Tissue Culture and Transformation of Rice (Oryza Sativa L.) Using Tobacco Nurse Cells
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Tissue Culture and Transformation of Rice (Oryza sativa L.) using Tobacco Nurse Cells.
Rajashekar M. Patil B.Sc. (Agri)
A Thesis submitted for the degree of Master of Agricultural Science
The University of Adelaide Department of Plant Science
February, 1991 Table of Contents
Table of Contents . .ll
List of Figures .....
List of Tables ...... vü Abstract .vüi f)eclaration .X Acknowledgments...... xl
Abbreviations...... xll
Chapter L Introduction 1
Chapter 2 Literature Review 4
2.1 INTRODUCTION 4
2.2 Acnopt crnnIUM MEDIATED TRANSFoRMATIoN 5 2.2.1 Introduction...... 5 2.2.2 AgrobacteriumBioIogy...... 6 2.2.3 TiPlasmid 1 2.2.4 Virulence Region..... 7 2.2.5 T-DNA 9 2.2.6 Plant Transformation and T-DNA Integration ..10
2.2.1 Factors Affecting Plant transformation .. 11
2.2.1 .l Agrobacterium Strains...... 12 2.2.1.2 Plant Genotype and Explant Selection .. t2 lll
2.2.1 .3 Co-Cultivation Parameters.. 2.2.8 Transformation of Monocotyledonous Plant Species
2.3 BIOLISTICS 2.3.I Introduction 2.3.2 Bombardment Device and Parameters 2.3.3 DNA Coating Methods 2.3.4 Plant Transformation....
2.4 fucp TRRNSFoRMATIoN ...... 2.4.I Introduction...... 2.4.2 GeneticTransformation 2.4.2.I Agrobacterium mediated Transformation 2.4.2., Protoplast Mediated Transformation...... 2.4.2.3 Biolistics..
2.5 DNA coNSTRUCTS...... 24
2.6 AN\4S OF THE PROJECT...... 26
Chapter 3 Materials And Methods .27
3.1 Pu,vT MATERIAL 21
3.2 Pu¡-r TSSUE CULTURS...... 21 3.2.1 Tissue Culture Media 27 3.2.1.1 Rice...... 28 3.2.I.2 Tobacco 28 3.2.2 Tissue Culture Conditions 28 3.2.2.1 Tobacco 29 3.2.2.2 Rice..... 29
3.3 PIeNr TRANSFoRMATION...... 31
3.3.1 Bacterial Strains and Plasmids ...... 31 3.3.2 SelectionMedia..... 32 3.3.3 Direct Gene Transfer by Microprojectile Bombardment...... '.'-"32 3.3.3.I Biolistic Gun and DNA Coating of Microprojectiles .....'.....32 3.3.3.2 Preparation of Explant.. JJ 3.3.3.3 Microprojectile Bombardmerrt and Selection of Potential Transformants. 34 lV
3 .3 .4 Agrobacterium Mediated Transformation ...... 34 3.3.4.1 Cloning Binary Vector.. 34
3.3.4.2 Co-cultivation ..., 36
3.3.4.3 Selection of Calli 31
3.3.5 Histochemical Staining 3l 3.3.6 Molecular Analysis of Potential Transformants 38
Chapter 4 Results 40
4.1 Rrce rssun CULTURE 40
4.2 MTcRopRoJECTILE BoMBARDMENT oF RICE 40
4.3 E¡'¡ecTs oF TOBACCO NLIRSE CULTURE... 44 4.3.I Regeneration of Rice Suspension Cells...... 44 4.3.2 AgrobacteriumMediated Transfotmation...... 50 4.3.2.1 Recovery of Embryogenic Calli... 50
4.3.2.2 Selection of Hygromycin Resistant Calli 55 4.3.2.3 PCR Analysis of Selected Calli 64
Chapter 5 Discussion .80
5.1 MTcRopROJECTILE BOMBARDMENT OF RICE .80
5.2 Er.¡'gcrs OF TOBACCO NURSE CULTURE...... 81 5.2.I Regeneration of Rice Suspension Cells 81 5.2.2 AgrobacteriumMediated Transformation of Rice.. 83
5.3 SulvtvtRRy 90
5.4 Furuns DrRECTIoNS...... 90
Appendix 1...... 92
Appendix 2...... 94
Appendix 3...... 96
References ...... 103 List of Figures
Figure 4.1 Culture of rice suspension cells (cv. 7l-I1O) on AA medium (A) without or (B) with tobacco nurse cells. 6l Figure 4.2 Regeneration of rice calli (cv. 77-170) cultured on AA medium (A) without oh (B) with tobacco nurse cells. 68 Figure 4.3 Regeneration of rice calü (cv. Nipponbare) cultured on AA medium (A) without or (B) with tobacco nurse cells. 68 Figure 4.4 Plantlet regeneration from rice callus (cv. 77-170), (A) Single shoot formation in rice callus cultured without nurse cells, (B) Multiple shoot formation in rice callus cultured with nurse cells. 69 Figure 4.5 Multiple shoot formation on rice calli (cv. 77-L70) cultured on tobacco nurse cells. 10 Figure 4.6 Percentage recovery of embryogenic calli of three cultivars of rice after co-cultivation with AGL1 (pIG121Hm) on three different media. 51 Figure 4.7 Percentage recovery of embryogenic calli of three cultivars of rice after co-cultivation with LBA4404 (pIG121Hm) on three different media. 52 Figure 4.8 Percentage of hygromycin resistant colonies of cv. Nipponbare obtained using different strains of Agrobacterium on three different co-cultivation media. 59 Figure 4.9 Transient expression of GUS in rice calli after one week of co-cultivation with LBA4404 (pIG121Hm) on tobacco nurse cells. 7l Figure 4.10 Rice callus culture (A) Control calli grown on NB medium (B) Control calli grown on NB-H50 medium, (C) Putatively transformed rice calli {cv.77-170, AGL1 (pIG121Hm)} on NB-H50 medium. 7I Figure 4.11 (A) Complete expression of GUS in the putatively transformed hygromycin resistant rice calli {cv.77-170, AGL1 (pIG121Hm)} stained with X-gluc along with (B) control calli. 72 Figure 4.12 Clnmeric expression of GUS activity showing different levels of blue coloration in different sectors of the rice calli. 12 Vl
Figure 4.13 GUS expression in putatively transformed hygromycin resistant calli of rice (cv. Nipponbare) obtained after co-cultivation with three Agrobacterium strun-plasmid combinations on three different media. 13 Figure 4.14 GUS expression in putatively transformed hygromycin resistant calli of rice (cv. 71-l1O) obtained after co-cultivation with three Agrobacterium strain-plasmid combinations on three different media. 14 Figure 4.15 GUS expression in putatively transformed hygromycin resistant calli of rice (cv. T-309) obtained after co-cultivation with three Agrobacterium strain-plasmid combinations on three different media. 75 Figure 4.16 Selection of putatively transformed calli of rice cultivar 77-l7O co-cultivated with three Agrobacterium strain-plasmid combinations on NB medium with tobacco nurse cells. 76 Figure 4.L7 Selection of putatively transformed calli of rice cultivar 77-I70 co-cultivated'on three media with three Agrobacterium súun-plasmid combinations. 76 Figure 4.L8 Selection of putatively transformed calli of rice cultivar Nipponbare co-cultivated with three Agrobacterium strain-plasmid combinations on NB medium with tobacco nurse cells. l1 Figure 4.19 Selection of putatively transformed calli of rice cultivar Nipponbare co-cultivated on three media with three Agrobacterium strain-plasmid combinations. 71 Figure 4.20 Selection of putatively transformed calli of rice cultivar T-309 co- cultivated with three Agrobacterium sftain-plasmid combinations on NB medium with tobacco nurse cells. 78 Figure 4.21 Selection of putatively transformed calli of rice cultivar T-309 co-cultivated on three media with three Agrobacterium strun-plasmid combinations. 78 Figure 4.22 The PCR analysis of hygromycin resistant rice calli for the presence of uidA gene fragment. 66 Figure 4.23 Transient expression of GUS in the immature embryos of rice (cv.77-I70) after bombarding with plasmid construct pACT-D. 19 Figure 4.24 ^hansient expression of GUS in suspension cells of rice (cv. 77-I70) after bombarding with plasmid constructpACT-D. 19 List of Tables
Table 4.1 Number of different explants of three rice cultivars bombarded with three plasmid constructs and number of blue spots obtained indicating transient GUS expression. 43 1 Table 4.2 Plant regenerãtion frequency (expressed as percentage) from the calli of two cultivars of rice cultured with or without tobacco nurse cells. 47 Table 4.3 Number of shoots per callus obtained (expressed as percentage) during regeneration of rice calli cultured with or without tobacco nurse cells. 48 Table 4.4 Percentage recovery of embryogenic calli of three rice cultivars after co-cultivation with tluee Agrobacterium strain-plasmid combinations on three different co-cultivation media. 53 Table 4.5 Percentage of GUS expressing hygromycin resistant rice calli of three cultiva¡s of rice co-cultivated with three Agrobacterium strain-plasmid combinations on three co-cultivation media. 60 Table 4.6 Percentage of hygromycin resistant calli of three rice cultivars obtained 16 weeks after co-cultivation with three Agrobacterium strain-plasmid combinations on three co-cultivation media. 6T Table 4.7 Analysis of Variance 62
vu Abstract
Plant genetic engineering involves delivery and integration of foreign genes into individual plant cells and subsequent regeneration of transformed cells into whole plants so that the newly acquired genes can be inherited in the progeny. Gene delivery systems such as PEG- mediated uptake of DNA by protoplasts, biolistics and quite recently the Agrobacterium- mediated gene transfer technique have been used in genetic engineering of cereals. However, regeneration of plants and low transformation frequency are still limiting factors in successful cereal transformation. In this project, using rice as a model plant, methods have been developed to improve regeneration frequency and to enhance the efficiency of
A g r o b ac t e r i um mediated tr an s formati o n tec hni que.
The use of fast growing cell lines as nurse culture is a common practice in regeneration of plants from protoplasts. In this project the heterologous system of tobacco nurse cells was utilised to improve the regeneration of suspension cells of two rice cultivars. In the nurse
culture treatment, more than 50To of green calli gave rise to plantlets, against 13-20Vo in the control. The overall plant regeneration frequencies in cv. 1l-fl} and cv. Nipponbare were 4lVo and 38Vo in the nurse culture treated calli and 5Vo and 4Vo in the control calli, respectively. The plant regeneration frequency of both rice cultiva¡s was increased by 8-9 times using tobacco nurse cells. Multiple shoot formation was commonly seen in the
tobacco nurse cell treated calli, while control calli mainly formed single shoots.
Subsequently, the effects of tobacco nurse cells on Agrobacterium mediated transformation
of rice callus were evaluated. Two strains of Agrobacterium namely AGL1 and LBÃ4404 containing the plasmid pIG121Hm or pPCV707+Gus were used. The calli used were derived from scutellum of immature embryos of three rice cultivars: Nipponbarc,l7-I70
vill Ix and T-309. The co-cultivation of rice calli with Agrobacterium was carried out on NB medium, NB medium with acetosyringone or NB medium with tobacco nurse cells. The
tobacco nurse cells enhanced the recovery of embryogenic properties of rice calli after co- cultivation w\th Agrobacterium. The recovery of embryogenic calli was as high as IOOVo
after co-cultivation on tobacco nurse cells, while it was reduced significantly (down to 27Vo in cv. T-309) when nurse cells were excluded from the co-cultivation medium. Co- cultivation on tobacco nurse cells increased the transformation frequency in all three rice cultivars. The number of hygromycin resistant rice calli and GUS expressing calli indicated
an interaction between the rice cultivars and the Agrobacterium slrain-plasmid combinations used. The Agrobacterium strain LBA4404 (pIG121Hm) was more effective with cv.l7-I70, while AGL1 (pIG121Hm) produced high transformation frequencies in cultivars Nipponbare and T-309. Transformed rice calli were obtained even when acetosyringone and tobacco nurse cells were excluded from the co-cultivation medium, albeit at a much lower frequency. The presence of uidA gene in potentially transformed rice calli was
confîrmed by PCR analysis.
In this study tobacco nurse cells enhanced the regeneration frequency of long term suspension cells and Agrobacterium mediated transformation of rice. These findings
suggest scope for utilising the tobacco nurse culture technique with other economically important cereal crops to improve regeneration frequency and Agrobacterium mediated
transformation il Declaration
This work contains no møteriøl which høs been øccepted for the award of any other degree or dþlomø in øny uniaersity or other tertiary institution ønd, to the best of my knowledge ønd belief, contøins no møterial preuiously published or written by another person, except where due reference høs been made in the text.
I giae consent to this copy of my thesis, when deposited in the Uniaersity Library, being øaøiløble for loan øndphotocopying. til r
Signed: Date: 7o.n.q+
I x Acknowledgments
I would like to thank all the people who have helped me during my masters program. I
wish to express my gratitude to my supervisors, Dr Arun Aryan and Dr Peter Langridge, for
their guidance and support during my studies. My special thanks to Dr Peter Langridge for
providing the f,rnancial support to complete this thesis, and to Dr Max Tate for critical
reading of this thesis. I also extend my special appreciation to Dr Javed Quareshi and Dr Narayana Upadhyaya, Plant Industry, CSIRO, Canberra, for their suggestions, helpful guidance and encouragement. I credit Miss Bronwyn MacDonald, Miss Rosalie Heppner and other friends in the laboratory for their support and co-operation. I extend my thanks ì[f to Mrs Jan Neild and other friends in Dr Peter Langridge's laboratory for their technical ,; advice and guidance.
I acknowledge the receipt of the Rotary Foundation's "Freedom From Hunger" scholarship
under 3H program. I am indebted to my sponsor and host Rotary Clubs, RC Haveri, India,
and RC Blackwood, Australia. I would like to extend my special thanks to my sponsor and
host counsellors, Dr M.V. Kamath and Dr John Jackson. I also thank my sponsor and host
RI districts, RI Dist 3170 and RI Dist 9520. I wish to thank all the Rotarians around the
world for their contribution to the program.
I particularly thank Miss Marcia Jackson and her parents, Mr & Mrs Jackson and their family, who not only provided me the accommodation but also their love, support and
encouragement. t I l I My special thanks to my family for their continued support, love and encouragement throughout my studies. I am ever grateful to my grandfather late Shri K.F. Patil, who has been source of inspiration in my life and to whom I dedicate this thesis.
