEWRC 30 3 e

Means followed by the same letter are not different (P =
0.05).


Table 3.16. Mean number of embryos per explant on different genotypes within each media system

Genotype Mediat

I II III

7-1-5-36 134 a 104 a 85 ab 7-1-5-34 62 c 86 a 69 ab C34TC 85 bc 108 a 86 ab C54TC 103 b 121 a 103 a
--------------------------------------------------------FLMR6-23-3 21 d 32 b 30 c C64TC 102 b 104 a 56 b C74TC 98 b 95 a 70 ab
--------------------------------------------------------NEWRC 1 8 d 4 b 2 d NEWRC 4 3 d 5 b 4 d NEWRC 30 2 d 5 b 1 d

Means followed by the same letter are not different (P =
0.05).
t I = B5C-LSP-LSP; II = L2C-LSE(6)-L2(20); III = B5C-B5EB5R.








47

Based upon the results of the four experiments, the optimum media system for plant regeneration in red clover was L2C(2)-LSP-L2(20). L2C(2) was an L2 basal medium supplemented with 2.0 mg L-' each of NAA, 2,4-D, and kinetin for callus induction. After four weeks, the calli were transferred onto L2 basal medium with 0.002 mg L- picloram + 0.2 mg L- BA for embryo induction for another four weeks. For plant regeneration, the L2 basal medium supplemented with 0.002 mg L- picloram + 0.1 mg L-1 BA for four weeks was superior.

The varying levels of regeneration of the genotypes

among the different media indicates that regeneration is at least partially dependent upon the culture environment. The L2 basal salt provided the optimum culture environment for callus induction and regeneration. This basal salt was optimized specifically for in red clover (Phillips and Collins, 1982). In the current study, the critical stage for embryogenesis and/or regeneration was culture on LSP medium using callus which had been induced on the medium containing 2.0 mg L-1 each of 2,4-D, NAA, and kinetin. A similar result was reported earlier by MacLean and Nowak (1989). They reported that somatic embryogenesis occurred on LSP medium after calli had been induced on B5C medium which contained a similar growth regulator composition.














CHAPTER 4
INHERITANCE STUDY OF PLANT REGENERATION IN RED CLOVER


Genetic studies of tissue culture response and regeneration capacity have been conducted by many researchers in numerous crop species; however, only limited studies have been conducted in red clover. An understanding of the genetics of regeneration capacity in red clover would assist in the selection and breeding of genotypes with high regeneration capacity which should facilitate improvement via genetic transformation.

Nuclear genes control in vitro response and plant

development from callus tissue culture and has been reported in various species (Frankenberger et al., 1981; Komatsuda et al., 1989; Chowdhury et al., 1991; Quimio and Zapata, 1990; Zhou and Konzak, 1992; Rakoczy-Trojanowska and Malepszy, 1993). In alfalfa, for example, regeneration was genotypespecific and only a few genotypes in certain cultivars have been isolated with the ability to regenerate plants from explant derived calli (Arcioni et al., 1990a). Two selected genotypes from the cultivar Adriana were shown to regenerate through somatic embryogenesis (Arcioni et al., 1990b).



48








49

Studies of the genetic control of somatic embryogenesis have been carried out in diploid (Reisch and Bingham, 1980) and tetraploid of alfalfa (Wan et al., 1988; HernandezFernandez and Christie, 1989). These studies found this trait to be controlled by at least two independent loci although with different interactions. Selection for regeneration capacity increased the level from 12 to 67% (Bingham et al., 1975) and from the 3% to 50% in cv. Adriana (Arcioni, 1990b).

In red clover, genotypic variation for in vitro plant

regeneration was shown to be conditioned by additive genetic variance (Keyes et al., 1980). This was also a significant source of variability for callus growth, chlorophyll production, and root initiation. Dominant genetic variance was significant only for the traits, callus morphology and root number. Recently, successful recurrent selection for plant regeneration of red clover from callus tissue culture has been achieved (Quesenberry and Smith, 1993). With five cycles of recurrent phenotypic selection for increased plant regeneration via somatic embryogenesis from callus tissue culture, they showed an increase in the percentage of regenerating plants from 4 to 72%. The number of regenerating plants per petiole explant was also increased to > 200 for selected individuals compared with <10 in the








50

base population. Narrow sense heritability estimates ranged from 40 to 50%.


Materials and Methods


Mating design


A diallel cross (without parents or reciprocals) of

nine genotypes of red clover, which had exhibited a range in level of plant regeneration response (low, medium and high regeneration capacity) was conducted (Table 4.1). All crosses were performed by hand without emasculation in a greenhouse. The thirty six crosses, each of forty progenies per cross, were evaluated for the response variables callus diameter at four weeks, and number of plants regenerated at 12 weeks on Beach and Smith (1979) media protocol using hypocotyl culture.


Table 4.1. Diallel cross of nine genotypes of red clover No. Parent Reg.t 1 2 3 4 5 6 7 8 9
Score

1 NEWRC1 Low - x x x x x x x x 2 NEWRC4 Low - - x x x x x x x 3 NEWRC30 Low - - - x x x x x x 4 NEWRC64 Medium- - - - x x x x x 5 NEWRC74 Medium- - - - - x x x x 6 FL23-3 Medium- - - - - - x x x 7 C34TC High - - - - - - - x x 8 C54TC High - - - - - - - - x 9 7-1-534 High - - - - - - - - x t Regeneration








51

Hypocotyl Culture


The FI seeds were scarified by soaking and stirring in 100% sulphuric acid for five minutes and then rinsed five times with distilled water. The seeds were then soaked and stirred in saturated calcium hypochlorite for five minutes. The seeds were rinsed with sterile, distilled water five times under aseptic conditions. The seeds were then placed on seed germination medium (SGL) at 250C � 20C under continuous light. The SGL medium contained one-tenth concentration of the major salts in the L2 medium. The minor salts and growth regulators were omitted. One-fourth concentration of thiamine, pyridoxine, and myo-inositol of the L2 medium were included (Phillips and Collins, 1982).

After germination (3-5 days), hypocotyls from

developing seedlings were excised into sections (3 to 5 mm long) and transferred to the B5 callus induction medium. A randomized complete block design with 36 F1s from each of 40 progenies was used in this experiment. All explants were incubated for four weeks to induce callus formation and growth. Cultures were maintained at 260C � 20C with a 16-h day, 85 pEm-2s-1. Irradiation was supplied by cool-white fluorescent light. After four weeks, the explants were transferred to B5 embryo induction medium for four additional weeks under the same environmental conditions.








52

The explants were then transferred to the B5 embryo development medium for another four week period.

Callus formation was measured as diameter of callus

(mm) after four weeks of culture on callus induction medium. Plant regeneration capacity was rated from 0 to 6 after twelve weeks of culture. Number of embryoids and/or plantlets were estimated separately for each explant and assigned a rating index number according to the scale:

0 = no embryoids or organized structures formed 1 = 1 to 3 embryos organized structures formed,

2 = 4 to 10 embryos or organized structures formed,

3 = 11 to 25 embryos or organized structures formed, 4 = 26 to 55 embryos or organized structures formed,

5 = 56 to 100 embryos or organized structures formed, and 6 = more than 100 embryos or organized structures formed.

Data for both callus diameter and plant regeneration capacity were analyzed using Griffing's Method IV, fixed model (Griffing, 1956). Differences among cross means and GCA effects were compared by Fisher's protected LSD. SCA effects were tested for difference from zero by the t test at P = 0.05.


Results and Discussions


Callus diameter means of 36 crosses of red cover genotypes 4 weeks after culture on B5 callus induction








53

medium are presented in Table 4.2. F1s with C54TC and 7-15-34 as one parent were among the combinations showing


Table 4.2. Means of callus diameter of 36 crosses of red clover genotypes 4 weeks after culture

Crosses Callus diameter (mm)

NEWRC 1 x NEWRC 4 9.0 NEWRC 1 x NEWRC 30 9.4 NEWRC 1 x NEWRC 64 10.7 NEWRC 1 x NEWRC 74 12.1 NEWRC 1 x FLMR6-23-3 14.4 NEWRC 1 x C34TC 10.7 NEWRC 1 x C54TC 13.5 NEWRC 1 x 7-1-5-34 11.8 NEWRC 4 x NERWC 30 10.8 NEWRC 4 x NEWRC 64 11.8 NEWRC 4 x NEWRC 74 12.2 NEWRC 4 x FLMR6-23-3 13.4 NEWRC 4 x C34TC 11.5 NEWRC 4 x C54TC 13.0 NEWRC 4 x 7-1-5-34 13.0 NEWRC 30 x NEWRC 64 11.1 NEWRC 30 x NEWRC 74 11.4 NEWRC 30 x FLMR6-23-3 10.8 NEWRC 30 x C34TC 11.7 NEWRC 30 x C54TC 12.8 NEWRC 30 x 7-1-5-34 12.3 NEWRC 64 x NERWC 74 12.6 NEWRC 64 x FLMR6-23-3 13.1 NEWRC 64 x C34TC 13.7 NEWRC 64 x C54TC 12.5 NEWRC 64 x 7-1-5-34 13.8 NEWRC 74 x FLMR6-23-3 12.0 NEWRC 74 x C34TC 12.6 NEWRC 74 x C54TC 14.9 NEWRC 74 x 7-1-5-34 15.0 FLMR6-23-3 x C34TC 12.4 FLMR6-23-3 x C54TC 13.4 FLMR6-23-3 x 7-1-5-34 12.7 C34TC x C54TC 13.7 C34TC x 7-1-5-34 13.7 C54TC x 7-1-5-34 16.9

LSD (0.05) 1.3








54

the largest callus diameter (>13.00 mm), and those with NEWRC 1, NEWRC 4, and NEWRC 30 as one parent were among the lowest callus diameter (<12.00mm). C34TC also showed high callus diameter in FI combination with several other parents.

The analysis of variance for general combining ability (GCA) and specific combining ability (SCA) is presented in Table 4.3. The diallel analysis showed highly significant GCA and SCA effects. These results suggest that both additive and non-additive effects may be important in the inheritance of callus diameter of red clover.


Table 4.3. Analysis of variance of general combining ability (GCA) and specific combining ability (SCA) effects for callus formation

Source Degrees of freedom Mean squares Crosses 35 4.8279**
GCA 8 14.4090** SCA 27 1.9891** Error 35 0.4731

** Significant at the P = 0.01


The contribution of the individual genotypes to the F1s' response was determined by comparing the general combining ability effects (Table 4.4). Positive values indicate a contribution towards a larger callus diameter, while negative values indicated a contribution towards a smaller callus diameter. The estimates of GCA effects








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Table 4.4. Estimates of general combining ability (GCA) effects for callus diameter of red clover Parent GCA NEWRC 1 -1.23 NEWRC 4 -0.77 NEWRC 30 -1.38 NEWRC 64 -0.10 NEWRC 74 0.39 FLMR6-23-3 0.31 C34TC 0.01 C54TC 1.52 7-1-5-34 1.28 LSD (0.05) 0.44


indicated that C54TC contributed the most to larger callus diameter. Although the contribution was less, 7-1-5-34 also contributed to large callus diameter. Conversely, NEWRC 1 and NEWRC 30 had relatively high negative effects on callus diameter.

The SCA effects for callus diameter per parental

combination are presented in Table 4.5. Estimates of SCA effects indicated there were five cross combinations that showed positive SCA different from zero. These include C54TC x 7-1-5-34, FLMR6-23-3 x 7-1-5-34, and NEWRC 64 x C34TC. These crosses involved all 7-1-5-34 or C34TC, both of which had high general combining ability for callus diameter. NERWC 1 x FLMR6-23-3 showed a positive and highly significant SCA effect and NEWRC 4 x FLMR6-23-3 showed a positive significant SCA effect, indicating that these crosses performed better than would have been predicted from








56

Table 4.5. Estimates of specific combining ability (SCA) effects for callus diameter for 4 weeks

Crosses SCA

NEWRC 1 x NEWRC 4 -1.54 * NEWRC 1 x NEWRC 30 -0.51 NEWRC 1 x NEWRC 64 -0.48 NEWRC 1 x NEWRC 74 0.41 NEWRC 1 x FLMR6-23-3 2.81 ** NEWRC 1 x C34TC -0.61 NEWRC 1 x C54TC 0.69 NEWRC 1 x 7-1-5-34 -0.77 NEWRC 4 x NERWC 30 0.40 NEWRC 4 x NEWRC 64 0.22 NEWRC 4 x NEWRC 74 0.11 NEWRC 4 x FLMR6-23-3 1.32 * NEWRC 4 x C34TC -0.17 NEWRC 4 x C54TC -0.28 NEWRC 4 x 7-1-5-34 -0.07 NEWRC 30 x NEWRC 64 0.12 NEWRC 30 x NEWRC 74 -0.09 NEWRC 30 x FLMR6-23-3 -0.57 NEWRC 30 x C34TC 0.60 NEWRC 30 x C54TC 0.16 NEWRC 30 x 7-1-5-34 -0.12 NEWRC 64 x NERWC 74 -0.19 NEWRC 64 x FLMR6-23-3 0.39 NEWRC 64 x C34TC 1.27 * NEWRC 64 x C54TC -1.39 * NEWRC 64 x 7-1-5-34 0.07 NEWRC 74 x FLMR6-23-3 -1.22 * NEWRC 74 x C34TC 0.30 NEWRC 74 x C54TC 0.44 NEWRC 74 x 7-1-5-3 0.85 FLMR623-3 x C34TC -0.41 FLMR6-23-3 x C54TC -0.91 FLMR6-23-3 x 7-1-5-34 1.40 * C34TC x C54TC -0.27 C34TC x 7-1-5-34 -0.12 C54TC x 7-1-5-34 1.55 *

*, ** Significant difference from 0 at P = 0.05 and P = 0.01 levels of probability, respectively.


the GCA effects of the parental genotypes. Of particular

interest is the combination of NEWRC1 x FLMR6-23-3, because








57

NEWRC1 had high negative GCA effects, but this cross showed high positive SCA. It may be of importance that FLMR6-23-3 is a selection out of 'Cherokee', a southern U.S. adapted red clover, and is more distantly related to all other plants which trace to the NEWRC germplasm. These results suggest that in these specific combinations dominant or epistatic effects or non-additive genetic variance were probably involved in the inheritance of callus diameter in red clover.

There were three cross combinations that showed

significant negative SCA effects. These included NEWRC 1 x NEWRC 4, NEWRC 64 x C54TC, and NEWRC 74 x FLMR6-23-3. Only one cross involved NEWRC 1 and NEWRC 4, which had poor GCA for callus diameter. The two other cross combinations performed more poorly than would have been predicted from the GCA effects of the parental genotypes; again suggesting involvement of dominant and epistatic effects.

This investigation indicated that both GCA and SCA effects were significant sources of variation for callus formation; however, GCA appeared to be more important than SCA. This study showed that useful sources of genetic variability for callus formation are available in this breeding material. Development of genotypes with good callus growth using methods that exploit additive types of gene action should be effective.








58

Plant regeneration capacity index of 36 crosses of red clover measured after 12 weeks in culture are presented in Table 4.6. Crosses with C54TC, 7-1-5-34, and C34TC as one parent were among the combinations showing the highest regeneration capacity index (>3.50), and those with NEWRC 1, NEWRC 4, and NEWRC 30 as one parent were among the lowest plant regeneration capacity index (<2.50).

The diallel analysis showed highly significant GCA and significant SCA effects (Table 4.7). These results revealed that both additive and non-additive effects were probably important in the inheritance of plant regeneration capacity of red clover, and that additive effects were probably more important than non-additive effects.

