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In vitro agrobacterium mediated transformation and regeneration of white clover (Trifolium repens L.)

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In vitro agrobacterium mediated transformation and regeneration of white clover (Trifolium repens L.)
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Goldman, Jason J
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English
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ix, 85 leaves : ill. ; 29 cm.

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Agrobacterium ( jstor )
Bacteria ( jstor )
Clover ( jstor )
Cotyledons ( jstor )
DNA ( jstor )
Genes ( jstor )
Genotypes ( jstor )
Plant roots ( jstor )
Plants ( jstor )
Plasmids ( jstor )
Agronomy thesis, Ph.D ( lcsh )
Dissertations, Academic -- Agronomy -- UF ( lcsh )
Genetic transformation ( lcsh )
Regeneration (Botany) ( lcsh )
White clover -- Genetics ( lcsh )
White clover -- Micropropagation ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph.D.)--University of Florida, 1998.
Bibliography:
Includes bibliographical references.
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Typescript.
General Note:
Vita.
Statement of Responsibility:
by Jason J. Goldman.

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University of Florida
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IN VITRO AGROBACTERIUMMEDIATED TRANSFORMATION AND
REGENERATION OF WHITE CLOVER (TRIFOLIUMREPENS L.)












By

JASON J. GOLDMAN


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


1998















ACKNOWLEDGMENTS


I wish to thank Dr. Paul Lyrene, Dr. Gloria Moore, Dr. Ken Quesenberry, Dr. Rex

Smith, and Dr. David Wofford. Their good advice and guidance was most helpful and

appreciated.

I wish to thank Dr. Maria Gallo-Meagher and Dr. Bob Shatters for supplying

helpful information about Southern blot DNA analysis.

I wish to thank Jeff Seib for aiding in the details and techniques of DNA

extraction and Southern blot analysis.

I wish to thank Gail Bryant and Jeremy Green for tissue culture and greenhouse

assistance.

I wish to thank my mother, father, and sister for your support and encouragement.














TABLE OF CONTENTS



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

TABLE OF CONTENTS .............................................. iii

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

LIST OF FIGURES .................................................. viii

AB STR A C T ........................................................ ix

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

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

W white C lover ......................................................... 2
Breeding and Genetics ............................................ 3
Regeneration ................................................... 4

Plant Transformation ................................................... 4
Virus Resistance ................................................ 5
Quality Enhancement ............................................. 8
An Elite Cultivar to Transform--Osceola .............................. 9
Vector for DNA Integration ........................................ 9
Agrobacterium Strains and Plasmids ................................ 11
Promoters .................................................... 13
Mannopine synthase (MAS) ................................. 15
Nopaline synthase (NOS) ...... ............................ 15
Cauliflower mosaic virus 35s (35s) ........................... 16
Tobacco basic chitinase .................................... 16
Selectable M arker Gene .......................................... 18
NPTII .................................................. ..19
bar .................................................... 20
Detectable Marker beta-glucuronidase (gus) ......................... 21
DNA Anaylsis ................................................. 23


iii









Southern blot ............................................ 23
Polymerase chain reaction-PCR ............................. 24

White Clover Transformation ........................................... 24

MATERIALS AND METHODS ......................................... 28
W hite Clover Regeneration ....................................... 28
Germinated Seedlings Preparation ............................ 28
Water Imbibed Pre-germinated Seeds .......................... 29
Data Collection & Analysis .................................. 30
White Clover Transformation ...................................... 30
Preliminary Lethality Tests .................................. 30
M edium Preparation ....................................... 31
Seed Sterilization H2S04, PPM .............................. 31
Cutting the Explant ........................................ 32
Agrobacterium Growth and Storage ........................... 32
Co-Cultivation ........................................... 33
Recovering Plants ......................................... 34
Evaluation of Transformed Plants ................................... 35
X-Gluc Stain Preparation ................ ................. 35
GUS Petiole Histochemical Assay ............................. 36
GUS Root Histochemical Assay .............................. 36
Leaf Painting Assay ....................................... _37
Herbicide Application Tests ................................. 37
Somaclonal Variation or Insertion Effect ....................... 38
Crossing and Segregation of Transgenes ........................ 38
Southern Blot ............................................ 39
Polymerase Chain Reaction .................................. 40

R E SU L T S .......................................................... 44
W hite Clover Regeneration ....................................... 44
Germinated Seedlings ...................................... 44
Water Imbibed Pre-Germinated Seeds ......................... 45
White Clover Transformation ...................................... .45
Preliminary Lethality Tests .................................. 45
Seed Sterilization ......................................... 46
Cutting the Explant ........................................ 47
Agrobacterium Growth and Storage ........................... 47
Co-Cultivation ........................................... 48
Recovering Plants ......................................... 49
Evaluation of Transformed Plants ................................... 50
GUS Histochemical Assays .................................. 50
Herbicide Painting and Spraying .............................. 51
Crossing and Segregation of the Transgene ...................... 53

iv









DN A Analysis ........................................... 53

DISCUSSION ....................................................... 67
White Clover Regeneration ....................................... 67
White Clover Transformation ...................................... 69
Breeding Transgenic White Clover .................................. 71
Patent Problems ................................................ _74

LITERATURE CITED ................................................ 76

BIOGRAPHICAL SKETCH ............................................ 85















LIST OF TABLES


Table page

1. Summary of cellular processes involved in Agrobacterium-plant interactions........... 26

2. Agrobacterium strains used in white clover transformation experiments.................. 27

3. Plant growth medium used to induce shoots and roots in non-transformed
partner genotypes .............................................................................................. 41

4. Medium used for liquid and solid phase Agrobacterium growth .............................. 41

5. Comparison for response to direct shoot induction among white clover
cultivars adapted to the southeast United States ................................................. 55

6. Effect ofcarbenicillin on direct shoot induction ...................................................... 55

7. White clover transformation experiments : 1996 1998 .......................................... 56

8. Response of transformed and non-transformed white clover to the
herbicide PPT .................................................................................................... 57

9. Seed yield from various white clover hand crosses .................................................. 58
















LIST OF FIGURES


Figure pag

1. Map of plasmid pCPOO 1 used with Agrobacterium strain AGL1 for
white clover transformation ............................................................................... 27

2. The three day old seedling and how to obtain the intact and split
cotyledons for tissue culture .............................................................................. 42

3. Solid and liquid phase Agrobacterium growth ........................................................ 43

4. Time frame required to regenerate transformed plants ............................................ 43

5. Effect of the hormones BAP and NAA on direct shoot induction ............................ 59

6. Direct shoots forming on 21 day old explants obtained from a
germinated white clover seedling ....................................................................... 60

7. Histogram comparing split and intact explants for response to
shoot induction .................................................................................................. 60

8. Stages of shoot development using the imbibed seed as an explant
source ................................................................................................................ 6 1

9. Root emerging from PPT resistant shoot while other non-transformed
shoots are killed ................................................................................................. 62

10. Rooted plant in peat pellet and many plants incubating in a mist-box ..................... 62

11. GUS histochemical staining of transformed white clover tissue ............................. 63

12. Effect of 200 mg/L PPT on partially transformed plant a5#16 .............................. 64

13. PCR amplification of the bar gene from transformed genomic DNA..................... 65

14. Southern blot of white clover genomic DNA........................................................ 66


vii

















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

IN VITRO AGROBACTERIUM MEDIATED TRANSFORMATION AND
REGENERATION OF WHITE CLOVER (TRIFOLIUMREPEIVS L.)


By

Jason J. Goldman

December 1998

Chairperson: Dr. David S. Wofford
Major Department: Agronomy

White clover is a high quality forage that fixes atmospheric nitrogen and can

improve animal fertility. Genetic transformation technology has the potential to enhance

existing varieties and to be used in the development of novel varieties for future use. A

genotype independent tissue culture regeneration system is required to use these genetic

technologies and this has proven to be a limiting factor for white clover. A new

regeneration protocol based on germinated seedlings was tested on five white clover

cultivars adapted to the USA. This system proved to be much less genotype specific than

previous reports, with 39 to 51% of the individuals in these five cultivars producing 1 or

more shoots. Attempts to use this system in combination with Agrobacterium-mediated

transformation techniques, however, proved fruitless. A different regeneration protocol


viii










was investigated that used imbibed seeds as the source of tissue for the cultures. Results

from this system showed equally high levels of prolific regeneration. The use of this

protocol with Agrobacterium proved successful and transgenic white clover plants were

recovered at a frequency of ca. 4%. PCR and Southern blot data provided molecular

evidence of integration of the novel genes in the transformed regenerated plants. Hand

pollination between transgenic and non-transgenic plants produced progeny which

segregated at the expected 1:1 ratio, which indicated the stable sexual transmission of the

novel genes. A greenhouse test was conducted to evaluate the response of transgenic and

non-transgenic plants to levels of the herbicide phosphinothricin (PPT). Results indicated

that some transformed plants were completely resistant to 200 mg/L PPT and could

tolerate 2000 mg/L while other transformants could not resist 200 mg/L. Use of this

protocol from a practical breeding point of view and concerns due to intellectual property

rights are also addressed.















INTRODUCTION


Plant genetic engineering is rapidly becoming a practical tool for cultivar

development and improvement. The first decade of research primarily involved the

development and optimization of proven transformation protocols for major experimental

and agronomic plant species. Improvements in vectors to integrate the chimeric DNA,

along with more effective selectable marker genes for recovering transgenic plants, has

made it possible to transform many different plant species. Refining transformation

protocols for greater convenience, higher efficiency, and broader genotype range is still a

high priority in some species. The following dissertation describes the requirements and

methods for a white clover (Trifolium repens L.) Agrobacterium transformation protocol.

The objectives of this experiment were to : (1) evaluate the effectiveness of genotype

independent direct shoot regeneration protocols using white clover explants from cultivars

adapted to the southeast United States (2) utilize the most effective regeneration protocol

to develop a white clover Agrobacterium-mediated transformation protocol in which both

transformed and non-transformed plants with the same genetic background are recovered

in vitro. The importance of recovering the non-transformed control is discussed. Initially

the transformation protocol was based on Voisey et al. (1994) and later changed to a

modified Larkin et al. (1996) protocol.















LITERATURE REVIEW


White Clover



White clover (Trifolium repens L.) is the most important pasture legume in many

parts of the temperate zones (Carlson et al. 1985). In addition to being a high quality

forage, white clover promotes growth of associated grasses and improves soil fertility by

fixing atmospheric nitrogen. Other benefits of white clover in pastures include

improvements in animal health, milk flow, calf weaning weight, daily gains, and conception

rates (Carlson et al. 1985). White clover is considered very nutritious and palatable at all

stages of development. All kinds of livestock, including dairy and beef cattle, sheep, hogs,

and poultry, relish the tender, succulent leaves. Cultivars of white clover can be classified

as small, intermediate, or large (Ladino), essentially the same except for size of leaf,

degree of flowering and plant height. White clover is usually planted with one or more

grass companions to reduce the risk of bloat in sheep and cattle, increase the chance of a

good stand, and aid in mowing and curing of hay.

Although a perennial, in the southeast United States white clover stands commonly

decline within two to three years after establishment (Dobson et al. 1976). Virus diseases

and nematodes have been suggested as the major factors in the weakening of white clover












plants, thus making them more susceptible to injury and death from other environmental

stresses and diseases (Gibson et al. 1981).


Breeding and Genetics


White clover (2n--4x=32) is an outcrossing species with disomic inheritance. A

strong gametophytic self-incompatibility system based on multiple oppositional alleles at

the S locus exits in white clover (Williams 1987). It is characterized by independent action

of S alleles in both pollen and style. Growth of pollen tube carrying a given S allele is

arrested in stigmas bearing an identical allele. Populations are, therefore, a heterogeneous

mixture of highly heterozygous individuals, resulting in high levels of genetic variation both

within and between populations. The large amount of genetic variability present in white

clover populations/cultivars enhances overall persistence in competitive, grazed swards,

where a wide variety of micro-environments are encountered. The goal of a white clover

breeder in developing cultivars is to increase the frequency of favorable genes while

avoiding inbreeding depression. Phenotypic recurrent selection within adapted germplasm

pools has been by far the most common means of population improvement in white clover

(Williams 1987). Improved production from grazing animals and persistence are major

breeding goals. Cultivars released to date have been synthetic varieties produced from

open pollination of selected elite clones or seed lines. These populations are maintained by

random pollination in isolation.