? xl Abbreviations
ABA abscisic acid BAP benzylaminopurine bp base pair
C centigrade
CaClz calcium chloride cm centimetre
CsCl cesium chloride cv. cultivar dNTP's deoxyribonucleoside triphosphates DNA deoxyribonucleic acid DNAase deoxyribonuclease ds double stranded E. coli Escherichia coli Naz-EDTA disodium ethylenediaminetetraacetate
Ç b grams
GUS B-glucuronidase IAA indole-3-acetic acid kbp kilo base pair L liter M molar mg milligrams
MgCl2 magnesium chloride
frìl milliliter
pg microgram
xll xlll
pl microliter pM micromolar NAA napthalene acetic acid NaCl sodium chloride NaOAc sodium acetate OD optical density
PCR polymerase chain reaction
psi pounds per square inch RNA ribonucleic acid
RNAase ribonuclease rpm revolutions per minute
ss iingle stranded Tris Tris (hydroxymethyl) aminomethane UV ultra violet
X-Gluc 5 -bromo-4-chloro- 3 -indolyl- p -D-glucuronide 2,4-D 2,4-dichlorophenoxyacetic acid
I t)¡:
S
Chapter 1
General Introduction
Plants can be classified à, *ono"otyledonous plant species and dicotyledonous plant species based on number of cotyledons. The monocotyledonous plant species include important group of cereals crops, such as wheat, rice,maize and barley. Cereals contribute a major share in human diet and this fact is also supported by a large area under cereal cultivation and their enormous yield production all over the world. In general, cereals have become important due to their high yields and easier methods of harvest
(Lazzeri and Shewry,1993). They can be grown in a variety of climatic conditions ranging from dry regions to deep water submerged environments and from temperate to tropical conditions (Lazzer\ and Shewry, 1993). Therefore improvement of cereals has attracted the major attention of plant breeders. Plant improvement started when neolithic age man initiated the settled agriculture and has come long way since then. Presently, different strategies have been developed for improvement of crop plants. Plant breeding is the oldest method of crop improvement that involves crossing two compatible genotypes followed by the selection and evaluation. There has been a significant improvement in the quality and quantity of the crop plants over the past 40-50 years using plant breeding techniques (Lazzeri and Shewry, 1993). However, there are some limitations that often restrict the scope of the plant breeding methods. The main disadvantage is that it is slow
and requires a long time in the selection and evaluation procedure. The non availability of genetic variation and sexual incompatibility among the plant species often limits the breeding program. The advent of tissue culture techniques has helped to overcome some of the problems involved in rescuing the wide crosses with wild relatives (Khush and Brar, 1992). The genetic engineering technology that allows precise transfer of genes into any
1 Chapter l. General Introduction 2 kind of plant is a novel tool for improvement of crop plants. Genetic engineering involves isolation and in vitro manipulation of genes, transfer of these genes to plant cells and regeneration of transgenic plants from the transformed cells (Ooms, 1992). The main advantage of this technology is that genes can be transferred across species, genera and even kingdoms. Furthel, in vitro selection of transgenic plants provides an efficient alternative to subsequent field selections.
The application of genetic engineering technology mainly depends on the fulfillment of basic requirements. These are, availability of economically important genes, methods to transfer these genes into plant cells and regeneration of plants from transformed cells.
There are a number of economically important genes which have been already isolated and characterised (Tischer et al., 1986; Thompson ¿/ aI., 1987) that can be used for crop
improvement. The methods to transfer these genes have also been developed, but most of them being efficient with dicotyledonous plants than monocotyledonous plants. The gene
transfer techniques such as Agrobacterium mediated transformation, biolistics and direct
DNA transfer into protoplasts are commonly used for plant transformation. Among these techniques Agrobacterium med\ated transformation is the most common and efficient technique for dicotyledonous plant species (Hooykaas and Schilperoort, 1985). Previously, it was believed that monocotyledonous'plants are out side the natural host range of Agrobacterium. However, the reports of transformation of Asparagus fficinalis (Bytebier et al., 1987), Dioscorea bulbifera (Schafer et al., 1987) and AIIium cepa (Dommisse et al., 1990) indicated the possibility of transformation of monocotyledonous plants using Agrobacterium. Recent reports of transformation of Zea mays (Gould et aI., 1991), Oryza sativa (Raineri et aL, 1990; Chan et al., 1992; Chan et aI., 1993 &.Hiei et aI., 1994), Sorghum bicolor (Godwin and Chikwamba, 1994), Triticum aestivum (Mooney et aI., 1991) and Hordeum vulgare (Deng et a\.,1990; Brettell, 1996, personal communication) have further strengthened the opinion. However, most of these results are not reproducible. In the case of biolistics, the DNA coated microparticles are bombarded into the plant cells. Although this technique is widely applicable to any kind of plant species, the efficiency of transformation is low (Brettell and Murray, 1995). Direct DNA
transfer technique into protoplasts is an efficient technique, but regeneration of plants from
protoplasts is the main problem in most of the plant species (Ayres and Park, 1994). Chapter I. General Introduction 3
Once the genes are stably inserted into the plant cell, the next step is to regenerate plants from the transformed cells. Presently, there are well developed protocols to regenerate plants from the single cells, again the efficiency is low in monocotyledons compared to dicotyledons. As most of the important food grains belong to the monocotyledonous plant species, it is important to improve the eff,rciency of regeneration of plants and the gene delivery techniques. This would enable the wider application of genetic engineering technology to improve the crop plants, particularly the cereals. Chapter 2
Literature Review
2.L INTRODUCTION
Genetic engineering of crop plants depends on the availability of isolated genes, techniques to transfer these genes into plant cells and methods to regenerate transformed cells into plants. The advancement in molecular biology has made it possible to isolate and characterise economically important genes. Currently there are several methods for gene
transfer into plant cells. They are Agrobacterium mediated transformation (Fraley, 1983), biolistics (Klein et al., 1981), direct gene transfer into protoplasts (Toriyama et al., 1988), micro injection (Neuhaus et al., 1981), tissue electroporation (D'Halluin ¿f aI., 1992) and DNA delivery via silicon carbide fibres (Frame et aI., 1994; Kaeppler et aI., 1990). The first three techniques aÍe commonly used for transfer of genes into plant cells. Agrobacteriumis the most commonly used vector in transformation of dicotyledonous plant species and it is a simple and an eff,rcient method (Hooykaas and Schilperoort, 1985). However, the use of this system in monocotyledonous plant species is not popular, as monocotyledons are not natural hosts for Agrobacterium. Biolistics, which involves bombardment of DNA coated gold particles into plant cells, is used in transformation of all types of crop plants including cereals, but the frequency of transformation is very low (Brettell and Murray, 1995). The other main requirement for using genetic engineering technology for crop improvement is availability of cell culture methods. The methods for
tissue culture and plant regeneration are cuffently available for most crop species including
rice. This review discusses the transformation systems in relation to cereal transformation
4 Chapter 2. Literatut e Review 5 and is primarily based on the reviews of Ooms (1992) and Kado (1991) together with my interpretation of the literature.
2.2 AGROBACTERIUM MEDIATED TRANSFORMATION
2.2.I Introduction
Agrobacterium is a plant pathogen in dicotyledonous plant species causing crown gall
disease. The discovery of mechanism of crown gall and hairy root formation has paved the way for today's plant genetic engineering technology. Agrobacterium has the ability to transfer genes into plant cells by virtue of possessing a specific plasmid called Ti-plasmid (tumour inducing plasmid). This Ti-plasmid is found in only a small percentage of the natural soil population of Agrobacterium (Zupan & Zambryski, 1995). Certain genes located in the Ti-plasmid (T-DNA) are transferred and integrated into plant genome and are
expressed to alter plant metabolism in favor of Agrobacterium. This fact has been exploited
in genetic engineering of plants by replacing genes on Ti-plasmid with genes of interest.
The natural host range of Agrobacterium was thought to be the limiting factor to use this system on most of the economically important crops such as cereals. The phenolic compounds released by wounded dicotyledonous plant cells are the main factors involved in identification of potential plants for infection by the pathogen. It has been suggested that the wound response in dicotyledonous plants makes them better hosts for Agrobacterium than monocotyledonous plants which lack wound response (Potrykus, 1990).
Agrobacterium Is a routine method of transformation in dicotyledonous plant species. It is used in transformation of few monocotyledonous plant species such as Asparagus fficialis and it has also been shown that gene transfer is possible in several cereals, for instance
rice(Raineriet aL, 1990; Chan et al., 1993; Hiei et al., 1994),maize (Gould et aL, l99I),
wheat and barley (Deng et al., 1990; Mooney et al., 1991). However in some instances, the results are not reproducible. Therefore, there is need for further investigation in this
system for better and complete understanding of all the events involved in the gene transfer
process. This would make the Agrobacterium transformation system more efficient and
routine on wider range of crops. Chapter 2. Literature Review 6
2.2.2 Agrobøcterium Biology
Agrobacteria are gram negative, rod shaped soil borne bacteria and are classif,red in the family Rhizobeaceae. At the beginning of the 20th century, Agrobacterium tumefaciens was identif,red as the causal organism of crown gall disease (Smith & Townsend, 1907). This finding instigated a detailed study of the organism and different isolates of Agrobacterium were identified. These isolates were shown to differ in their host range, growth rate and morphology of the tumour they induce (Smith & Townsend, l90l). Interestingly, plants and plant organs differed in their responses to infection by the same
Ag rob ac t e rium isolate (Smith, 19 17 ).
Initially, differences in the morphology of tumors induced formed the basis for the classification of Agrobacterium isolates. They were grouped as A. tumefaciens (crown gall), A. rhizogenes (harry root), A. rubi (cane galls) and A. radiobacter (non pathogenic) (Ooms, 1992). Tumorous tissues of infected plants produce certain low molecular weight nitrogenous or phosphorous compounds called opines
@iemann et aI., 1960). The type of opine produced is dependent on the Agrobacterium isolate inducing the tumour formation. Based on the opine produced, additional classification was introduced: the octopine class (octopine, lysopine, octopinic acid and histopine) and the nopaline class (nopaline and nopalinic acid).
The mechanism by which Agrobacterium infects and causes crown gall disease is the foundation for the development of transformation techniques. Agrobacterium infects a wounded dicotyledonous plant cell and transfers a segment of DNA (T-DNA) into plant cells. T-DNA which carries genes coding for phytohormones and opines, integrates into the plant nuclear genome. Expression of these bacterial genes in plants leads to unlimited cell division resulting in tumour formation. Agrobacterium uttlizes the opines produced by tumorous cells as the source of carbon and nitrogen. The regulatory genes that govern the infection and transfer of genes are located on both chromosomes and the Ti-plasmid. T- DNA, which is physically transferred to plants, is also located on the Ti-plasmid. These genetic components involved in the gene transfer will be discussed in the following sections. Chapter 2. Literature Review 7
2.2.3 Ti-Plasmid
The pathogenicity of Agrobacterium depends on the type of plasmid it is carrying. These plasmids are classihed as Ti-plasmid (tumour inducing) and Ri-plasmid (induce hairy root) flMhite and Nester, 1980). The ability of Agrobacterium to transform plant cells is correlated with the presence of Ti or Ri plasmids. The two important regions in the Ti or Ri plasmids for the transfer of genes are T-DNA (Chilton et al., 1987) and the virulence region
(Garfinkel and Nester, 1980 ; Ooms et aI., 1980). These regions are adjacent to each other and thus vir genes operate in cls. However, it has also been shown that vir genes can also operate in trans when the uir region and T-DNA are on different plasmids (Hoekema et al.,
1983). This important finding has paved the way for construction of binary vectors. These binary vectors are one of two groups of plant transformation vectors based on Agrobacterium. In the case of binary vectors, the vector plasmids replicate autonomously tn Agrobacterium (or E CoIi) and need not co-integrate into resident Ti-plasmid; while in co-integrating vectors, the other group, the vector plasmid integrates into the resident Ti- plasmid. The use of binary vectors is preferred because of their small size and their independence of any specific Ti-plasmid. They can be introduced into any Agrobacterium host containing disarmed Ti or Ri plasmid, as long as vir functions are provided (Hoekema et al., 1983).
2.2.4 Virulence Region
Pathogenicity of Agrobacterium tumefaciens is regulated by the genes present on the Ti-plasmid as well as those present on the chromosomes (Ooms, 1992). Chromosomal virulence genes specific to Agrobacterium were identified by studying the activities of
Ti-plasmid in both heterologus and homologus chromosomal backgrounds (Holsters et al.,
i978). It is suggested that these genes have a role to play in attachment of Agrobacterium to the plant cells (Douglas et a1.,1985). A large segment of the Ti-plasmid is occupied by
the virulence region (vir) consisting of several vir genes. It consists of certain loci whose products are directly involved in T-DNA processing and transfer. Several vir loci from different types of Ti-plasmids have been identified: virA, virB, vitt, virD, virB, virG Chapter 2. Literature Review 8
(Zambryski, 1988), virF (Otten et al., 1985), tzs (Beaty et aL, 1986 ; John and Amasino,
1988), pinF (Stachel & Nester, 1986) and virH (Kado,l98l ; Rogowsky et aI., 1990).
Induction of vir genes is related to exudates released from wounded plant cells. A wounded dicotyledonous plant cell releases certain low molecular weight phenolic compounds such as acetosyringone (AS), a-hydroxyacetosyringone and sinapinic acid. These cell exudates act as chemo-attractants at low concentrations (Ashby et aI., 1987) and at higher concentrations they induce the vir genes. Although a two component regulatory system of
virA. and vlrG is constitutively expressed, it is possible to induce vlrG together with virB, vitt, virD and virB by these phenolic compounds (Stachel et aI., 1985). In response to phenolic compounds VirA protein undergoes autophosphorylation and in turn phosphorylates VirG, which activates other vir genes.
Following activation of vir genes, two polypeptides are induced: VirDl and VirD2, coded by virD locus. These polypeptides are ss-endonucleases that recognize and cleave at the 25 bp direct repeat border sequences flanking the T-DNA region of the bottom strand (Yanofsky et aI., 1936). The nicks at the right and left borders, representing the 5' and 3'
ends respectively, result in an ss-T-DNA strand. The efficiency of T-strand synthesis is
known to be increased by certain enhancer sequences called overdrive; these are outside T- DNA but close to the right border (Peralta et al., 1986; Toro et aI., 1988; Van Hareen ¿t aI., I987b). VirD2 attaches covalently and tightly at the right border or 5' end, giving the T-strand polar character. The integration of T-DNA into plant DNA is precise at the right border while it is rearranged at the left border (Gheysen et aL, 1987). This suggests a possible role of VirD2 protein in directing and integrating T-DNA into the plant nuclear 'When genome (Durrenberger et al., 1989; Steck et a1.,1990). VirD proteins are limiting,
VirCl increases T-strand production (DeVos andZambryski, 1989). The virC expression
has been shown to be necessary for T-DNA transfer to monocotyledonous plants (Grimsley
et al., 1989).
The T-strand is completely bound by ss-DNA binding proteins coded by virB locus, thus
protecting the T-DNA against any plant and bacterial nucleases (Citovsky et al., 1988; Gieti et aI., 1987). The "VirD2-VirE-T-DNA strand" complex is suggested to be the intermediate in the gene transfer process. This T-complex has to traverse a bacterial Chapter 2. Literature Review 9 membrane, a plant cell wall and membranes before it can integrate into the nuclear genome of plants. The virB locus codes for proteins of which seven are associated with the membrane (Thorstenson et al., 1993; Thorstenson &. Zarrbryski, 1994) and the VirB4
(Berger & Christie, 1993) and the VirB 1 1 provide energy for transporting the T-complex.
Although an enorrnous amount of work has been done on understanding events of T-DNA transfer into plant cells, some aspects are still not clear. These include the site for gene integration, the copy number and those events involved when T-DNA traverses membranes. A more detailed understanding of how the vlr region regulates T-DNA transfer is needed to enable the wider use of Agrobacterium as a tool of genetic engineering.