The contribution of individual genotypes to the F, response can be seen by comparing the general combining ability effects (Table 4.8). Positive values indicate a contribution towards greater regeneration capacity of red clover, while negative values indicate a contribution towards smaller regeneration capacity. The estimates of GCA effects indicated that C54TC contributed the most to greater regeneration capacity. Although its contribution was smaller, 7-1-5-34 also contributed to regeneration capacity. Conversely, NEWRC 1, NEWRC 4, and NEWRC 30 had a similar and negative effect on regeneration capacity.








59

Table 4.6. Regeneration capacity index of 36 crosses of red clover genotypes 12 weeks after culture

Crosses Regeneration capacity index

NEWRC 1 x NEWRC 4 0.9 NEWRC 1 x NEWRC 30 1.1 NEWRC 1 x NEWRC 64 2.5 NEWRC 1 x NEWRC 74 2.4 NEWRC 1 x FLMR6-23-3 2.6 NEWRC 1 x C34TC 2.1 NEWRC 1 x C54TC 3.4 NEWRC 1 x 7-1-5-34 2.4 NEWRC 4 x NERWC 30 1.3 NEWRC 4 x NEWRC 64 2.1 NEWRC 4 x NEWRC 74 2.4 NEWRC 4 x FLMR6-23-3 2.8 NEWRC 4 x C34TC 2.4 NEWRC 4 x C54TC 3.5 NEWRC 4 x 7-1-5-34 3.0 NEWRC 30 x NEWRC 64 1.9 NEWRC 30 x NEWRC 74 2.3 NEWRC 30 x FLMR6-23-3 2.3 NEWRC 30 x C34TC 2.3 NEWRC 30 x C54TC 3.6 NEWRC 30 x 7-1-5-34 3.1 NEWRC 64 x NERWC 74 3.1 NEWRC 64 x FLMR6-23-3 3.0 NEWRC 64 x C34TC 3.5 NEWRC 64 x C54TC 2.9 NEWRC 64 x 7-1-5-34 3.4 NEWRC 74 x FLMR6-23-3 2.7 NEWRC 74 x C34TC 2.7 NEWRC 74 x C54TC 4.3 NEWRC 74 x 7-1-5-34 3.7 FLMR623-3 x C34TC 2.5 FLMR6-23-3 x C54TC 3.5 FLMR6-23-3 x 7-1-5-34 2.6 C34TC x C54TC 3.9 C34TC x 7-1-5-34 3.4 C54TC x 7-1-5-34 4.9

LSD (0.05) 0.6








60

Table 4.7. Analysis of variance of general combining ability (GCA) and specific combining ability (SCA) effects for regeneration capacity Source Degrees of freedom Mean squares Crosses 35 1.5177 **
GCA 8 5.7782 **
SCA 27 0.2755 * Error 35 0.0926

*, ** Significant at the P = 0.05 and P = 0.01, respectively


Table 4.8. Estimates of general combining ability (GCA) effects for regeneration capacity of red clover Parent GCA NEWRC 1 -0.82 NEWRC 4 -0.59 NEWRC 30 -0.68 NEWRC 64 -0.01 NEWRC 74 0.19 FLMR6-23-3 0.25 C34TC -0.01 C54TC 1.10 7-1-5-34 0.56 LSD (0.05) 0.19


Estimates of SCA effects indicated three cross

combinations FLMR6-23-3 x 7-1-5-34, NEWRC 64 x C34TC, and NEWRC 64 x C54TC showed significant positive SCA effects (Table 4.9). Two of these three crosses involved 7-1-5-34 and C34TC, which also had good general combining ability for plant regeneration capacity index. These two parents also had high GCA for callus growth. The cross of NERWC 1 x NEWRC 4 showed a significant negative SCA effect, indicating that this cross performed more poorly than would have been








61

been predicted from the GCA effects of the parental genotypes, although both parents also had highly negative GCA effects. These results suggest that in this specific combination SCA effects due non-additive genetic variance are probably involved in the inheritance of plant regeneration capacity of red clover.

This investigation indicated that GCA and SCA were

significant sources of variation, and SCA was a significant source of variation for both callus diameter and plant regeneration. Although SCA effects were significant for a few crosses, the results generally showed that additive genetic effects were most important in conditioning inheritance of these responses. This study showed that useful sources for plant regeneration are available in this breeding material for the development of responsive plant regeneration genotypes.








62

Table 4.9. Estimates of specific combining ability (SCA) effects for regeneration capacity

Crosses SCA

NEWRC 1 x NEWRC 4 -0.75 * NEWRC 1 x NEWRC 30 -0.37 NEWRC 1 x NEWRC 64 0.39 NEWRC 1 x NEWRC 74 0.22 NEWRC 1 x FLMR6-23-3 0.42 NEWRC 1 x C34TC -0.12 NEWRC 1 x C54TC 0.36 NEWRC 1 x 7-1-5-34 -0.18 NEWRC 4 x NERWC 30 -0.19 NEWRC 4 x NEWRC 64 -0.09 NEWRC 4 x NEWRC 74 0.05 NEWRC 4 x FLMR6-23-3 0.32 NEWRC 4 x C34TC 0.24 NEWRC 4 x C54TC 0.15 NEWRC 4 x 7-1-5-34 0.24 NEWRC 30 x NEWRC 64 -0.22 NEWRC 30 x NEWRC 74 0.02 NEWRC 30 x FLMR6-23-3 -0.09 NEWRC 30 x C34TC 0.14 NEWRC 30 x C54TC 0.38 NEWRC 30 x 7-1-5-34 0.32 NEWRC 64 x NERWC 74 0.15 NEWRC 64 x FLMR6-23-3 -0.03 NEWRC 64 x C34TC 0.69 * NEWRC 64 x C54TC 0.96 * NEWRC 64 x 7-1-5-4 0.04 NEWRC 74 x FLMR6-23-3 -0.53 NEWRC 74 x C34TC -0.28 NEWRC 74 x C54TC 0.26 NEWRC 74 x 7-1-5-34 0.09 FLMR623-3 x C34TC -0.64 FLMR6-23-3 x C54TC -0.63 FLMR6-23-3 x 7-1-5-34 1.06 ** C34TC x C54TC -0.08 C34TC x 7-1-5-34 0.02 C54TC x 7-1-5-34 0.49

*, ** Significant difference from 0 at P = 0.05 and P = 0.01 levels of probability, respectively.














CHAPTER 5

SUMMARY AND CONCLUSION


The overall objectives of this research were to

optimize a tissue culture media sequence for in vitro plant regeneration of red clover, and to obtain information concerning the genetic basis of callus formation and plant regeneration response of nine red clover genotypes.

Four experiments were conducted to optimize the culture system for red clover regeneration in vitro. In Exp. I, six genotypes of red clover were tested for tissue culture responses in six previously published media systems. The best regeneration response was observed on the sequence B5C-LSP-LSP. The B5C medium contained B5 basal salts supplemented with 2.0 mg L-' each of 2,4-D, NAA, and kinetin for callus induction. After four weeks, calli were then transferred to LSP medium (L2 basal medium with 0.002 mg L-1 picloram plus 0.2 mg L-1 BAP) for embryo induction and development. The explants were again transferred to LSP medium for four weeks for plant regeneration .

In Exp. II, six genotypes were tested on media which had different combinations of auxin and cytokinin sources 63








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and levels. Optimum regeneration in vitro was observed on the sequence L2C(2)-LSP-L2(20) with four weeks interval. L2C(2) medium contained L2 basal salt supplemented with 2.0 mg L-1 each of 2,4-D, NAA, and kinetin for callus induction. L2(20) contained L2 basal salt with 0.002 mg L-' of picloram and 1.0 mg L'1 of BA.

In Exp. III, the above two media systems selected in Exps. I, and II, and the B5 protocol were tested on six genotypes of NEWRC. The media system L2C(2)-LSP-L2(20) from Exp. II gave the best plant regeneration response over genotypes.

In Exp. IV, the same procedure as Exp.III was applied to ten genotypes of red clover which had exhibited a range in levels of plant regeneration capacity (high, medium, and low regeneration capacity) on B5 protocol. The media system L2C(2)-LSP-L2(20) was again the best for plant regeneration in vitro in red clover. With this protocol genotypes which had exhibited an intermediate regeneration response on the B5 protocol showed increased regeneration.

Progenies of 36 crosses of nine red clover genotypes were evaluated for callus formation and plant regeneration capacity using hypocotyl culture on B5 protocol. Diallel analysis was performed on callus diameter after 4 weeks in culture and plant regeneration response after 12 weeks of








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culture. Based upon results obtained from the diallel analysis, crosses with C54TC, 7-1-5-34, or C34TC as one parent were among the combinations showing the highest callus diameter and regeneration capacity, and those with NEWRC1, NEWRC4, or NEWRC30 as one parent were among the lowest for callus diameter and regeneration capacity. The diallel analysis showed highly significant GCA and SCA effects for callus diameter. Estimates of GCA effects indicated that C54TC had a significant, positive GCA effect, suggesting that C54TC contributed the most to high callus diameter in its progenies. Estimates of SCA effects indicated that there were five cross combinations that showed significant positive effects for callus diameter.

Highly significant GCA effects for regeneration

capacity were observed, as well as significant SCA effects. Estimates of GCA effects indicated that C54TC and 7-1-5-34 contributed the most to high regeneration capacity in their progenies. Estimates of SCA effects indicated there were three cross combinations that showed significant positive effects for regeneration capacity of red clover.

The conclusions drawn based upon the results of these studies were as follows: (1) The optimum media system for plant regeneration capacity in red clover was an L2 basal medium supplemented with 2.0 mg L-1 each of NAA, 2,4-D, and kinetin for callus induction for four weeks, with 0.002 mg








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L-1 picloram + 0.2 mg L' BA for embryo induction and regeneration for four weeks, and for another four weeks in medium supplemented with 0.002 mg L-' picloram + 0.1 mg LBA for plant regeneration; (2) Both GCA and SCA were significant sources of variation for response of red clover genotypes in tissue culture, both for callus diameter and regeneration capacity; however, GCA appeared to be more important than SCA and; (3) Useful sources of plant regeneration in vitro were observed in the red clover genotypes tested.















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BIOGRAPHICAL SKETCH


Yuyu Suryasari Poerba was born on November 23, 1961, in Sukabumi, West Java, Indonesia. She graduated from high school in 1980 in Sukabumi. She then attended the University of Padjadjaran in Bandung, Indonesia from 1980 to 1984, to study agriculture. She received a bachelor of science degree in agriculture, 'Sarjana Pertanian', in December 1984. In April 1985, she began working in the Indonesian Institute of Sciences, at the Plant Genetics Laboratory in Bogor. During the first year, she received a scholarship to continue her study. In August 1986, she enrolled in Mississippi State University to begin working towards her Master of Science degree in Genetics. During her study, she was married to Desendi Poerba on August 18, 1988. After completing her study in 1989, she went back to her country and continued working in the same institution. On April 18, 1991, the couple were fortunate to have their first son, Yudson, enter their lives. During her son's first year of age, she again received a scholarship to pursue her Ph.D. degree. In August 1992, she came to the University of Florida to continue working towards her Ph.D


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in Agronomy. Her area of specialization is plant breeding and genetics, with a minor in environmental horticulture. On February 1993, she was reunited with her family. The family was again fortunate to have Deyson, who was born prematurely on April 13, 1994. She plans to return to her institution in Indonesia, upon completion of her degree.








I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



Kenneth H. Quesenberry, Chair Professor of Agronomy

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



David S. Woffo pd
Associate Professor of
Agronomy

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequatg9 in scope and quality, as a dissertation for the degree of 'qdctor of P4sopy.



Paul L. Pfahle
Professor of Agronomy

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



Mighael E. Kane
Associate Professor of
Horticultural Science








I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



e W. Grosser
rofessor of Horticultural Science

This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor Philosophy.


May 1996
Dean, College of Agriculture




Dean, Graduate School




Full Text

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OPTIMIZATION OF TISSUE CULTURE MEDIUM FOR PLANT REGENERATION AND INHERITANCE STUDIES OF PLANT REGENERATION IN RED CLOVER By YUYU SURYASARI POERBA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1996

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ACKNOWLEDGMENTS I wish to express my deepest gratitude to Dr. Kenneth H. Quesenberry, supervisory committee chairman, for his support, guidance, help, and encouragement throughout my Ph.D. program. I would like also to sincerely thank my supervisory members, Dr. Paul L. Pfahler, Dr. David S. Wofford, Dr. Michael E. Kane, and Dr. Jude W. Grosser, for their advice, suggestions, guidance, and support throughout my program. I am very grateful to all my committee members for showing such interest . I would also like to extend my special thanks to Ms. Loan Ngo and Mr. David Moon for their help in the laboratory and in the greenhouse; to my fellow students for their help, support, and encouragement; and to all the staff of the Agronomy Department for helping me throughout my study. I certainly appreciates the opportunity for this graduate study given by the Indonesian Institute of Sciences and the Agency for the Assessment and Application of Technology. I would like to deeply thank my parents, brothers, sisters, and all my in laws, for their encouragement and support of my study. Finally, I would like to express my ii

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deepest gratitude and appreciation to my husband, Desendi Poerba, for being here throughout the study, help, love, support, encouragement, and making life so much easier and enjoyable; and to our sons, Yudson and Deyson, for making life so precious and joyful.

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS ii LIST OF TABLES vi ABSTRACT viii CHAPTERS 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 4 Red Clover Genetics 4 Red Clover Tissue Culture Medium Development. 5 Genetic Studies of Tissue Culture Response... 13 Quantitative Genetic Studies 14 Mendelian Genetic Studies 15 Forage Legumes In vitro Selection Response... 17 3 OPTIMIZATION OF TISSUE CULTURE MEDIUM FOR PLANT REGENERATION OF RED CLOVER 22 Materials and Methods 25 General Procedure 25 Experiment 1 26 Experiment II 28 Experiment III 32 Experiment IV 32 Results and Discussion 33 Experiment 1 33 Experiment II 36 Experiment III 41 Experiment IV 43 4 INHERITANCE STUDY OF PLANT REGENERATION IN RED CLOVER 48 Materials and Methods 50 Mating Design 50 Hypocotyl Culture 51 Results and Discussion 52 iv

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5 SUMMARY AND CONCLUSION 63 REFERENCE LIST 67 BIOGRAPHICAL SKETCH 73 v

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LIST OF TABLES Xahle. page 3.1 Media used in red clover regeneration as proposed in the literature 27 3.2 Media systems used in regeneration study of red clover 27 3.3 Callus fresh weight gain on different media with fixed genotypes 33 3.4 Number of embryos per explant on different media with fixed genotypes 35 3.5 Number of developing embryos per explant on different media with fixed genotypes 36 3.6 Callus fresh weight gain for 4 weeks of culture with fixed growth regulator combinations 37 3.7 Mean squares of callus weight gain in different auxin and cytokinin sources with fixed basal salts 38 3.8 Mean number of embryos per explant for 8 weeks of culture with fixed callus induction 39 3.9 Mean number of embryos per explant for 12 weeks of culture with fixed embryo induction media 40 3.10 Mean number of embryos per explant on different media at 8 weeks of culture 42 3.11 Mean number of embryos per explant over genotypes on different media at 12 weeks of culture 42 3.12 Mean number of embryos per explant at 8 weeks of culture 44 3.13 Mean number of embryos per explant on different genotypes at 8 weeks of culture 44 vi