Regeneration


A regeneration system independent of genotype is desirable for a highly out-

crossing species like white clover. White clover is responsive to a wide range of

regeneration protocols. Plants have been regenerated from callus (Mohapatra and

Gresshoff 1982; White 1984; Bhojwani et al. 1984; Webb et al. 1987a; Yamada 1989),

protoplasts (Bhojwani and White 1982; Webb et al. 1987b; Yamada 1989), directly from

stolon segments (Bond and Webb 1989), and through somatic embryogenesis (Parrot 1991;

Weissinger and Parrott 1993). All of these regeneration protocols were limited, however,

to very few genotypes with high regeneration capacity. White and Voisey (1994) reported

a method to regenerate shoots directly from cotyledonary stalk tissue of germinated

seedlings that reduced the strict dependency on genotype. White and Voisey (1994) split

the cotyledon in half and obtained numerous direct shoots with an average of 20/cotyledon.

Beattie and Garrett (1995) obtained adventitious shoot production from immature embryos

of single cross T. repens cultivar Haifa plants using a technique that also may be

independent of genotype. Most recently, Larkin et al. (1996) obtained high frequency

direct shoot regeneration from the same cotyledon-hypocotyl region using pre-germinated,

water-imbibed seeds.



Plant Transformation


In order to successfully introduce foreign DNA into the plant nuclear genome,

many different tools are required. A desirable gene for agronomic or experimental










5
purposes, promoters, selectable markers, and an efficient vector for stable DNA integration

are a few of the essential tools. The next few sections will describe in more detail the

components required for white clover transformation. Although there are many different

options for what components are combined in an overall transformation protocol, only

tools that were used in this study or may be valuable for future white clover transformation

experiments will be described. Currently single gene traits are most commonly inserted

into a plant genome. Virus, insect, and nematode resistance are desirable traits that may be

used to increase stand persistence in white clover. White clover transformed with a

herbicide resistance gene could enable new weed control options for broad leaf weeds in

clover pastures. Less common, but also desirable, are genes that alter quality or produce

substances that are too complex or expensive for laboratory synthesis.


Virus Resistance


Plant virus infection can cause severe damage, resulting in significant yield

reduction. Since there is no known way to completely eradicate a virus from commercial

production of many agronomic crops, a number of preventative strategies are typically

implemented. Some of these strategies include planting certified virus free cultivars,

extensive spraying of chemical insecticides to eliminate vectors for transmission, and

breeding for host plant resistance. Of these strategies, host plant resistance is the only

feasible option for a forage legume such as white clover. Breeding for resistance to

viruses, however, is normally complicated because resistance genes are either unknown or

found in wild-type germplasm. Extensive backcross programs are usually required to










6

eliminate other undesirable wild type genes that are linked to the resistance genes. This can

be very expensive, require many years to complete, and the occurrence of resistance

breaking virus biotypes is always a possibility. Clover yellow vein virus, peanut stunt virus

(PSV), and alfalfa mosaic virus are the predominant viruses affecting white clover in the

southeast United States (McLaughlin et al. 1992). Trifolium ambiguum may contain

genetic resistance to peanut stunt virus (Pederson and McLaughlin 1989), however,

resistance to the most common viruses including PSV is lacking in domesticated

germplasm (Pederson and McLaughlin 1994). The expression of virus-derived genes in

plants, either in a functional or mutated form, can protect the host plant from the virus in

which the genes were isolated. This appears to overcome many of the drawbacks of

breeding for resistance. With the advent of improved cell culture and molecular biology

techniques, virus genes can be introduced and expressed in a wide variety of crops without

interfering with important agronomic traits. The virus coat protein (CP) gene, the virus

replicase gene, and movement protein (MP) genes are the most common sequences used

for transgenic protection. Integration of virus gene in the antisense orientation or mutating

the sequence to render the mRNA transcript untranslatable or the mature protein non-

functional are common resistance strategies. The coat protein gene of PSV has been

cloned (Naidu et al. 1991) and may be useful for inducing stable long-term resistance in

white clover. There is currently no model that explains how coat protein derived resistance

is achieved. Gene silencing via RNA:RNA hybridization or an in vivo dsRNA degradation

system that is only active when a certain threshold of dsRNA is achieved are current

theories (Goodwin et al. 1996; Tanzer et al. 1997; Jorgensen et al. 1998; Wassenegger and












Pelissier 1998). Interference of the invading virus life cycle by a mutated virus protein to

stop replication or movement may be involved in some resistance situations.


The Bt Gene(s) and Insect Resistance


Insect pests are a major cause of damage to the world's important agricultural

crops. Common control strategies include the application of chemical pesticides to

eliminate the pest. Integrated pest management (IPM), a more environmentally sensitive

approach, uses multiple control measures to reduce the insect damage below the economic

threshold. Trap crops, resistant cultivars, crop rotation, spraying, and insect phermones are

frequently used techniques for IPM control. The use of resistant varieties is an effective

control method, however, genes for insect resistance are not always available or easily

integrated into existing cultivars. The development of insect resistant biotypes along with

multiple insect pests can quickly lead to major crop damage. A new strategy for insect

control involves transforming plants with a resistance gene(s) from another species that

conveys resistance Most of the insecticidal genes currently in use are of bacterial origin, in

particular from Bacillus thuringiensis (Bt). The soil microorganism Bt has proven to be a

rich source for insecticidal proteins and genes. During the sporulation phase, Bt produces

parasporal crystals that consist of about 130 kDa proteins know as 6-endotoxins (Koziel et

al. 1993). They exert their toxicity by binding to the midgut epithelial cells and ultimately

causing osmotic lysis through pore formation in the cell membrane (Gill et al. 1992). Bt

has been used as an insecticide for more than 40 years, but it was the cloning and

sequencing of the insecticidal protein genes (Schnepfand Whiteley 1981) that raised the












prospects of using insecticidal proteins in transgenic plants. Early experiments indicated

that the gene was expressed in plants but at a level too low to convey adequate field

resistance. The gene was modified by increasing the GC content of the sequence for

increased plant expression. Currently, there are a few cultivars available that exhibit high

levels of insect resistance by expressing a Bt gene (Estruch et al. 1997). Known Bt strains

contain a great variety of 6-endotoxin encoding genes. A total of 96 genes have been

described so far and more are being reported routinely. Based on recent USA patents that

have been filed, some of these genes appear to convey high levels of resistance against the

major root knot nematodes that infest root systems of some important crops including

white clover.


Quality Enhancement


Most of the transgenic plants currently in production were developed for insect,

virus, or herbicide resistance. Other possibilities are altered plant fats and oils, methionine-

and lysine-enhanced grain and legume proteins, plant foods that can deliver immunizing

antigens, and alteration of metabolic processes such as fruit ripening (Day 1996). White

clover already possesses a high protein content and many of the enhancements previously

mentioned are catered toward human consumers. Since it is a perennial and produces high

levels of vegetative growth per unit time, it may make an ideal species for pharmaceutical

or biodegradable polymer production (Haq et al. 1995; Nawrath et al. 1995).

Bioremediation by expressing genes for heavy metal and mercury accumulation may be

possible with white clover. It could be beneficial to transform white clover with genes)












that are involved with synthesis of an organic molecule or protein that is too complex for

commercial laboratory production. In this way the active ingredient could be produced in

the leaves and extracted at multiple harvests throughout the growing season.


An Elite Cultivar to Transform-Osceola


Osceola is a ladino-type, medium-blooming white clover cultivar adapted to Florida

and other white clover producing areas of the United States (Baltensperger et al. 1984). It

was developed over a 30 year period to provide a longer-lived, more productive, and better

reseeding ladino-type for Florida conditions. Seeds from 35 selected intercrossed clones

were bulked in 1973 to produce the breeder's seed for the synthetic cultivar Osceola white

clover. Osceola combines better summer stolon persistence and fall regrowth with

sufficient seed production to ensure a stand.


Vector for DNA Integration


Assuming a desirable gene is available, a vector to integrate the foreign DNA into

the plant nuclear genome is required. There are many published protocols that describe a

novel way to integrate the DNA. These methods may or may not be reliably reproduced or

feasible for a variety of plant species. Agrobacterium and a biolistic approach are the most

common and reproducible methods for DNA integration used to date. The biolistic

approach is literally a shot gun approach in which the foreign DNA is coated onto micro

size gold or tungsten beads. The coated beads, propelled by a helium gas blast and

regulated by rupture disks, are directed at plant tissues that can be regenerated (Sanford










10
1988). The biolistic approach is best suited for monocots and dicotyledons species that are

recalcitrant to Agrobacterium transformation.

The Agrobacterium plant cell interaction is the only known natural example of

DNA transport between kingdoms (Sheng and Citovsky 1996). Agrobacterium

tumefaciens is a soil plant pathogen that genetically transforms host plant cells. Wild type

strains of Agrobacterium transfer genes that code for plant hormone-like compounds that

cause rapid undifferentiated cell division resulting in a tumor-like structure known as a gall.

Another set of genes that code for amino acid-like molecules termed opines are also

integrated into the plant genome. These genes function in association to provide a place

for the invading bacteria to live, the gall, and food to ingest, opines. Other soil

microorganisms can not metabolize opines, therefore, creating a favorable biological niche

for Agrobacterium. The DNA that is transported from the bacteria (T-DNA) and inserted

in the plant genome is located on a large tumor inducing (Ti) plasmid. T-DNA is defined

by two 25-bp imperfect direct repeats known as the left and right borders. All other genes

required for transfer of the T-DNA to the plant nucleus, known as the virulence (vir)

genes, are also located on the Ti plasmid. Seven major vir loci (virA, virB, virC, virD,

virE, virG, and virH) and a few genes located on the Agrobacterium chromosome (chv)

are involved in the chain of events from excision of T-DNA to binding of bacteria to host

plant cell surface receptors, to integration of T-DNA into plant genomic DNA. Table 1

contains a summary of major events and genes involved in the T-DNA transfer process. To

exploit Agrobacterium as a vector for plant transformation, the wild type bacteria must be

disarmed by removing the wild-type tumor and opine genes. Because the T-DNA is












defined by its borders, the coding region can be replaced by a DNA sequence without a

deleterious effect on transfer from Agrobacterium to the plant. Original vectors introduced

the chimeric gene between the left and right T-DNA boarders of the Ti plasmid by

homologous recombination. It was later discovered that the vir genes will work in trans

and that the T-DNA region could be located on a separate plasmid. The binary vector

system (Hoekema et al. 1983), uses two compatible plasmids, one containing the vir-

region, the other carrying the T-DNA on a wide host-range replicon. This system has

many advantages over homologous recombination. The Ti plasmid is large and difficult to

manipulate. By placing T-DNA on a separate, smaller plasmid, it can be easily genetically

manipulated using Escherichia coli as a host. Transfer of this plasmid into an A.

tumefaciens strain harboring the the disarmed Ti plasmid with the vir-region allows

introduction of manipulated T-DNA into plant cells. Binary plasmids are usually 10-20kb

with the T-DNA region somewhat smaller. Recent evidence indicates that use of a binary

bacterial artificial chromosome vector, with helper plasmids enhancing production of VirG

and VirE proteins, can allow efficient Agrobacterium mediated transfer of at least 150kb of

foreign DNA into the plant nuclear genome (Hamilton 1997). This may allow large regions

of chromosomes that are thought to contain certain genes or quantitative trait loci to be

transformed into the plant genome.


Agrobacterium Strains and Plasmids


Different strains ofAgrobacterium can vary greatly in their virulence to specific

plant species. Inoculating a wounded plant with a variety of wild type Agrobacterium











12

strains and then evaluating tumor formation is a common way to determine if a particular

strain is a good candidate for transformation. Many confirmed virulent wild-type strains

have been engineered to disarm the T-DNA and enhance virulence. Agrobacterium strains

EHA101 and AGL1 are both derived from wild type A281, a L,L-succinamopine strain

(Hood et al. 1986). A281 contains a C58 chromosomal background and is hypervirulent

on several solanaceous plants. The Ti plasmid of A281, pTiBo542, was disarmed

producing strain EHA101. The disarmed Ti plasmid of EHA101 was further modified to

produce AGL1 (Lazo et al. 1991). Both EHA101 and AGL1 are hypervirulent in trans

when a binary plasmid containing the T-DNA borders is present. Table 2 contains a

summary of how the Agrobacterium strains were derived and what binary plasmid they

contain.

The Agrobacterium plant transformation vector is most commonly exploited using

a binary vector approach. The binary plasmid that contains the T-DNA borders may be

quite variable. The genes that are located between the left and right border and their

arrangement are common differences. The T-DNA is thought to be transferred in a right

border to left border, 5' to 3', orientation (Zambryski 1988; Gleave 1992; Becker et al.