2.2.5 T.DNA
Transfer-DNA, also abbreviated as T-DNA, is the segment of the Ti-plasmid that is actually transferred to the plant cells. It caries genes that play a direct role in tumorogenesis: iaaM
(auxl, tmsl), iaaH (aux2, tmsT) and ipt (cyt, tmr). These genes encode for enzymes that catalyze the synthesis of auxin and cytokinins (Akiyoshi ¿/ aI., 1983; Barry et aI., 1984;
Inze et al., 1984; Schroder et aL, 1984), which are plant growth hormones. These two plant growth hormones can induce unlimited cell division leading to tumorous growth on plants bearing T-DNA. Along with these genes T-DNA also carries genes for synthesis of opines. However, the genes for catabolism of opines reside outside the T-DNA and are not transferred to the plants.
The T-DNA is flanked by two 25bp imperfect direct repeat border sequences (Barker et al., 1983; Simpson et al., 1982; Yadav et al., 1982; Zambryskj et al., 1982). They are
designated as right and left borders, indicating the 5' and 3' ends of T-DNA respectively.
These border repeats are the only cis acting sequences for T-DNA recognition and excision
by vir gene products. The right border in its original orientation is essential for transfer of DNA to plants (Caplan et al., 1985; Holsters et al., 1983; Jen and Chilton, 1986a; VanHareen et aI., I981a; Wang et aL, 1984; Jen and Chilton, 1986b) and reversing the
orientation of border repeats restricts the transfer of DNA (Wang et aL, 1984). Although only the right border is required for DNA transfer, the left border can decide the length of Chapter 2. Literature Review 10
DNA to be transferred. Therefore, the left border might serve as a terminator (Wang et al',
1984).
The border sequences are the only recognition sites for T-strand formation. No other gene on T-DNA is required for its transfer. Therefore, any gene placed between these two
border sequences will be transfered to plants. This fact has been widely utilized in genetic
engineering of plants. The genes on T-DNA responsible for tumour formation are replaced
with genes of agronomic importance. Thus foreign genes ca.n be transferred to plants.
2.2.6 Plant Transformation and T-DNA Integration
Once the T-DNA has been transferred into plant cells, it mostly integrates in the plant
nuclear genome (Ooms, 1992), although there are reports of integration in chloroplast DNA (De Block et al., 1935). The transient expression of T-DNA in the nucleus is very high 3-4
days after transfer and subsequently declines (Jansenn and Gardner, 1989). This has been attributed to the presence in the plant nucleus of ds-T-DNA which is transcriptionally active. Although this contradicts the ss-T-DNA intermediate as described in the previous
sections, there is a pcssibility of conversion of ss T-DNA to ds T-DNA (Rodenberg et al., 1989). VirD2 protein which is bound to the right border might play a role in directing T- DNA integration in the plant nuclear genome. Site of T-DNA integration seems to be random with no specific preference for any chromosome or chromosomal region (Ooms, 1992). Often multiple copies of T-DNA are found in the nuclear genome (Hiei et al., t9e4).
It has been suggested that DNA insertion in the plant nuclear genome is mediated by host nuclear enzymes (Kado, 1991). This is further supported by integration of foreign DNA which is introduced by microinjection, biolistics and other direct gene transfer techniques. Integration of T-DNA can cause rearrangement of adjoining plant DNA sequences (Gheysen et aL, 1987). These rearrangements show that T-DNA integration is not by
simple recombination and might be accompanied by replication and repair activities. Chapter 2. Literature Review 11
2.2.7 Factors Affecting Plant transformation
There are several factors that influence plant transformation by Agrobacterium. The steps involved in T-DNA transfer and integration have been defined (Zambryski, 1988) and are: (1) Identiffing a susceptible host plant cell and attachment, (2) Induction of virulence genes, (3) Synthesis of T-strand, (4) Transfer of T-DNA complex to bacterial membrane,
(5) transfer of T-DNA complex through bacterial membrane, plant cell wall and plant cell membrane, (6) Transfer of T-DNA complex through plant cytoplasm and nuclear membrane, (7) Integration of T-DNA into plant nuclear genome.
Identifîcation of susceptible host cell and attachment of bacteria is a primary step in the transformation process (Lippincott and Lippincott, 1969). Earlier it was suggested that inability of Agrobacterium to attach to monocots (Lippincott and Lippincott, 1978), or attach at a low rate (Ohyama et al., 1979) is due to methylation of binding sites on cell walls (Rao er at., 1982). Recently it has been shown that there is preferential attachment of bacteria to wounded sites of wheat cells (Mooney and Godwin, 1991). In another instance, Musa indica cells were bombarded with gold particles to wound the cells before co-cultivation with Agrobacterium (May et aI., 1995).
Once the bacterial cell is attached to the plant cells, the next step is induction of virulence genes. The conditions required for vir induction are, a pH of < 5.1 (Stachel et aL, 1985), temperature below 30" C (Stachel et al., 1935) and a carbon source such as sucrose (Alt-Moerbe et aL, 1988). In the presence of these above conditions, another important
requirement is the signal molecules released by wounded piant cells such as acetosyringone
(Stachel et aI., 1986) or phenolics like vanillin, caffeic acid and sinapinic acid (Bolton et al',
1986 ; Melchers et al., 1989). Dicotyledonous plants like tobacco produce enough signal molecules for vir induction and T-DNA transfer (Stachel et a1.,1986). In the case of monocotyledonous plants which lack wound response, vlr induction can be done by the addition of acetosyringone into the bacterial culture medium (Sheikholeslam and Weeks, 1987) or during co-cultivation (Owen and Smigocki, 1988). After the induction of bacteria and T-DNA transfer, the next step is the ability of the plant cells to grow and regenerate back into plants. Thus by knowing the requirements and behaviour of Agrobacterium, it is Chapter 2. Literatu¡'e Review t2 possible to create the conditions needed for cereal cells to be identified as hosts for infection and T-DNA transfer
2.2.7.1 AgrobøcteriumStrains
The choice of Agrobacterium strains in plant transformation is important due to two main reasons. The first reason is that the Agrobacterium strains vary in their virulence. The vlr
genes (Yanofsky et al., 1985) and the border sequences (Paulus et al., 1991) determine the virulence of Agrobacterium. It was shown in Beta vulgaris that the virulence was low in octopine strains (Ach5, LBA4404 and C58pB6S3), intermediate in nopaline strains (T37 and N2/73) and high in T,,L-succinamopine (4231) strains (Godwin et aI., 1992). The strain 4281 is known as a broad range super-virulent strain (Hood et aL, 1986) and has
been used in transformation of recalcitrant crops, such as soybean (Owens and Smigouki,
1988), Picea abies (Hood et al., 1990) and rice (Raineri et al., 1990). A number of other
strains are used in cereal transformation such as EHA101 (Hiei et aL, L994; Rashid et al.,
1996) and LBA44O4 in rice (Hiei et aL, 1994). The other reason is that Agrobacterium
strains exhibit plant host specificity. It has been reported that genetic elements located on the Ti-plasmid might be responsible for determination of host range (Loper and Kado, 1919). The Agrobacterium strain and host specificity have been reported in beet (Krens er aL, 1988), tomato (Komari, 1939) and chrysanthemum (van Wordragen et al., 1991). Therefore, in cereal transformation it might be necessary to evaluate different strains of Agrobacterium to suit the crop plant.
2.2.7.2 Plant Genotype and Explant Selection
The plant genotypes and explants vary in their response to Agrobacterium infection.
Differences in the interaction of plant genotypes and Agrobacterium have been observed in
pea (Puonti-Kaerlas et al., 1989), soybean (Owens and Smigocki, 1988), willow (Yahala et aI., 1989) and rice (Raineri et al., 1991). It has been shown that transfer of T-DNA also depends on the age and physiological state of plant tissues (An et al., 1986, Chang and Chan, 1991, Dale et aL, 1989, Gould et al., 1991 and Hernalsteens et aI., 1984). In the
case of rice, 3 week old calli derived from scutellum was found to be an excellent starting Chapter 2. Literature Review 13 material (Hie\ et aI., 1994; Rashid et aI., 1996). In Beta vulgaris, explants from plants grown in vitro were more responsive than those from the glass house (Godwin et al., Igg2). Therefore, it might be necessary to start the plant transformation
experiments with a genotype evaluation for both response to tissue culture and susceptibility to Agrobacterium. A compromise might be necessary in selecting a genotype for transformation as an economically important cultivar may not necessarily be susceptible to Agrobacterium. Ãfter optimizing the important parameters involved in T-DNA transfer and integration, these conditions can be extended to genotypes of interest with modifications.
2.2.7.3 Co-CultivationParameters
Co-cultivation of the explant with Agrobacteriumis a very important step in transformation. The conditions provided during co-cultivation should be optimum for host plant cell proliferation and induction of vir genes for T-DNA transfer (Godwin et aI., 1992) Tissue culture media includes inorganic and organic salts, vitamins, plant hormones, sucrose as a
carbon source and a pH of 5.8-6.0 (Murashige and Skoog , 1962) and temperature of 25o C.
A bacterial induction medium includes a carbon source such as sucrose, an optimum pH 5.1
(AlrMoerbe et a1.,1988), a temperature below 30" C (Stachel et al., 1985) and a sufficient
amount of signal (phenolic compounds) molecules (Melchers et aI., 1989).
Addition of phenolics: Although a number of vir inducing phenolic compounds released by
the wounded plant cells have been identifred, acetosyringone is most commonly used in the transformation experiments. It has been reported that inclusion of acetosyringone in co- cultivation media enhanced gene transfer in Antirrhinum majus, soybean (Godwin et al., 1991), mustard (Hadfi and Bastschauer, 1994), orange (Kaneyoshi,etaI.,1994), apple (James et aI:, lg93), pea (Davies et aI., 1993) and it was necessary in rice transformation (Hiei et al., 1994; Rashid et al., 1996). Different amounts of acetosyringone have been
used, for instance 200 pM acetosyringone was necessary in Antirrhinum majus and soybean (Godwin etaL,1991), l00pMinJaponicarice(Hiei etaI., 1994) and50pMin Indicaice
(Rashid et aI., Lgg6). Among different phenolics tested, acetosyringone and syringaldehyde were reported to enhance virulence of Agrobacterium strains C58 and 4281 in transformation of Antirrhinum majus (Holford et aI., 1992). Chapter 2. Literature Review l4
Extracts of dicotyledonous plants: In order to achieve the transformation of plant cells, extracts or inducing factors released from dicotyledonous plants or cell cultures have been used for vir gene induction. In number of cases, the suspension cells of tobacco have been used as feeder cells during co-cultivation of explant with the Agrobacterium- In the transformation of pea, Lulsdo rf et aL , 199 1 , obtained highest transformation frequency with the use of tobacco nurse cells during the co-cultivation. In the transformation of peanuts, addition of tobacco leaf extract to the bacterial culture enhanced the transient GUS expression, while inclusion of acetosyringone did not have any significant effect (Cheng et al., 1996). Similarly, the transient expression of GUS was obtained when co-cultivation of Moricandia arvensis was done on tobacco feeder cells, while acetosyringone had no effect in transformation (Rashiô et aI., 1996). The tobacco nurse cells enhanced the Agrobacterium mediated transformation of Anthurium (Chen and Kuehnle, 1996). The tomato explants pre-cultured on tobacco feeder cells before co-cultivation with Agrobacterium resulted in increased transformation frequency (Hamza and Chupeau, 1993). It is also reported that pre-culture of tomato explants on feeder cells for more than 2 days reduced the regeneration ability of the transformed cells (Hamza and Chupeau, 1993). In rice transformation, 3-4 days old seedlings were co-cultivated with Agrobacterium rn peftr
dishes containing filtrate from potato suspension cultures (Chan et al., 1992). In the similar way, extracts from other easily transformable dicotyledons might be exploited for higher vlr gene induction. These reports indicate that induction of virulence genes is a complex
procedure which needs more than acetosyringone alone. A more detailed investigation in this regard could widen the knowledge of vir induction methods and lead to higher
transformation frequency.
2.2.8 Transformation of Monocotyledonous Plant species
Agrobacterium is commonly used to transform dicotyledonous plant species. However, 'When monocotyledons include the most important group of crops, cereals. dicotyledons
are wounded, they release some phenolic compounds as a wound response. These phenolic
compounds act as chcmo-attractants and vir gene inducers. Therefore, they are necessary Chapter 2. Literature Review 15 for Agrobacterium to identify and infect the plant cell. Cereals lack a wound response and wounding leads to death of cell. This was the main reason to classiff monocots outside the natural host range of Agrobacterium. The hrst report of evidence of T-DNA transfer to species of monocot orders, Liliales and Arales (Decleene and Deley, 1976) expanded the host range of Agrobacterium. Since then the host range has been under constant expansion with addition of new crop plants including cereals. The reports of integration of T-DNA in monocots such as Asparagus fficinalis (Bytebier et a1,., 1987), Dioscorea bulbiftra (Schafer et a1.,1987), Allium cepa (Dommisse e/ al., I99O) and Musa accuminata (May et al., 1995) have further proved that monocotyledons have the potential for transformation by Agrobacterium. Earlier, tumour formation on infected plants was the criteria to decide the host range. This is not entirely correct, although tumorogenesis indicates the integration and expression of T-DNA; it is because there is a possibility of T-DNA integration without any phenotypic expression. This could be due to either inadequate expression of genes on T-DNA or the inability of host tissues to proliferate after infection. The host range could also differ with host genotype and Agrobacterium strains. Therefore it is incorrect to decide host range of Agrobøcterium based on interaction between few strains and host
genotypes.
Recently, the host range of Agrobacterium tumefaciens has been extended to cereals. The reports of transformation of Zea mays (Gould et al., 1991), Oryza sativa (Raineri et a1.,1990; Chan et al., 1992; Chan et al., 19% e.Í\ei et aI., 1994) and wheat
and barley (Deng et aI., 1990; Mooîey et aI., I99I) have given scope for using this natural
vector for introducing foreign genes in important crops. Raineri et al., (1990) reported the transformation of rice callus derived from mature embryos of japonica rice. Selection was
imposed using kanamycin and the gus gene was used as a reporter gene. Although a very
high frequency of transformation of callus was reported by them, no transgenic plants were regenerated. The co-cultivation of immature embryos, scutellum and suspension cells with
Agrobacterium gave rhe stable transformants (Hiei et al., l)94). The results obtained were confirmed by Southern hybridization and analysis of the boundary sequences. The progenies were also tested positive for the inheritance of the genes. The frequency of transformation obtained by them was between l2-29%o, which is similar to that of
dicotyledonous species (Hiei er aI., 1994). This detailed report by Hiei et a1.,1994 indicate Chapter 2. Literature Review 16 the potential of use of Agrobacterium in rice transformation. It would be beneficial to extend this technique to other genotypes of rice with improved co-cultivation practices.