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3.14 Mean number of embryos per explant on different media sequences at 12 weeks of culture 45 3.15 Mean number of embryos per explant on different genotypes at 12 weeks of culture 46 3.16 Mean number of embryos per explant on different genotypes within each media system 4 6 4.1 Diallel cross of nine genotypes of red clover 50 4.2 Mean callus diameters of 36 crosses of red clover genotypes 4 weeks after culture 53 4.3 Analysis of variance of general combining ability (GCA) and specific combining ability (SCA) effects for callus formation 54 4.4 Estimates of general combining ability (GCA) effects for callus diameter red clover 55 4.5 Estimates of specific combining ability (SCA) effects for callus diameter for 4 weeks 56 4.6 Regeneration capacity index of 36 crosses of red clover genotypes 12 weeks after culture 59 4.7 Analysis of variance of general combining ability (GCA) and specific combining ability (SCA) effects for regeneration capacity 60 4.8 Estimates of general combining ability (GCA) effects for regeneration capacity of red clover... 60 4.9 Estimates of specific combining ability (SCA) effects for regeneration capacity of red clover. . . 62 vii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy OPTIMIZATION OF TISSUE CULTURE MEDIUM FOR PLANT REGENERATION AND INHERITANCE STUDIES OF PLANT REGENERATION IN RED CLOVER By Yuyu Suryasari Poerba May 1996 Chairman: Dr. Kenneth H. Quesenberry Major Department: Agronomy Red clover (Trifolium pratense L.) is an important forage species grown in temperate climates around the world. One of the problems encountered in red clover tissue culture is low plant regeneration frequency. A series of experiments were conducted to optimize tissue culture media for plant regeneration and to investigate the genetic basis of in vitro regeneration of red clover. An experiment to evaluate previously published plant regeneration protocols showed that the superior treatment was B5C (B5 basal medium plus 2.0 mg L" 1 each of NAA , 2,4-D, and kinetin) for callus induction, LSP (L2 basal medium plus 0.002 mg L" 1 picloram and 0.2 mg L" 1 BA) for shoot induction, and LSP for plant regeneration, each with a 4-week interval. An experimentally selected optimum media protocol for red clover regeneration was L2 basal viii

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medium supplemented with 2 . 0 mg IT 1 NAA, 2,4-D, and kinetin for callus induction, with 0.002 mg IT 1 picloram + 0.2 mg IT 1 BA for embryo induction, and L2 basal medium supplemented with 0.002 mg If 1 picloram + 0.1 mg IT 1 BA for regeneration. With this protocol genotypes with an intermediate regeneration response on the B5 protocol showed increased regeneration. Nine genotypes of red clover, with a range in level of plant regeneration (low, intermediate, and high regeneration) on the B5 protocol, were used in the inheritance study of in vitro plant regeneration. Progenies of 36 crosses were tested for callus formation at four weeks of culture and plant regeneration at 12 weeks of culture using hypocotyl explants. Diallel analysis was performed on these data using Griffing's Method 4, Model I. Results obtained from the diallel experiment showed that crosses with C54TC, C34TC, or 7-1-5-34 as one parent were among the combinations showing the highest callus diameter and regeneration capacity. Those crosses with NEWRC1, NEWRC4, or NEWRC30 as one parent were among the lowest callus diameter and regeneration response. Both general combining ability and specific combining ability (SCA) were significant sources of variation for callus diameter and for plant regeneration. Although SCA effects were significant for a few crosses, the results generally showed that additive genetic effects were most important in controlling inheritance of these responses. ix

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CHAPTER 1 INTRODUCTION Plant regeneration is a vital objective of tissue culture, and is necessary for the application of molecular and somatic cell genetics (genetic transformation and somatic hybridization) to crop improvement. An understanding of the role of the genotype in the regeneration is important to allow selection of clones with superior regeneration capacity for specialized experiments. This understanding can be used to select clones that regenerate, and to enhance the efficiency of regeneration. Selection of suitable genotypes as well as tissue culture procedures, therefore, is essential in regeneration and genetic analysis of plants following in vitro selection methods. Desirable variation can be separated from undesirable by genetic analysis and breeding. Knowledge of the inheritance of regeneration capacity and of somaclonal variation should aid in finding and exploiting variation and in conducting transformation experiments. Red clover is one of the few major crop species where most all phases of tissue culture, plant regeneration, and molecular genetic engineering can be performed. Although

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2 considerable research has been conducted on tissue culture of red clover, most of the media protocols for red clover are genotype-dependent; not all genotypes respond similarly to a particular media system. By developing tissue culture media on which a broad spectrum of genotypes will respond, in vitro plant regeneration would not be an obstacle in plant transformation studies. Genetic studies of tissue culture response and regeneration capacity have conducted by many researchers in crop species; however, these studies have lagged behind in red clover. Understanding the genetic mechanism of tissue culture response and regeneration capacity in red clover would assist in the selection and breeding of genotypes with high regeneration capacity. This would facilitate improvement via genetic transformation to be achieved effectively and efficiently. Genotypic variation for in vitro plant regeneration in red clover was ascribed to the occurrence of additive genetic variance (Keyes et al., 1980). This trait was reported to be highly heritable and was responsive to recurrent selection (Quesenberry and Smith, 1993) . In this study, the optimization of tissue culture media and the role of genetics in plant regeneration of red clover were investigated using petiole and hypocotyl callus

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cultures. A three step media protocol (i.e., callus induction media, embryo induction media, and embryo development media, each of four-week intervals) was used throughout these studies. The first part of the research was a series of tissue culture media sequence optimization experiments to improve plant regeneration of red clover. These studies were conducted using genotypes which had been selected for high regeneration capacity on the B5 media protocol (Quesenberry and Smith, 1993) . The second part was a genetic study of plant regeneration from hypocotyl callus of nine red clover genotypes which had a range in levels of regeneration (high, medium, and low regeneration capacity) on B5 media protocol.

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CHAPTER 2 LITERATURE REVIEW Red Clover Genetics Red clover is an important cross-pollinated forage species grown in temperate climates around the world. In the natural form, it is a highly self-incompatible diploid (2n=2x=14) . Red clover has very stable meiotic associations and is apparently sexually isolated from all other species of the genus Trifolium (Cope and Taylor, 1985) . There are no naturally occurring polyploid forms, although chemically and sexually induced tetraploids (2n=4x=28) exist. The chemically induced tetraploids are grown predominantly in Europe, where they were first developed (Smith et al., 1985) . They produce more forage and are superior in quality, persistence, and disease resistance compared with their diploid counterparts in Europe. Chemically induced red clover tetraploids are produced using either colchicine or N 2 0 (Taylor et al., 1976). Sexually induced tetraploids have been produced by the union of gametes in which one gamete (unilateral, 2x-4x or 4x-2x) or both gametes (bilateral, 2x-2x) contribute the sporophytic chromosome number (2n) (Meglic and Smith, 1992).

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Red clover is a short-lived perennial and its usefulness is limited by its lack of persistence (Phillips et al., 1982). A recent report by Phillips et al.(1992) indicated that interspecific hybridization between red clover and T. alpestre L. (diploid strongly perennial species) using in vitro embryo rescue techniques was successful, although the hybrids (2n=2n+l=15) were functionally sterile. The hybrid resembled T. alpestre more closely than the paternal parent, red clover. If the hybrid infertility barriers could be overcome through backcrossing, these wide hybrids potentially offer new genetic variability for the improvement of red clover (Phillips et al., 1992). The perennial growth habit of red clover causes plants to be exposed to pathogens and other pests in the environment for several years. Several different fungal, bacterial, and viral diseases have been identified as factors limiting production and persistence. Incorporation of pest resistance genes by genetic transformation appears to be a valuable technique that can be used in red clover (Quesenberry et al., 1992). Red Clover Tissue CnlhirP Medium Development One of the problems encountered in genetic transformation experiments is a low frequency plant

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6 regeneration either from callus or cell suspensions. As early as 1979, research showed that whole plant regeneration could be achieved from tissue cultures of various Trifolium species (Collins et al., 1979; Collins and Phillips, 1980). Beach and Smith (1980) demonstrated whole plant regeneration from tissue cultures of T. pratense L. and T. incarnatum L. using Gamborg's B5 media (Gamborg et al., 1976) with growth regulator modifications. However, plant regeneration using most of these systems was at a low frequency and primarily from unadapted plant introduction material or wild Trifolium species (Quesenberry et al., 1992). The capacity of gametophytic or sporophytic cells to form in vitro shoots or embryos which are competent to develop i.e. to regenerate, is a particular feature of plants. Plant regeneration via in vitro somatic embryogenesis and organogenesis has been used to regenerate a wide variety of plant species (Tisserat et al., 1979; Williams and Maheswaran, 1986) . However, many agronomically-important crops still cannot be regenerated from culture, and others regenerate only at low frequency (McGee et al., 1989; Williams and Maheswaran, 1989). The levels of shoot or embryo induction and plant regeneration from in vitro tissue cultures are basically influenced by the genotype and the physiological status of

PAGE 16

7 the donor plant, the source of explant tissues within the plant, the culture medium and culture conditions, and the interaction between all these factors (McGee et al., 1989; Tisserat et al., 1979; Williams and Maheswaran, 1986; Mathias and Simpson, 1986; Lazar et al., 1984a; Bregitzer, 1992) . In red clover, protocols have been developed for somatic embryogenesis and organogenesis that involve incubation on a series of different media for callus initiation, embryo induction, embryo development or shoot development, and plant regeneration (Beach and Smith, 1979, Grosser and Collins, 1984; Phillips and Collins, 1979, 1980, 1982; Collins and Phillips, 1982, Bhojwani et al., 1984; MacLean and Nowak, 1989) ; and for direct somatic embryogenesis from immature embryos (Maheswaran and Williams, 1984, 1986) or from petiole segments derived from established shoot cultures (McGee et al., 1989). Beach and Smith (1979) developed a protocol specific for callus induction, and plant regeneration of red clover from seedling hypocotyls and excised pistils of two cultivars on B5 medium (Gamborg et al., 1968). The callus induction medium was B5 medium supplemented with 10 uM each of 2,4-dichloro phenoxy acetic acid (2,4-D), a-naphthaline acetic acid (NAA) , and 6-furfuryl amino purine (kinetin) . Hypocotyls produced more callus than pistils. Shoot buds

PAGE 17

formed on this medium in some cases. After four to five weeks, callus was transferred to B5 medium containing twice the normal concentration of thiamine, 10 uM NAA, and 15 uM adenine. Numerous shoots developed. Rooting of shoots was accomplished on B5 medium containing the higher concentration of thiamine and 1.1 uM NAA. The SH medium (Schenck and Hildebrandt, 1972) was a suitable substitute for B5 basal medium in callus induction. Most of the regenerated plants were normal, fertile diploids. A few abnormal, sterile plants were recovered. Phillips and Collins (1979) investigated the growth of callus from seedling sections of five cultivars. Visual ratings of various combinations of NAA, indole-3-acetic acid (IAA), p-chlorophenoxy acetic acid (CPA), 2,4-D, 4-amino3, 5, 6-trichloro picolinic acid (picloram) , kinetin, 6(y,Y dimethylallylamino) purine (2iP) , and 6-benzylamino purine (BAP) on the SH (Schenk and Hildebrandt, 1972), B5, and Murashige and Skoog (MS) (1962) basal media were carried out. The combination of 0.25 uM of picloram and 0.44 uM of BAP was optimal for callus initiation and cell proliferation. However the basal media were unsatisfactory. An improved basal medium (L2) was experimentallydeveloped (Phillips and Collins, 1979) . The L2 medium contained a lower concentration of NH 4 + and higher concentrations of P0 3 ", K + , Mg 2 + , and Ca 2 + than that of MS

PAGE 18

9 medium. The minor salts formulation was similar to that of SH medium, with adjustment in the concentration of several salts. The organic formulation (vitamins and sucrose) was similar to that of Linsmaier and Skoog (1965), with higher concentrations of thiamine and myo-inositol . Fresh weight increases were evaluated comparing the L2, MS, SH, and Miller inorganic formulation using the growth regulators and organic formulation of the L2 medium. No statistical differences were detected between the L2 and MS inorganic formulation; however, the L2 formulation was superior to the others tested. Vigorous calli from both mature and immature vegetative and reproductive explant sources were obtained on L2 medium. Plant regeneration from callus culture was studied by Phillips and Collins (1979) . Meristem-derived callus regenerated from 30 to 80% of the genotypes while nonmeristem-derived callus regenerated from only 1%. Plants regenerated from calli cultured on various combinations of 2,4-D, NAA, CPA, picloram, BAP, and kinetin. Combinations including 2,4-D yielded more plants than a combination of picloram and BAP. Plants were recovered efficiently when callus grown on medium containing 2,4-D was transferred to a medium containing a low concentration of picloram and a high concentration of BAP. Plants were rooted on a half -strength L2 medium supplemented with nicotinic acid and a vitamin-

PAGE 19

10 like analog, 3-amino pyridine. All regenerated plants were diploid and normal in appearance. Phillips and Collins (1980) evaluated various combinations of IAA, NAA, picloram, 2,4-D, BAP, and adenine in solidified L2 basal medium to regenerate plants from cell suspension cultures and to satisfy the conditions for the application of genetic selection procedures at the cellular level. Concentrations of 2,4-D at 23nM to 0.14uM were statistically superior to other concentrations. L2 medium containing 45nM 2,4-D and 15uM adenine was optimal for initiating plant regeneration. Plants regenerated via somatic embryogenesis, as evidenced by the developmental pattern of regeneration. Some somatic embryos developed complete shoots and roots on the callus induction medium. Shoot development of somatic embryos was most efficient on a L2 medium containing a low concentration of picloram and a moderate concentration of BAP. Most regenerated plants were normal, fertile diploids. Phillips and Collins (1982) summarized the in vitro culture protocols and the composition of media used for red clover. Explant preparation (including seedling germination) , callus induction and proliferation, induction of somatic embryogenesis and plant regeneration, rooting of shoots, and establishment of plants in soil were described.

PAGE 20

Bhojwani et al. (1984) studied intra-varietal variation for in vitro plant regeneration in Trifolium. Plant regeneration of red clover was successfully accomplished when calli derived from anthers (somatic tissues of the anther) was transferred to embryo development medium. The callus induction medium was MS basal medium supplemented with 0.1 mg IT 1 2,4-D and 0 . 5 mg IT 1 BAP or 1 mg IT 1 2,4-D and 2 mg IT 1 2iP with or without casein hydrolysate. The embryo development medium was MS basal medium containing 0.05 mg IT 1 2,4-D and 0.5 mg L" 1 2iP. MacLean and Nowak (1989) studied plant regeneration of red clover using media sequences described by Beach and Smith (1979) and Collins and Phillips (1982), and a combination of media from both sequences. They observed that plant regeneration was best accomplished when callus tissues derived from B5 callus initiation medium (Beach and Smith, 1979) were incubated on SPL medium (Phillips and Collins, 1982) using regenerative genotypes. Bhojwani and White (1982) developed a method for the culture of mesophyll protoplast of T. repens that gave a high frequency of cell division. The protoplast-derived calli were transferred to B5 solid medium or to MS medium supplemented with various growth regulators to obtain plant regeneration. Callus initiation medium contained B5 basal

PAGE 21

salt supplemented with 2 mg IT 1 each of 2,4-D, NAA, and kinetin. Plant regeneration medium was MS basal medium supplemented with 0.5 mg IT 1 2iP, and 0.1 mg L" 1 IAA. Maheswaran and Williams (1984, 1986) developed direct somatic embryogenesis of red clover using immature embryos as explants. Embryoids were induced directly from the hypocotyl region of these embryos on EC6 basal medium (Maheswaran and Williams, 1984) containing 0.05 mg L" 1 BAP and 1.0 mg L" 1 yeast extract. Lowering of the yeast extract level to one half or zero at a BAP level of 0.05 mg IT 1 gave precocious germination of the immature embryo explant or abnormal growth of the explanted zygotic embryo without initiation of embryoids. Increasing the BAP level above 0.05 mg IT 1 gave a shift from direct somatic embryogenesis to the formation of a nodular morphogenic callus. McGee et al. (1989) developed a more rapid, one-step regeneration system for Trifolium petiole segment of shoots cultured in vitro. They observed that petioles from in vitro Trifolium shoot cultures excised and placed on regeneration medium (L2 basal medium supplemented with 0.015 mg L" 1 picloram and 0.06 mg IT 1 BAP), provided an improved system for studying the cellular and molecular events of somatic embryogenesis as compared with callus cultures. This system has been used to select isogenic cell lines of

PAGE 22

T. rubens showing highor low-frequency somatic embryogenesis, and to examine polypeptide translational profiles during culture of high-frequency, low-frequency or non-embryonic cell lines of T. rubens and T. pratense (McGee et al., 1989) . Comparing the methods above, it is important to consider types and concentrations of growth regulators, and to determine the timing of changes in growth regulator ratios in order to have an efficient system of plant regeneration of red clover. Genetic Studies of Tissue Culture Response Plant development from callus tissue culture has been shown to be under genetic control in various species and numerous experiments have shown that nuclear genes control the in vitro response (Frankenberger et al., 1981; Komatsuda et al., 1989; Chowdhury et al., 1991; Quimio and Zapata, 1990; Zhou and Konzak, 1992; Rackocszy-Trojanowska and Malepszy, 1993) . Plant tissue culture and regeneration capacity has been studied using both quantitative and qualitative genetic approaches.