1992). Selectable marker genes are, therefore, better located closest to the left border

making it the last gene to be integrated, assuming complete T-DNA transfer. If T-DNA

transfer from the plasmid is prematurely aborted and only partial T-DNA starting from the

right border is integrated into the plant genome, resistant cells lacking the novel gene will

not be recovered. If the selectable marker gene is closer to the right border than the novel

gene and T-DNA transfer is aborted prematurely, plants may be recovered that express the










13
selectable marker gene but lack the novel gene. Other differences between binary plasmids

include their size in base pairs, restriction sites available for cloning, and which antibiotic

resistance genes they carry for maintenance of the binary plasmid.

Binary vector plasmid pCPOO1 contains a 1764 bp tobacco (Nicotiana tabacum cv.

Samsun) basic chitinase promoter sequence derived from FB7-1 (Neale et al. 1990). This

had been PCR-engineered to allow cloning into the HindIIlI and BamHI sites of the binary

transformation vector pBIlOl.3 (Clonetech, Palo Alto, CA). The FB7-1 promoter: 3-

glucuronidase:nopaline synthase terminator (chitinase:gus:NOS) cassette was excised with

EcoRI and HindIII. EcoRI linkers were added, and the cassette was cloned into a unique

EcoRI site in the binary transformation vector pTAB 1O (Kahn et al. 1994) to create

pCPOO1 (Figure 1). This plasmid also contains a 35S:bar selectable marker.

Binary vector plasmid pMON9793 (McKently 1995) is a derivative ofpMON505

(Monsanto Co., St. Louis, MO) in which a chimeric gene containing a mannopine synthase

promoter(MAS), the coding region for 0-glucuronidase (gus) (Jefferson 1987), and the

nopaline synthase (NOS) 3' polyadenylation signal was cloned into the multilinker (Rogers

et al. 1987). Plasmid pMON9793 also contains a chimeric neomycin phosphotransferase

II (NPTII) selectable marker gene with the NOS promoter and NOS 3' polyadenylation

signal.


Promoters


Plant promoters are regulatory elements and constitute one of the major factors that

determines the temporal and spatial expression of a plant gene(s). A promoter can be












constitutive and allow for transcription in all cells, or it can be tissue specific and only

transcribe the gene in that particular cell type. Strong promoters induce high levels of

transcription producing high levels of steady state mRNA, in contrast to weak promoters

that result in low levels of mRNA synthesis. Eukaryote promoters contain conserved

regions in the 5' leader sequence. These include a CAAT and TATA box which aids in

recognition and of the promoter and binding by transcription factors (TF's). These TF

proteins are essential because RNA polymerase II cannot bind directly to eukaryotic

promoter sites and initiate transcription without their presence. RNA polymerase II can

initiate transcription from a promoter in vitro in the presence of five factors: TATA-binding

protein (TBP), TFIIB, TFIIE, TFIIE, TFIIF, and TFIIH (Emili and Ingles 1995).

Fusing the beta-glucuronidase (gus) reporter gene (Jefferson 1987) to an uncharacterized

promoter and then assaying enzymatic GUS activity is a common way to study promoter

specificity and strength. This can be misleading, however, due to post-transcriptional

processes that may result in gene silencing, although the promoter is fully active and

functioning. Early promoters used for transformation experiments were thought to be

constitutive were and used to control transcription of selectable marker genes. Now with

advancements in tissue specific promoter isolation, along with more desirable agronomic

foreign genes that require specific temporal expression, many new plant promoters are

available. The following sections contain a description of all promoters used in this study.

Agrobacterium tumefaciens, the wild type plant pathogen, carries eukaryotic-like

promoters that, when integrated into the host plant genome, transcribe genes for amino

acid-like molecules termed opines. Opines serve as a food source for the invading bacteria.










15

The promoters for two opine production genes, mannopine synthase and nopaline synthase,

were some of the first promoters used in plant transformation experiments. Initially it was

thought that these promoters were constitutive, thus making them good candidates to

control transcription of selectable marker genes, detectable marker genes, or any other

gene that should be transcribed in all cells.


Mannopine synthase (MAS)


The promoter for mannopine synthase, originally thought to be constitutive, is now

considered inducible and, in some cases, tissue specific. Strongest expression is usually

seen in the phloem and roots. This promoter is also wound-inducible, which increases

transcription and alters tissue specificity, leading to expression in leaves (Guevara-Garcia et

al. 1993). The addition of the plant growth regulators 2,4-dichlorophenoxyacetic acid

(2,4-D) and indole-3-acetic acid (IAA) also enhanced expression (Saito et al. 1991).


Nopaline synthase (NOS)


In nopaline-type tumor tissues, the nopaline synthase gene (nos) is one of the most

abundant transcripts (Dai and An 1995). Since the nos gene was considered to be

constitutively active in a variety of plant tissues, the promoter has been used to control

transcription of plant selectable and detectable marker genes. It was later shown, however,

that the nos promoter activity is organ-specific and developmentally regulated (An et al.

1988). In seedlings, lower parts exhibit a higher activity compared to upper parts. In older

plants, the promoter activity is very low throughout the entire plant except in roots and










16

certain reproductive organs (An et al. 1988). Promoter activity is enhanced or induced by

wounding, auxin (An et al. 1990), 1H202 (Dai and An 1995), methyl jasmonate and salicylic

acid, many of which are thought to be involved in plant defense related signal transduction

pathways (Kim et al. 1993).


Cauliflower mosaic virus 35s (35s)


The 35S promoter region of the cauliflower mosaic virus (CaMV) provides a model

plant nuclear promoter system, since its double-stranded DNA genome is transcribed by

host nuclear RNA polymerase II from a CaMV minichromosome (Oiszewski et al. 1982).

When the 35S promoter fused to a chloramphenicol acetyltransferase reporter gene was

transformed into tobacco, expression was approximately 30 times stronger than the

nopaline synthase promoter (Nagy et al. 1985). Moreover, it was constitutively expressed

in all organs oftransgenic plants. The relatively high promoter strength and constitutive

type of expression has made the 35S promoter a good candidate for controlling the

transcription of chimeric genes used as selectable markers in plant transformation

experiments.


Tobacco basic chitinase


The tobacco basic chitinase gene was isolated when Meeks-Wagner et. al (1989)

were attempting to clone genes transcribed specifically during flower development in

tobacco. A RNA differential display approach to identify mRNA that is transcribed in

early floral but not vegetative meristems was implemented. A thin cell layer (TCL) in vitro










17

tissue culture system that could be induced through hormones to develop into vegetative or

floral meristems was used for the poly A mRNA source of early genes in floral

development. Floral bud transcripts at day seven (FB7) were isolated from three different

gene families. FB7-1, the first group of transcripts isolated, were detected in stem and

internode segments and leaves of plants possessing an immature inflorescence. The highest

levels of FB7-1 transcript were found in the roots, reaching a maximum level at the stage

immediately prior to the transition of the shoot apex to its reproductive state

(Meeks-Wagner et. al 1989). Using DNA sequence data, Neale et al. (1990) determined

that the FB7-1 sequence belonged to the chitinase gene family. In tobacco, this gene

family comprises both acidic and basic chitinases (Legrand et al. 1987). All FB7-1

sequences show extensive homology to the basic chitinase sequence isolated from tobacco

Havana 425 (Shinshi et al. 1987). White clover cv. Haifa, an intermediate type, and

tobacco cv. Samsun, were transformed with the tobacco basic chitinase promoter fused to

the gus reporter gene to compare the temporal and spatial expression of this promoter

between species (Pittock et al. 1997). Transcription from the promoter was induced by

similar developmental and environmental signals in each species. In white clover, no

staining was observed in leaf, floral or stem tissue unless mechanically wounded. The

strongest expression was in root meristems of all main and emerged lateral root tips. This

root specific expression was dramatically reduced during the period of floral initiation and

flowering.













Selectable Marker Gene


A selectable marker gene is a key requirement for any plant transformation

protocol. This gene must be constitutively transcribed in the initially transformed cell(s). It

must then be expressed in all further cell divisions and ultimately in the entire plant. The

selectable marker gene codes for a protein that can detoxify, degrade, or inhibit a

phytotoxic chemical. After the DNA integration step, the plant material is placed on

selection medium containing the phytotoxic chemical in order to eliminate non-transformed

cells and permit the growth and proliferation of only transformed cells. An ideal selectable

marker gene should not permit plant material to escape the selection screen, resulting in

recovery of plants that do not express the desired transgenes. Two main classes of

selectable marker genes have been used in plant transformation experiments. One class

encodes proteins that confer resistance to antibiotics such as kanamycin and hygromycin B

(Waldron et al. 1985). The other class encodes proteins that confer tolerance or detoxify a

herbicide. Resistance to atrazine (Cheung et at. 1988), glyphosate (Comai et al. 1985), and

sulfonyl-urea herbicides (Haughn et al. 1988) have been achieved by the introduction of

foreign genes encoding modified insensitive target proteins. Resistance to phosphinotricin

(PPT) (DeBlock et al. 1987), bromoxynil (Stalker et al. 1988), and 2,4-

dichlorophenoxyacetic acid (2,4-D), (Streber and Willmitzer 1989) are based on expression

of detoxifying enzymes originally isolated from microorganisms. Selectable marker genes

vary in their effectiveness depending on which gene is used (Witrzen et al. 1998), the plant

species in which it is expressed, and the concentration needed to kill non-transformed cells.












Since the selectable marker gene is integrated into genomic DNA, these genes are active

throughout the life of the plant and in progeny. The nature of these genes, antibiotic /

herbicide resistance, and their persistence is a major reason for some groups to reject the

use of plant transformation technology. The next section will describe the two plant

selectable marker genes used in this study.


NPTII


The most commonly used selectable marker is the gene from transposon 5 (Tn5)

from Escherichia coli K12 encoding aminoglycoside 3-phosphotransferase II (APH(3') II).

This enzyme commonly known as neomycin phosphotransferase II (NPTII), inactivates

kanamycin and neomycin by phosphorylation (Flavel et al. 1992). Kanamycin inhibits

protein synthesis by targeting the ribosome and interfering with translocation and elicits

miscoding. The original gene housed in a large segment of Tn5 DNA was sequenced

(Beck et al. 1982) and an open reading frame that codes for a 264 amino acid protein was

confirmed to be the exact NPTII gene. This gene acts as a dominant selectable marker

when transformed into eukaryotic cells. To facilitate the safety assessment of the NPTII

protein, the same coding sequence used for plant transformation was introduced into

Escherichia coli to produce gram quantities of this protein (Fuchs et al. 1993a). The

NPTII protein was shown to degrade rapidly under simulated mammalian digestive

conditions (Fuchs et al. 1993b). It was concluded (Fuchs et al. 1993b) that ingestion of

genetically engineered plants expressing NPTII protein poses no safety concerns and Flavel

et al. (1992) comment that overall, the ubiquity of the gene in nature and its benign












properties make it ideal as a selectable marker in plant transformation. NPTII is the

selectable marker gene on plasmid pMON9793.


bar


Bar and pat are similar genes with the same function. The bialaphos resistance

gene (bar) confers resistance to the commercial herbicide bialaphos and codes for a

phosphinothricin acetyltransferase (PAT). The bar gene, which shows significant sequence

homology to the pat gene, was isolated from Streptormyces hygroscopicus (Thompson et al.

1987). The pat gene which codes for a PAT protein was cloned from Streptomyces

viridochromogenes Tu494 (Strauch et al. 1988). Bialaphos and phosphinothricin (PPT)

are potent non-selective herbicides. Bialaphos is a tripeptide antibiotic produced by

Streptomyces hygroscopicus. It consists of PPT, an analogue of L-glutamic acid, and two

L-alanine residues. In both bacteria and plants, intracellular peptidases remove the alanine

residues and release active PPT. PPT is a potent inhibitor ofglutamine synthase (GS).

This enzyme plays a central role in the assimilation of ammonia and the regulation of

nitrogen metabolism in plants (Miflin and Lea 1977). It is the only enzyme in plants that

can detoxify ammonia released by nitrate reduction, amino acid degradation, and

photorespiration. Inhibition of GS by PPT causes rapid accumulation of ammonia which

leads to death of the plant cell (Tachibana et al. 1986). PPT, also known as glufosinate, is

chemically synthesized (BASTA, FinaleTM, LIBERTYM, IGNITE, RELY*), while

bialaphos is produced by fermentation of Streptomyces hygroscopicus (Herbiace, Meiji

Seika Ltd.). To summarize, both the bar and pat genes encode a phosphinothricin










21

acetyltransferase (PAT), which acetylates the free NH2 group of PPT, thereby preventing

autotoxicity in the producing organism (Murakami et al. 1986). Therefore, in addition to

its value as a selectable marker, bar's ability to confer resistance to PPT makes it a useful

gene for production of herbicide resistant plants. The bar gene is the selectable marker on

plasmid pCPOO1.