2.3 BIOLISTICS
2.3.1 Introduction
When it became apparent that Agrobacterium does not easily transform atl the monocotyledons and both protoplast transformation and regeneration were difficult, a mechanical method for transferring genes into intact plant cells was discovered. Biolistics is
comparatively a new method of plant transformation, where genes are transferred to intact tissues. Biolistics is acceleration of DNA coated gold or tungsten micro-particles into plant
cells, where the DNA becomes integrated into plant nuclear genome. This system neither
exhibits a host range nor needs delicate handling of plant cells. Apart from being a tool for transformation, it is also used for rapid testing of gene constructs, so very soon it became
popular. Despite its popularity and a large amount of work and published achievements, the efficiency of transformation is still very low (Brettell and Murray, 1995).
The microparticles used are as small as lpm in size (Robertson et aI., 1992) and the velocity with which the particles penetrate should be regulated so as to maintain the
integrity and viability of cells (Southgate et aI., 1995). The microparticles deliver DNA into
cells in surface layers of the target tissue (Vasil et aI., 1985). The DNA is integrated stably in few of those thousands of cells containing the gene. The stably transformed cells can be
recovered by using a selection pressure corresponding to the selectable markers used. The commonly used selection agents are antibiotics such as kanamycin, hygromycin, geneticin, paromomycin and herbicides such as basta and bialaphos. The coating of DNA to microparticles and delivery of genes into plant cells can be conltrmed immediately after
bombardment by using reporter genes. B-glucuronidase gene is commonly used as reporter
gene as the product in cells can be visualized by addition of substrate X-gluc (Daniell et al.,
1991). Flurometric assays of GUS enzyme activity (Jefferson, 1987) and chloromphenicol
acetyl transferase activity (Kartha et al., 1939) can also be used to detect the presence of DNA after transformation. Transgenic plants have been obtained by using biolistics in many Chapter 2. Literature Review n crops. These plants can contain multiple copies of genes at any single locus. However, efforts can be made to regulate delivery and integration of genes in target cells by actually manipulating the parameters involved in the technique.
There are three basic components involved in using this technique:
1. A system or device to accelerate particles & optimization of parameters 2. Microparticles and DNA coating 3. Plant tissue and regeneration
Each of the components will be discussed briefly.
2.3.2 Bombardment Device and Parameters
The first successful plant transformation via microprojectile bombardment (Klein et a1.,1937) was done using a BiolisticsrM PDS 1000 accelerator. The explosive force of gun powder was used to accelerate a polypropylene cylindrical microprojectile loaded with DNA coated microparticles down a 0.22 callibre barrel towards target cells. The polycarbonate disk with small holes stops the macroprojectile but allows only the microprojectiles to penetrate the plant tissue. The vacuum in the chamber reduces the air resistance and thereby increases the velocity of the particles (Klein et aI', 1988;
Sanford et aI., l9g3). In the following models, the acceleration force was generated using different sources such as, compressed air or gas (Iida et al., 1990), electric discharge
(Christou et aI., 1988; Mc Cabe et al., 1988) and compressed helium (Kikkert, 1993). The biolistic PDS-1000/He device uses helium pressure released by rupture disk to accelerate macrocarriers loaded with DNA coated gold particles. The stopping screen stops the
macrocarrier but allows the microcarriers to travel towards the target tissue. The vacuum is
maintained in the chamber to avoid air resistance, there by increasing the velocity of the particles. The velocity can also be changed by varying the distance between the rupture
disk and macrocarriers; and varying distance between macrocarrier and stopping screen and helium pressure (referred in Biorad PDS 1000 He device manual). The impact with which the microparticles hit the target plant tissue depends on velocity and distance between the
macro carrier and the plant tissue. Chapter 2. Literature Review 18
The microprojectile bombardment method of plant transformation involves interaction of
physical and biological parameters. Therefore it is very important to optimize the levels of
both sets of parameters to generate a particle velocity just sufficient to penetrate into target tissue, without causing irreversible damage (Sanford et al., 1993). This enables the
transformed plant tissues or cells to regenerate back to plants. This is especially important in monocotyledonous plant species, where wounding leads to death of cells. Different combinations of helium pressures, distances between rupture disk and macro carrier and
distances at which target tissues are placed, should be established for different crop species.
These combinations may vary with plant species, genotypes and explants.
2.3.3 DNA Coating Methods
Microcarriers are high density particles that carry DNA into plant tissues. Gold and tungsten (both with similar density) are the commonly used microcarriers. The size of
particles used vary from 1-5pM in diameter depending on size of plant cells i.e., smaller the (Klein et 1988; Robertson ¿/ al. 1992). 1 size of the plant cell, smaller the size of particles al., rt soon became less popular as it 'ìJ Earlier tungsten was widely used as it is cost effective, but it I oxidizes readily and forms aggregates quickly (Hunold et al., 1994). Therefore, gold particles which have a similar density as tungsten, but inert in reaction, came into use. Gold, which does not form aggregates, is widely used as a microcarrier despite its cost factor I
The DNA is coated on to microcarriers using different methods. In one method, DNA is precipitated on to gold or tungsten particles by CaCl2 and spermidine (free base)
(Ktein et a1.,1988). In some instances, (CaNO:)z is used alone (Perl et al., 1992). Ethanol is also used to precipitate DNA on to microparticles (Iida et aI., 1990). The efficient precipitation also depends number of other factors including human skills. High quality of DNA should be used as impurities coming along may cause aggregation of particles. The
concentration of DNA used in most cases is lpg/pl, higher concentration may lead to
aggregation and lower concentration result in poor coating (Oard, 1991). The DNA coated microcarriers are used for bombardment either as a drop of liquid or dried on to
macrocarriers depending on the design of the particle gun. ! Chapter 2. Literatut'e Review t9
2.3.4 Plant Transformation
The plant material used in transformation should always be embryogenic i.e., capable of
regenerating back into whole plants. The suspension cells are used for initial optimization experiments as it needs a large quantity of material. Immature embryos, young inflorescence and embryogenic calli are commonly used for actual transformation experiments. The target plant material is likely to splash due to the release of high pressure
and the impact of microparticles. This may hinder targeting of explants. Therefore, ltlter
papers are used in the case of suspension cells and calli and embryos are firmly pressed in the agar medium. Better,transformation is also obtained by placing the target tissue at
different distances from the macrocarriers. Pretreatment of the explant with osmoticum is
shown to increase transient expression (Petl et al., L992)
Embryogenic suspension cells were used for transformation of muze (Gordon-Kamrrt et al., 1990; Fromm etal., 1990),rice(Cao etal., 1992),oats(Sometsetal., 1992)and wheat
(Vasil et al., Ig92). The low regeneration from suspension cells is the main disadvantage of this system. The recent method is to bombard the scutellum of immature embryos. This
has been used in rice (Christou et al., I99I), sorghum (Casas et aI., 1993), muze (Kozell et aI., 1993), barley (Ritala eÍ al., 1994; Wan and Lemaux, 1994), triticale (Ztmmy et al.,
1995), rye (Castillo et al., 1994) and wheat (Vasil et al., 1993; Weeks et al., 1993; Nehra ¿r aI., 1994; Becker et aI., 1994). The cells in the surface layers of the scutellum will give rise to somatic embryos on an appropriate culture medium. However, obtaining a sufficient number of immature embryos throughout the year is the limiting factor for most of the laboratories. Immature inflorescence has been used for the initiation of embryogenic callus
in many monocotyledons such as sorghum (Brettell et aI., 1980), wheat (Ozias and Vasil, { lg82), rice (Aryan, unpublished), Lolium rigidum (Patil, unpublished) and Lolium perenne (Molenaar et al., 1992). The immature inflorescence is also used directly for bombardment
(Barcelo et al., 1994).
I
I Chapter 2. Literature Review 20
2.4 RICE TRANSFORMATION
2.4.1 Introduction
Rice is an important cereal crop and serves as primary source of calories for nearly half of the world's population. Rice out crosses wheat and maize nutritionally, generating more
calories per unit area. Therefore, improvement of rice has a place of priority in the breeding
programs. Although much has been done through conventional breeding methods, a lack of variation among the species has slowed down the process. Genetic engineering and tissue
culture techniques can assist breeders in either incorporating variabilities from other species
or in creating variabilities ámong the existing species. Oryza sativa is the largely cultivated rice consisting of two subspecies namely, Indica and Japonica. Indica is tropical and a long grain rice and it accounts for more than three quarters of cultivated rice. While
Japonica is a short grained rice and is restricted to temperate climates.
Rice is a cereal crop which is easily manipulated under in vitro conditions, and it can also be
considered as model cereal crop for most of the basic studies. This can be attributed to the comparatively smaller genome of rice, which makes it convenient for genetic studies and manipulation. This is supported by the amount of work done and achievements in rice
genetic studies such as mapping and genetic transformation.
2.4.2 Genetic Transformation
2.4.2.1 Agrobacteriummediated Transformation
As discussed in the previous sections of this chapter, Agrobacterium mediated transformation remains the preferred method due to its simplicity and accuracy. Dicotyledonous plant species are routinely transformed using Agrobacterium by virtue of their wound response. Absence of wound response in cereals puts them out of the natural host range of Agrobacterium. Initially, some efforts were made to fuse Agrobacterium spheroplasts to rice protoplasts using PEG (Baba et aI., 1936). Although expression of
nopaline synthase was detected in calli, transgenic plants were not regenerated. Production Chapter 2. Literature Review 2l of a kanamycin resistant transgenic calli (Raineri et al., 1990) made researchers reconsider
Agrobacterium as tool of transformation for monocotyledonous plant species. This lead to further studies on the factors influencing the Agrobacterium infection in rice
(Li et al., 1992a). Different explants were tested for their ability to show transient expression of GUS after co-cultivating with Agrobacterium. In their experiments, all the rice lines tested showed at least some GUS expression and level of expression was higher in Indica types (Li et aL, 1992). Surprisingly,vir gene induction prior to co-cultivation did
not enhance the transient GUS expression (Li et al., 1992).
Several attempts were made to enhance the infection by using additional copies of virG
genes (Liu et aI., 1992). They showed that additional copies of vlrG genes from octopine-
type enhanced transient exþression than an agropine-type, and vitlj- genes from a nopaline-
type had no effect (L\u et at., 1992). However, agropine-type vitG genes were shown to be
more effective than those of nopaline or the octopine-type (Li et aI., 1992). Transgenic rice
plants were produced using Agrobacterium (Chan et al., 1993) and presence of transferred
genes was shown by Southern hybridization and enzyme assays. The potato suspension
culture rich in acetosyringone andsinapinic acid was used for preinduction. Only one of the four transgenic plants survived, the progeny of which also contained the T-DNA
(Chan et al., 1993).
The complete and convincing evidence of rice transformation was shown (Hiei et aI., 1994) by molecular and genetic analysis of transgenic plants. Various explants such as shoot
apices, segments of roots from young seedlings, scutellum, immature embryos, calli induced
from young roots and scutellum, and suspension cells induced from scutellum were used for co-cultivation with Agrobacterium. The expression of GUS was mainly observed in scutellum (Hiei er aL, 1994; Rashid et al., 1996). H\ei et al., (1994) also confirmed this result by obtaining highest transformation frequency with scutellum and calli derived from scutellum. They used a super-binary vector which gave them highest frequency of transformation. The intron-gus was used as reporter gene which expresses in rice and does not show any activity in bacteria (Ohta et aL, 1990). Such constructs would help in
eliminating false transient expression results. The necessity of addition of acetosyringone n co-cultivation medium was confirmed (Hiei et aI., 1994; Rashid et aI., 1996). However, Ifiei et al., (1994) did not find potato suspension culture necessary for preinduction Chapter 2. Literature Review 22
(Chan et aL, lgg}). The T-DNA integration was shown to be random based on the bands obtained in Southern hybridization. Hte\ et aI., (1994) also analyzed the progeny of transgenic plants which also contained the T-DNA. Importantly, they demonstrated that T-DNA boundaries in transgenic rice plants were similar to that shown in the dicotyledonous plants. The frequency of transformation reported in rice is as high as in dicotyledonous plants (Hiei et al., 1994; Rashid et al., 1996)-
Such a detailed report has made most of the researchers to re-evaluate the utility of the Agrobacterium system, which was once considered to be unsuitable for cereals. The improvement and wider adaptation of this system to other cereals can be done by using new
vectors. This mainly includes use of plant promoters that express well in cereals.
2.4.2.2 ProtoplastMediated Transformation
Among cereals, rice was the first crop to be transformed using protoplasts' The main limitation for wide application of this technique is availability of regeneration protocols.
Plant regeneration has been possible in some of the cultivars and while in others it remains to be resolved. The first successful regeneration of plants from rice protoplasts (Fujimura et al., 1935) was a major break through in cereal technology. The source of
protoplasts was suspension cells derived from immature and mature embryos of Japonica
cultivars.
The regeneration of plants from protoplasts from suspension cultures derived from mature
seeds and leaf base tissue (Abdullah et al., 1986), was also shown possible. The haploid
calli derived from anthers of Japonica cultivars was used to isolate haploid protoplasts and both haploid and diploid plants were regenerated (Toriyama et al., 1986). This was later
extended to indica cr-ritivars (Kyozuka et al., 1988), where plant regeneration was possible from four cultivars of the fourteen tested. The use of nurse cultures from japonica cultivar
was shown to be useful in regenerating plants from indica types (Lee et al., 1989). Plants
were regenerated from protoplasts of Javonica cultivar Gulfmont flMang et al., 1989). The
regeneration has been reported in several other cultivars of Japonica, Indica and Javonica types. The regeneration of plants from the protoplasts, which is genotype dependent Chapter 2. Literature Review 23
(Kyozuka et a1.,1988), is the main limiting factor for the wide application of this technique.
The use of nurse culture of fast growing cell lines has helped to overcome some of the problems associated with the regeneration of plants from protoplasts.
The most common source of protoplast is suspension cells derived from different sources.
The callus is induced from explants on an appropriate medium. The friable calli are selected
and partly immersed in liquid media. The suspension cells are regularly subcultured with a
fresh medium. The explants commonly used for initiation of calli are immature embryos, immature inflorescence, mature embryos and microspores. The microspore derived
suspension cells are haploid and the plants regenerated from them will consist of both haploid and deploid (Guiderdoni and Chair, 1992). This gives scope for development of
homozygous transgenic plants. Homozygous transgenic plants have been obtained in rice by transformation of protoplasts of microspore derived suspension cells (Datta et al., 1990).
Protoplasts are devoid of the cell wall which makes them easy to manipulate in vitro conditions. The protoplasts can be made to take up external DNA by creating pores in the cell membrane. This is achieved by chemical treatment (PEG) or electric shock (electroporation). The first gene transfer in rice was done by electroporation
(Ou-Lee et al., 1936) and a transient expression of CAT gene was obtained. Transgenic calli resistant to kanamycin was obtained by PEG mediated transformation of protoplasts (Uchimiya et al., 1986). Subsequently, transgenic rice plants were obtained following protoplast transformation of Japonica cultivars (Toriyama et al., 1988; Zhang et al., 1988).
This has also been achieved in Indica (Peng et al., 1990; Piong et al., 1992) and Javonica cultivars (Li et al., Lggzb). Transgenic plants are produced routinely by protoplast transformation in rice cultivars amenable to easy regeneration. However, the regeneration of fertile normal plants from protoplasts remains the main hurdle for wide application of this
technique.