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14 Quantitative Genetic Studies Diallel analysis of shoot formation from cauliflower tissue culture was performed as early as 1974 (Buiatti et al., 1974), indicating that gene action was additive, but with a low heritability . Additive gene action also accounted for a large part of the shoot-forming variation between tomato FxS in a diallel cross ( Frankenberger et al., 1981) . For maize somatic embryogenesis, additive variance was high compared to non-additive or dominance variation (Nesticky et al., 1983). Additive genetic variance was also the most significant source of variation for somatic embryogenesis in rice (Peng and Hodges, 1989) and wheat (Chevrier et al., 1990). Most of these results suggested that parents producing highly responsive hybrids can be identified. In rice anther culture, reciprocal effects on percent of explants producing callus and percent green-plant regeneration were not significant, indicating that both characters were under strict nuclear control and were not influenced by chloroplast and mitochondrial genomes. Further analysis showed gene action for both characters to be partially dominant with the high response being highly recessive and controlled by a few genes. There were no indications of inter-allelic interaction (Quimio and Zapata, 1990) .

PAGE 24

15 Genetic variation for maize callus growth and shoot regeneration ability were observed, although a large proportion of the variation was additive type, positive heterosis increased the tissue culture response in maize somatic embryogenesis (Tomes and Smith, 1985) . Although many differences between genotypes for in vitro tissue culture responses were reported to be additive and heritable (Keyes et al., 1980; Tomes and Smith, 1985), some genetic studies indicated that dominance, additive x additive and dominance x additive genetic effects were important (Chu and Croughan, 1990) . Studies designed to analyze the inheritance of anther culture ability in maize demonstrated that general combining abilities were highly significant (Petolino and Thompson, 1988), with a high heritability . A predominantly additive gene action exists for microspore-derived embryo induction (Koba et al., 1993) and green plant regeneration (Zhou and Konzak, 1992; Shimada et al., 1993). Estimated heritability was high for anther culture ability (Lazar et al., 1984b). Mendel i an Gen^ir st.nriips Genotypes which produce callus, somatic, or androgenic embryos in vitro have been tested for their ability to sexually transmit these traits, and sometimes reciprocal

PAGE 25

16 crosses have been performed. All the analyses of segregating populations demonstrated that there was a genetic component for tissue-culture traits. In wheat anther culture, the green plant regeneration trait segregated among backcross populations (Zhou and Konzak, 1992), and this trait was attributed to nuclear genes. Similar evidence was obtained from somatic embryogenesis experiments, and it has been demonstrated that callus induction rate and regeneration ability are controlled by independent genetic systems in wheat (Chowdhury et al., 1991) and barley (Komatsuda et al., 1989). Most studies conducted to determine the number of genes controlling the various components of tissue culture responses have indicated that one, two, or a few nuclear genes were involved. Recessive nuclear genes were responsible for regeneration capacity either from leaf disc in tomato ( Frankenberger et al., 1981), or from immature inflorescence callus in rye (Rakocszy-Trojanowska and Malepszy, 1993) . On the contrary for alfalfa (Reisch and Bingham, 1980; Wan et al., 1988; Ray and Bingham, 1989; Hernadez and Christie, 1990; Kielly and Bowley, 1992; Yu and Pauls, 1993) , dominant genes seem to control somatic embryogenesis or anther culture ability (Taylor and Veilleux, 1992) . A few reports suggest that complementary genes are necessary for the whole tissue culture process

PAGE 26

(Wan et al., 1988; Hernandez and Christie, 1990; Yu and Pauls, 1993) . Forage Legumes In vitro Selection Response Genetic variation for in vitro response has been reported for a number of forage legumes, especially in alfalfa (Bingham et al., 1975), red clover (Keyes et al., 1980) and Desmodium (Wofford et al., 1992). In red clover, genotypic variation was ascribed to the occurrence of additive genetic variance (Keyes et al. 1980) whereas in diploid alfalfa, the capacity for somatic embryogenesis was controlled by two dominant genes (Reisch and Bingham, 1980) . In alfalfa, regeneration was genotype-specific and only a few genotypes in certain cultivars have been isolated with the ability to regenerate plants from explant derived calli (Arcioni et al., 1990a). Two selected genotypes from the cultivar 'Adriana' were shown to regenerate through somatic embryogenesis (Arcioni et al., 1990b). Studies of the genetic control of somatic embryogenesis have been carried out in diploid (Reisch and Bingham, 1980) and tetraploid alfalfa (Wan et al., 1988; Hernandez-Fernandez and Christie, 1989) and all the studies showed that this trait was under the control of at least two independent loci although with different interactions. The high heritability of

PAGE 27

18 embryogenesis allowed an increase in the regeneration capacity of a cultivar from 12 to 67% (Bingham et al., 1975) . Plant regeneration from callus was found to be highly heritable in alfalfa (Bingham et al., 1975; Wan et al., 1988) and red clover (Quesenberry and Smith, 1993) . Regeneration was increased from 12% in a standard alfalfa cultivar to 67% after three generations (Bingham et al., 1975) . Genetic analysis of regeneration in diploid alfalfa confirmed that regeneration was relatively simply inherited. Segregation for regeneration in F lr F 2 , and backcross generations involving crosses between the high regenerator and genotypes with very low regeneration indicated that regeneration was dominant and fitted a two-locus model (Reisch and Bingham, 1980) . A dominant allele at each locus appeared necessary to explain high regeneration in which >75% of callus colonies produced buds. However, it is not known how many different modes of genetic control may exist for regeneration in alfalfa. Recently, Crea et al. (1995) studied the genetic control of somatic embryogenesis in the alfalfa cultivar Adriana. The results indicated that the somatic embryogenesis observed in the cv. Adriana was under genetic control. The segregation ratio of the progenies from intercrossing or selfing embryogenic genotypes gave a

PAGE 28

19 population which segregated following the two dominant loci model proposed by Wan et al.(1988). The possibility of increasing the frequency of embryogenic plants has been demonstrated. From the 3% frequency of regenerable genotypes observed in the cv. Adriana (Arcioni et al., 1990b), they were able to obtain by selfing and intercrossing a seed population in which 50% of plants were embryogenic. Keyes et al (1980) performed genetic studies evaluating variation in callus cultures of red clover. The callus induction medium used was KB2 medium, consisting of the L2 basal medium supplemented with Beach and Smith (1979) callus growth regulators i.e 2 mg IT 1 each of 2,4-D, NAA, and kinetin. Plant regeneration media used were LSE (Phillips and Collins, 1980) and the plant regeneration medium of Beach and Smith (1979) . Additive genetic variance was a significant source of variability for most in vitro traits evaluated, including rapid callus growth, colony vascularization, root initiation, chlorophyll production, and somatic embryogenesis . These traits were highly heritable. Dominance genetic variance was significant for only a few in vitro characters such as callus morphology, percentage of colonies initiating roots, and total number of roots. Maternal and cytoplasmic factors were significant

PAGE 29

20 primarily in early subcultures. No significant differences for embryogenesis were attributable to differences in the regeneration media used. Furthermore, no interaction of additive genetic effects with regeneration media were observed. These results indicated that improvement in the frequency of plantlet regeneration from callus of red clover could effectively be achieved by breeding and selection for embryonic types (Keyes et al., 1980). Recently, Quesenberry and Smith (1993) reported successful recurrent selection for plant regeneration of red clover from callus tissue culture. After five cycles of recurrent phenotypic selection for increased plant regeneration via somatic embryogenesis from callus tissue culture, the percentage of regenerating plants increased from 4 to 72%. Number of regenerating plants per petiole explant was > 200 for selected individuals compared with <10 in base population individuals. They reported that plant regeneration from callus tissue of red clover was a highly heritable trait. Narrow sense heritability estimates ranged from 40 to 50%. Although much research has been conducted on tissue culture of red clover, most of the media developed for red clover are genotype-dependent. Not all genotypes responded similarly in a particular media system. By developing tissue culture media which support regeneration from a broad

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spectrum of genotypes, in vitro plant regeneration would not be an obstacle in transformation experiments. Genetic studies of this trait have been conducted extensively in many species; however, in red clover, this area of research has received limited attention. Understanding the genetic mechanism of the tissue culture and regeneration response of red clover would be helpful in the selection and breeding for these traits so that the improvement could be achieved efficiently and effectively.

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CHAPTER 3 OPTIMIZATION OF TISSUE CULTURE MEDIUM FOR PLANT REGENERATION OF RED CLOVER One of the problems encountered in genetic transformation experiments is a low frequency of plant regeneration either from callus or cell suspensions. Much research has been conducted on tissue culture of red clover; however, the frequency of plant regeneration using most of these systems was at low (McGee et al., 1989) and primarily from unadapted plant introduction materials or wild Trifolium species (Quesenberry et al., 1992). Red clover protocols for somatic embryogenesis and organogenesis that involve incubation on a series of different media for callus initiation, embryo induction and embryo development or shoot development, and plant regeneration have been presented (Beach and Smith, 1979, Phillips and Collins, 1979, 1982; Collins and Phillips, 1982, Bhojwani et al., 1984; MacLean and Nowak, 1989; Myers et al., 1989). Other research has demonstrated direct somatic embryogenesis from immature embryos (Maheswaran and Williams, 1984, 1986) or from petiole segments derived from established shoot cultures (McGee et al., 1989). 22

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23 A protocol specific for callus induction, and plant regeneration of red clover from seedling hypocotyls and excised pistils using B5 basal salt (Gamborg et al., 1968) has been reported. The callus induction medium was B5 medium supplemented with 10 uM each of 2,4-D, NAA, and kinetin. Calli were then transferred at 4 to 5 weeks to B5 medium containing twice the normal concentration of thiamine, 10 uM NAA, and 15 uM adenine. Rooting of developed shoots was accomplished on B5 medium containing the higher concentration of thiamine and 1.1 uM NAA. Other research led to development of a new basal medium called L2 (Phillips and Collins, 1979) . This medium contained a lower concentration of NH 4 + and higher concentrations of P0 3 ~, K + , Mg 2 + , and Ca 2 + than MS medium. The minor salts formulation was similar to SH medium, with adjustment in the concentration of several salts. The organic formulation (vitamins and sucrose) was similar to that of Linsmaier and Skoog (1965), with higher concentrations of thiamine and myo-inositol . Phillips and Collins (1982) described explant preparation (including seedling germination) , callus induction and proliferation, induction of somatic embryogenesis and plant regeneration, rooting of shoots, and establishment of plants in soil. MacLean and Nowak (1989) studied plant regeneration of red clover using media sequences described by Beach and

PAGE 33

Smith (1979) and Collins and Phillips (1982), and a combination of media from both sequences. They concluded that optimum plant regeneration was obtained when callus was initiated on B5 callus medium (Beach and Smith, 1979) but then transferred after four weeks to LSP medium (Phillips and Collins, 1982) . Working with white clover {T. repens L.), Bhojwani and White (1982) developed a method for the culture of mesophyll protoplasts that gave a high frequency of cell division. Protoplast-derived calli were transferred to B5 solid medium or to MS medium supplemented with various growth regulators to obtain plant regeneration. The callus initiation medium contained B5 basal salt supplemented with 2 mg IT 1 each of 2,4-D, NAA, and kinetin. Plant regeneration medium was MS basal medium supplemented with 0.5 mg L" 1 2iP, and 0.1 mg L" 1 IAA. Comparing the methods above, it is important to consider types and concentrations of growth regulators, and to determine timing of changes in growth regulators ratios in order to have an efficient system of plant regeneration of red clover.

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Materia] s and Methods 25 General Procedure Three-step media protocols (callus induction embryo induction plant regeneration) were used throughout these studies. For callus induction, petiole sections (3 to 5 mm long) were excised from sterile plant cultures maintained in Magenta boxes on B5 medium without growth regulators and transferred to the callus induction media. All explants were incubated for 4 weeks to induce callus formation and growth in a growth chamber. Cultures were maintained at 26 ± 2°C C with a 16-h day, 85 uEm" 2 s" 1 . Irradiation was supplied by cool-white fluorescent light. Factorial experiments in Randomized Complete Block (RCB) were used as an experimental design throughout these studies. Six replicates of 4 to 6 explants per replicate were initially established. After 4 weeks, two replicates of each medium treatment were sacrificed for fresh callus weight gain data. The remaining 4 replicates were transferred onto embryo media under the same environmental conditions. After 4 additional weeks, two replicates of each medium treatment were sacrificed for determination on number of embryos per explant. The remaining two replicates were transferred into embryo germination media for a 4-week period. The number of developing embryos for each genotype

PAGE 35

26 cultured on the various media systems were recorded at the end of 8and 12-week culture periods. Analyses of variance were performed as a factorial experiment in a randomized block design for callus fresh weight gain (4 weeks of culture), number of embryoids (8 weeks of culture), and number of developing embryos per explant (12 weeks of culture) , respectively. Before analysis, data for both number of embryos per explant and number of developing embryos per explant were transformed using square root of (x + 0.5). Differences among genotype means and among media system means were compared by Duncan's Multiple Range Test (DMRT), and were declared different at P < 0.05. Experiment I Six genotypes (7-1-5-36, 7-1-5-43, C34TC, 8-2-16-5, 82-16-39, FLMR6-23-3) of red clover that previously had been determined to be good regenerators on the protocol of Beach and Smith (1979) were tested for plant regeneration capacity using six media sequences proposed in the literature. Three callus induction media (L2, B5C, and KB2) were used in this study. L2 medium contained L2 basal salt plus 0.06 mg IT 1 picloram and 0.1 mg IT 1 BAP (Phillips and Collins, 1979); B5C medium contained B5 basal salt supplemented with 2 . 0 mg IT 1 each of NAA, 2,4-D mg IT 1 , and kinetin (Beach & Smith,