Detectable Marker beta-glucuronidase (gus)


Detectable marker genes, more commonly known as reporter genes, are very useful

for plant molecular biology studies. Fusion of the reporter gene to an uncharacterized

promoter offers an effective way to study gene regulation. If the reporter gene tolerates

amino terminus fusion and can pass across membranes, it can be used to study protein

transport and organelle targeting (Jefferson 1987). Other factors related to post

transcriptional and post translational processing may interfere with quantification of the

reporter gene and should be accounted for if possible. By using a reporter gene that

encodes an enzyme activity not found in plants, the sensitivity with which chimeric gene

activity can be measured is limited only by the properties of the reporter enzyme and the

quality of the available assays for the enzyme (Jefferson 1987). Some older plant reporter

genes include neomycin phosphotransferase, chloramphenicol acetyl transferase (Herrera-

Estrella et al. 1983), nopaline synthase (Depicker et al. 1982) and firefly luciferase

(DeLuca and McElroy 1978). Difficulty in assaying or high endogenous activity are some

reasons why these genes are seldom used at this time. The E. coli beta-glucuronidase (gus)

gene (Jefferson 1987) is the most popular plant reporter system currently implemented.












GUS is a hydrolase that catalyzes the cleavage of a wide variety of beta-glucuronides,

many of which are available commercially as spectrophotometric, flourometeric and

histochemical substrates. GUS is very stable, and will tolerate many detergents, widely

varying ionic conditions, and general abuse. GUS has no cofactors or ionic requirements,

although because it is inhibited by some heavy divalent metal ions, EDTA should be added

when assaying. Detection of GUS activity depends on the availability of substrates for the

enzyme which, when acted on by the enzyme, liberate a product that is distinguishable from

the enzyme. The substrate should be cleaved only by the enzyme under study with minimal

spontaneous cleavage. Although GUS has fluorogenic substrates for better quantitative

analysis, only the histochemical substrate used in this experiment will be described. The

best substrate currently available for histochemical localization of GUS activity in tissue

and cells is 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc). The substrate gives a blue

precipitate at the site of enzyme activity. The product of GUS action on X-Gluc is not

colored. Instead, the indoxyl derivative produced must undergo an oxidative dimerization

to form the insoluble and highly colored indigo dye. The dimerization is stimulated by

atmospheric oxygen, and can be enhanced by using oxidation catalyst such as K+

ferricyanide/ferrocyanide mixture. Without such a catalyst, the results are often very good

and Jefferson (1987) suggested infrequent use of a catalyst for routine work. The gus gene

is fused downstream from the tobacco basic chitinase and MAS promoter in plasmids

pCPOO1 and pMON9793, respectively.













DNA Anaylsis


Southern blot


This technique, first reported by Southern (1975), enables one to detect the

presence of a certain DNA sequence (target) based on the complementarity between the

target and a labeled DNA fragment known as the probe. The method is used to detect a

particular DNA fragment in a mixture of DNA fragments such as those obtained when

genomic DNA is digested with a restriction endonuclease. After restriction, DNA is

fractionated by gel electrophoresis. The gel is then treated to make the DNA single

stranded and then the DNA is transferred to a nylon membrane by capillary action. The

hydrogen bonding sites on the nitrogenous bases in the single-stranded genomic DNA are

available to hydrogen bond with the single-stranded probe. The probe labels are generally

separated into two classes: radioactive and non-radioactive. An isotope frequently used to

label probes is phosphorous 32 esterified to the C5' ofdeoxyribose. The decay of the

radioisotope within the probe emits energy in the form of beta particles which identifies the

location of the probe when detected by exposure of the membrane to X-ray film. This

allows for determination of the presence and size of a particular genomic DNA fragment

that is complementary to the probe. Most of the non-radioactive labeling and detection

systems utilize enzyme linked probes (e.g. biotin, alkaline phosphatase or horseradish

peroxidase) and a chemiluminescent substrate to induce an in situ chemiluminescence that

is detectable with X-ray film.












Polymerase chain reaction-PCR


The polymerase chain reaction (Mullis and Faloona 1987; Saiki et al. 1988) is a

technique used to amplify a specific sequence of DNA known as the target. A

thermostable DNA polymerase, nucleotides, Mg+, DNA primers, and a thermocycler are

the components required for the amplification reaction. The theory for the reaction is as

follows: (1) high temperature to denature the double stranded DNA containing the target

sequence (2) optimized temperature for DNA primers to anneal to target sequence (3)

DNA synthesis at the required temperature for the thermostable polymerase (4) Repeat 1-

3. Millions of copies of the target sequence can be generated when the reaction is repeated

multiple times. The amplified product is then run on a electrophoresis gel and viewed by

ethidium bromide staining.



White Clover Transformation


Previously, White and Greenwood (1987) described a white clover transformation

system dependent on a highly regenerable genotype, WR8. In this system, stolons were

incubated with Agrobacterium. The very low frequency of transformation and the strict

genotype dependence made this approach impractical. White and Voisey (1994) split 3-day

old cotyledons in half and obtained numerous direct shoots with an average of

20/cotyledon, however, Voisey et al. (1994), in the same journal issue, did not use the split

cotyledons, instead using intact cotyledons to achieve Agrobacterium-mediated

transformation of white clover with a frequency of less than 1%. Transgenic plants










25

appeared to be recovered from the apical meristem and not as direct shoots from the cut

region. Furthermore, in another publication, this group reported white clover transformed

with the pea albumin gene using genotype WR8 and the older stolon transformation system

(Ealing et al. 1994). Most recently, Larkin et al. (1996) combined their imbibed seed

regeneration protocol with Agrobacterium-mediated transformation and regenerated

transformed plants at a frequency varying from 5-80% with the bar gene. The efficiency of

transgenic plant recovery was consistently an order of magnitude better with PPT than with

kanamycin selection.







Table 1. Summary of cellular process that occur between Agrobacterium and a plant cell

Cellular process Agrobacterium role Agrobacterium genes involved


Cell-cell recognition



Signal transduction


Transcriptional activation


Conjugal DNA metabolism



Intercellular transport


Nuclear import


T-DNA integration


Binding of Agrobacterium to the host
cell surface receptors


Recognition of plant signal molecules


Expression of vir genes after
phosphorylation of the transcriptional
activator

Nicking at the T-DNA borders and
mobilization of the transferable single
stranded copy of the T-DNA (T-strand)

Formation ofprotein-DNA T-complex;
formation of a transmembrane
channel; export of the T-complex into
the cytoplasm of the host plant cell

Interaction with the host cell NLS
receptors and transport of the
T-complex through the nuclear pore

Integration into the plant cell genome;
synthesis of the second strand of the
T-DNA


ChvA, ChvB, PscA, Att



ChvE, VirA, VirG


VirG


VirDl, VirD2, VirCI



VirE2, VirEl, VirD2, VirD4
VirB4, VirB7, VirB9
VirB10, VirBI 1


VirD2, VirE2










Table 2 Agrobacterium strains used in white clover transformation experiments.

Strain Genotype and / or Bacterial Phenotype_ Binary Plasmid

EHA 101 C58 pTiBo542 T-region::aph, Km'n pMON9793

AGLO* EHA 101 pTiBo542DT-region Mop+

AGLI AGLO recA::bla pTiBo542DT Mop+ CbR pCP001
* Strain AGLO was not used but was included here to indicate how AGLI was formed
Abbreviations: pTiBo542 hpenrirulence; aph aminoglycoside phosphotransferase
Km' kanamycin resistance; Mop mannopine utilization; CbR carbenicillin resistance









SpTAB10
11.15 Kb

L LB RB

EcoRl f
R35S Unique Pstl site


EA iiibiw\io


Figure 1. Map of plasmid pCPOO 1 used with Agrobacterium strain AGL1 for
white clover transformation.
















MATERIALS AND METHODS


White Clover Regeneration


Germinated Seedlings Preparation


Seeds of four white clover cultivars adapted to the southeastern U.S.A ('Osceola',

'Regal Ladino', 'California Ladino', 'Lousiana S ') and one experimental population

(Florida Red Leaf) were surface-sterilized by submerging them into 50% sulfuric acid for 2

minutes followed by four, 1-minute washes in glass distilled deionized water. Seeds were

then plated on 0.8% (w/v) gum agar (Sigma Chemicals Co., St. Louis, MO.) and incubated

at 26"C; 16-h photo period, to germinate. A five-day incubation period was required to

achieve the proper seedling stage (Figure 2). Explants were obtained by making an

excision at the dark green band that separates the hypocotyl from the apical portion of the

seedling containing the two cotyledons and an extremely small apical meristem (Figure 2).

Explants were plated horizontally on shoot inducing medium. Split explants were obtained

in like manner with the addition of a second symmetrical excision resulting in a pair of

cotyledons still containing their stalk and a small portion of the hypocotyl. Individual

cotyledons were plated with the abaxial surface in contact with the medium Fifteen

different media containing MS salts (Murashigi & Skoog 1962), and B5 vitamins












(Gamborg 1968), 30 g/L sucrose, 8 g/L agar formulated from five levels of 6-

benzylaminopurine (BAP) (0.1, 0.25, 0.5, 1.0, 2.0 mg/L) and three levels of cc-

naphthaleneacetic acid (NAA) (0, 0.05, 0.1 mg/L) were tested for shoot inducing efficiency

by counting number of shoots per explant at 21 days after plating.

In experiments to determine the optimum shoot inducing medium and to evaluate

for cultivar differences in induction, explants were not split and twenty explants were

plated per dish. Four replications, each containing 900 genotypes, were evaluated. To

determine the effect of the second excision resulting in a pair of cotyledons still containing

the hypocotyl, 160 split and 208 intact cv Osceola explants were compared on the medium

found to be optimum in the shoot induction experiments.


Water Imbibed Pre-germinated Seeds


Seeds ofcv Osceola were surface sterilized in 0.5% sulfuric acid for 30 seconds

followed by four, 1-minute washes in sterile water. Seeds were left in the last wash and

incubated for 15 hours at 15"C in the dark. Using a binocular microscope, explants were

obtained by making a single slice in the seed coat with a #10 blade and removing the two

separate cotyledons still containing a small piece of the hypocotyl region by squeezing.

Explants were plated abaxial side down on B5 medium (Gamborg 1968), containing 20 g/L

sucrose, 7 g/L agar, 12 nM Picloram and 2.2 mM N6-benzylaminopurine. One week after

plating, explants were reoriented so the hypocotyl region was in good contact with the

medium. A random sample of 100 cv Osceola seeds (200 cotyledons) was evaluated by

observing shoot induction 21 days after plating.













Data Collection & Analysis


Twenty-one days after plating on shoot inducing medium, direct shoots were

counted on all explants using a binocular microscope. Only shoots originating from the cut

end of the explant (opposite the apical end) were counted. An analysis of variance

(ANOVA) using a completely randomized design was performed on the four replications in

the germinated seed protocol to detect cultivar and medium differences for shoot induction.




White Clover Transformation


Preliminary Lethality Tests


A few preliminary tests were conducted to determine if kanamycin, carbenicillin, or

PPT effected shoot induction. Carbenicillin was tested on both split and intact germinated

cotyledons at 300 mg/L. Thirty eight dishes each containing 11 genotypes were evaluated.

Four levels ofkanamycin, 0, 25, 50, 100 mg/L were tested for effect on shoot induction on

non-split germinated cotyledons. Fifty genotypes per/treatment, replicated twice, were

evaluated. PPT at 10 mg/L was tested on 24 non-transformed imbibed genotypes. PPT

was also tested at 20 mg/L in the rooting medium on 24 non-transformed explants that

contained many vigorous green shoots without roots.













Medium Preparation


Recovering both a transformed and non-transformed plant from the same genotype

required four different mediums (Table 3). Basal medium powder stocks for shoot

induction (SH) and root induction (RI) were purchased from Sigma. All hormones,

antibiotics, and PPT were filter sterilized and added after autoclaving. Medium was stored

at 8C. Media were prepared and used within a two-week period.