2.4.2.3 Biolistics
Invention of the particle gun to introduce foreign genes in plant cells helped in extending transformation technique to the cereal crops that are difhcult to transform by other Chapter 2. Literature Review 24 methods. In case of rice, the cultivars where regeneration of protoplasts was diff,tcult, were the main beneficiaries. Different explants are used as target tissue. Suspension cells were used for bombardment with uidA and CAT genes and a transient expression was obtained (Wang et aL, 1988). Herbicide resistant transgenic plants were recovered following bombardment of suspension cells with the bar gene (Cao et al., 1992). Immature embryos are used for transformation, by directly shooting on the scutellar region, or used for inducing embryogenic calli which are used for bombardment. Transgenic plants were obtained after bombardment of immature embryos of Indica and Japonica cultivars (Christou et aL, 1991). Despite the low transformation frequency, the technique is routinely used for obtaining transgenic rice plants in many laboratories.
2.5 DNA CONSTRUCTS
Choice of DNA construct depends on the aims of the transformation project. In the process of parameter optimization, one needs gene constructs that can be assayed rapidly and easily.
In the GUS reporter system, enzyme B-glucuronidase coded by the qus gene, cleaves a
colorless substrate X-gluc (5 bromo-4 chloro-3 indolyl-B-D-glucuronic acid) into a product
that upon oxidation ¡iroduces an indigo colored dye (Jeffei'son ¿t aL, L987). Presence of genes in the plant cells can be visualized by the blue colored spots. This system is widely used in initial experiments of transformation (Klein et al., 1988; Perl et aI., 1992; Aragao et al., 1993; Charest et at., 1993). Other similar genes that produce anthocyanin (Ktein et al., 1939) or luminescence (Fromm et al., 1990) have been used. The cat gene which encodes for the enzyme chloromphenicol acetyltransferase is another reporter gene
but requires extensive analysis after transformation (Kartha et al., 1989).
Selection of transformed cells from thousands of untransformed cells is as important as the transformation procedure. Among the many cells that have received the DNA, in only a
few cases it is integrated stably in the nuclear genome. It is very important to identiff these few cells and culture them to obtain transgenic plants. This can be achieved by using efhcient selectable marker genes that allow stringent selection of transformed cells. Antibiotic and herbicide resistance genes are the commonly used selectable markers. The
nptlI gene (Bevan et al., 1983) coding for neomycin phosphotransferase confers resistance Chapter 2. Literature Review 25 to kanamycin, geneticin and paramomycin. This has been used as a selectable marker in many crops including soybean (Christou et al., 1988) and rice (Uchimiya et al., 1986;
Yang et al., 1988; Raineri et al., 1990). Tlte hptll gene coding for enzyme the hygromycin phosphotransferase confers resistance to hygromycin (V/aldron et al., 1985), and has been extensively used in rice transformation (Hayashimoto et aI., 1990; Li and Murai, 1990;
Murai et al., 1991; Datta et al., 1992;Hiei et aI., 1994; Rashid et al., 1996). The bar gene codes for the enzyme phosphinothricin acetyl transferase (PAT) conferring resistance to herbicide bialaphos. This has been successfully used in wheat (Vasil et al., 1992;
Becker et al., 1994), maize (Gordon-Karcm et al., 1990; Spencer et aI., 1990) and rice
(Uchimiya et aL, 1993; Toki et al., 1992; Cao et aI., 1992). The use of herbicide resistant genes is attractive as it not only acts as selective agent, but also confers herbicide resistance to the transgenic plant, which could be a desirable character. The genes transferred to plant cells require time for stable integration and production of enough enzyme to combat selective agents. Antibiotics or herbicides corresponding to the selectable markers, differ in their toxicity to plants. The size and developmental state of the plant cells also influence the toxicity. Therefore, it necessary to work out the actual concentration of the selective agent required to kill the untransformed cells without harming the transformed cells (Wilmink and
Dons, 1993).
The foreign genes that are introduced' into plant cells require a promoter for their expression. The Cauliflower Mosaic Virus CaMV 35S promoter has been used extensively (Bekkaoui et aI., 1990; Charest et al., 1993). Thts CaMy 35S promoter is efficient in dicotyledons, but has some limitations and may not be as effective in monocotyledons (Christensen et al., 1992). The rice Act I promoter (Mc Elroy et aI., 1991) is known to be efficient in monocotyledons. Other promoters used for monocotyledons include muze alcohol dehydrogenase (Adh I) and maize ubiquitin (Ubil). The CaMV 35S promoter is
used in binary vectors for Agrobacterium mediated transformation. This promoter shows
expression in bacteria which make it difficult to identiff the actual transformed cells' In order to overcome this problem plant introns are used in the binary vectors (Ohta et al., 1991) which avoids expression of genes in bacteria. Another strategy for plant
improvement could be isolation of efficient promoter from particular plant species and put it back into the same plant with the genes of interest. This may help in a higher stable
expression and complete expression of the gene introduced. Chapter 2. Literatut'e Review 26
2.6 AIMS OF THE PROJECT
The survey of the literature indicates that transformation of cereal crops could be possible by optimising the parameters involved in the techniques. Each individual crop species or even cultivar of a species must be considered independently as some species or cultivars might be recalcitrant to particular transformation techniques. It is always important to establish plant regeneration protocols before using transformation procedures in any crop species. With this ba':kground, the aims of the project were :
1. To establish and improve the regeneration protocols for rice (Oryza sativaL.), 2. To evaluate different techniques of transformation in cereals with rice as a model system,
3. To enhance the efficiency of these techniques of transt'ormation. Chapter 3
Materials and Methods
3.1 PLANT MATERIAL
Three Japonica rice cultivars,lT-\J0, Nipponbare and T-309 were used in the experiment and the seeds wdre kindly provided by Rice breeder Dr Laurie Lewin, and rice Dr Upadhyaya, N.M. Division of Plant Industry, CSIRO, Canberra, Australia- The seeds were surface sterilised by immersing in 70Vo ethanol for 2 minutes and then transferred to lTo NaoCl for 20 minutes. They were thoroughly washed 3 times in sterile
nanopure water. The seeds were germinated on a sterile moist filter paper in a petridish' After 8-10 days, the germinated plants were transferred to the soil pots and placed in a growth charnber. In order to provide submerged conditions for the rice plants, pots were 20o C placed in a plastic tray filled with the water. The plants were grown at 30o C day and night temperature. The plants were watered regularly and the water level in the plastic tray
was always maintained. The plants were supplied with urea 30 and 45 days after planting. in The tobacco plants (Nicotiana xanthi and V/-38) were grown aseptically and maintained MSO (Murashige and Skoog, 1962) medium without any growth hormones'
3.2 PLANT TISSUE CULTURE
3.2.1 Tissue Culture Media
Different tissue culture media were used in different experiments for various pu{poses. The detailed composition of the media is listed in the Appendix 1. 2l Chapter 3. Materials And Methods 28
3.2.I.1 Rice
Three different media containing 2,4-D (2 mg/I-) were used for callus induction; NB, MS (Murashige and Skoog, 1962) and AA (Toriyama and Hinata, 1985). NB medium
(Appendix 1) contains N6 organic salts (Chu et al., 1915), B5 micro elements and vitamins (Gamborg et al., 1968), casein hydrolysate (300 mglI-), proline (500 mg/L), glutamine (500 mg/L) and sucrose (30 g/L). Two pre regeneration media, NBPR (Appendix 1) and AN6 (Appendix 1) were used. For regeneration two media, NBR (Appendix 1) and MS9 (Appendix 1), were used. For root initiation, MSO medium (Appendix 1), which is MS medium without any growth hormones, was used. The nitrogen is available readily in
AA medium as the sole source of nitrogen is amino acids. AA liquid medium was used for initiation and maintenance åt.i"" suspension cells.
3.2.L.2 Tobacco
The tobacco plants were maintained in jars containing MSO medium which is MS basal medium (Appendix 1) without any growth hormonies. The suspension cells were maintained in cSV medium (Appendix 1) containing 2,4-D (2 mgfi-) and kinetin (0'5 mg/L).
3.2.2 Tissue Culture Conditions
All the tissue culture procedures were performed in a sterile laminar flow hood. The temperature in the tissue culture laboratory was maintained at 25o C. The glassware and other material used for the tissue culture were sterilised by autoclaving at l2lo C for
20 minutes. These materials were transferred to laminar flow hood for cooling after autoclaving. The calli were induced from explant in the dark at 25o C. For regeneration the
calli were transferred to the light at 25o C. Chapter 3. Materials And Methods 29
3.2.2.1 Tobacco
Tobacco plants Nicotiana xanthi and cv. Wisconsin 38 were maintained on MSO medium in tissue culture pots and placed in the light at 25o C. Tobacco suspension cells of tobacco \¡/ere provided by Dr Aryan and were maintained in CSV (Appendix 1) medium. The suspension cells were subcultured every week in fresh 250 ml flasks containing 10 ml CSV medium. These flasks were placed on a rotary shaker at 100 rpm and incubated at
Z5o C. The tobacco suspension cells were subcultured at 4 days interval for 2 weeks before using as nurse culture. The tobacco cells were spread in the center of petri plates containing NB (Appendix 1) or AA (Appendix 1) solid medium and were incubated at25o
C for 1-2 days. A sterile filter paper was placed on the tobacco cells and these nurse culture plates were used in the rice regeneration and Agrobacterium mediated transformation
experiments.
3.2.2.2 Rice
Surface Sterilisation: Different explants were used in the tissue culture of rice. In case of cultivars 17-170 and Nipponbare, immature seeds were collected at milky grain stage and
surface sterilised. The immature seeds were immersed in TOVo ethanol for 2 minutes and
then transferred to 17o NaOCI for 2O minutes. They were thoroughly washed 3 times in
sterile nanopure water.
CaIIus Induction: Immature embryos were excised aseptically and placed on the different callus induction media based on NB (Chu er al., 1975), MS (Murashige and Skoog, 1962) and AA (Toriyama and Hinata, 1985) salts with 2,4-D (2 mg/I-) (for details see Appendix 1), incubated in the dark at 25o C. After 5-6 days the emerging shoots were clipped to facilitate the callus formation from the scutellum. The first sub culture of callus was
performed after 3 weeks and thereafter subculturing was done every 4 weeks. Due to poor
seed set and grain filling, mature seeds were used in the case of cultivar T-309. The seeds
were dehusked, surface sterilised as above, and placed on MSO medium in the dark for
7 days at 25o C. The embryos were excised from the softened endosperm after 7 days and 30 Chapter 3. Materials And Methods placed on NB medium for callus initiation. The callus induced from scutellum was subcultured after 3 we;eks and then regularly every 4 weeks cn fresh NB medium'
the Suspension Cells: Suspension cells of cv.7l-Il0 and cv. Nipponbare used in calli regeneration experiment were provided by Dr A. Aryan and were initiated using derived from immature inflorescences and immature embryos respectively' They were years- the maintained in liquid AA medium with regular subculturing for more than two In 2O-25 smal' case of cultivar Nipponbare, the suspension cultures were initiated by placing friable calli derived from immature embryos on AA medium, in a large petri dish containing (100 rpm) 5 rnl AA liquid medium. The cultures were incubated at25o C on a rotary shaker in the dark and the medium was changed weekly. After the suspension cells started multiplying, they were ftânsferred to 250 ml flasks. These fresh suspension cells of as an cv. Nipponbare were not used in the regeneration experiments, but were started cells were exercise to learn the technique of establishing suspension cells. The suspension at transferred to fresh liquid AA medium every 7 days. Suspension cells were subcultured calli were 4 day interr¡als for two weeks before using them for any experiment. Healtþ on selected and used in the experiments. The suspension cells were also maintained
AA solid medium as backup material.
Regeneration of Suspension Cells with and without tobacco nurse cells: For the
regeneration of plants, the suspension cells of two rice cultiva¡s,71-I70 and Nipponbare' were tested. This experiment was conducted in three replicates for each treatment. Each tobacco treatment had 20 calli clusters in the beginning of experiment. On day one, 2 ml of no' 1 suspension cells was transferred to AA solid medium. Next day, a sterile Whatman filter paper was placed on the cells. After two days, 20 calli clusters derived from ml of suspension cells were placed on the filter paper which had been pre-wetted with 1-2 liquid AA medium. The plates were sealed with parafilm and incubated at25o C in the dark solid for 10-14 days. For the control treatment, the calli were transferred directly on to AA medium without the nurse culture. After 10-14 days, one half of the calli from each all treatment was transferred to AN6 medium and the other half to NBPR medium, and were were then incubated at25o C in the dark for 14 days. The calli from the AN6 medium
then transferred to MS9 medium and those from NBPR to NBR medium. At this time each
calli cluster had at least l0 calli and these calli were then incubated at25o C in the dark for Chapter 3. Materials And Methods 3t
7 days before transferring them to the light (16 hr day/8 hr night). The embryogenic calli with shoot primordia were subcultured on to fresh NBR plates every two weeks to promote shoot formation. Although the initiation of shoot primordia started as early as 1 week in some calli, the count was taken after 3 weeks and 4 weeks of incubation in the light at
25o C. The number of shoots were counted after 8 weeks in the light. The shoots were 'Well-rooted transferred to MSO jars for root initiation. plants were transferred to soil pots and placed in the growth chamber.
3.3 PLANT TRANSFORMATION
3.3.1 Bacterial Strains and Plasmids
Strains of Bacteria: Two strains of E. coli used were, DH5a and JM 101 and both were provided by Dr Aryan. Two different strains of Agrobacterium were used: AGL1 and Lp.A44O4. Both were obtained from Dr Clare, 8., The Department of Crop Protection, The Universiry of Adelaide. Both the strains were maintained on YEP medium containing rifampicin (50 mg/L).
Bacterial Medium: Different bacterial media were used for growing Agrobacterium and E. coli. Agrobacterium were grown in YEP medium, while E. coli were grown on LB medium. Both the strains of Agrobacterium were grown on medium containing rifampicin (50 mglL). The Agrobacterium strains containing binary vector (section 3.5) pPCV7|7+Gus were grown in medium containing ampicillin (20 mgfl-), while hygromycin
(50 mg/L) and kanamycin (50 mg/L) were included in the media to grow strains containing binary vector pIG121Hm. The E. coli strains harbouring pUC based plasmid vectors were
selected on the medium containing ampicillin (100 mg/L).
Plasmid constructs: Four different plasmid vectors: p'/'ctl-D (McElroy et aI'' 1991)' pAHC25, p35S-Hygro, and pGli-Gus, were used for DNA coated microprojectile bombardment experiments. The vector pAct-D has GUS reporter gene with rice actin promoter and first intron (McElroy et al., 1991). The vector pAHC25 contains the bar gene and GUS reporter gene, both under the regulation of the maize ubiquitin promoter. Chapter 3. Materials And Methods 32
The bar gene confers resistance against the herbicide basta. Both p35s-Hygro and pGliGus were constructed by Dr Aryan. The vector p35S-Hygro contains hptll gene with
CaMV 35S promoter and nos terminator (Appendix 3), where as vector pGIïGus has GUS
reporter gene under the regulation of ø-gliadin gene promoter of wheat (Appendix 3).