PAGE 36

1979). KB2 contained L2 basal medium supplemented with 2.0 mg L" 1 each of NAA, 2,4-D, and kinetin (Keyes et al., 1980). The various media to which calli were transferred are listed in Table 3.1, and the six media sequences are listed in Table 3.2. Table 3.1. Media used in red clover regeneration as proposed in the literature Growth regulators Medium Salts & vitamins Auxin Cytokinin Proposed use (mg L" •i) L2 L2 PIC, 0 .06 BAP, 0.1 Callus induction B5C B5 NAA, 2 .0 KIN, 2.0 Callus induction 2,4-D, 2.0 and regeneration KB 2 L2 NAA, 2 .0 KIN 2.0 Callus induction 2,4-D, 2.0 and regeneration LSE L2 2,4-D, 0.01 Adenine, Somatic embryo 2.0 induction B5E B5 NAA, 2 . 0 Adenine, 2.0 Embryo germination MSR MS IAA, 0 .1 2iP, 0.5 Embryo germination LSP L2 PIC, 0 .002 BAP, 2.0 Shoot development B5R B5 NAA, 0 .2 Root development Table 3.2. Media systems used in regeneration study of red clover No. Media Callus sequences Embryo Shoot Reference 1 L2 — — LSE — LSP Phillips and Collins (1980) 2 B5C ~ ~ B5E — B5R Beach and Smith (1979) 3 KB2 — — LSE — LSP Keyes et al. (1980) 4 KB 2 — — B5E — B5R Keyes et al. (1980) 5 B5C — — LSP — •LSP MacLean and Nowak (1989) 6 B5C — — MSR — MSR Bhojwani and White (1982)

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28 Data were analyzed as a 3 x 6, 6x6, and 6x6 factorial in a RCB design for: callus fresh weight gain (4 weeks of culture) , number of embryos or organized structures per explant (8 weeks of culture) , and number of developing embryos per explant (12 weeks of culture) , respectively. Experiment TT Six genotypes (7-1-5-36, FLMR6-23-3, C25TC, C34TC, C51TC, and C54TC) of red clover, which had exhibited a high level of plant regeneration capacity on B5 protocol (Beach and Smith, 1979) , were used to study the effects of various combinations and concentrations of auxin and cytokinin on different basal media for callus formation and plant regeneration response. Four basal media (L2, B5, SH, and MS) and three combinations of auxin and cytokinin (0.06 mg IT 1 picloram + 0.10 mg L" 1 BAP; 2 . 0 mg IT 1 NAA + 2.0 mg L" 1 2,4-D + 2.0 mg IT 1 kinetin; and 2 . 0 mg IT 1 NAA + 2.0 mg IT 1 2,4-D + 0.10 mg If 1 BAP) were used for callus initiation. The twelve treatments were: 1. L2 basal salt plus 0.06 mg IT 1 picloram + 0.10 mg L" 1 BAP 2. L2 basal salt plus 2 . 0 mg IT 1 NAA + 2.0 mg L" 1 2,4-D + 2.0 mg IT 1 kinetin 3. L2 basal salt plus 2 . 0 mg IT 1 NAA + 2.0 mg IT 1 2,4-D + 0.10 mg L" 1 BAP 4. B5 basal salt plus 0.06 mg IT 1 picloram + 0.10 mg IT 1 BAP 5. B5 basal salt plus 2 . 0 mg L _1 NAA + 2.0 mg IT 1 2,4-D + 2.0 mg IT 1 kinetin 6. B5 basal salt plus 2 . 0 mg If 1 NAA + 2.0 mg IT 1 2,4-D + 0.10 mg L" 1 BAP 7. SH basal salt plus 0.06 mg IT 1 picloram + 0.10 mg IT 1 BAP

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29 8. SH basal salt plus 2.0 mg L' 1 NAA + 2.0 mg IT 1 2,4-D + 2.0 mg L" 1 kinetin 9. SH basal salt plus 2 . 0 mg L" 1 NAA + 2.0 mg IT 1 2,4-D + 0.10 mg IT 1 BAP 10. MS basal salt plus 0.06 mg If 1 picloram + 0.10 mg If 1 BAP 11. MS basal salt plus 2 . 0 mg IT 1 NAA + 2.0 mg IT 1 2,4-D + 2.0 mg L" 1 kinetin 12. MS basal salt plus 2 . 0 mg If 1 NAA + 2.0 mg IT 1 2,4-D + 0.10 mg IT 1 BAP Data for callus weight gain were analyzed as 6 x 4 x 3 factorial experiment in Randomized Complete Block Design with two replications. The four media producing the best quantity and quality of callus were chosen (i.e. number 2, 5, 6, and 8) from the 12 callus induction media. These four media were then used for somatic embryo induction in combination with six different auxin-cytokinin concentrations. The six combination of auxin and cytokinin were : 1 . none 2. 0.01 mg IT 1 2,4-D + 2.0 mg L* 1 adenine 3. 0.01 mg IT 1 2,4-D + 0.1 mg If 1 BAP 4 . 2.0 mg L" 1 NAA + 2.0 mg L" 1 adenine 5. 2.0 mg IT 1 NAA + 2.0 mg If 1 kinetin 6. 0.002 mg L" 1 picloram + 0.2 mg IT 1 BAP. The 24 media combinations were: 1. B5C(5) — > B5 basal salt without growth regulators 2. B5C(5)— > B5 basal salt + 0.01 mg L" 1 2,4-D + 2.0 mg L 1 adenine 3. B5C(5) — > B5 basal salt + 0.01 mg IT 1 2,4-D + 0.1 mg L" 1 BAP 4. B5C(5) — > B5 basal salt + 2.0 mg L" 1 NAA + 2.0 mg L" 1 adenine 5. B5C(5)— > B5 basal salt + 2.0 mg L _1 NAA + 2.0 mg If 1 kinetin 6. B5C(5)--> B5 basal salt + 0.002 mg If 1 picloram + 0.2 mg mg L" 1 BAP 7. B5C(6) — > B5 basal salt without growth regulators

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30 8. B5C(6)~> B5 basal salt + 0.01 mg If 1 2,4-D + 2.0 mg L" 1 adenine 9. B5C(6)~> B5 basal salt + 0.01 mg L* 1 2,4-D + 0.1 mg If 1 BAP 10. B5C(6)~> B5 basal salt + 2.0 mg If 1 NAA + 2.0 mg If 1 adenine 11. B5C(6)— > B5 basal salt + 2.0 mg IT 1 NAA + 2.0 mg L" 1 kinetin 12. B5C(6)— > B5 basal salt + 0.002 mg L" 1 picloram + 0.2 mg L _1 BAP 13. L2C(2) — > L2 basal salt without growth regulators 14. L2C(2)~> L2 basal salt + 0.01 mg L _1 2,4-D + 2.0 mg L" 1 adenine 15. L2C(2)--> L2 basal salt + 0.01 mg IT 1 2,4-D + 0.1 mg IT 1 BAP 16. L2C(2) — > L2 basal salt + 2.0 mg If 1 NAA + 2.0 mg IT 1 adenine 17. L2C(2)— > L2 basal salt + 2.0 mg IT 1 NAA + 2.0 mg IT 1 kinetin 18. L2C(2)— > L2 basal salt + 0.002 mg IT 1 picloram + 0.2 mg IT 1 BAP 19. SHC(8) — > SH basal salt without growth regulators 20. SHC(8) — > SH basal salt + 0.01 mg L" 1 2,4-D + 2.0 mg L" 1 adenine 21. SHC(8)--> SH basal salt + 0.01 mg IT 1 2,4-D + 0.1 mg L" 1 BAP 22. SHC(8) --> SH basal salt + 2.0 mg If 1 NAA + 2.0 mg IT 1 adenine 23. SHC(8)— > SH basal salt + 2.0 mg If 1 NAA + 2.0 mg If 1 kinetin 24. SHC(8)— > SH basal salt + 0.002 mg L" 1 picloram + 0.2 mg IT 1 BAP The number embryoids or organized structures per explant were analyzed as 6 x 4 x 6 factorial in a randomized block design with two replications. Five best media based on number and quality of embryos were chosen from these 24 somatic embryo induction media (i.e. number 1, 4, 8, 12, and 18) . These were combined with the four different concentrations of auxin and cytokinin below for embryo development i.e.:

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31 1 . none 2. 0.2 mg L" 1 NAA 3. 0.002 mg L" 1 picloram + 0.2 mg L" 1 BAP 4. 0.002 mg IT 1 picloram + 0.1 mg IT 1 BAP The 20 media combinations were: 1. B5C(5) — > B5 — > B5 basal salt without growth regulators 2. B5C(5)~> B5--> B5 basal salt + 0.2 mg IT 1 NAA 3. B5C(5) — > B5 — > B5 basal salt + 0.002 mg L" 1 picloram + 0.2 mg IT 1 BAP 4. B5C(5) — > B5 — > B5 basal salt + 0.002 mg L" 1 picloram + 0.1 mg L" 1 BAP 5. B5C(5) — > BSE (4) — > B5 basal salt (no growth regulators) 6. B5C(5)--> BSE(4)--> B5 basal salt + 0.2 mg L" 1 NAA 7. B5C(5)--> BSE(4)--> B5 basal salt + 0.002 mg L" 1 picloram + 0.2 mg IT 1 BAP 8. B5C(5)--> BSE(4)--> B5 basal salt + 0.002 mg IT 1 picloram + 0.1 mg IT 1 BAP 9. B5C(6) — > BSE (2) — > B5 basal salt (no growth regulators) 10. B5C(6)~> BSE(2)-> B5 basal salt + 0. 2 mg L" 1 NAA 11. B5C(6)— > BSE(2)-> B5 basal salt + 0. 002 mg L' 1 picloram + 0.2 mg L" 1 BAP 12. B5C(6)~ > BSE(2)-> B5 basal salt + 0. 002 mg IT 1 picloram + 0.1 mg L" 1 BAP 13. B5C(6)~> BSE(6)-> B5 basal salt (no growth regulators) 14. B5C(6)~> BSE(6)-> B5 basal salt + 0. 2 mg L" 1 NAA 15. B5C(6)~> BSE(6)+ 0.2 mg IT 1 BAP -> B5 basal salt + 0. 002 mg L" 1 picloram 16. B5C(6)~> BSE(6)+ 0.1 mg IT 1 BAP -> B5 basal salt + 0. 002 mg IT 1 picloram 17. L2C(2)— > LSE(6)-> L2 basal salt (no growth regulators) 18. L2C(2)~> LSE(6)-> L2 basal salt + 0. 2 mg L' 1 NAA 19. L2C(2)~> LSE(6)+ 0.2 mg IT 1 BAP -> L2 basal salt + 0. 002 mg If 1 picloram 20. L2C(2)— > LSE(6)+ 0.1 mg IT 1 BAP -> L2 basal salt + 0. 002 mg L" 1 picloram The number of developing embryos at 12 weeks was analyzed as 6 x 5 x 4 factorial in an RCB design with two replications .

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32 Experiment III Six genotypes (NEWRC 12, NEWRC 25, NEWRC 32, NEWRC 37, NEWRC 56, and NEWRC 98) of red clover which had a high plant regeneration response on the B5 protocol (Beach and Smith, 1979) were used. Three media systems, i.e the best media system from Exp. I and II, and the standard media system of Beach and Smith (1979) , were compared for their callus formation and plant regeneration response. The three media systems were: 1. B5C B5E B5R (standard) 2. B5C LSP LSP (Exp. I) 3. L2C(2) LSE(6) L2(20) (Exp. II) All data were analyzed as 3 x 6 factorial in a randomized complete block design with two replicates. Experiment TV Ten genotypes (NEWRC1, NEWRC4, NEWRC30, NEWRC 64, NEWRC74, FLMR6-23-3, C34TC, C54TC, 7-1-5-36) of red clover which had a range in levels of plant regeneration capacity on B5 protocol (Beach and Smith, 1979) were used. The same three media systems used in Exp. Ill were evaluated. Response variables measured were callus formation and plant regeneration capacity. All data were analyzed as 3 x 10 factorial in an RCBD with two replications.

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Results and Discussions Experiment 1 The criteria used to select the optimal red clover culture media for tissue culture responses included growth in culture and regeneration capacity because cell division should allow for the rapid proliferation of callus from petiole tissues. Growth was determined by measuring fresh weight gain (g) of callus over 4 weeks. The analysis of variance showed highly significant effects of red clover genotypes for callus fresh weight gain. The genotype by media interaction was also significant for this trait. There was no effect of media for this variable. Because of the interaction between genotypes and media, further analysis was conducted for each genotype to test the differences in callus fresh weight gain among different media (Table 3.3). Table 3.3. Callus fresh weight gain on different media with fixed genotypes Callus Genotype induction Mean media 1 2 3 4 5 6 g L2 KB2 B5C 0.21 a 0.10 a 0.08 b 0.09 a 0.10 a 0.09 b 0.11 ab 0.16 ab0.ll a 0.09 b 0.09 a 0.07 a 0.11 ab 0.10 b 0.15 b 0.11 a 0.15 a 0.13 a 0.07 a 0.13 a 0.12 a Means followed by the same letter within a column are not different (P 0.05) .

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34 With genotypes 2(7-1-5-34), 4(8-2-16-5), and 5(8-2-1639) no significant differences were observed in callus fresh weight gain on the three different callus induction media (CIM) . However with genotypes 1(7-1-5-36), 3 (C34TC) , and 6 (FLMR6-23-3) significant differences among three media were observed. L2 medium gave the highest callus fresh weight gain for genotype 1, whereas B5C medium showed the highest callus weight gain for genotype 3 and 6. Over all genotypes, callus weight gain was highest on B5C and lowest on KB2. Highly significant differences were observed among genotypes, media, and their interactions for number of embryos at eight weeks. For this reason, further analyses were conducted on each genotype to determine the differences in response on different media of a given genotype (Table 3.4). In all genotypes (except 6) significant differences were observed in number of embryoids per explant on the six media tested. In all genotypes, media system 1 (L2-LSE) resulted in lowest number of embryos per explant. Averaged over all genotypes, media system 5 (B5C-LSP) gave the highest number of embryos per explant.