Seed Sterilization H2S04, PPM


Initially a tea strainer was used to contain seeds while being sterilized. With this

method, the strainer containing white clover Osceola seeds was submerged into 50%

H2SO4 for 1 minute, followed by four 1 minute washes in sterile water. Seeds were

incubated in the dark at 15C for 16 hours and rinsed once in sterile water immediately

prior to use. The protocol was revised by eliminating the strainer and reducing the percent

of H2SO,. Seeds were poured into a beaker containing 0.25% H2S04 for 30 seconds

followed by four 1-minute sterile water washes. Seeds were incubated in the dark at 15 C

for 16 hours and rinsed in water immediately prior to use. The sterilization procedure was

later changed to a PPM (plant preservative mixture; Plant Cell Technology, Inc,

Washington, D.C.) based method. PPM contains antibacterial and antifungal properties.

Seeds were poured into a beaker containing 1 ml/L PPM and incubated in the dark at 15C

for 15 hours. Seeds were used directly from the PPM solution.













Cutting the Explant


Since the co-cultivation tray used contained 24 wells, individual experiments

contained 24 genotypes. A binocular scope with a fiber optic light source for enhanced

visibility, two dishes of SH medium, a large number of imbibed seeds and sterilized tools

were arranged in the laminar flow hood. Initially petri dishes were labeled on the bottom

with a felt tipped marker to indicate explant number (1-24). For speed and convenience, a

generic template made out of transparency film containing a laser printed 1-24 spot grid

was developed. The template was placed under the petri dish providing a uniform grid for

explant placement and identification. It was essential that the seeds were at the ideal stage

for explant removal; imbibed but not ruptured. The two cotyledon explants were obtained

the same way as described in the regeneration section. A #10 scalpel and a very thin

spatula were used to handle and transport individual cotyledons. The first cotyledon from

the first genotype was plated in a specific numbered location on SH medium. The other

cotyledon was plated in same location on the other SH dish. One dish of 24 cotyledons

was labeled with a test number and incubated to induce shoots. The other plate containing

the 24 cotyledon partners was used for Agrobacterium co-cultivation (see below).


Agrobacterium Growth and Storage


Agrobacterium strain EHA101 was originally grown by placing a single colony

from an agar streak (Figure 3A) into 20 ml Luria-Bertani broth (LB) supplemented with

100 mg/L spectinomycin and 50 mg/L kanamycin. The flask was placed on an orbital










33

shaker at 300 rpm overnight at room temperature. Cells were measured at optical density

620 nm (OD620) to determine the concentration using 5 X 108 cell / ml for 1 OD62o. Four

flasks of bacteria at OD620 0.9 1.2 were centrifuged at 2000 x g for 10 min and the pellets

re-suspended in 80 ml 10 mM MgSO4 supplemented with 100 uM acetosyringone. After

many spectrometer readings, a distinct color (Figure 3B) and aroma were characteristic of

bacteria at OD620 0.8 1 and all further bacteria cultures were not read with the

spectrometer. This protocol was adjusted by using 10 ml LB and removing the centrifuge

and resuspension step. Agrobacterium strain AGL1 I was grown in a similar manner using

20 mg / L tetracycline. Table 4 summarizes the recipes for solid and liquid phase bacteria

growth. Long-term storage of bacteria was achieved by adding 5% (v/v) DMSO to log

phase liquid cultures and dispensing 1 ml into small centrifuge tubes. Tubes were frozen at

-80C. Approximately one year later, a frozen stock of strain AGLI was tested by

streaking on fresh LB medium supplemented with the appropriate antibiotics.


Co-Cultivation


Initial transformation experiments used the re-suspended Agrobacterium strain

EHAI01 and germinated split cotyledons. The bacteria were loaded into a 10cc syringe

(Figure 3C) and a single drop was placed at the cut region of the explant. The explant was

co-cultivated with the bacteria for three days on shoot inducing (SH) medium and then

rinsed and transferred to shoot inducing medium for selection (SH+). With the imbibed

seed method using strain EHA101:pMON9793 and AGL1 :pCP001, the bacteria were

applied in the same way, covering the entire cotyledon. Ultimately, strain AGLI:pCP001












was dispensed into a Costar 24 well cell culture cluster (Figure 3D), at a rate of 1

ml/well. One of the two cotyledons from a single genotype was then placed into a filled

well. After all wells were filled, the container was covered and periodically agitated for 1

hour. Explants were then removed, blotted on filter paper and plated on (SH) medium.

Three days later the explants were rinsed in sterile water, blotted and plated on (SH+)

medium.


Recovering Plants


Seven stages, starting with seed preparation were required to recover rooted plants

in the greenhouse (Figure 4). Three days after placement of non-transformed explants on

SH medium, explants were reoriented by pushing the enlarged hypocotyl into the medium.

Explants that were co-cultivated were transferred to new SH+ medium 10-14 days after

initial plating. After well established green shoots were present, plantlets on SH and SH+

were transferred to root inducing media with and without selection (RI+, RI). Explants

were left on RI and/or RI+ until a main and lateral root tips were present. Rooted plants

were transplanted into individual peat pellets and incubated in a plastic mist box with 24

hours offlourescent light at 70F. When many healthy roots tips were seen protruding

through the side of the pellet, plants were transplanted into 6 inch plastic pots and placed

on concrete blocks on the floor of the greenhouse. A large piece of wood supported by

two benches was placed over the plants to block out any direct sunlight. After two days,

plants were moved onto the greenhouse benches in direct sunlight.












Evaluation of Transformed Plants


As soon as green transformed shoots can be putatively identified in stage one, a

variety of confirmation assays can be performed. These can continue through the first

cross seed generation or any later generation seed related to the original transformed

parental genotype. The different assays that can be implemented depends on what genes

were integrated into the plant genome. Only assays that can be performed with plasmid

pCPOO1 and pMON9793 will be described.


X-Gluc Stain Preparation


It was previously mentioned that 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc)

is a histochemical substrate for the gus gene product. Although many literature citations

indicate that this stain was formulated with all components including catalysts to enhance

dimer formation, the author discovered that in actuality many researchers do not include

these catalysts (GA Moore, M. Gallo-Meagher, personal communication). For this

experiment, X-Gluc stain was generated by dissolving 5 mg of 5-bromo-4-chloro-3-indolyl-

beta-D-glucuronic acid, cyclohexylammonium salt into 50 ul NN-dimethyl formamide. A

solution containing 2.5 ml 0.2 M NaPO4 and 100 ul 0.5 M Na2EDTA was added to the

dissolved X-Gluc. Subsequently, 2.4 ml D.I. H20 was added to obtain a volume of 5.0 ml.

The stain was then transferred to five micro-centrifuge tubes each containing 1.0 ml and

used immediately or frozen for storage.













GUS Petiole Histochemical Assay


This test was performed after shoot induction when sufficient plant material was

available to sacrifice and not limit opportunity to recover complete plants. The assay

required one to five, half-millimeter petiole slices and was performed by initially adding 45

ul X-Gluc stain to empty 1.5 ml micro centrifuge tubes. Petiole slices were then inserted

into tubes (1 tube/genotype), making sure that all petiole tissue was fully submerged into

the stain. At least one transformed and non-transformed control tube, if available, were

also included. All tubes were placed in the dark at 37C for 12 to 24 hours. Tissues were

cleared of chlorophyl to aid in stain detection by pipetting out X-Gluc stain and adding 70

ul 3:1 ethyl alcohol (95%) : glacial acetic acid. The clearing step was repeated if tissue was

not adequately cleared in two hours. Stained tissue was recorded immediately after the

incubation period, however, it was noticed that the tissue could be stored at room

temperature in the clearing agent for months.


GUS Root Histochemical Assay


This test was best performed with plants containing the tobacco basic chitinase

promoter fused to the GUS reporter gene because it had been shown to produce main root

tip and lateral root tip specific expression (Pittock et al. 1997). Small root tips were

excised from plants rooting in PPT or by carefully removing them from the sides of plants

rooted in peat pellets. The assay required one to three two-millimeter lateral or main root

tip cuttings. Forty-five pl X-Gluc stain were added to empty 1.5 ml micro centrifuge












tubes. Clean root tips were inserted into the tubes insuring that the tip end was fully

submerged in the stain. At least one transformed and non-transformed control tube, if

available, were included. All tubes were incubated in the dark at 37C for 12 to 24 hours.

Stained tissue was recorded immediately after the incubation period, however, as with

petioles, the root tips could be stored at room temperature in the clearing.


Leaf Painting Assay


This assay was best performed on plants in the greenhouse that contained at least

three green healthy young leaves. The assay tests for effectiveness of the bar gene and,

therefore, requires plants transformed with plasmid AGL1 :pCP001. On a sunny day,

crossing labels were attached to single leaflets indicating the date of application. A small

paint brush was used to apply 20 mg/L glufosinate (PPT) to the adaxial side of the leaf,

making sure that at least one or two drops of herbicide remained adhered to the leaf

surface. Five to seven days after application leaves were observed for herbicide damage.


Herbicide Application Tests


To determine how well the 35S promoter fused to the bar gene functions in white

clover, a replicated herbicide resistance test was performed. A randomized complete block

design was used to test for differences in PPT resistance. Four treatments using the pure

ammonium salt form of glufosinate (PPT) at 0 mg/L, 20 mg/L, 200 mg/L and 2000 mg/L

were sprayed on both transformed and non-transformed partners. Multiple plants of each

genotype were vegetatively propagated from each plant produced in vitro. Each block












contained all treatments and was replicated twice. Data were collected as a plant health

rating of 0 (completely resistant) to 5 (completely dead) at seven days after PPT

application. Analysis of variance (ANOVA) was used to test for differences in PPT

resistance between transformed and non-transformed partners and between different

transformed genotypes.


Somaclonal Variation or Insertion Effect


Aside from gross phenotype differences such as mutant morphology or color

alterations, whole plant differences between transformed and non-transformed partners will

likely be hard to detect. In the herbicide application test, each block contained each

transformed and non-transformed partner genotype in quadruple. Prior to spraying PPT,

the two replications provided eight clones at a similar stage of development for evaluation.

Data were collected as a plant health / vigor score (1 -5); 1 weak and poor growth to 5

healthy and vigorous growth.


Crossing and Segregation of Transgenes


Controlled crosses were made in both directions between transformed plants and

either a white clover plant with the dominant red leaf mark (Red Leaf) or a four leaf variety

with the dominant red leaf mark (Four Leaf). Seeds of individual flower heads were

germinated in metal trays and grown to a young stage when the red leaf mark could be

clearly identified. All plants were sprayed with 50 mg/L PPT and counts for number of

resistant and susceptible plants were taken after one week.










39

The x2 analysis was used to test for disomic inheritance of a single copy T-DNA insertion

in the T, generation (1:1).


Southern Blot


White clover genomrnic DNA was extracted using the method ofDellaporta et al.

(1983) that utilizes a SDS (sodium dodecyl sulfate), potassium acetate precipitation of

proteins, and carbohydrates. Both plant leaves dehydrated overnight (Tai and Tanksley

1990) and liquid nitrogen frozen leaves were used for DNA extraction. Approximately 25

pig of DNA was digested with highly concentrated (70 units/pl) EcoRI which cuts the

introduced T-DNA fragment once to yield unique fragment sizes. Undigested DNA and

DNA cut with EcoRI and PstI to release the intact bar gene were also included. Digested

genomic DNA and a purified 0.60-kb bar sequence was separated by gel electrophoresis

(0.8% agarose) at 33 volts for 18 hours. DNA was transferred to Hybond-N nylon

membrane (Amersham, Arlington Heights, IL) by capillary action and then cross-linked to

the membrane by exposure to UV. Blots were prehybridized for 5 hours at 65C in a

NaP04 buffer containing 7% SDS (sodium dodecyl sulfate), BSA, and 100 ug/ml

sonicated/denatured salmon sperm. The probe consisted of a gel purified 0.60-kb PstI

fragment of the bar coding region labeled with [32P]dCTP by the random primer technique

according to the Prime-a-Gene protocol (Promega Corp., Madison, WI). Hybridization

was performed at 65 C for 20 hours and then washed three times for 20 minutes with 0. 1X

SSC. The blot was exposed to X-ray film for 7 and 14 days.










40


Polymerase Chain Reaction


A 418 bp internal sequence of the bar gene was amplified using two 18-nucleotide

primers homologous to the upper and lower DNA strands (upper: 5'

7GGCGGTCTGCACCATCGT74 3', lower: 5' 458GCCAGTTCCCGTGCTTGA475 3').