Two binary vectors were used in the Agrobacterium mediated transformation experiments. The binary vector pIGI2lHm (Hiei et at., 1994) was provided by Prof. Nakamura, K., Nagoya University, Japan. This binary vector contains hptll gene with the CaMV 35S
promoter and GUS reporter gene with the CaMV 35S promoter (Appendix 3).
3.3.2 Selection Media
For selection of potentially transformed cells of rice different antibiotics were used in the normal tissue culture medium. In the case of Agrobacterium mediated transformation, the calli after co-cultivation were cultured on NB-T150, which is NB medium containing timentin @ 150 mgll,.The timentin was used to avoid the growth of Agrobacterium on the explant after the co-cultivation. NB-TH50 (Appendix 1) was used for selection of calli which were transformed with plasmids containing hptll gene. The pre-regeneration medium used was NBPR-TH50 (Appendix 1) and the regeneration medium was NBR-TH3O (Appendix 1). The concentration of hygromycin was reduced to 30 mg/L in the regeneration medium to facilitate higher plant regeneration. The selection and regeneration
media used in rice transformation were similar in both the techniques, except that timentin
was used in Agrobacterium mediated transformation.
3.3.3 Direct Gene Transfer by Microprojectile Bombardment
3.3.3.1 Biolistic Gun and DNA Coating of Microprojectiles
The Biolistic PDS-1000/He particle gun was used for delivering plasmid DNA into plant tissues. The rupture disks, macrocarriers, stopping screens and gold particles were purchased from Bio-Rad Laboratories. Gold particles of size 1.1 pm (Bio-Rad Laboratories) were prepared in sterile nanopure water as explained in Biorad PDS 1000/He Chapter 3. Materials And Methods JJ manual. Sixty mg of gold particles were weighed out in an eppendorf tube and I rnl of thorough IOOVo ethanol was added. The tube was placed on vortex for 30 minutes. After vortexing, the tube was centrifuged at 4 K for 1 minute and the ethanol was removed. The gold particles were washed three times in sterile nanopure water and f,rnally resuspended in 1 ml of sterile nanopure water. Sterilisation of macrocarriers, rupture disks, stopping screens, macrocarpa rings and bombardment equipment was done by soaking or spraying withlOVo ethanol for 10 minutes. Gold particle suspension was vortexed thoroughly for an hour before using them for DNA coating. An aliquot of 50 pl particle suspension was centrifuged at 4K for 30 seconds and most of the water was removed. Then, 6 pl of plasmid DNA (or 6-8 pl of 1:1 mix in case two plasmids) was added to the pellet and thoroughly mixed. Then 50 pl of 2.5 M CaClz (pH 10) was added slowly and vortexed. This was followed by adciition of 20 pl of 0.1M spermidine and thoroughly mixed by vortexing. The mix was left on ice for 5-i0 minutes and then centrifuged at 4K for
30 seconds. All the supernatant was removed and the particles were washed with 150 pl of
70Vo ethanol. The particles were then resuspended in 200 pl absolute ethanol by continuous vortexing and 10 pl suspension was spread in the centre of macro carriers. Much cafe was given to avoid particle aggregation during any of the steps. Eppendorf tube was vortexed during every sampling while spreading the suspension on to the macro carriers,' The
suspension was spread starting from the center of the macro carrier and extending towards outside. The suspcnsion on the macro carriers was dried for 15 minutes before bombardment.
3.3.3.2 Preparation of ExPlant
Different explant tissues of rice were used for microprojectile bombardment: scutellum derived calli, immature embryos and suspension cells. After 2-3 subcultures, 40-50 small
pieces of scutellum derived calli or 30 immature embryos were alranged on NB plates in the centre. In the case of rice suspension cells, 1 rnl of hne cells were sPread in the centre of 'Whatman No. I filter paper placed on the NB solid medium. Half rnl of osmoticum (mannitol 3O gmlL and sorbitol gm/L) was added to the explants 3-4 hours before microprojectile bombardment and dried in a laminar flow hood. After 15-16 hours of
microprojectile bombardment 1 ml of liquid NB medium was added to the explants. Chapter 3. Materials And Methods 34
3.3.3.3 Microprojectile Bombardment and Selection of Potential Transformants
Transformation of rice and tobacco explants was mainly performed at 1100 psi pressure level. For optimisation experiments, different pressures 1350 psi and 1550 psi were used. The plant material was placed 6 cm from the microcarriers. To avoid the splashing of the material, the calli or the embryos were firmly pressed into the agar' The petri plates jerk carrying the explant were frmrly placed on the base plate using blue stick, to avoid
movement during release of pressure. To increase the velocity of the particles, the distance
between macrocarrier and rupture disk was reduced. The calli were shot two times, once on each side and embryos'four times, twice on each side. The catli or immature embryos
were turned upside down after one or two bombardments and were shot again.
After one week of núcroprojectile bombardment, the call were transferred to NB-H30 medium for two weeks. The control calli that were not shot were also placed on similar medium. proliferating calli or portions of calli that survived were transferred to fresh NB-H50 for two weeks. All the selected calli or portions of individual callus were maintained separately. This mainly helped in isolating independent transformation events
and their clones. The calli were multiplied on selection medium NB-H50 before transferring to pre-regeneration medium NBPR-H50. After 2 weeks on NBPR-H5O in the dark, the calli were transferred to regeneration medium NBR-H30 and incubated for one week in the
dark and then transferred to the light.
3.3.4 AgrobøcteriumMediatedTransformation
3.3.4.1 Cloning Binary Vector
Two binary vectors were used in the experiment pPCV7}7+Gus and pIGl2lHm (Hiei et aI., lgg4). Binary vector pPCV7ï7+Gas was constructed using two plasmids namely, pPCV707 which is a binary vector and contains hptll gene under CaMV 35S promoter
(Appendix 3), and pPRTL2Q which is a pUC based plasmid containing uidA gene under 35 Chapter 3. Materials And Methods dual caMV 35S promoter (Appendix 3). The ptasmid pPRTL2Q was digested with Hind lll restriction enzyme to isolate 4 kbp GUS gene cassette and analysed on a l7o a$arose flel' the The area of the gel with GUS gene cassette (4 kb) was cut from the gel under low UV light. The piece of gel was transferred to a dialysis tube containing TE buffer. The DNA was extracted from the agalose gel fragment using the gene cleaning procedure (Sambrook et al., 1989). The TE buffer with the DNA fragment obtained from the gene cleaning procedure was treated with phenol : chloroform (1:1). The DNA was precipitated using 0.1 volume of 3M NaOAc (pH 4.8), 2 volumes of ethanol and carrier RNA for 3 hours at 37o C and (0.5 ¡rg/rnl). The plasmi d pPCV707 was linearised with Hind III phosphatased according to Sambrcok et al., 1989. The ligation reaction was carried out with phosphatased plasmid pPCV707 and 4 kbp GUS fragment according to the procedure of Sambrook et al., 1989. 'The eppendorf tube with ligation reaction mix was incubated at room temperature for 2 hours.
Transþrmation of E. coli: The ligation mix was used for transforming E. coli
(DH56¿ strain) using the heat-shock method (Sambrook et al., 1989). The frozen
competent cells (prepared according to Sambrook ¿/ aI., 1989) of DH5cr strain (50p1) were
thawed before addition of 50pl TCM buffer and 4 pl ligation mix. The bacteria were placed
on ice for 20 minutes and transferred to 42o C water bath for 2 minutes. The LB medium
(a00pl) was added to the bacteria and the mixture incubated at 31o C for one hour on a
rotary shaker. The bacteria were plated on LB solid plates containing 100p1 ampicillin and
incubated at 3Jo C overnight. The presence of plasmid in resistant colonies that appeared on selection medium was conhrmed by small scale plasmid isolation and restriction digestion analysis (Sambrook et al., 1989) with Hind III enzyme'
A large scale plasmid isolation by equilibrium centrifugation in CsCl-ethidium bromide gradients was done using the procedure published in Sambrook et aI., (1989)- This new plasmid was tested for GUS expression by DNA coated microprojectile bombardment of
tobacco suspension cells.
Transþrmation and Screening of Agrobacterium: The binary vectors were transferred into Agrobacterium usíng freeze-thaw method as described by Gelvin et al., (1988) with some modifications. TCM (50 pl) buffer (Appendix 2)was added to 50 pl competent cells (Gelvin Chapter 3. Materials And Methods 36
were and Schilperoort, 1988) before adding 5 pl of plasmid DNA (1 pg/pl). The bacteria mixed well with the pippetman and immediately frozen in liquid nitrogen for 5 minutes. The mixture was then placed on ice for few seconds before incubating them at 37o C for shaker 5 minutes. The YEP medium (1 rrìl) was added to the tube and placed on a rotary for 2-4 hours at 28o C. The bacteria were plated on YEP plates containing rifampicin (50 mg/L) and other rntibiotics depending on the binary vertor re., hygromycin (50 mgtL)
and kanamycin (50 mgtL) for pIGt2tHm and ampicillin (20 mgfi-) fot pPCV7}7+Gus. The
colonies of Agrobacterium that appeared on the selection medium containing the antibiotics were grown in l-2 rnl YEP medium with selective agents and incubated at 25o C for 48 hours. The plasmid DNA was extracted from the culture of selected colonies of Agrobacterium us\ng the procedure in Gelvin and Schilperoort, 1988. The presence of plasmid DNA in the Agiobacterium sftains was confirmed using restriction digestion
analysis all the times before starting co-cultivation.
3.3.4.2 Co-cultivation
Single colonies of Agrobacterium strains were used to start 2 ml cultures in YEP medium conraining antibiotics; hygromycin (50 mg/L) along with kanamycin (50 mgtL) for pIG12lHm and ampicillin (20 mgl-L) for bacteria harbouring binary vector pPCV7}7+Gus,
These cultures were grown overnight at 25o C and were used to inoculate 10 rnl of AAM induction medium (Hiei et aI., 1994). The bacteria were grown in AAM medium for 12-14 hours on a rotary shaker at 25o C. The density of Agrobacterium grown for 72-14 hours
was measured and the ODeoo was adjusted to 0.3 with induction medium before using them for co-cultivation.
Agrobacterium cultures (5 nrl) were pipetted into a small petri dish. The rice calli were dipped in the bacterial cultures and incubated for 5-10 minutes. These tissues were blotted on a sterile Whatman No 1 filter paper to drain the excess culture. The calli were then transferred to three co-cultivation media; NB, NB with acetosyringone (100 pM) and NB with nurse cells. These plates were sealed with parafilm and incubated in the dark at in Z5o C for 2 days. The rice calli were then washed twice with timentin (150 mg/l) lx MS macro salts solution (Murashige and Skoog, 1962) and three times with sterile Chapter 3. Materíals And Methods 37 nanopure water. The tissues were then placed on a sterile Whatman No 1 paper to drain all the water. The rice calli were then transferred to NB medium with timentin (150 mg/L) and incubated 250 C in the dark for 2-3 weeks for recovery of embryogenic calli. The experiment was conducted in three replicates with 10 calü in each replicate. Three Agrobacterium strain-plasmid combinations were used to transform three cultivars rice on three co-cultivation media. At all levels of selection and regeneration of transformed calli, the timentin (150 mgil,) was used to avoid contamination fromA grobacterium.
3.3.4.3 Selection of Calli
After three weeks, the rice calli were transferred to selection medium NB-TH50
(Appendix 1) and incubated at 25o C for 2-3 weeks. The calli or segments of calli surviving on the first selection medium were transferred to the fresh selection medium every 2-
3 weeks. The proliferating resistant calli were then transferred to NBPR-TH5O medium (Appendix l)for two weeks in the dark. The calli were then transferred to NBR-TH30 medium (Appendix 1) and incubated at 25o C in the dark for 1 week before transferring them to the light.
3.3.5 Histochemical Staining
For testing the transient expression of GUS activity in the explants after microprojectile
bombardment, one or two pieces of calli or immature embryos were selected randomly for X-gluc staining. After 48 hours of microprojectile bombardment the selected calli were
stained in X-gluc solution (Appendix 2) and incubated at37o C for 24 hours. The calli or
embryos were observed under microscope and the number of blue spots were counted and recorded. In some instances, incubation was carried out for more than 24 hours. In the case of Agrobacterium mediated transformation, small pieces of calli were selected randomly I week after co-cultivation, and stained with X-gluc solution. After incubation at 3lo C for 24 hrs, the calli were observed under microscope and the blue spots were scored. After 8 weeks of selection, small portions of hygromycin resistant calli were stained with
X-gluc, subjected to mild vacuum and incubated at 370 C for 24 hrs. The blue colored calli
were scored to determine the frequency of transformation. Chapter 3. Materials And Methods 38
3.3.6 Molecular Analysis of Potential Transformants
The hygromycin resistant calli were selected randomly to verify the presence of uidA gene. Both GUS expressing calli and the ones that did not show any GUS expression including the control calli were selected for the analysis. Two plasmid DNA's pIGl2lHm and pPCV7\7+Gøs were also used for PCR reaction as positive controls. Approximately 200 mg calli were transferred to 2 ml eppendorf tubes for DNA isolation. The tubes were
dipped in liquid nitrogen before crushing the callus into a fine powder. The finely crushed
calli were homogenised with 600 pl of DNA extraction buffer (Appendix 2) and then 600 pl phenol (25): chloroform(24): isoamyl alcohol (1) was added. After 5 minutes of extraction by gentle shaking, the tubes were centrifuged at 8K for 5 minutes. The clear supernatant was transferred to a fresh tube and the phenol: chloroform extraction step was repeated.
The DNA was precipitated by adding 60 pl NaOAc (pH 4.8) and 600 pl isopropanol. The
tubes were incubated on ice for 15 minutes and then centrifuged at 10K for 8 minutes. The
supernatant was removed and the pellet was washed with'r.}Vo ethanol The pellet was air
dried in a laminar flow hood and furally resuspended in 40 pl of TE buffer with DNAase
free pancreatic RNAase (2Opglmt). The genomic DNA samples were run on a l7o agarose
gels to test the presence of DNA in the solution.