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35 Table 3.4. Number of embryos per explant on different media with fixed genotypes Genotype Media Mean It 2 3 4 5 6 1* 0 d 0 c 0 c 0 c 0 d 0 a 0 c 2 38 b 25 a 62 ab 60 b 53 c 7 a 37 a 3 16 c 0 a 85 a 53 b 99 a 1 a 29 b 4 34 b 7 b 45 b 95 a 67 b 4 a 35 ab 5 45 ab 20 ab 59 ab 63 b 73 b 3 a 39 a 6 57 a 17 ab 61 ab 54 b 66 b 0 a 36 a Means followed by the same letter within a column are not different (P = 0.05) . t 1 = 7-1-5-36, 2 = 7-1-534, 3 = C34TC, 4 = 8-2-16-5, 5 = 82-16-39, 6 = FLMR6-23-3. * 1 = L2-LSE-LSP, 2 = B5C-B5E-B5R, 3 = KB2-LSE-LSP, 4 = B5CB5E-B5R, 5 = B5C-LSP-LSP, 6 = B5C-MSR-MSR. Highly significant differences in number of developing embryos at 12 weeks were observed for genotypes, media, and for the interaction between genotype by media. Further analyses were conducted to evaluate differences in number of developing embryos among different media for each genotypes (Table 3.5) . Over all genotypes, significant differences were observed in number of developing embryos on the six different media systems tested (Table 3.5). Averaged over all genotypes, media system 1 (L2-LSE-LSP) had lowest number of developing embryos or shoots per explant, whereas the five other media systems were not different for mean number of developing embryos or shoots per explant with media system 5 (B5C-LSP-LSP) having the numerically highest number

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36 of developing embryos per explant. Genotype 6 generally had the lowest number of developing embryos per explant of any media systems tested, except in media 5 (B5C-LSP-LSP) . Genotype 3 generally had the highest mean number of developing embryos per explant. Table 3.5. Number of developing embryos per explant on different media with fixed genotypes Genotype Media Mean It 2 3 4 5 6 It 0 c 0 c 13 b 1 b 0 d 0 c 2 b 2 65 b 60 a 137 a 131 a 82 a 10 be 73 a 3 159 a 5 be 131 a 93 a 135 a 3 be 69 a 4 82 b 17 b 87 a 109 a 109 b 15 b 63 a 5 96 ab 26 ab 110 a 92 a 96 be 46 a 74 a 6 99 ab 27 ab 105 a 102 a 86 cd 8 be 63 a Means followed by the same letter within a column are not different (P = 0.05) t 1 7-1-5-36, 2 = 7-1-534, 3 = C34TC, 4 = 8-2-16-5, 5=82-16-39, 6 = FLMR6-23-3. t 1 = L2-LSE-LSP, 2 = B5C-B5E-B5R, 3 = KB2-LSE-LSP, 4 B5CB5E-B5R, 5 = B5C-LSP-LSP, 6 = B5C-MSR-MSR. Experiment TT Callus fresh gain weight (g) after 4 weeks in culture showed that genotypes were significantly different. However, since the main focus of this work was to develop media satisfactory for many genotypes, data were analyzed over genotypes. The analysis of variance showed highly significant effects for basal salts, growth regulators and basal salt by

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37 growth regulator interaction. These results revealed that both basal salt and growth regulator, as well as their interactions were important for callus fresh weight gain. Thus, further analysis was necessary to understand the differences in response related to basal salt and/or growth regulator combinations (Table 3.6). Table 3.6. Callus fresh weight gain for 4 weeks of culture with fixed growth regulator combinations Growth regulator combination Basal salt It 2 3 g L2 0.087 a 0.072 b 0.067 ab B5 0.072 be 0.096 a 0.079 a SH 0.076 ab 0.079 ab 0.063 b MS 0.061 c 0.071 c 0.067 c Mean 0.074 A 0.080 B 0.067 C Means within a column followed by the same lower case letter are not different (P = 0.05). Means within a row followed by the same upper case letter are not different (P = 0.05). t 1 = 0.06 mg IT 1 picloram + 0.10 mg L" 1 BAP 2 2.0 mg IT 1 NAA + 2.0 mg L" 1 2,4-D + 2.0 mg IT 1 kinetin 3 = 2.0 mg L" 1 NAA + 2.0 mg IT 1 2,4-D + 0.10 mg L' 1 BAP. Within growth regulator combination 1 (0.06 mg IT 1 picloram + 0.10 mg L" 1 BAP) L2 basal salts had the best callus growth. However, within growth regulator 2 (2.0 mg IT 1 each of 2,4-D, NAA, and kinetin) the B5 basal salts had the highest callus production. With growth regulator combination 3 (2.0 mg L" 1 2,4-D, 2.0 mg L' 1 NAA and 0 . 1 mg IT 1

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38 BAP) the best callus fresh weight gain was obtained with B5 basal salt although this treatment was lower than treatment 2 with B5. In general, callus fresh weight gain was lowest using MS basal salt with any growth regulator combination. Over all basal salts, the growth regulator combination 3 had the lowest mean of callus fresh weight gain. A contrast procedure was used to investigate differences between auxin and cytokinin sources in callus production (Table 3.7). Auxin source significantly influenced callus production in the L2 basal salt with 0.06 mg L" 1 picloram being superior to 2.0 mg IT 1 NAA and 2,4-D. Table 3.7. Mean squares of callus weight gain in different auxin and cytokinin sources with fixed basal salts Growth regulator Basal salt L2 B5 SH MS Auxin (1 vs 3) Cytokinin (2 vs 3) 0.074* 0.001 0.010 0.070* 0.043 0.065* 0.005 0.049* * Significant at P = 0.05 level of probability Cytokinin source was significantly different for callus production in B5, SH, and MS basal salts but not L2 . The 2.0 mg IT 1 kinetin treatment was superior to 0.10 mg L" 1 BAP. Analysis of variance showed highly significant effects of callus induction media (CIM) , embryo induction media (EIM), and CIM by EIM interaction. These results revealed that both CIM and EIM as well as the CIM by EIM interaction

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39 were important in the number of embryos per explant. Because there was an interaction between CIM and EIM, further analysis was necessary to determine the differences in response related to CIM and EIM combination (Table 3.8). Table 3.8. Mean number of embryos per explant for 8 weeks of culture with fixed callus induction media Embryo Callus induction media (CIM) induction Mean media (EIM) B5C(5) B5C(6) L2C(2) SHC(8) It 36 a 43 a 28 a 19 a 32 a 2 22 ab 41 a 43 a 18 a 31 ab 3 30 a 47 a 21 a 23 a 30 ab 4 21 ab 23 a 33 a 18 a 24 c 5 23 ab 36 a 25 a 10 a 23 c 6 3 b 33 a 45 a 21 a 26 be Mean 23 B 37 A 32 A 18 B Means within a column followed by the same lower case letter are not different (P = 0.05). Means within a row followed by the same upper case letter are not different (P = 0.05). t 1 = basal salt without growth regulators 2 = basal salt with 0.01 mg L" 1 2,4-D + 2.0 mg IT 1 adenine 3 = basal salt with 0.01 mg IT 1 2,4-D + 0.1 mg If 1 BAP 4 = basal salt with 2.0 mg IT 1 NAA + 2.0 mg L" 1 adenine 5 = basal salt with 2 . 0 mg L" 1 NAA + 2.0 mg L" 1 kinetin 6 = basal salt with 0.002 mg L" 1 picloram + 0.2 mg L" 1 BAP. When EIM were evaluated within each CIM, the number of embryos was significantly different only within B5C(5). Over all CIM, EIM 1 (without growth regulators) had the highest number of embryos per explant, although it was not significantly different from EIM 2(0.01 mg IT 1 2,4-D and 2.0 mg IT 1 adenine) and EIM 3(0.01 mg L" 1 and 0.1 mg If 1 ) .

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40 The analysis of variance for number of embryos per explant after 12 weeks in culture showed significant effects (P = 0.01) EIM and EIM by embryo development media (EDM) interaction. No significant main effects for EDM were observed. Because there was interaction between EIM and EDM, further analysis was conducted to examine differences within each EIM (Table 3.9). Table 3.9. Mean number of embryos per explant for 12 weeks of culture with fixed embryo induction media Embryo Embryo induction media (EIM) Mean media (EDM) It 2 3 4 5 1* 55 a 36 a 39 a 41 a 62 a 47 a 2 17 b 28 a 39 a 39 a 73 a 39 a 3 26 b 39 a 51 a 34 a 69 a 44 a 4 30 b 44 a 33 a 50 a 69 a 45 a Mean 32 B 37 B 40 B 41 B 68 A Means within a column followed by the same lower case letter are not different (P = 0.05). Means within a row followed by the same upper case letter are not different (P = 0.05). t 1 = B5C(5)-B5 basal salt without growth regulators 2 = B5C(5)-B5 basal salt + 2.0 mg IT 1 each of NAA and adenin 3 = B5C(6)-B5 basal salt + 0.01 mg IT 1 2,4-D + 2.0 mg IT 1 adenin 4 = B5C(6)-B5 basal salt + 0.002 mg IT 1 picloram + 0.2 mg IT 1 BAP 5 = L2C(2)-L2 basal salt + 0.002 mg IT 1 picloram + 0.2 mg IT 1 BAP $ 1 = basal salt without growth regulators 2 = basal salt + 0.2 mg IT 1 NAA 3 = basal salt + 0.002 mg L" 1 picloram + 0.2 mg If 1 BAP 4 = basal salt + 0.002 mg If 1 picloram + 0.1 mg L" 1 BAP

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41 When EDM were evaluated within each EIM, number of embryos were significantly different only within EIM 1 [ B5C(5)-B5 without growth regulators]. Over all EIM, EDM 1 (without growth regulators) had the highest number of embryos per explant, although it was not significantly different from three others. Over all EDM, EIM 5 i.e. L2C (2 ) -LSE ( 6) had the highest number of developing embryos per explant. The protocol L2C (2) -LSE (6) -L2 (20) was selected from this experiment for further testing. Experiment ITT For the response variable callus fresh gain weight (g) after 4 weeks in culture, the analysis of variance showed significant genotypes effects. No significant effects were observed for media or for the interaction of media by genotype. However, since the focus of this research was medium optimization, these differences are only of marginal interest. It is important that the genotype by media component was not significant. The analysis of variance for number of embryos at eight weeks showed highly significant differences among media. After 4 weeks, there were no genotype by media interaction effects. There were also no significant differences among genotypes. Media sequence L2C (2 ) -LSE ( 6) yielded the highest number of embryos per explant (Table 3.10).

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42 Table 3.10. Mean number of embryos per explant on different media at 8 weeks of culture Media Number of embryo per explant B5C-LSP 78 b L2C(2)-LSE(6) 120 a B5C-B5E 84 b Means followed by the same letter are not different (P = 0.05) . For the variable number of developing embryos at twelve weeks of culture, differences were observed among genotypes, and among media system. The medium combination L2C(2)LSE (6) -L2 (20) gave the highest number of developing embryos per explant, while B5C-B5E-B5R was the poorest in number of developing embryos per explant (Table 3.11). Table 3.11. Mean number of embryos per explant over genotypes on different media at 12 weeks of culture Media Number of embryo per explant B5C-LSP-LSP 126 b L2C(2)-LSE(6)-L2(20) 175 a B5C-B5E-B5R 88 b Means followed by the same letter are not different (P = 0.05) The finding of no genotype by media interaction again suggests that this group of genotypes responds similarly on the three media. Mean number developing embryos per explant varied among genotypes from 112 to 163 (data not shown) .

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43 Ex periment IV No significant differences among media or genotypes for callus fresh weight gain at four weeks of culture were observed. This finding is of interest since the genotypes were selected for variable regeneration response. This finding suggests that callus mass may not be related to regeneration response in this group of genotypes. This result is similar to Myers et al. (1989), who found that cell growth and protoplast division frequency in vitro were not associated with regeneration potential in the red clover genotypes which they tested. A high rate of callus growth in culture could be ideal for regeneration to take place, but not entirely necessary. Some of their genotypes which produced the most callus had a low frequency of regeneration, and their most highly regenerable genotype showed only average division frequencies in protoplast culture . After eight weeks of culture, highly significant differences in number of embryos per explant were observed among genotypes and among media, but there were no genotype by media interactions. Media system L2C (2) -LSE (6) yielded the highest number of embryos per explant although it was not significantly different to media system 1 (B5C-LSP) (Table 3.12) .

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44 Table 3.12. Mean number of embryos per explant at 8 weeks of culture Media Number of embryo per explant B5C-LSP-LSP 45 ab L2C(2)-LSE(6)-L2(20) 53 a B5C-B5E-B5R 37 b Means followed by the same letter are not different (P = 0.05) . The fact that the standard B5 medium on which this germplasm was developed yielded the lowest number of embryos shows that either of the newly selected media may be superior to the B5 medium. Genotype C54TC resulted in the highest number of embryos per explant (Table 3.13), while NEWRC 4 was the lowest in the number of embryos per explant. Table 3.13. Mean number of embryos per explant on different genotypes at 8 weeks of culture Genotype Number of embryo per explant 7-1-5-36 66 b Selected as high regeneration 7-1-5-34 61 b C34TC 65 b C54TC 89 a FLMR6-23-3 22 c Selected as intermediate C64TC 71 ab regeneration C74TC 69 b NEWRC 1 4 cd Selected as low regeneration NEWRC 4 2 d NEWRC 30 4 cd Means followed by the same letter are not different (P = 0.05) .

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45 The analysis of variance at twelve weeks of culture for number of developing embryos per explant showed highly significant differences among genotypes and among the media systems tested. No significant differences were observed for genotype by media interaction. Media L2C (2 ) -LSE ( 6) L2(20) again yielded the highest number of embryos per explant, although it was not significantly different from media system B5C-LSP-LSP (Table 3.14). Table 3.14. Mean number of embryos per explant on different media sequences at 12 weeks of culture Media Number of embryos per explant B5C-LSP-LSP 62 a L2C(2)-LSE(6)-L2(20) 66 a B5C-B5E-B5R 51 b Means followed by the same letter are not different (P = 0.05) . Genotype 7-15-36 had the highest number of developing embryos per explant, while genotype NEWRC 30 was the lowest in the number of developing embryos per explant (Table 3.15). When genotypes were evaluated within each medium, the number of embryos per explant was significantly different within each medium (Table 3.16). Furthermore, genotype C64TC and C74TC, which previously was rated as intermediate regeneration on media III (B5 protocol) showed increased regeneration on either media I (B5C-LSP-LSP) or media II [L2C (2) -LSP-L2 (20)].

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46 Table 3.15. Mean number of embryos per explant on different genotypes at 12 weeks of culture Genotype Number of embryos per explant 7-1-5•36 107 a Selected as high regeneration 7-1-5•34 74 c C34TC 93 ab C54TC 109 a FLMR6•23-3 26 d Selected as intermediate C64TC 87 be regeneration C74TC 88 be NEWRC 1 5 e Selected as low regeneration NEWRC 4 4 e NEWRC 30 3 e Means followed by the same letter are not different (P = 0.05) . Table 3.16. Mean number of embryos per explant on different genotypes within each media system Genotype Mediat I II III 7-1-5-36 134 a 104 a 85 ab 7-1-5-34 62 c 86 a 69 ab C34TC 85 be 108 a 86 ab C54TC 103 b 121 a 103 a FLMR6-23-3 21 d 32 b 30 c C64TC 102 b 104 a 56 b C74TC 98 b 95 a 70 ab NEWRC 1 8 d 4 b 2 d NEWRC 4 3 d 5 b 4 d NEWRC 30 2 d 5 b 1 d Means followed by the same letter are not different (P = 0.05) . t I = B5C-LSP-LSP; II = L2C-LSE ( 6) -L2 (20) ; III = B5C-B5EB5R.

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47 Based upon the results of the four experiments, the optimum media system for plant regeneration in red clover was L2C (2) -LSP-L2 (20) . L2C(2) was an L2 basal medium supplemented with 2 . 0 mg L" 1 each of NAA, 2,4-D, and kinetin for callus induction. After four weeks, the calli were transferred onto L2 basal medium with 0.002 mg If 1 picloram +0.2 mg L" 1 BA for embryo induction for another four weeks. For plant regeneration, the L2 basal medium supplemented with 0.002 mg LT 1 picloram + 0.1 mg IT 1 BA for four weeks was superior. The varying levels of regeneration of the genotypes among the different media indicates that regeneration is at least partially dependent upon the culture environment. The L2 basal salt provided the optimum culture environment for callus induction and regeneration. This basal salt was optimized specifically for in red clover (Phillips and Collins, 1982) . In the current study, the critical stage for embryogenesis and/or regeneration was culture on LSP medium using callus which had been induced on the medium containing 2 . 0 mg L" 1 each of 2,4-D, NAA, and kinetin. A similar result was reported earlier by MacLean and Nowak (1989) . They reported that somatic embryogenesis occurred on LSP medium after calli had been induced on B5C medium which contained a similar growth regulator composition.