Amplification conditions using Taq polymerase were as follows : denaturation, 94C for 1

min; annealing, 60C for 1 min; extension, 72C for 1.5 min. Reactions were run for 30

cycles. PCR products were analyzed on a 1.2% agarose gel.















Table 3. Plant growth medium used to induce shoots and roots in non-transformed and transformed
partner genotypes.


Non Transformed


SH B5 basal salts and vitamins
(Gamborg et al., 1968)
0.7% Agar (Sigma gum)
2% Sucrose
pH 5.8
Autoclave
2.2 uM BAP
12 nM Picloramn

RI M & S w/B5 vitamins
0.8% Agar (Sigma gum)
2% Sucrose
pH5.7
Autoclave
1.2 uMIBA


SH=Shoot Inducing: Rl=Root Inducing


Transformed


SH+

Same as SH with
Autoclave
20 mg / L PPT
250 mg / L Cefotaxime



RI+ Same as RI with
Autoclave
20 mg / L PPT
250 mg L Cefotaxime


Table 4 Medium used for liquid and solid phase Agrobacterium growth.

Strain EHA101 : pMON 9793 AGLI : pCPOOI

LBAgar pH 7 LBAgar pH 7
Solid 100 mg / L Spectinomycin 20 mg / L Tetracycline
50 mg / L Kanamycin 20 mg / L Rifampicin


LB Broth pH 7 LB Broth pH 7
Liquid 100 mg / L Spectinomvcin 20 mg / L Tetracycline
50 mg / L Kanamycin










42

















.4.





















Figure 2. The Three day old seedling and how to obtain the intact and split cotyledons
for tissue culture.

























Figure 3. Solid and liquid phase Agrobacterium growth.
A) Streak of single colony bacteria; B) Flask containing log phase bacteria; C) Syringe used
For micro-drop co-cultivation; D) Co-cultivation 24 well container.


Figure 4. Time frame required to regenerate transformed plants.














RESULTS


White Clover Regeneration


Germinated Seedlings


The analysis of variance revealed significant differences (a=.05) for media effect on

direct shoot induction. Medium containing 0.05 mg/L NAA and 1 mg/L BAP was most

effective at inducing direct shoots, however, two of the remaining fourteen media tested

were not significantly different for shoot induction (Figure 5). The least numbers of direct

shoots were produced on media supplemented with 0.1 mg/L BAP. Increasing the

concentration of BAP increased the number of direct shoots up to 1 mg/L. This trend was

not observed at 2 mg/L BAP, which produced results similar to 0.25 mg/L. Direct shoots

appeared to originate from the cut end of the explant after a swelling phase, approximately

20 days after placement on shoot inducing medium (Figure 6). In some cases, shoots were

also observed emerging from the apical end prior to the formation of direct shoots.

Genotypes in all cultivars produced from 0 to 173 shoots/explant. The percentage of

genotypes producing one or more direct shoots ranged from 39% for Florida Red Leaf to

51% for California Ladino. Mean number of shoots per explant ranged from 1.6 for Red

Leaf to 2.3 for California Ladino. Only Louisiana S 1 (1.8) and Red Leaf (1.6) differed












significantly (a=-.05) from other entries according to Duncan's new multiple range test

(Table 5). The additional splitting step used to initiate cultures reduced the number of un-

responsive explants by 50%, and increased the number of genotypes that produced more

than ten shoots per explant from 8.6% to 20% (Figure 7).


Water Imbibed Pre-Germinated Seeds


One hundred percent response was obtained after the cutting technique had been

mastered. Although this method was not statistically compared with the germinated seed

protocol, it appeared to have a 100% explant response rate for direct shoot induction and

explants producing greater than ten shoots per explant (personal observation). Individual

explants produced clusters of direct shoots all originating from the cut end opposite the

apical shoot meristem region. Two weeks after plating most explants contained greater

than 15 shoots, with some cotyledons producing more then 50 shoots (Figure 8).




White Clover Transformation


Preliminary Lethality Tests


No significant difference (a = 0.5) in shoot induction or proliferation could be

detected on medium supplemented with carbenicillin compared to control medium minus

carbenicillin (Table 6). Kanamycin, however, completely inhibited shoot induction at all

levels tested (data not shown). When using 25 mg/L kanamycin, some germinated intact












explants were able to develop a pale green apical meristem. On one occasion, very pale

green shoots were observed from the cut end of an explant. The apical meristem and/or

any direct shoots produced, however, did not persist. Glufosinate (PPT) completely

inhibited imbibed explants from developing direct shoots. No pale green or non-direct

shoots were observed at any time and complete decay usually occurred within 10 days. In

the experiment to test if PPT inhibited root induction on non-transformed explants

containing numerous direct shoots, none of the 24 explants tested produced roots on 20

mg/L PPT. Plants started to brown in five days, and most were completely dead within

two weeks.


Seed Sterilization


The tea strainer worked well initially for containing and handling seeds while

sterilizing. Corrosion of the strainer while in the acid and incubating in the last water rinse,

however, had a negative effect on seed imbibition. There appeared to be a correlation

between the age of the strainer, the amount of corrosion present, and the premature rupture

of the seeds. The older the strainer, the more corrosion seemed to occur, resulting in

premature seed rupture (personal observation) This condition made it difficult to obtain

seeds at the correct stage over a defined period of time. It also was not uncommon to have

the strainer components disengage due to acid damage. Eliminating the strainer and

reducing the acid concentration enabled more consistent and reproducible seed

preparation. Decanting the acid and water solutions without losing seeds was difficult. By

using 1 ml/L PPM, consistently uniformly imbibed seeds were obtained. This was by far










47

the easiest and most effective protocol. The seeds were sterilized simply by placing them in

lml/L PPM and incubating for 15 hours. The seeds could then be removed directly from

this initial container and immediately used for obtaining the explant.


Cutting the Explant


Obtaining two split cotyledons, each with an intact hypocotyl, after a single slice

with a #10 blade required practice. The slice must be fast, smooth and in the exact spot

required. If the seed was not fully imbibed and wet, a smooth cut was difficult to obtain. If

the seed coat had already ruptured, it was very difficult or impossible to cut in the required

location. Since individual explants were less than 1 mm in size, a binocular microscope

was necessary to excise the tissue. Initially an incandescent light source was used, but it

was later found that a brighter fiber optic light source reduced eye fatigue. Although

labeling each petri dish with a marker was effective, the transparency template greatly

enhanced speed and accuracy of cotyledon plating. All petri dishes contained a uniform

plastic notch on the bottom dish. Lining up the template with the notch on the dish

produced 24 uniform spots for individual cotyledons to be plated. It also made for easy

record collection of non-transformed and transformed partners at later dates.


Agrobacterium Growth and Storage


Strain EHA101 was centrifuged and re-suspended with acetosyringone before co-

cultivating with germinated cotyledons. Due to the lack of positive results and the fact that

many protocols do not re-suspend bacteria, the resuspension step was eliminated. It was










48

easier to inoculate a single colony of either EHA101 or AGL1 into a flask containing 20 ml

LB. The culture was usually ready in 17 hours. Multiple single colonies were sometimes

added to increase the rate of growth. Both EHA 101:pMON9793 and AGL 1 :pCP001

appeared and grew similarly on LB agar and LB liquid. Agar streaks grew quickly and

produced thick shiny growth. Liquid cultures of both strains contained the same foul odor

present at log phase stage. Strain AGL1 that was in -80 C storage for a year grew well

after plating on LB agar with the appropriate antibiotics.


Co-Cultivation


Dispensing a micro-drop of Agrobacterium was a fast, easy way to introduce the

bacteria to the wounded explant. With germinated explants, the drop was applied at the

cut region and left for three days. This produced sufficient bacteria growth that explants

were rinsed off before plating on selection medium. With the imbibed explants, the micro-

drop enclosed the whole explant. The small size of the imbibed cotyledon explants

compared to the large volume of stationary inoculmn caused difficulty in removing excess

bacteria from explants at the end of co-cultivation. Co-cultivation in cell culture wells

alleviated this problem. This approach was more like that ofLarkin et al. (1996) in which

all cotyledon explants were placed in a petri dish with Agrobacterium and periodically

agitated. With the wells, each transformed partner genotype received its own well of

bacteria. Cotyledons were relatively easy to transport into and out of the wells with

practice. After blotting off excess bacteria, the co-cultivating cotyledon explants plated on

SH medium did not develop as thick a bacterial growth compared to the micro-drop












method. Cotyledons appeared to have a glaze of thin bacterial growth around them that

was rinsed off.


Recovering Plants


Many white clover transformation tests were performed over a 2 V year period

(Table 7). Non-transformed cultures usually developed green shoots within three weeks.

Once placed on root inducing medium, roots formed approximately two weeks later. The

transformed cultures were slower in response. Within 7 10 days on selection medium,

dead explants could be removed. Initially 10 mg/L PPT was used for selection, but no PPT

was used in the root inducing medium. Although 10 mg/L PPT completely killed explants

not Agrobacterium co-cultivated (data not shown), this system produced shoots using co-

cultivated explants that appeared to be escapes. The SH+ medium was increased to 20

mg/L PPT and the RI medium was supplemented with 20 mg/L PPT (RI+). In some cases

cultures that produced many shoots on SH+, when placed on RI+, developed roots from

some shoots but other shoots were killed (Figure 9). Plants that developed roots in either

RI or RI+ medium were transplanted into peat pellets and incubated in a mist box. Most

plants produced lateral roots that protruded from the sides of the pellet in 5 to 10 days

(Figure 10). Although plants rooted quickly in pellets, they were left in the mist box until

many roots and new shoot growth had appeared and the plants appeared to be healthy and

vigorous. This enabled a gradual adjustment to the greenhouse environment. The plants

that were repotted and placed in the greenhouse showed little signs of stress when placed in

a normal bench top spot. At the time of transfer to the greenhouse, plants varied in terms












of vigor or amount of growth present. Therefore plants were shaded for two days, in an

attempt to temporarily retard transpiration before they were moved to the top of the bench.

One genotype using EHAI01:pMON9793, the micro-drop and imbibed cotyledons, and ten

genotypes using AGLl :pCP001, wells, and imbibed cotyledons were putatively

transformed (Table 7). The pMON9793 recovered plant produced aberrant shaped flowers

and would not set seed after many hand-crosses. Seed were not produced when this plant

was used as either the male or female parent.




Evaluation of Transformed Plants


GUS Histochemical Assays


The gus gene was helpful in determining if explants that contained green shoots on

selection medium were possible escapes. Both tobacco basic chitinase and mannopine

synthase promoters fused to the gus gene were effective in detecting enzyme activity when

using a thin petiole slice. The single putatively transformed plant (L10) with

EHAI01 :pMON9793 stained positive for the petiole assay in the lab but failed the root

assay. Expression was also variable in this plant under greenhouse conditions with

complete loss of activity in some tests. Chitinase:gus in AGL1 :pCP001 produced expected

results when using the petiole test, although inconsistent results from the same plant in later

tests lead to doubt of stable gene expression and/or stain quality. It was later discovered

that clusters of shoots in transformed explants contained non-transformed shoots that

developed normally during the whole plant recovery procedure. The GUS root tip assay












with the chitinase promoter was the most reliable indicator of a putatively transformed

plant. All white, actively-growing roots on 20 mg/L PPT stained positive in the root tip

assay. Staining for three hours at 37C usually was enough to detect expression of GUS.

If the roots were incubated overnight at this temperature, expression was extreme in some

situations (Figure 11 A). Ten random stolon cuttings were rooted from entry a5#16, the

first plant regenerated from the transformation protocol using pCPOO I, and also thought to

contain both non-transformed and transformed sections. Roots from three cuttings stained

positive and resisted 200 mg/L PPT, while roots from the other seven cuttings did not stain

and were killed by 200 mg/L PPT. Similar results were obtained with many transgenic

plants regenerated via tissue culture. A petiole staining test that included progeny from an

ax5#16 cross, delta-2-#4, delta-1-#1 1, L10, and controls indicated variable GUS expression

from transformants in the greenhouse. Genotype L 10 containing the MAS:gus construct

produced lighter color staining compared to most plants transformed with chitinase:gus.

Genotype delta-2-#4 contained the weakest GUS expression (Figure 11B) which was also

later found to be correlated with reduced resistance to PPT.