The PCR primers for uidA gene were provided by Dr Aryan and they were, 1. CTG TAG AAA CCC CAA CCC GTG 2. CAT TAC GCT GCG ATG GAT CCC The PCR reaction was set up with total volume o120 pl including the sample DNA. A
master mix (Appendix 2) was prepared for all the reactions and then aliquots of 19 pl was transferred to each tube. All the DNA samples including two plasmids, pIGl2lHm and pPCV7T7+Gus, were then mixed with the reaction mix. The solutions in the tubes were
thoroughly mixed by sucking up and down with the pippetman. Then, 20-25 pl of paraffin
oil was added to avoid any evaporation of reaction mix during the PCR. The PCR program
used in this experiment was as follows; Chapter 3. Materials And Methods 39
Step Temperature 10 C) Time ji 1 94 4 min I ,) 94 1 min
3 55 2 min
4 72 2 min
5 step 2 35 times
6 l2 5 min
1 25 5 min
8 End
J
A 517 bp sized band was expected to be amplified from the uidA gene using the above mentioned primers. The PCR products were analysed on a IVo agarose gels along with low molecular weight DNA markers. The polaroid photograph of the gel was taken under UV transilluminator. rlr ll
r
I Chapter 4
Results
4.I RICE TISSUE CULTURE
Response of both the rice cultivars, 11-n0 and Nipponbate, for callus induction was similar on three media; MS, AA and NB. The callus obtained from scutellum after the second
subculture was yellowish, compact with an embryogenic appearance. Regular subculturing ri of the callus onto fresh medium ensured the maintenance of embryogenic properties of the 1 callus. Callus derived from immature embryos was found to be better than the callus derived from mature embryos. Callus derived from mature embryos of cultivar T-309 was
used for all the experiments. The satellite calli formed on the mother callus that were friable
and compact, were picked to start the suspension cells. Suspension cells were f,rnally plated
on solid medium to obtain uniform sized calli for the experiments'
4.2 MICROPROJECTILE BOMBARDMENT OF RICE
Three cultivars of rice were used in the microprojectile bombardment experiment; cv.7l -170, cv. Nipponbare and cv. T-309. Different explants of rice were used; suspension cells, immature embryos and scutellum derived calli. The explants were treated with the microprojectile bombardment. The I osmoticum 4 hours before and until 16 hours after bombardment parameters were optimised by various trials in the laboratory. Different helium pressures, i.e.,900 psi, 1100 psi, 1500 psi and 1800 psi, were tested using the
suspension cells. The pressure level 1100 psi was selected based on the transient expression ! 40 Chapter 4. Results 4l of GUS in the suspension cells. The distance between the explant and the microprojectile was maintained at 6 cm.
The explant were transferred to the selection medium 7 days after the bombardment. This one week period without any selection was provided to allow the transgenes, to produce sufficient levels of enzymes to counteract the corresponding selective agent. Proliferating calli were transferred to the fresh selection medium every 2 weeks. The untransformed calli turned brown in about 2 weeks on the selection medium. The selected calli were multiplied on the selection medium before transferring them to the regeneration medium' Table 4.1 shows the number of different explants bombarded with three plasmid constructs. The selected calli of cv.7l-llO on NB-H50 containing hygromycin (50 mgtL) after bombarding
with pAct-D and pjíS-Hygro,were grown along with the control calli on the same selection plate. The control calli stopped the growth on the selection medium and turned brown in two weeks, while the selected calli continued to grow normally. No hygromycin resistant calli were obtained after bombardment of explants of cv. Nipponbare and cv. T-309 with
pAct-D and p35S-Hygro. Addition of bialaphos (4 mg/L) to the calli bombarded with pAHC25 failed to kill the untransformed cells and all the calli continued the normal growth.
A number of independently selected calli from cv. 11-ll0 ( 1 1 calli using pAct-D with p35S- Hygro,30 calli using pGll-Gr,¿s with pj\S-Hygro) were transferred to the regeneration medium, NBRH-3g containing hygromycin (30 g/L). The calli did not exhibit any signs of
regeneration on NBRH-3O medium.
H i s t o ch emi c al Analy s i s :
The pieces of explants after the bombardment were selected randomly for histochemical analysis. The X-gluc solution was added to the pieces of calli, embryos or the suspension cells and incubated at 3Jo C overnight. The number of blue spots were counted on each
piece of explant under the stereo microscope. The number of blue spots showing the level of rransient GUS expression indicated the efficiency of DNA delivery. From the Table 4.1, the immature embryos of cv. 71-l7O showed maximum number of blue spots after bombarding with pAct-D (Figure 4.23), followed by the scutellum derived calli and
suspension cells (Figure 4.24). The level of GUS expression also depended on the plasmid
construct used for the microprojectile bombardment. The plasmid pAct-D produced highest Chapter 4. Results 42 transient GUS expression followed by pAHC25, and the pGli-Gus with the least expression. Number of hygromycin resistant calli were tested for the GUS expression before transferring them to the regeneration medium. However, none of the hygromycin resistant calli showed the GUS expression. Both the plasmid constructs pAcrD and pGIïGu.s were co-transformed along withp35S- Hygro, which resulted in the hygromycin resistant calli. Chapter 4. Results 43
Table 4.L Number of different explants of three rice cultivars bombarded with three plasmid constructs and number of blue spots obtained indicating transient GUS expression
Plasmid Calli Immature embryos Suspension cells pActDl 340 90 100 (80 spots) (250 spots) (25 spots) pGli-Gus r82 204 (25 spots) (25 spots) pAHC25 360 (80 spots) Chapter 4. Results 44
4.3 EFFECTS OF TOBACCO NURSE CULTURE
4.3.1 Regeneration of Rice Suspension Cells
Suspension cells were grown on the solid AA medium for two weeks before transferring them onto the filter paper above the nurse cells. The calli clusters were grown on the tobacco nurse cells for 10-14 days. As shown in the Figure 4.1, the calli clusters were uniformly distributed over the filter paper covering the tobacco nurse cells, while the control calli were placed directly on the AA solid medium. After 7 days on the nurse culture, 1 ml of liquid AA,medium was added to the Whatman No. 1 paper to ensure the supply of nutrients to the calli. The calli on the nurse cells appeared more embryogenic and compact than the control calli. The division of the calli continued on both the treatment and the control. Each calli cluster had at least ten calli at the time of transferring to NBR or MS9 medium. The calli cultured with or without nurse cells regenerated into plants on NBR medium, while no regeneration was seen on MS9 medium.
The calli cultured on tobacco nurse cells had typical embryogenic properties with more compact and yellowish appearance. These calli were transferred on to NBPR or AN6 pre-
regeneration media where the visual differences between the nurse cell treated calli and the control calli were observed. These were then transferred to NBR or MS9 regeneration media and placed in the dark for one week before transferring to the light. On NBR medium, the formation of chlorophyll in the minute shoot primordia on the calli grown on the nurse cells was seen as early as 2-3 days in the light, compared to 6-7 days in the control calli. Liquid NBR medium was added once a week to the plates to maintain the regular supply of nutrients to the regenerating calli. The calli continued the multiplication and this resulted in overcrowding on the plates. Therefore, green calli were transferred to the fresh NBR medium every two weeks. The small calli which appeared to be the break offs from the main calli also turned green in the light. The maximum number of green
calli was seen after 4 weeks in the light in both the treatment and the control. However, in the case of calli placed on MS9 medium for regeneration, no further changes were observed. These calli appeared to have the normal yellowish colour, but were drying. After 4 weeks on MS9 medium, the calli started turning brown and eventually died. In the Chapter 4. Results 45 case of cultivar 77 -I10 (Figure 4.2), after 4 weeks in the light, thcre were 84 and 34 green calli in the treatment and control, respectively (Table 4.2). In the case of cultivar Nipponbare (Figure 4.3), the number of green calli was 54 and 17 in the treatment and control, respectively (T able 4.2).
The differentiation of shoot primordia, in both cv. 77 - 170 and cv. Nipponbare, occurred as early as 1 week in the calli cultured on the nurse cells. However, in general the differentiation of shoot primordia in the light occurred after second and fourth week in the treatment and control, respectively. There was not much difference in the regeneration pattern of the two cultivars tested. Both the cultivars passed through the similar stages of regeneration at the same time. However, there was a difference observed in the number of greencalliwhichformedshoots. Inthecaseof cultivarTT-I10, morethan SOVoof green calli gave rise to the shoots in the nurse cell treated calli, while only l37o of green calli gave rise to the shoots in the control (Table 4.2). The remaining calli initially turned brown and later black on NBR medium. In the case of cultivar Nipponbare, more than 70Vo of the green calli gave rise to the shoots in the nurse culture treated calli, while only 20Vo of the green calli gave rise to the shoots in the control (Table 4.2). The remaining green calli turned brown initially and eventually black as in cv. 17-I10.
The overall regeneration frequencies in cv.'sJJ-170 and Nipponbare were 4lVo and3SVo in the nurse cell treated calli, against 57o and 4Vo in the control, respectively (Table 4.2).
The tobacco nurse cells increased the regeneration frequency of these two cultivars of rice by 8-9 times. Although the percentage of green calli eventually forming the shoots was higher in the case of cv. Nipponbare, the overall regeneration frequency was found to be slightly higher in cv. 77 -110, with the use of tobacco nurse cells (Table 4.2). There was not much difference in the overall regeneration frequency of control calli of both the cultivars (Table 4.2).
The effect of tobacco nurse culture was not just on the number of calli that formed the shoots, but it also influenced the number of shoots per callus. Where no nurse culture treatment was provided, mainly single shoots were formed with the control calli. In contrast, multiple shoot formation was coÍìmonly observed on calli cultured with the nurse cells. In the case of cv. 7l-110, the use of tobacco nurse culture resulted in Chapter 4. Results 46
4lVo reseneration, out of which 2Vo had more than 20 shoots per callus, another 27o had more than 5 shoots (Figure 4.4), llTo had 1-5 shoots and 26Vo had single shoot (Table 4.3). In contrast, the regeneration frequency was 5Vo in the control calli, out of which only l%o had 1-5 shoots, while rest of them had only a single shoot. Results were similar in the case of cv. Nipponbare, where tobacco nurse cells resulted in 37Vo regeneration frequency, out of which l7ohad more than 20 shoots, 2Vohad more than 5 shoots, 9Vo had 1-5 shoots while 257o had only single shoot (Table 4.3). Multiple shoot formation was not observed
with the control calli of cv. Nipponbare (Table 4.3).
For root initiation the shoots were transferred to MSO medium which lacks growth hormones. Root initiation was complete in two weeks after transferring the regenerated shoots to MSO medium.' Multiple shoots were maintained separately with the main shoot (Figure 4.5). The plants were then transferred to the soil pots and placed in the growth chamber. Initially the plants were covered with plastic jars to conserve sufficient humidity and to acclimatise the plants to the glass house conditions. The plants were fertile and normal, and no albinos were produced during the process of regeneration. Chapter 4. Results 47
Table 4.2 Plant regeneration frequency (expressed as percentage) from the calli of two cultivars of rice cultured with or without tobacco nurse cells.
Treatment After 3 weeks After 4 weeks After 8 weeks Green calli Green calli Plants 77-170 l3 34 5 Control 77-170 54 84 4l (with Nurse cells) Nipponbare tl t7 4 Control Nipponbare 34 54 37 (with nurse cells) Chapter 4. Results 48
Tabte 4.3 Number of shoots per callus o¡øini¿ (expressed as percentage) during regeneration of rice calli cultured with or without tobacco nurse cells.
Treatment 1 shoot 1-5 >5 >20 shoots shoots shoots 77-710 4 I Control 71-170 26 11 2 ) (with Nurse cells) Nipponbare 4 Control Nipponbare 25 9 2 I (with nurse cells) Chapter 4. Results 49
4.3.2 Agrobøcterium Mediated Transformation
4.3.2.1 Recovery of Embryogenic Catli
Pre induction of Agrobacterium and Co-cultivation: The Agrobacterium cultures were grown in the AAM medium and incubated at 25o C for 12-16 hours. The bacterial culture at density ODeoo = 0.3 was used for co-cultivation with the rice calli. The growth of bacteria on the rice calli was observed after 2 days of co-cultivation. The calli co-cultivated
on the tobacco nurse cells had minimum growth of bacteria, while calli co-cultivated on NB medium (appendix 1) and.NB+As medium (appendix 1) had higher bacterial growth on them. In some of these cases, the entire callus was covered with the bacteria. After co-cultivation of 2 days on different media, the calli were washed with timentin (150 mg/L) in lX MS macro salts solution. Extensive washing was required where the overgrowth of bacteria was observed. The calli were washed 2 times in the timentin solution and 3 times
in sterile nanopure water. These calli were placed on a sterile filter paper to drain all the water. The calli were then placed on the recovery medium NB-T150 (appendix 1) for 2-3 weeks without any selective agent.
Recovery of Embryogenic Properties: The calli placed on the recovery medium NB-T150 (appendix 1) seemed to have undergone shock from the co-cultivation with
Agrobacterium and the washing procedures. The calli appeared dull, shrivelled and soft. However, after one week, the calli co-cultivated on the tobacco nurse cells had already started showing the signs of recovering their embryogenic properties. These calli were compact, yellowish and resuming the normal growth. The calli that were entirely covered
by the Agrobacteriumhad reached point of no recovery. These calli turned brown on the recovery medium and died, while there was no browning observed in any of the calli co-cultivated on the tobacco nurse culture. The recovery of embryogenic calli after co-cultivation with Agrobacteriumwas enhanced by tobacco nurse cells. This is indicated
by the results that are mentioned in the following paragraphs of this sections. Chapter 4. Results 50
The recovery of embryogenic rice calli co-cultivated with AGLI (pIGl2lHm) strain (Figure
4.6) on the tobacco nurse culture was as high as 1,O0Vo in cv.'s 17-110 and Nipponbare, and gOVo incv. T-309, while it was reduced to 87Vo n cv.77-I1O,83Vo in Nipponbare and 5J7o in cv. T-309, when co-cultivated on NB with acetosyringone (100¡t"M) (Table 4.4). The recovery of embryogenic calli after co-cultivation on NB medium was as low as J4Vo in cv. l7-l7o,8OVo n cv. Nipponbare and 43Vo in T-309 (Table 4.4). The overall recovery of embryogenic calli after co-cultivation with LBA4404 (pIG12lHm) was similar (Figure 4.7) to AGL1 (pIG12lHm) in all the three cultivars of rice.
After co-cultivation of rice calti with AGL1 (pPCV707+Gus) strain, the overall recovery of embryogenic calli was reduced particularly in cv. T-309 (Table 4.4). Co-cultivation on tobacco nurse culture resirlted in highest recovery of calli, IO)Vo in cv.'s 7l-I7O and Nipponbare,74Vo in cv. T-309, while it was reduced to 87Vo in cv. l7-LlO,7JVo n
Nipponbare and3TVo in cv. T-309 when cg-cultivated on NB with acetosyringone (I00¡tM) (Table 4.4). The recovery of embryogenic calli was as low as TJVo in cv.'s 7l-l7o and
Nipponbare, and 277o inT-309, when the co-cultivation was carried out on NB medium.