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CHAPTER 4 INHERITANCE STUDY OF PLANT REGENERATION IN RED CLOVER Genetic studies of tissue culture response and regeneration capacity have been conducted by many researchers in numerous crop species; however, only limited studies have been conducted in red clover. An understanding of the genetics of regeneration capacity in red clover would assist in the selection and breeding of genotypes with high regeneration capacity which should facilitate improvement via genetic transformation. Nuclear genes control in vitro response and plant development from callus tissue culture and has been reported in various species (Frankenberger et al., 1981; Komatsuda et al., 1989; Chowdhury et al., 1991; Quimio and Zapata, 1990; Zhou and Konzak, 1992; Rakoczy-Trojanowska and Malepszy, 1993) . In alfalfa, for example, regeneration was genotypespecific and only a few genotypes in certain cultivars have been isolated with the ability to regenerate plants from explant derived calli (Arcioni et al., 1990a). Two selected genotypes from the cultivar Adriana were shown to regenerate through somatic embryogenesis (Arcioni et al., 1990b). 48

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Studies of the genetic control of somatic embryogenesis have been carried out in diploid (Reisch and Bingham, 1980) and tetraploid of alfalfa (Wan et al., 1988; HernandezFernandez and Christie, 1989) . These studies found this trait to be controlled by at least two independent loci although with different interactions. Selection for regeneration capacity increased the level from 12 to 67% (Bingham et al., 1975) and from the 3% to 50% in cv. Adriana (Arcioni, 1990b) . In red clover, genotypic variation for in vitro plant regeneration was shown to be conditioned by additive genetic variance (Keyes et al., 1980). This was also a significant source of variability for callus growth, chlorophyll production, and root initiation. Dominant genetic variance was significant only for the traits, callus morphology and root number. Recently, successful recurrent selection for plant regeneration of red clover from callus tissue culture has been achieved (Quesenberry and Smith, 1993) . With five cycles of recurrent phenotypic selection for increased plant regeneration via somatic embryogenesis from callus tissue culture, they showed an increase in the percentage of regenerating plants from 4 to 72%. The number of regenerating plants per petiole explant was also increased to > 200 for selected individuals compared with <10 in the

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50 base population. Narrow sense heritability estimates ranged from 40 to 50%. Materials and Methods Mating design A diallel cross (without parents or reciprocals) of nine genotypes of red clover, which had exhibited a range in level of plant regeneration response (low, medium and high regeneration capacity) was conducted (Table 4.1). All crosses were performed by hand without emasculation in a greenhouse. The thirty six crosses, each of forty progenies per cross, were evaluated for the response variables callus diameter at four weeks, and number of plants regenerated at 12 weeks on Beach and Smith (1979) media protocol using hypocotyl culture. Table 4.1. Diallel cross of nine genotypes of red clover No. Parent Reg.t 1 2 3 4 5 6 7 8 9 Score 1 NEWRC1 Low X X X X X X X X 2 NEWRC4 Low X X X X X X X 3 NEWRC30 Low X X X X X X 4 NEWRC64 Medium X X X X X 5 NEWRC74 Medium X X X X 6 FL23-3 Medium X X X 7 C34TC High X X 8 C54TC High X 9 7-1-534 High X t Regeneration

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51 Hypocotyl Culture The Fj seeds were scarified by soaking and stirring in 100% sulphuric acid for five minutes and then rinsed five times with distilled water. The seeds were then soaked and stirred in saturated calcium hypochlorite for five minutes. The seeds were rinsed with sterile, distilled water five times under aseptic conditions. The seeds were then placed on seed germination medium (SGL) at 25°C ± 2°C under continuous light. The SGL medium contained one-tenth concentration of the major salts in the L2 medium. The minor salts and growth regulators were omitted. One-fourth concentration of thiamine, pyridoxine, and myo-inositol of the L2 medium were included (Phillips and Collins, 1982) . After germination (3-5 days) , hypocotyls from developing seedlings were excised into sections (3 to 5 mm long) and transferred to the B5 callus induction medium. A randomized complete block design with 36 F x s from each of 40 progenies was used in this experiment. All explants were incubated for four weeks to induce callus formation and growth. Cultures were maintained at 26°C ± 2°C with a 16-h day, 85 uEm~ 2 s _1 . Irradiation was supplied by cool-white fluorescent light. After four weeks, the explants were transferred to B5 embryo induction medium for four additional weeks under the same environmental conditions.

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52 The explants were then transferred to the B5 embryo development medium for another four week period. Callus formation was measured as diameter of callus (mm) after four weeks of culture on callus induction medium. Plant regeneration capacity was rated from 0 to 6 after twelve weeks of culture. Number of embryoids and/or plantlets were estimated separately for each explant and assigned a rating index number according to the scale: 0 = no embryoids or organized structures formed 1 = 1 to 3 embryos organized structures formed, 2 = 4 to 10 embryos or organized structures formed, 3 = 11 to 25 embryos or organized structures formed, 4 = 26 to 55 embryos or organized structures formed, 5 = 56 to 100 embryos or organized structures formed, and 6 = more than 100 embryos or organized structures formed. Data for both callus diameter and plant regeneration capacity were analyzed using Griffing's Method IV, fixed model (Griffing, 1956) . Differences among cross means and GCA effects were compared by Fisher's protected LSD. SCA effects were tested for difference from zero by the t test at P = 0.05. Results and Discussions Callus diameter means of 36 crosses of red cover genotypes 4 weeks after culture on B5 callus induction

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medium are presented in Table 4.2. F x s with C54TC and 7-15-34 as one parent were among the combinations showing Table 4.2. Means of callus diameter of 36 crosses of red clover genotypes 4 weeks after culture Crosses Callus diameter (mm) NEWRC 1 X NEWRC 4 9.0 NEWRC 1 X NEWRC 30 9.4 NEWRC 1 X NEWRC 64 10.7 NEWRC 1 X NEWRC 74 12.1 NEWRC 1 X FLMR6-23-3 14.4 NEWRC 1 X C34TC 10.7 NEWRC 1 X C54TC 13.5 NEWRC 1 X 7-1-5-34 11.8 NEWRC 4 x NERWC 30 10.8 NEWRC 4 x NEWRC 64 11.8 NEWRC 4 x NEWRC 74 12.2 NEWRC 4 x FLMR6-23-3 13.4 NEWRC 4 x C34TC 11.5 NEWRC 4 x C54TC 13.0 NEWRC 4 x 7-1-5-34 13.0 NEWRC 30 x NEWRC 64 11.1 NEWRC 30 x NEWRC 74 11.4 NEWRC 30 x FLMR6-23-3 10.8 NEWRC 30 x C34TC 11.7 NEWRC 30 x C54TC 12.8 NEWRC 30 x 7-1-5-34 12.3 NEWRC 64 x NERWC 74 12.6 NEWRC 64 x FLMR6-23-3 13.1 NEWRC 64 x C34TC 13.7 NEWRC 64 x C54TC 12.5 NEWRC 64 x 7-1-5-34 13.8 NEWRC 74 x FLMR6-23-3 12.0 NEWRC 74 x C34TC 12.6 NEWRC 74 x C54TC 14 . 9 NEWRC 74 x 7-1-5-34 15.0 FLMR623-3 x C34TC 12.4 FLMR623-3 x C54TC 13.4 FLMR623-3 x 7-1-5-34 12.7 C34TC x C54TC 13.7 C34TC x 71-5-34 13.7 C54TC x 71-5-34 16.9 LSD (0.05) 1.3

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54 the largest callus diameter (>13.00 mm), and those with NEWRC 1, NEWRC 4, and NEWRC 30 as one parent were among the lowest callus diameter (<12.00mm). C34TC also showed high callus diameter in F 1 combination with several other parents . The analysis of variance for general combining ability (GCA) and specific combining ability (SCA) is presented in Table 4.3. The diallel analysis showed highly significant GCA and SCA effects. These results suggest that both additive and non-additive effects may be important in the inheritance of callus diameter of red clover. Table 4.3. Analysis of variance of general combining ability (GCA) and specific combining ability (SCA) effects for callus formation Source Degrees of freedom Mean squares Crosses 35 4.8279** GCA 8 14 .4090** SCA 27 1.9891** Error 35 0.4731 ** Significant at the P = 0.01 The contribution of the individual genotypes to the FiS 1 response was determined by comparing the general combining ability effects (Table 4.4). Positive values indicate a contribution towards a larger callus diameter, while negative values indicated a contribution towards a smaller callus diameter. The estimates of GCA effects

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55 Table 4.4. Estimates of general combining ability (GCA) effects for callus diameter of red clover Parent GCA NEWRC 1 -1.23 NEWRC 4 -0.77 NEWRC 30 -1.38 NEWRC 64 -0.10 NEWRC 74 0.39 FLMR6-23-3 0.31 C34TC 0.01 C54TC 1.52 7-1-5-34 1.28 LSD (0.05) 0.44 indicated that C54TC contributed the most to larger callus diameter. Although the contribution was less, 7-1-5-34 also contributed to large callus diameter. Conversely, NEWRC 1 and NEWRC 30 had relatively high negative effects on callus diameter. The SCA effects for callus diameter per parental combination are presented in Table 4.5. Estimates of SCA effects indicated there were five cross combinations that showed positive SCA different from zero. These include C54TC x 7-1-5-34, FLMR6-23-3 x 7-1-5-34, and NEWRC 64 x C34TC. These crosses involved all 7-1-5-34 or C34TC, both of which had high general combining ability for callus diameter. NERWC 1 x FLMR6-23-3 showed a positive and highly significant SCA effect and NEWRC 4 x FLMR6-23-3 showed a positive significant SCA effect, indicating that these crosses performed better than would have been predicted from

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56 Table 4.5. Estimates of specific combining ability (SCA) effects for callus diameter for 4 weeks Crosses SCA NEWRC 1 x NEWRC 4 -1.54 * NEWRC 1 x NEWRC 30 -0.51 NEWRC 1 x NEWRC 64 -0.48 NEWRC 1 x NEWRC 74 0.41 NEWRC 1 x FLMR6-23-3 2.81 * * NEWRC 1 x C34TC -0.61 NEWRC 1 x C54TC 0.69 NEWRC 1 x 7-1-5-34 -0.77 NEWRC 4 x NERWC 30 0.40 NEWRC 4 x NEWRC 64 0.22 NEWRC 4 x NEWRC 74 0.11 NEWRC 4 x FLMR6-23-3 1.32 * NEWRC 4 x C34TC -0.17 NEWRC 4 x C54TC -0.28 NEWRC 4 x 7-1-5-34 -0.07 NEWRC 30 x NEWRC 64 0.12 NEWRC 30 x NEWRC 74 -0.09 NEWRC 30 x FLMR6-23-3 -0.57 NEWRC 30 x C34TC 0.60 NEWRC 30 x C54TC 0.16 NEWRC 30 x 7-1-5-34 -0.12 NEWRC 64 x NERWC 74 -0.19 NEWRC 64 x FLMR6-23-3 0.39 NEWRC 64 x C34TC 1.27 * NEWRC 64 x C54TC -1.39 * NEWRC 64 x 7-1-5-34 0.07 NEWRC 74 x FLMR6-23-3 -1.22 NEWRC 74 x C34TC 0.30 NEWRC 74 x C54TC 0.44 NEWRC 74 x 7-1-5-3 0.85 FLMR6233 x C34TC -0.41 FLMR623 -3 x C54TC -0.91 FLMR623 -3 x 7-1-5-34 1.40 C34TC X C54TC -0.27 C34TC X 7-1-5-34 -0.12 C54TC X 7-1-5-34 1.55 * * ** Significant difference from 0 at P = 0.05 and P = 0.01 levels of probability, respectively. the GCA effects of the parental genotypes. Of particular interest is the combination of NEWRC1 x FLMR6-23-3, because

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NEWRC1 had high negative GCA effects, but this cross showed high positive SCA. It may be of importance that FLMR6-23-3 is a selection out of 'Cherokee', a southern U.S. adapted red clover, and is more distantly related to all other plants which trace to the NEWRC germplasm. These results suggest that in these specific combinations dominant or epistatic effects or non-additive genetic variance were probably involved in the inheritance of callus diameter in red clover. There were three cross combinations that showed significant negative SCA effects. These included NEWRC 1 x NEWRC 4, NEWRC 64 x C54TC, and NEWRC 74 x FLMR6-23-3. Only one cross involved NEWRC 1 and NEWRC 4, which had poor GCA for callus diameter. The two other cross combinations performed more poorly than would have been predicted from the GCA effects of the parental genotypes; again suggesting involvement of dominant and epistatic effects. This investigation indicated that both GCA and SCA effects were significant sources of variation for callus formation; however, GCA appeared to be more important than SCA. This study showed that useful sources of genetic variability for callus formation are available in this breeding material. Development of genotypes with good callus growth using methods that exploit additive types of gene action should be effective.

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58 Plant regeneration capacity index of 36 crosses of red clover measured after 12 weeks in culture are presented in Table 4.6. Crosses with C54TC, 7-1-5-34, and C34TC as one parent were among the combinations showing the highest regeneration capacity index (>3.50), and those with NEWRC 1, NEWRC 4, and NEWRC 30 as one parent were among the lowest plant regeneration capacity index (<2.50). The diallel analysis showed highly significant GCA and significant SCA effects (Table 4.7). These results revealed that both additive and non-additive effects were probably important in the inheritance of plant regeneration capacity of red clover, and that additive effects were probably more important than non-additive effects. The contribution of individual genotypes to the F 1 response can be seen by comparing the general combining ability effects (Table 4.8). Positive values indicate a contribution towards greater regeneration capacity of red clover, while negative values indicate a contribution towards smaller regeneration capacity. The estimates of GCA effects indicated that C54TC contributed the most to greater regeneration capacity. Although its contribution was smaller, 7-1-5-34 also contributed to regeneration capacity. Conversely, NEWRC 1, NEWRC 4, and NEWRC 30 had a similar and negative effect on regeneration capacity.

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59 Table 4.6. Regeneration capacity index of 36 crosses of red clover genotypes 12 weeks after culture Crosses Regeneration capacity index IN Ej W 1 V 1 A IN Ej W E\v^ 4 q y in Hi w 1 V LN Ej Vy r\v^ OU 1 1 X LNEjVVJa*^ 1 V J. X HPMDP (LA LN Ej VN t\\^ D 4 0 z • c,
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60 Table 4.7. Analysis of variance of general combining ability (GCA) and specific combining ability (SCA) effects for regeneration capacity Source Degrees of freedom Mean squares Crosses 35 1.5177 ** GCA 8 5.7782 ** SCA 27 0.2755 * Error 35 0.0926 *, ** Significant at the P 0.05 and P = 0.01, respectively Table 4.8. Estimates of general combining ability (GCA) effects for regeneration capacity of red clover Parent GCA NEWRC 1 -0.82 NEWRC 4 -0.59 NEWRC 30 -0.68 NEWRC 64 -0.01 NEWRC 74 0.19 FLMR6-23-3 0.25 C34TC -0.01 C54TC 1.10 7-1-5-34 0.56 LSD (0.05) 0.19 Estimates of SCA effects indicated three cross combinations FLMR6-23-3 x 7-1-5-34, NEWRC 64 x C34TC, and NEWRC 64 x C54TC showed significant positive SCA effects (Table 4.9). Two of these three crosses involved 7-1-5-34 and C34TC, which also had good general combining ability for plant regeneration capacity index. These two parents also had high GCA for callus growth. The cross of NERWC 1 x NEWRC 4 showed a significant negative SCA effect, indicating that this cross performed more poorly than would have been

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61 been predicted from the GCA effects of the parental genotypes, although both parents also had highly negative GCA effects. These results suggest that in this specific combination SCA effects due non-additive genetic variance are probably involved in the inheritance of plant regeneration capacity of red clover. This investigation indicated that GCA and SCA were significant sources of variation, and SCA was a significant source of variation for both callus diameter and plant regeneration. Although SCA effects were significant for a few crosses, the results generally showed that additive genetic effects were most important in conditioning inheritance of these responses. This study showed that useful sources for plant regeneration are available in this breeding material for the development of responsive plant regeneration genotypes.