Herbicide Painting and Spraying


The PPT painting assay was a good indicator of bar gene activity. When the pure

ammonium salt was used, it was difficult to keep the solution in sufficient contact with the

leaf. As long as a few drops adhered to the tissue, non-transformed leaves developed

yellow splotches in those locations and most completely died in a few days. Labeling

individual leaves and multiple leaves on the same plant was tedious and usually led to












results that did not indicate a definite status of the plant (transformed, escape, mixed).

Misting 20 2000 mg/L PPT was an efficient method to determine if a plant was resistant.

It was also useful to eliminate non-transformed sections from a transformed plant. When

the a5#16 transformed plant contained a full green canopy with many leaves, 200 mg/l PPT

was applied. Within one week it was evident that portions of this plant were completely

resistant to PPT at this level, while other portions were completely destroyed (Figure 12).

After one week, much of the dead plant material was removed, all of which could be traced

back to individual crown stolon segments. This scenario of recovering mixed plants was

evident in most transformed plants regenerated via tissue culture. The replicated herbicide

test performed on entries delta-2#4, a5#16, and delta-2#18, indicated that delta-2#18 was

an escape, a5#16 was the most resistant, and delta-2#4 was not as resistant as a5#16. All

the control non-transformed partners and delta-2# 18 were tolerant to 20 mg/L PPT, but

200 and 2000 mg/L proved to be lethal. None of the treatments were lethal for ct5#16 or

delta-2#4, however, a5#16 was completely resistant to 200 mg/L and delta-2#4 was not

(Table 8). It was later discovered that the delta-2#4 source plant contained portions that

were either not transformed or expressing a very low level of resistance. In a later

herbicide spray test, delta 1#11, another putatively transformed genotype, appeared almost

undamaged when 2000 mg/L PPT was applied indicating this genotype may be expressing

the bar gene at a higher level than a5# 16.













Crossing and Segregation of the Transgene


Eighteen hand crosses were made using a5#16 as a transformed parent. Three

other crosses involving delta# 11 and one cross using delta8#16 were also performed.

Seed yield per individual flower head ranged from 9 for (Red-Leaf X ax5#16) to 87 for

(a5#16 X Four-Leaf). Crosses involving Four-Leaf tended to produce the most seed

(Table 9). Seven days after application of 50 mg/L PPT to 30 progeny from a5#16 X Four

Leaf, a ratio of 17 resistant: 13 lethal ratio was obtained. The null hypothesis of expected

Mendelian segregation ratio for disomic inheritance of a single dominant gene (1:1) was

tested using the x2. A x2 value of 0.53; p=0.48 was evidence for a good fit of the data

resulting in failure to reject the null hypothesis. The 17 resistant progeny all stained

positive for the GUS root tip assay, indicating that both linked genes from the T-DNA

insert (bar, gus) were segregating together. The intensity of staining in the progeny

appeared the same as the parent in most cases.


DNA Analysis


The polymerase chain reaction (PCR) was successful at amplifying the internal 418

bp sequence of the bar gene from genomic DNA extracted from sample a5#16 (Figure 13)

No non-specific DNA amplification was present. Southern blot analysis revealed single

positive hybridization bands using DNA extracted from liquid nitrogen frozen plant samples

and cut with EcoRI. Undigested DNA and DNA cut with Pstil yielded hybridization bands

of high molecular weight. When DNA was cut with both EcoRI and PstI in an attempt to










54
splice out the intact bar gene, two bands were present (Figure 14). Since neither band is

the expected size of the bar gene (600bp), and the slower band appears to be the same size

as DNA cut with EcoRI alone, these two bands may be the result of incomplete DNA

digestion and are likely the EcoRI and PstI fragments expected to contain the bar gene.

The control containing non-transformed DNA cut with EcoRI did not hybridize with the

bar probe. The lanes that included DNA that was isolated from dehydrated leaves did not

produce definite hybridization bands.












Table 5. Comparison for responses to direct shoot induction among white clover
cultivars adapted to the southeast United States
Cultivar N Percent Response* Mean # of shoots**

California Ladino 625 51% 2.3A

Osceola Ladino 520 47% 2.2A

Regal Ladino 635 48% 2.1A

Louisiana S1 433 41% 1.8B

Florida Red Leaf 646 39% 1.6B

* Number of genotypes that produced >0 shoots
** Means include zero values. Means with the same letter are not
significantly different (a =.05) according to Duncan's LSD test




Table 6. Effect of carbenicillin on direct shoot induction
Treatment N Mean

300 mg / L Carbenicillin 155 3.71

0 mg/ L Carbenicillin 176 3.52













Table 7. White clover transformation experiment
Test Date Explant
3/6/96 6/6/96 germinated-split


Test J
Test K
Test L
Test M
Test N
Test 0
Test I
Test Z
Test A
Test H
Test PI
Test P2
Test P3
Test P4
Test P5
Test P6
Test P7
Test LI
Test A3
Test A4
Test A6
Test A8
Test A9
Test AlO
Test Bl I
Test Cl
Testal
Test a2
Test a3
Test a4
Test a5
Test delta 1
Test delta 2
Test delta 3
Test delta 4
Test delta 5
Test delta 6
Test delta 7
Test delta 8
Test delta 9
Test delta 10
Test W2
Test W3
Test W4
Test W5


2/16/97
2/19/97
2/25/97
3/26/97
3/31/97
4/8/97
5/5/97
5/29/97
7/7/97
7/21/97
8/17/97
8/19/97
8/22/97
8/25/97
9/9/97
9/11/97
9/16/97
9/23/97
9/30/97
9/30/97
10/8/97
10/15/97
10/30/97
11/5/97
11/12/97
11/12/97
12/13/97
12/14/97
1/20/98
1/22/98
1/28/98
3/10/98
3/10/98
3/11/98
3/11/98
3/18/98
3/24/98
3/24/98
3/25/98
3/25/98
3/31/98
4/7/98
4/7/98
4/8/98
4/8/98


Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed
Imbibed


3its : 1996 1998


Strain:Plasmid Inoculation
EHA 101 :pMON9793 Micro-Drop
EHA101O:pMON9793 Micro-Drop
EHA101 :pMON9793 Micro-Drop
EHA101O:pMON9793 Micro-Drop
EHA101 :pMON9793 Micro-Drop
EHA 101 :pMON9793 Micro-Drop
EHA 101 :pMON9793 Micro-Drop
EHA1O1 :pMON9793 Micro-Drop
EHA 101 :pMON9793 Micro-Drop
EHA 101 :pMON9793 Wells
EHA101 :pMON9793 Wells


EHA 101 :pPSV
EHA101:pPSV
EHA 101 :pPSV
EHA 101 :pPSV
EHA101:pPSV
EHAI01:pPSV
EHA 101 :pPSV
AGLl :pCP00I
AGLI :pCP00l
AGLIl:pCP00i
AGLIl:pCP001
AGLI :pCP001
AGLI :pCP001
AGL 1 :pCP001
AGL 1 :pCP001
AGLI :pCP001
AGLI :pCP001
AGLI :pCP001
AGLI :pCP001
AGLI:pCP001
AGLI :pCP001
AGLI :pCP001
AGLI :pCP001
AGL I :pCP001
AGLI :pCP001
AGL 1 :pCP001
AGLI :pCP001
AGL :pCP001
AGLI :pCP001
AGL I :pCP001
AGLI :pCP001
AGLI :pCP001
AGL I :pCP001
AGL I :pCP001
AGLI :pCP001


Wells
Wells
Wells
Micro-Drop
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells
Wells


N # Transformed


1-#16







2-#4,24
5-#I 1,13,16,21,22












Table 8. Response of transformed white clover to the herbicide PPT.
Entry ID Replication mg/L PPT Rating (0-5)*
1 delta2#4-NT 1 0 0
2 delta2#4-NT 2 0 0
3 delta2#4-NT 1 20 2
4 delta2#4-NT 2 20 2
5 delta2#4-NT 1 200 5
6 delta2#4-NT 2 200 5
7 delta2#4-NT 1 2000 5
8 delta2#4-NT 2 2000 5
9 delta2#4-T 1 0 0
10 delta2#4-T 2 0 0
11 delta2#4-T 1 20 1
12 delha2#4-T 2 20 0
13 delta2#4-T 1 200 4
14 delta2#4-T 2 200 1
15 delta2.4-T 1 2000 2
16 delta2#4-T 2 2000 1
17 a5#16-NT 1 0 0
18 a5#16-NT 2 0 0
19 a5#16-NT 1 20 4
20 a5#16-NT 2 20 3
21 a5#16-NT 1 200 5
22 a5#16-NT 2 200 5
23 a5#16-NT 1 2000 5
24 a5#1 6-NT 2 2000 5
25 a5#1 6-T 1 0 0
26 a5#16-T 2 0 0
27 aS#16-T 1 20 0
28 a5#16-T 2 20 0
29 a5#16-T 1 200 0
30 a5#16-T 2 200 0
31 a5#16-T 1 2000 2
32 a5#16-T 2 2000 1
33 delta2#18-NT 1 0 0
34 delta2#18-NT 2 0 0
35 delta2#18-NT 1 20 4
36 delta2#18-NT 2 20 4
37 delta2#18-NT 1 200 5
38 delta2#18-NT 2 200 5
39 delta2#18-NT 1 2000 5
40 delta2#18-NT 2 2000 5
41 delta2#18-T 1 0 0
42 delta2#18-T 2 0 0
43 delta2#18-T 1 20 3
44 delta2#18-T 2 20 3












Table 8-continued.
Entry ID Replication mg/L PPT Rating (0-5)*
45 delta# I 8-T 1 200 5
46 delta2#l 8-T 2 200 5
47 delta2#18-T 1 2000 5
48 delta2# 18-T 2 2000 5
* 0 = no visable leaf damage, 1-4 = increasing levels of damage, 5 = Complete plant death





Table 9. Seed yield from various white clover hand crosses
Cross Number of Seed/Inflorescence

Four Leaf Xa5#16 82
Four Leaf X o5#16 40
Red Leaf X 5#16 19
Red Leaf X a5#16 9
Red LeafX a5#16 36
Red LeafX a5#16 44
Red Leaf X a5#16 38
Red LeafX a5#16 33
a5#16 X Red Leaf 35
a5#16X Four Leaf 46
a5#16X Red Leaf 34
a5#16X Red Leaf 50
a5#16X? 50
a5#16X Four Leaf 87
a5#16 X Red Leaf 52
a5#16 X Red Leaf 61
a5#16X Red Leaf 56
a5# 16X Red Leaf 32
DeltaS# I IX Four Leaf 20
Delta8#1 IX Four Leaf 23
Four Leaf XDelta8# 11 18
Four Leaf XDelta8#16 17




















Hormone Effect on Shoot Induction


NAAmgAL


BAPmgIL


Figure 5. Effect of the hormones BAP and NAA on direct shoot induction.
Mean # of shoots includes genotypes that produced zero shoots. Treatments
with different letters are significantly different (a=.05) according to DNMRT.



















/*^ "S^" "S
*._... ... ..;





Figure 6. Direct shoots forming on 21 day old explants obtained from germinated
white clover seedlings.


0.6
0.5





Total Shoots I explantexplant

0 5 10 15 20 25
Total Shoots / explant


Figure 7. Histogram comparing split and intact explants for response
to shoot induction.










1mm A


1mmr


4D


e


Imbibed


1mm


Day4


Q


Day 6


imm
;MM


b


Day 8


1mmr


Day 17.
.. ,*" :... ":
Day 17


Figure 8. Stages of shoot development using the imbibed seed as an explant source


Day 10










62




























Figure 9. Root emerging from PPT resistant shoot while other non-transformed
shoots are killed.




4 '.,:....:_'_.__._ __._.'._ __'_..













Figure 10. Rooted plant in peat pellet and many plants incubating
in a mist-box.






















T#16 Control LIO T,48


T,43


DM&a2-0 '*





-VI



Figure 11. GUS histochemical staining of transformed white clover tissue.
Root assay (Top) with plants transformed with AGL1 :pCP001. Petiole
assay (Bottom) on genotype L10 transformed with EHA101 :pMON9793 and
an assortment of genotypes transformed with AGL LpCPOO1.

















































Figure 12. Effect of 200 mg/L PPT on partially transformed plant a5#16. A) Full plant
with partially resistant and susceptible segments. B), C) Close view of susceptible and
resistant leaves respectively. D) Susceptible segments tracing back to a single stolon.


f






































Figure 13. PCR amplification of the bar gene from transformed genomic DNA. Lanes
1,2,3 transformed a5#16 genomic DNA diluted 1/100,1/500, 1/1000 respectively. Lane 4
and 5 a5#16 non-transformed genomic DNA diluted 1/500, 1/1000. Lanes 6 and 7 BAR
gene diluted 1/100 and 1/10,000. The identical monochrome image on the right was
included for enhanced visibility of amplification bands.















3z,
-Wl.. t


V


2 3 4 5 6 7 S 9


Figure 14. Southern blot of white clover genomic DNA. a5#16 DNA cut
with EcoRI (lanes 2 and 5), PstI (lane 3), EcoRl and PstI (lane 4), and
undigested (lane 8). DNA extracted from dehydrated leaves and cut with
EcoRI (lane 6) and non-transformed DNA (lane 7). The 600 bp bar gene
(lane 9) ran off the gel.


32kb--
















DISCUSSION


White Clover Regeneration



Results indicated that medium containing 0.05 mg/L NAA and 1 mg/L BAP was

most effective at inducing direct shoots with germinated seedling explants. These

experimental results indicated that the cotyledon stalk, specifically the region that unites

with the hypocotyl, is the primary tissue responsible for shoot induction. Splitting the

cotyledons and plating them abaxial side down allowed direct contact between this region

and the medium. Intact explants cut slightly proximal of the hypocotyl region had a much

lower chance of responding due to lack of contact between medium and receptive tissue.

The fact that Voisey et al (1994) did not use this more responsive expjant for

Agrobacterium transformation, instead using the intact explant to recover a transformed

apical meristem at low frequency, leads to uncertainty of the compatibility of these

regeneration systems with Agrobacterium transformation. Although it was more difficult

to obtain and manipulate explants from imbibed seeds, this protocol was more effective and

faster in time required for direct shoot induction compared to germinated explants.

Another benefit was the shorter seed preparation time, 15 hours compared to five days on

germination medium. Both protocols utilize the hypocotyl region that connects with the












cotyledon stalk, which seems to be more responsive to shoot induction at an embryonic

stage. It is not clear whether it is the specific stage of the explant tissue, the medium, the

growth regulators, or interactions that are responsible for the high frequency genotype

independent shoot induction with imbibed seeds.

Unlike inbred lines, open-pollinated cultivars are a composite of genotypes with all

desirable genes (traits) theoretically maintained at constant frequencies over generations.

Single copy integration of a transgene into a large number of agronomic genotypes

followed by selection of the best transformed genotype in terms of transgene expression

will be prerequisites for cultivar development in open-pollinated species. If many

independent transformed genotypes with different genome integration sites or greater than

single copy per genome plants are used as parents, problems in trait stabilization and

genetic load may occur in future generations. Genotypes derived from material selected for

regeneration usually lack agronomic potential. Although transformation may be efficient,

these genotypes would not make suitable founding parents. The real utility with methods

that are genotype independent will be the ability to transform many genotypes followed by

selection of the best individual in terms of overall agronomic potential, insert copy number,

and transgene expression. This desirable transformed plant can then be used as a parent in

cultivar development or improvement.













White Clover Transformation



The majority of transformed plants were recovered from tests using Agrobacterium

strain AGL1 harboring plasmid pCPOO1. AGL1 and EHA101 are very similar strains of

Agrobacterium, both derived from the wild-type C58. Plasmid pCPOOl and pMON9793

differ in certain key aspects. The 35S promoter controlling transcription of the bar

selectable marker gene is thought to be stronger than the NOS promoter controlling nptll

on pMON9793. Although both strains produced escapes, kanamycin selection produced

more shoots that slowly died in contrast to PPT. Most escapes with strain AGL1 occurred

before PPT was added to the root inducing medium and 10 mg/L PPT was used in the

shoot inducing medium. Since PPT is not thought to be systemic within the plant, a few

non-transformed cells surrounded by transformed cells may allow shoot initials to form

under selection. If these shoots are not in good contact with the medium, a cluster of

shoots from a single explant may contain some non-transformed initial shoots. This

situation was evident in all transgenic plants regenerated via tissue culture. It is not clear if

the mixed plants were recovered due to transformed shoots detoxfying PPT around non-

transformed shoots, lack of efficient contact of shoots and PPT containing medium,

concentration of PPT or other unknown factors. Problems could arise if these initial

laboratory derived plants not only contain portions that are not transformed but if

transformed sections contain multiple different T-DNA insertions. Root specific GUS

staining was linked to herbicide resistance in mixed plants. Entry a5#16 was recovered










70

without PPT in the rooting medium and contained mixed non-transformed and transformed

segments originating from distinct crown sections. Since mixed plants still occurred with

PPT in the rooting medium, unless individual shoots are removed and maintained at a very

early age, mixed plants may be inevitable. Spraying PPT on mixed plants will eliminate

non-transformed segments but will not solve the problem of multiple, different integration

sites. After being screened by spraying 200mg/L PPT, entry a5#16 indicated single copy

integration based on Southern blot data and sexual transmission segregation data. Although

single copy integration was present in a5#16, it is unknown if this situation will occur in all

other transgenic plants. If this escape scenario consistently occurs at 100% frequency, it

may be possible to recover the non-transformed control plants by detecting them with a leaf

painting or mild PPT spraying assay from source plants. This would eliminate the extra

handling and maintenance required in the lab to recover the non-transformed control plant.

Once the timing for seed sterilization and swelling was more predictable, it was

easier to synchronize log phase bacteria and explant preparation. The cell culture well

plates were beneficial in maintaining the identity of individual cotyledons while also

providing liquid swirling type exposure to Agrobacteriim. Recovering the non-

transformed control plant required more handling of the partner explant being co-

cultivated. The extra handling at a young stage, however, may lower the chances for

direct shoot induction. Assuming setup timing of all required components for cutting and

co-cultivation is achieved and the cotyledons are obtained intact with an undamaged

hypocotyl, this transformation method appears to be suitable for transgenic cultivar

development in white clover.













Breeding Transgenic White Clover



Most of the literature on plant genetic engineering pertains to new protocols for

gene integration or novel genes that may have great agronomic potential. Rarely is the

transfer of the novel gene, now located in a single genome, into an existing cultivar

discussed. If the cultivar is released as an inbred line or is asexually propagated then, in

theory, a single elite transformed plant from that cultivar can be amplified via self

pollination and/or cloning and released. For outcrossing, highly heterozygous species like

white clover, a different breeding strategy may be required. Voisey et al., 1994 described a

method of breeding transgenic white clover. Their first step was to produce plants

homozygous for the new insert by crossing primary transformants with elite genotypes and

then intermating transgenic plants from the F, population. Transgenic white clover

cultivars can be produced by selecting intermating F2 plants homozygous for the transgene

and with good general combining ability to intermate for 5-6 generations. They state that

the transgenic cultivar will, therefore, be comprised of individuals which are homozygous

for the introduced gene and should deliver the agronomic performance which characterized

the genotypes from which they were derived. Two problems may occur with this proposed

breeding method. First, by crossing more than a single primary transformed plant, the

transgene will most likely be housed at more than one locus. In later generations of

random mating, this could cause problems with genetic load and trait stability. To ensure

stable permanent expression of the new gene, it should be confirmed that the founding

parent has a single hemizygous locus with the insert. It should then be confirmed that in












the F2 generation the insert segregates in a Mendelian fashion and that homozygous and

hemizygous individuals do not differ in expression of the new transgene. Then, in theory,

all individuals in later generations should only contain the insert at a single locus in either a

hemizygous or homzygous functioning state. If multiple independent transformed plants

are used, the number of unlinked repetitive loci containing the transgene will increase with

random mating. Due to new homology in different regions of the genome, genetic

recombination during meiosis may occur between non-homologous chromosomes causing

cytogenetic alterations such as duplications and deletions. With a high copy number of the

transgene, the chances of a gene silencing effect at the DNA, RNA, or protein level may

increase. Secondly, the Voisey et al., 1994 breeding approach does not appear to

maximize heterozygosity which is essential for optimum white clover population

performance. A different breeding strategy that may address these drawbacks is discussed

in the next paragraph.

The main objectives of this proposed breeding method are to maintain all beneficial

qualities of the original cultivar, maximize heterozygosity, and ensure that the new

transgene is maintained at a sufficient frequency in future generations. This breeding

strategy is basically a backcross approach for an open pollinated species. A single

transformed plant would be used to move the new gene into the existing cultivar. It is

critical that this founding parent be free of defects due to DNA insertion effects or

somaclonal variation. The availability of a non-transformed control plant that has the same

genetic background and was regenerated in vitro substantially aids in the selection process.

Although it is more tedious in the lab to transform and maintain material when recovering










73

control plants, the assurance that this crucial selection decision is made correctly makes it

worthwhile. It should be beneficial to compare a population of transformed/non-

transformed partners and then select the single best transformed plant rather than to select

the best plant from a random group of 200 transformants, considering that after the first

cross, all the progeny are half-sibs related to this initial parent. This breeding strategy,

described here with cultivar Osceola, but applicable to any white clover cultivar, requires a

minimum of 50 paired genotypes containing a gene for easy large scale selection of

transgenic plants in later generations, such as herbicide resistance. Replicated experiments

of transformed and non-transformed plants to compare for differences in yield, vigor,

flowering characteristics, and persistence should be completed immediately after plants are

available. The single best transformed plant should be selected and multiplied asexually. In

an ideal white clover seed production area that also provides an environment suitable for

selection, randomly plant 30 transformed clones in the field in a 1 transformed clone : 10

random Osceola plants proportion, with all seed harvested (T). This is done in to avoid

the development of a population containing a single cytoplasm. If seeds were solely

harvested from transformed clones, then all future generations would contain an identical

cytoplasm. The remainder of the breeding strategy is designed to reduce the inbreeding

coefficient of the population being selected, recover the Osceola phenotype, and increase

the gene frequency of the new gene(s). The T, generation should be grown as spaced

plants and sprayed with herbicide to eliminate any non-transformed individuals before

selection. Re-select the best transformed plants, at least 50 from no less than 1000

genotypes, if space and labor are available. Place these transformed plant selections back












into Osceola at random locations and harvest seed (T2). Repeat the selection and

reintroduction of transformed plants back into Osceola cultivar until the group of

transformed plants under selection exhibits all desirable traits of Osceola in addition to the

transgene. Further selections, if required, should be in the form of mass selection and

originate from seed produced by intercrossing the last transformed generation evaluated.



Patent Problems



The future is uncertain in terms of who will be able to develop and release

transgenic cultivars. There are now many confirmed and efficient transformation protocols

for most valuable species, however, apparently each component required in the complete

transformation system has been patented. For example, the biolistic bombardment and

Agrobacterium transformation protocols are patented. The patent source depends on the

crop. For example, if you want to transform turf grass with a biolistic approach, then

Scotts (The Scotts Company, Ohio, USA) holds the rights. Patents for many of the best

plant promoters, including the 35S, are held by Monsanto. The trend appears to be that a

few large companies are purchasing smaller bio-tech and seed companies in order to

produce transgenic cultivars without any patent concerns. University scientists are using

many of these components in order to develop new transformation protocols and

investigate transgenic gene expression. Currently it would be very difficult to develop a

system that does not rely on any patented components. Universities may soon discover

that it is too costly to develop transgenic cultivars because of licensing costs to holders of









75

patents. The convoluted legal quagmire of this situation may have an impact in the use of

technology and the development ofcultivars in the university system.
















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BIOGRAPHICAL SKETCH


Jason James Goldman was born to Robert and Johanna Goldman in Pequannock,

New Jersey. He graduated from Wayne Valley High School in 1988. Jason attended

Rutgers University, Cook College in New Brunswick, New Jersey majoring in plant

science. He graduated in 1992 from Rutgers University receiving a Bachelor of Science

degree. In 1994, Jason completed his master's degree in plant breeding at Texas A&M

University, College Station, Texas. In 1998, he will complete work on his doctorate

degree in plant breeding.








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 thesis
for the degree of Doctor of Philosophy.

David S. Wofford, Ch&CF /
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 thesis
for the degree of Doctor of Philosophy.

^^2^>^^' 4 e2.a
Kenneth H. Quesenbery J--
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 thesis
for the degree of Doctor of Philosophy. K9 .

Rex L. Smi
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 thesis
for the degree of Doctor of Philosophy.


Gloria A. Moore
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 thesis
for the degree of Doctor of Philosophy.


Paul M. Lyrene
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 of Philosophy.


December, 1998


6Dean, College'of Agriculture


Dean, Graduate School
















LD

1996
6'^/














UNIVERSITY OF FLORIDA
1262111 5099ll IIII3lllllllllili
3 1262 08555 0993




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