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Table 4.9. Estimates of specific combining ability (SCA) effects for regeneration capacity Crosses SCA NEWRC 1 x NEWRC 4 -0.75 * NEWRC 1 x NEWRC 30 -0.37 NEWRC 1 x NEWRC 64 0.39 NEWRC 1 x NEWRC 74 0.22 NEWRC 1 x FLMR6-23-3 0.42 NEWRC 1 x C34TC -0.12 NEWRC 1 x C54TC 0.36 NEWRC 1 x 7-1-5-34 -0.18 NEWRC 4 x NERWC 30 -0.19 NEWRC 4 x NEWRC 64 -0.09 NEWRC 4 x NEWRC 74 0.05 NEWRC 4 x FLMR6-23-3 0.32 NEWRC 4 x C34TC 0.24 NEWRC 4 x C54TC 0.15 NEWRC 4 x 7-1-5-34 0.24 NEWRC 30 x NEWRC 64 -0.22 NEWRC 30 x NEWRC 74 0.02 NEWRC 30 x FLMR6-23-3 -0.09 NEWRC 30 x C34TC 0.14 NEWRC 30 x C54TC 0.38 NEWRC 30 x 7-1-5-34 0.32 NEWRC 64 x NERWC 74 0.15 NEWRC 64 x FLMR6-23-3 -0.03 NEWRC 64 x C34TC 0.69 * NEWRC 64 x C54TC 0.96 * NEWRC 64 x 7-1-5-4 0.04 NEWRC 74 x FLMR6-23-3 -0.53 NEWRC 74 x C34TC -0.28 NEWRC 74 x C54TC 0.26 NEWRC 74 x 7-1-5-34 0.09 FLMR6233 x C34TC -0.64 FLMR623 -3 x C54TC -0.63 FLMR6•23 -3 x 7-1-5-34 1.06 ** C34TC X C54TC -0.08 C34TC X 7-1-5-34 0.02 C54TC X 7-1-5-34 0.49 * * * Significant difference from 0 at P = 0.05 and P = 0.01 levels of probability, respectively.

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CHAPTER 5 SUMMARY AND CONCLUSION The overall objectives of this research were to optimize a tissue culture media sequence for in vitro plant regeneration of red clover, and to obtain information concerning the genetic basis of callus formation and plant regeneration response of nine red clover genotypes. Four experiments were conducted to optimize the culture system for red clover regeneration in vitro. In Exp. I, six genotypes of red clover were tested for tissue culture responses in six previously published media systems. The best regeneration response was observed on the sequence B5C-LSP-LSP. The B5C medium contained B5 basal salts supplemented with 2 . 0 mg L" 1 each of 2,4-D, NAA, and kinetin for callus induction. After four weeks, calli were then transferred to LSP medium (L2 basal medium with 0.002 mg L" 1 picloram plus 0.2 mg If 1 BAP) for embryo induction and development. The explants were again transferred to LSP medium for four weeks for plant regeneration . In Exp. II, six genotypes were tested on media which had different combinations of auxin and cytokinin sources 63

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64 and levels. Optimum regeneration in vitro was observed on the sequence L2C (2) -LSP-L2 (20) with four weeks interval. L2C(2) medium contained L2 basal salt supplemented with 2.0 mg L" 1 each of 2,4-D, NAA, and kinetin for callus induction. L2(20) contained L2 basal salt with 0.002 mg L" 1 of picloram and 1.0 mg L" 1 of BA. In Exp. Ill, the above two media systems selected in Exps. I, and II, and the B5 protocol were tested on six genotypes of NEWRC. The media system L2C (2 ) -LSP-L2 (20) from Exp. II gave the best plant regeneration response over genotypes . In Exp. IV, the same procedure as Exp. Ill was applied to ten genotypes of red clover which had exhibited a range in levels of plant regeneration capacity (high, medium, and low regeneration capacity) on B5 protocol. The media system L2C (2) -LSP-L2 (20) was again the best for plant regeneration in vitro in red clover. With this protocol genotypes which had exhibited an intermediate regeneration response on the B5 protocol showed increased regeneration. Progenies of 36 crosses of nine red clover genotypes were evaluated for callus formation and plant regeneration capacity using hypocotyl culture on B5 protocol. Diallel analysis was performed on callus diameter after 4 weeks in culture and plant regeneration response after 12 weeks of

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65 culture. Based upon results obtained from the diallel analysis, crosses with C54TC, 7-1-5-34, or C34TC as one parent were among the combinations showing the highest callus diameter and regeneration capacity, and those with NEWRC1, NEWRC4, or NEWRC30 as one parent were among the lowest for callus diameter and regeneration capacity. The diallel analysis showed highly significant GCA and SCA effects for callus diameter. Estimates of GCA effects indicated that C54TC had a significant, positive GCA effect, suggesting that C54TC contributed the most to high callus diameter in its progenies. Estimates of SCA effects indicated that there were five cross combinations that showed significant positive effects for callus diameter. Highly significant GCA effects for regeneration capacity were observed, as well as significant SCA effects. Estimates of GCA effects indicated that C54TC and 7-1-5-34 contributed the most to high regeneration capacity in their progenies. Estimates of SCA effects indicated there were three cross combinations that showed significant positive effects for regeneration capacity of red clover. The conclusions drawn based upon the results of these studies were as follows: (1) The optimum media system for plant regeneration capacity in red clover was an L2 basal medium supplemented with 2.0 mg IT 1 each of NAA, 2,4-D, and kinetin for callus induction for four weeks, with 0.002 mg

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66 If 1 picloram +0.2 mg If 1 BA for embryo induction and regeneration for four weeks, and for another four weeks in medium supplemented with 0.002 mg IT 1 picloram + 0.1 mg If 1 BA for plant regeneration; (2) Both GCA and SCA were significant sources of variation for response of red clover genotypes in tissue culture, both for callus diameter and regeneration capacity; however, GCA appeared to be more important than SCA and; (3) Useful sources of plant regeneration in vitro were observed in the red clover genotypes tested.

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68 Chu, Q. R., and Croughan, T. P. 1990. Genetics of plant regeneration in immature panicle culture of rice. Crop Sci. 30:1194-1197. Collins, G. B., and Phillips, G. C. 1982. In vitro tissue culture and plant regeneration in Trifolium pratense L. In : Variability in Plants Regenerated from Tissue Culture. E.D. Early and Y. Demarly (eds.). Praeger, New York. Page 22-34. Cope, W. A., and Taylor, N. L. 1985. Breeding and Genetics. Xa: Clover Science and Technology. N. L. Taylor (ed.). Agron. Monogr. 25. ASA, CSSA, and SSSA, Madison, WI . Page 383-400. Crea, F. , Bellucci, M. , Damiani, F. , and Arcioni, S. 1995. Genetic control of somatic embryogenesis in alfalfa {Medicago sativa L.cv. Adriana) . Euphytica 81:151-155. Frankenberger, E. A., Hasegawa, P. M., Tighelaar, E. C. 1981. Diallel analysis of shoot-forming capacity among selected tomato genotypes. Z. Pf lanzenphysiol . 102:233242. Gamborg, 0. L., Miller, R. A., and Ojima, K. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50:151-158. Grosser, J. W., and Collins, G. B. 1984. Isolation and culture of Trifolium rubens protoplasts with whole plant regeneration. Plant Sci. Lett. 37:165-170. Hernandez-Fernadez, M. M., and Christie, B. R. 1989. Inheritance of somatic embryogenesis in alfalfa (Medicago sativa L.). Genome 32:318-321. Keyes G. J., Collins, G. B., and Taylor, N. L. 1980. Genetic variation in tissue cultures of red clover. Theor. Appl. Genet. 58:265-271. Kielly, G. A., and Bowley, S. R. 1992. Genetic control of somatic embryogenesis in alfalfa. Genome 35:474-477. Koba, T., Shimada, T., and Otani, M. 1993. Diallel analysis of the performances on pollen embryo formation and plant regeneration in anther culture of common wheat (Triticujn aestivum L.). Bull RIAR Ishikawa Agr. Coll. 3:8-13.

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69 Komatsuda, T., Enomoto, S., and Nakajima, K. 1989. Genetic of callus proliferation and shoot differentiation in barley. J. Heredity 80:345-350. Lazar, M. D., Baenziger, P. S., and Schaeffer, G. W. 1984a. Combining abilities and heritability of callus formation and planlet regeneration in wheat (Triticum aestivum L.) anther cultures. Theor. Appl . Genet. 68:131-134. Lazar, M. D., Schaeffer, G. W., and Baenziger, P. S. 1984b. Cultivar and cultivar x environment effects on the development of callus and polyhaploid plants from anther cultures of wheat. Theor. Appl. Genet. 67:273-277. Linsmaier, E. M. , and Skoog, F. 1965. Organic growth factor requirement of tobacco tissue cultures. Physiol. Plant. 18:100-127. MacLean, N. L., and Nowak, J. 1989. Plant regeneration from hypocotyl and petiole callus of Trifolium pratense L. Plant Cell Rep. 8:395-398. Maheswaran, G., and Williams, E. G. 1984. Direct somatic embryoid formation on immature embryos of Trifolium repens, T. pratense and Medicago sativa, and rapid clonal propagation of T. repens. Ann. Bot. 54:201-211. Maheswaran, G., and Williams, E. G. 1986. Clonal propagation of Trifolium pratense, T. resupinatum and T. subterraneum by direct somatic embryogenesis on cultured immature embryos. Plant Cell Rep. 3:165-168. Mathias, R. J., and Simpson, E. S. 1986. The interaction of genotype and culture medium on the tissue responses of wheat (Triticum aestivum L.em.Thell) callus. Plant Cell Tissue Organ Culture 7:31-37. McGee, J. D., Williams, E. G., Collins, G. B., and Hildebrand, D. F. 1989. Somatic embryogenesis in Trifolium: Protein profiles associated with highand low-frequency regeneration. J. Plant Physiol. 135:306312. Meglic, V., and Smith, R. R. 1992. Self-incompatibility and seed set in colchicine-, nitrous oxide-, and sexually derived tetraploid red clover. Crop Sci. 32:1133-1137.

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70 Murashige, T., and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497. Myers, J. R., Grosser, J. W., Taylor, N. L., and Collins, G. B. 1989. Genotype-dependent whole plant regeneration from protoplasts of red clover (Trifolium pratense L.) Plant Cell Tissue Organ Culture 19:113-127. Nesticky, M . , Novak, F. J., Piovarci, A., and Dolezelova, M . 1983. Genetic analysis of callus growth of maize {Zea mays L.) in vitro. Z. Pf lanzenzuchtg 91:322-328. Petolino, J. F., Jones, A. M . , and Thompson, S. A. 1988. Selection for increased anther culture response in maize Theor. Appl . Genet. 76:157-159. Phillips, G. C, and Collins, G. B. 1979. In vitro tissue culture of selected legumes and plant regeneration from callus cultures of red clover. Crop Sci. 19:59-64. Phillips, G. C, and Collins, G. B. 1980. Somatic embryogenesis from cell suspension cultures of red clover. Crop Sci. 20:323-326. Phillips, G. C, and Collins, G. B. 1982. Red clover and other forage legumes. In: Handbook of Plant Cell Culture. W. R. Sharp, D. A. Evans, P. V. Amirato, and Y. Yamada (eds.). MacMillan, New York. Page : 169-210 . Phillips, G. C, Grosser, J. W., Berger, S., Taylor, N . , and Collins, G. B. 1992. Interspecific hybridization between red clover and Trifolium alpestre using in vitro embryo rescue. Crop Sci. 32:1113-1115. Quesenberry, K. H., and Smith, R. R. 1993. Recurrent selection for plant regeneration from red clover tissue culture. Crop Sci. 33:585-589. Quesenberry, K. H. , Wofford, D. S., Krottje, P. A., and Smith, R. L. 1992. Production of transgenic red clover using Agrobacteriuin-mediated DNA transfer. Proc. Twelfth Trifolium Conference. UF Gainesville. Page 5-10. Quimio, C. A., and Zapata, F. J. 1990. Diallel analysis of callus induction and green-plant regeneration in rice anther culture. Crop Sci. 30:188-192.

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71 Rakoczy-Trojanowska, M., and Malepszy, S. 1993. Genetic factors influencing regeneration ability in rye {Secale cereale L.) immature inflorescences. Theor. Appl. Genet. 86:406-410. Ray, I. M . , and Bingham, E. T. 1989. Breeding diploid alfalfa for regeneration from tissue culture. Crop Sci . 29:1545-1548. Reisch, B., and Bingham, E. T. 1980. The genetic control of bud formation from callus cultures of diploid alfalfa. Plant Sci. Lett. 20:71-77. Schenk, R. U., and Hildebrandt, A. C. 1972. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot . 50:199-204 . Smith, R. R., Taylor, N. L., and Bowley, S. R. 1985. Red Clover. In: Clover Science and Technology. N. L. Taylor (ed.). Agron. Monogr. 25. ASA, CSSA, SSSA, Madison, WI. Page 458-468. Taylor, N. L., Anderson, M. K., Quesenberry, K. H., and Watson, I. 1976. Doubling the chromosome number of Trifolium species using nitrous oxide. Crop Sci. 16:516518. Taylor, T. E., and Veilleux, R. E. 1992. Inheritance of competences for leaf disc regeneration, anther culture and protoplast culture in Solanum phureja and correlations among them. Plant Cell Tissue Organ Culture 31:95-103. Tisserat, B., Esan, E. B., and Murashige, T. 1979. Somatic embryogenesis factors in angiosperms . Hort. Rev. 1:1-78. Tomes, D. T., and Smith, 0. S. 1985. The effect of parental genotype on initiation of embryogenic callus from elite maize (Zea mays L.) germplasm. Theor. Appl. Genet. 70:505-509. Wan, Y., Sorensen, E. L., and Liang, G. H. 1988. Genetic control of in vitro regeneration in alfalfa {Medicago sativa L.) . Euphytica 39:3-9.

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72 Williams, E. G., and Maheswaran, G. 1986. Somatic embryogenesis factors influencing coordinated behaviour cells as an embryogenic group. Ann. Bot. 57:443-462. Wofford, D. S., Baltensperger, D. D., and Quesenberry, K. H. 1992. In vitro culture responses of alyceclover genotypes on four media systems. Crop Sci. 32:261-265. Yu, K., and Pauls, K. P. 1993. Identification of a RAPD marker associated with somatic embryogenesis in alfalfa. Plant Mol. Biol. 22:269-277. Zhou, H., and Konzak, C. F. 1992. Genetic control of green plant regeneration from anther culture of wheat. Genome 35:957-961.

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BIOGRAPHICAL SKETCH Yuyu Suryasari Poerba was born on November 23, 1961, in Sukabumi, West Java, Indonesia. She graduated from high school in 1980 in Sukabumi. She then attended the University of Padjadjaran in Bandung, Indonesia from 1980 to 1984, to study agriculture. She received a bachelor of science degree in agriculture, 'Sarjana Pertanian', in December 1984. In April 1985, she began working in the Indonesian Institute of Sciences, at the Plant Genetics Laboratory in Bogor. During the first year, she received a scholarship to continue her study. In August 1986, she enrolled in Mississippi State University to begin working towards her Master of Science degree in Genetics. During her study, she was married to Desendi Poerba on August 18, 1988. After completing her study in 1989, she went back to her country and continued working in the same institution. On April 18, 1991, the couple were fortunate to have their first son, Yudson, enter their lives. During her son's first year of age, she again received a scholarship to pursue her Ph.D. degree. In August 1992, she came to the University of Florida to continue working towards her Ph.D 73

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74 in Agronomy. Her area of specialization is plant breeding and genetics, with a minor in environmental horticulture. On February 1993, she was reunited with her family. The family was again fortunate to have Deyson, who was born prematurely on April 13, 1994. She plans to return to her institution in Indonesia, upon completion of her degree.

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. L\ , ' ;^T/: 7^. Cuts ,4 Cu U±. AKenneth H. Quesenberry, Chair Professor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. David S. Woffopd' Associate Professor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate^ in scope and quality, as a dissertation for the degree of /Doctor of PhfTbsophy . use < "J^ti^ Paul L. Pfahlei Professor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Miohael E. Kane Associate Professor of Horticultural Science

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. JfoAe W. Grosser Professor of Horticultural Science This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor Philosophy. May 1996 Dean, College of Agriculture Dean, Graduate School


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