The tobacco nurse culture enhanced the recovery of embryogenic calli fromZTVo to 74Vo n the case of cv. T-309 after co-cultivation with AGL1 (pPCV707+Gus) (Table 4.4). The effect of nurse culture was also observed in cv.'s Nipponbare artd 71-170, but to a less extent compared to cv. T-309. Among the three cultivars, calli of the cv.'s 77-170 and Nipponbare showed higher ability to regain embryogenic properties after co-cultivation compared to cv. T-309. The nurse cells helped the calli to regain their embryogenic properties after co-cultivation in all the three cultivars of rice tested. After 2-3 weeks, the embryogenic calli were transferred to the selection medium NB-TH50 (appendix 1) to screen the potential transformants. 51 Chapter 4. ResuLts
100
90
cÉ I BO e¿ (¡) 70 ä0 >. 60 Ê q) 50
40 Ð, c) 30 (Jo ê) ú 7A o\ 10
0 Control NB NB+As NBfNurse cells
Co-cultivation media
177-l7O trNipponbare IT-309
Figure 4.6 Percentage recovery of embryogenic calli of three cultivars of rice after co-cultivation with AGLI (pIG121Hm) on three different media. Chapter 4. Results 52
100 f/
90 ñ CJ BO CJ É 6l 70 â0 f ¡ 60 I q¿ 50 o >ì 40 ü 30 e) (¡) ú )^ ê\ 10
0 Control NB ¡g+As NB*Nurse cells
Co-cultivation media
a77'fiA trNipponbare IT-309
Figure 4.7 Percentage recovery of embryogenic calli of three cultivars of rice after co-cultivation with LB'L4404 (pIG121Hm) on three different media. Chapter 4. Results 53
Table 4.4 Percentage recovery of embryogenic calli of three rice cultivars after co- cultivation with three Agrobacterium strain-plasmid combinations on three different co-cultivation media
TREATMENT RICE CULTIVARS No Strain Medium 77-170 Nipponbare T-309
1 Control NB 100 100 100 2 AGLI (pPCV707+Gus) 77 77 27 3 AGLI (plG 121 Hm) NB 74 80 43 4 LBA4404 (plG121Hm) 70 73 40 5 AGLI (pPCV707+Gus) 87 77 37 6 AGLI (plG121 Hm) NB+As 87 83 57 7 LBA44O4 (plG121 Hm) 83 77 57 I AGLI (pPCV707+Gus) 100 100 74 I AGLI (plG121 Hm) NB+ 100 100 90 10 LBA44O4 (plG121 Hm) Nurse cells 100 100 90 Chapter 4. Results 54
4.3.2.2 Selection of Hygromycin Resistant Calli
The selection of the putatively transformed rice calli after co-cultivation with Agrobacterium was carried out on the NB-TH50 medium containing hygromycin (50 mg/L). The calli from recovery medium were placed on the selection medium 2-3 weeks after co-cultivation. These calli appeared yellowish, compact and with typical embryogenic properties. The calli were spread all around the selection plate to ensure proper selection. The large calli were broken into small pieces before placing them on the selection medium. The non-transformants started turning brown within 2 weeks on the
selection medium. In the event of transformed cells, small proliferating yellowish calli were observed on the main calli which were turning brown. These proliferating calli were transferred to fresh selection medium every 2-3 weeks. In order to confirm the selection procedure, the control calli were placed on the selection medium as well as on NB medium without hygromycin. The hygromycin resistant calli grew normally with typical embryogenic appearance on the hygromycin selection medium (Figure 4.10), as did the control calli growing on the NB medium without hygromycin (Figure 4.10). However, the control calli on the selection medium turned brown and died (Figure 4.10) within 2-3 weeks. After 8 weeks, a small portion from each independently selected hygromycin
resistant callus was tallen to test for GUS expression. The number of hygromycin resistant calli that showed the GUS expression was recorded to determine the frequency of transformation. Although uniform expression of GUS was detected in most of the hygromycin resistant calli (Figure 4.lI), a few of the calli showed chimaeric
expression (Figure 4.I2).
After 8 weeks of selection, in all the three cultivars of rice, higher number of hygromycin
resistant colonies were obtained in the treatments where co-cultivation was carried out on the tobacco nurse cells. This was followed by co-cultivation treatments on NB medium
with acetosyringone (100 pM). A low frequency of transformation was also obtained when co-cultivation was carried out without tobacco nurse cells or acetosyringone in the co- cultivation medium (Table 4.5). The highest number of hygromycin resistant colonies (93Vo) were obtained in the case of cultivar Nipponbare using AGL1 (pIG121Hm) and co-cultivated on NB medium with tobacco nurse cells (Table 4.5). This was followed by Chapter 4. Results 55 the treatments where co-cultivation was carried out on NB medium with acetosyringone (77Vo) and NB medium (51Vo) (Table 4.5). The lowest number of hygromycin resistant colonies were obtained in the case of cultivar T-309 using LBAMO4(pIG121Hm). In cv. T-309, the transformation frequency was l4%o when co-cultivated on NB rvith tobacco nurse cells,JVo on NB v¡ithacetosyringone and4Vo on NB medium (Table 4.5). However, in the case of cv. T-309, highest hygromycin resistant calli (34Vo) were obtained after co-cultivation with AGL1 (pIG121Hm) on tobacco nurse cells (Table 4.5). This was followed by the treatments where co-cultivation was carried out on NB medium with acetosyringone (34Vo) and NB medium (207o) (Table 4.5). In the case of cv. 'll -I'7 O, the strain LB (pIG 12 l Hm) produced highest hygromycin resistant rice ^4404 calli (SOVo) after co-cultivation on tobacco nurse cells (Table 4.5). This was followed by the treatments co-cultiVated on NB medium with acetosyringone (27Vo) and NB medium (ZOVo) (Tabte 4.5). However, the transformation frequency determined is based on the number of GUS expressing calli, rather than the number of hygromycin
resistant calli.
Histochemical analysis showed that not all the hygromycin resistant calli exhibited the GUS
expression (Figure 4.13, Figure 4.14, Figure 4.t5). Small pieces of calli were taken from
each hygromycin resistant calli and were placed in the wells of the micro titter plates. The X-gluc solution was added to the wells and incubated at 37o C. Some of the calli started turning blue within 15 minutes of addition of X-gluc solution. The blue dye coloration was
also secreted into the incubated solution. In the Figure 4.13 (also Figure 4.14 and Figure
4.15), it can be seen in some of the wells that not only the calli, but also the X-gluc solution is blue colored. The number of blue calli was recorded to determine the transformation frequency. The number of calli showing the GUS expression primarily indicated the
interaction between tl,e rice cultivars and the Agrobacterium strain-plasmid combinations
and the positive effect of tobacco nurse culture.
In the case of cultivar Nipponbare, the strain AGL1 (pIG121Hm) was more effective in transformation on all the three co-cultivation media; NB with tobacco nurse cells (90%), NB with acetosyringone (70Vo) and NB medium (57Vo) (Figure 4.I3). In the case of cultivar 1'7-170, the strain LBA44O4 (pIGl2lHm) produced highest GUS expressing rice calli on all the three co-cultivation media; NB with tobacco nurse cells (5O7o), NB with Chapter 4- Results 56 acetosyringone (27Vo'¡ and NB medium (2OVo) (Figure 4.I4). In the case of cultivar T-309, AGL1 (pIG121Hm) produced highest number of rice calli expressing GUS gene after co-cultivation on tobacco nurse cells (307o) (Figure 4.15). This was followed by treatments co-cultivated on NB medium with acetosyringone (IO7o) (Figure 4.15). In case of cv. T-309, there were no GUS expressing rice calli obtained when co-cultivated on NB medium (Figure 4.15). The hygromycin resistant calli obtained using AGL1 (pPCV707+Gus) did not show GUS expression in any of the three rice cultivars. The strain LBA4404 (pIG121Hm) produced GUS expressing rice calli in the cv. Nipponbare (IOVo) on NB medium with acetosyringone and in cv. T-309 on NB medium. In these two cases neither the nurse culture nor the interaction with the Agrobacterium strain irad any effect.
The hygromycin resistant calli were then transferred to fresh NB-TH5O medium which was solidif,red using 4Vo agarose. Escapees that had survived during the first round of selection died on this fresh medium. The number of rice calli that proliferated on the fresh NB-TH5O medium were counted and used for statistical analysis. Overall, the two way interaction between rice cultivar and Agrobacterium strain-plasmid combination, and co-cultivation
media and Agrobacterium strain-plasmid combination was found to be significant (Table 4.7), while the interaction between the rice cultivar and the co-cultivation medium was non- significant (Table 4.7). The three way interaction between Agrobacterium strain-plasmid combinations, co-cultivation media and rice cultivars was found to be marginally significant
(Table 4.7).
In the interaction between rice cultivar and Agrobacterium strains, AGL1 (pIG12lHm) was
more effective in cv. Nipponbare and cv. T-309, while LBA4404 (pIG12lHm) effective in cv.7l-170 (Table 4.6). However, the hygromycin resistant colonies were also obtained using LBA4404 (pIG121Hm) in cv. Nipponbare and cv. T-309 and using AGL1 (pPCV707+Gus) in all the three rice cultivars, but at low frequency (Table 4.6).
These hygromycin resistant colonies obtained at low frequency have influenced the results,
but no particular interaction has been indicated by them (Table 4.7).
In the case of cv. ''t7-I70, LBA4404 (pIGlZIHm) produced highest transformation frequency on all the three co-cultivation media followed by AGLI (pPCV707+Gus) (Table Chapter 4. Results 57
4.6). The Figure 4.16 shows the effect of tobacco nurse cells in transformation of cv.ll-ll0 when co-cultivated with three different Agrobacterium strain-plasmid combinations. Figure 4.16 clearly shows thatLBA4404 (pIG12lHm) is more effective than other two Agrobacterium strain-plasmid combinations tested. Among the three co- cultivation media, tobacco nurse culture produced highest transformation frequency (50Vo) (Figure 4.17), followed by NB medium with acetosyringone (44Vo) (Table 4.6). However, hygromycin resistant calli were also obtained on NB medium without acetosyringone or tobacco nurse cells, but at comparatively lower frequency (23Vo) (Table 4.6). The Figure 4.16 clea¡ly indicates that tobacco nurse cells have positive effect only when there is effective interaction between the Agrobacterium strain-plasmid combination and the rice cultivar. The tobacco nurse cells had no significant influence when AGL1 (pPCV707+Gus) was used for co-cultivation'in cv.71-llO (Table 4.6).
In the case of cv. Nipponbare, AGL1 (pIG121Hm) produced the highest transformation frequency (Figure 4.19) on all the three co-cultivation media followed by LAB4404 (pIG12lHm). The Figure 4.18 clearly indicates that strain AGLl (pIG12lHm) is more effective in transformation of rice cultivar Nipponbare than the other two Agrobacterium strain-plasmid combinations when co-cultivated on tobacco nurse cells. The co-cultivation of AGL1 (pIG121Hm) on tobacco nurse cells produced highest transformation frequency (94Vo), followed by NB medium with acetosyringone (7OVo) and NB medium (57Vo) (Figure 4.t9). Hygromycin resistant calli were also obtained by co- cultivation of LBA44(uø- (pIG121Hm) on NB wíth acetosyringone (L4Vo) and tobacco nurse culture (4Vo) (Table 4.6). However, with LBA44M (pIGlzlHm) tobacco nurse cells did not enhance the transformation frequency. AGL1 (pPCV707+Gus) also produced hygromycin resistant cdlt (4Vo) after co-cultivation on NB medium wbile acetosyringone or tobacco nurse culture had no effect (Table 4.6).
In the case of cv. T-309, AGL1 (pIG121Hm) produced highest transformation frequency when co-cultivated on tobacco nurse cells (Figure 4.20) compared to other two strains tested. The transformation frequency using AGL1 (pIG12lHm) was higher on NB medium (I7Vo) than on NB medium with acetosyringone (IOVo) (Table 4.5). However, co-cultivation on tobacco nurse cells produced 37Vo transformation frequency using AGL1 (pIG121Hm) (Figure 4.2I). Hygromycin resistant calli were also obtained using Chøpter 4. Results 58
LBA44O4 (pIGl2lHm) on NB medium (4Vo) and AGL1 (pPCV707+Gus) on NB medium
with acetosyringone (1¡Vo) (Table 4.6).
:t, t' In the Agrobacterium mediated transformation, it was observed that interaction between the
rice cultivars and the Agrobacterium strain-plasmid combination is complex. The tobacco
nurse cells had positive effect in transformation of calli of rice cv.'s 77-170, Nipponbare and T-309. However, this positive effect was mainly dependent on the interaction of the rice cultivar and the Agrobacterium sûain-plasmid combination. In the event of positive interaction between rice cultiva¡ and Agrobacterium strain-plasmid combination, the
tobacco nurse cells enhanced the transformation frequency.
tü l Chapter 4. Results 59
100
90
BO ñ (.,) 70
60 O) A) c) 50 a h 40 Èt
Þ0 30 H ,l, 2.O r,l r,li 10
0 NB NB+As NB*Nurse cells Co-cultivation media
I AGL1 (pPCV707+Gu s) trAGL1 (pIGl2rlIrn) I LBA4404 (PIGl2lIIm) obtained Figure 4.8 Percentage of hygromycin resistant calli of cv' Nipponbare using different strains of Agrohacterium on three co-cultivation media'
I I
;
! Chapter 4. Results 60
Table 4.5 Percentage of GUS expressing hygromycin resistant rice calli of three cultivars of rice co-cultivated with three Agrohacterium strain-plasmid combinations on three co-cultivation media.
Agrohøcteríum Co-cultivation 77-170 Nipponbare T-309 strain medium GUS+* Æested* GUS+* /Tested* GUS+ *lTested*
AGLl NB 017 0t20 0lt4 (pPCV707+Gus) NB+As 0tl o/30 0/fl
Nurse cells 0/10 0i30 0/40
AGLl NB ot14 57t57 0/20
(pIG121Hm) NB+As 0/10 70t77 t0t34
Nurse cells 0tfl 90t93 30134 LBAMO4 NB 20t20 0/24 4t4
(pIG121Hm) NB+As 27127 10/30 011
Nurse cells 50/50 0/20 0/14
GUS+ Rice calli showing GUS expression
Tested Hygromycin resistance rice calli tested for GUS expression
I Chapter 4. Results 6T
Table 4.6 Percentage of hygromycin resistant calli of three rice cultivars obtained 16 weeks after co-culti';ation with three Agrobacterium straìn-plasmid combinations on three co-cultivation media.
Agrobacterium Co-cultivation 77-170 Nipponbare T-309 strain medium AGLl NB l0 4 0
(pPCV707+Gus) NB+As T4 0 4
NB+Nurse cells 4 0 0
AGLl NB 0 5l t7
(pIGl21Hm) NB+As 0 70 10
NB+Nurse cells 0 94 37
LB NB 24 0 4 ^4404 (pIG121Hm) NB+As 44 I4 0
NB+Nurse cells 50 4 0 Chapter 4. Results 62
I
i
I i I
Table 4.7 Analysis of Variance
Degrees of Sum of Mean Variance F Freedom Squares Square Ratio ProbabiliW Cultivar. Treatment 4 2.88420 o.72to5 62.t3 <.001 Cultivar. Medium 4 0.03975 0,00994 0.86 o.496 Treatment. Medium 4 0.16568 0.4t42 3.57 o.oI2 Cultivar. Treatment. Medium 8 0.19358 o.02420 2.O9 0.05
Cultivar = Three rice cultivars. Medium = Three co-cultivation media. Treatment = Three A grob act e rium strun-plasmid combinations. Chapter 4. Results 63
4.3.2.3 PCR Analysis of Selected Calli
PCR analysis was performed to test for the presence of uidA gene. The hygromycin resistant calli were selected randomly which included GUS expressing rice calli and also the ones that did not show any GUS expression. Control calli grown on the NB medium were also used for the analysis. Two plasmids pIGl2lHm and pPCV7}7+Gus were used as positive controls for the PCR reaction. The PCR products were analysed on a lVo agarose gel and a GUS fragment of 517 bp was expected to be amplified by the PCR reaction. The two plasmids plGl2lHm and pPCV7}7+Gus gave the expected size band on lVo agarose
gel (lane 2 and 3 of Figure 4.22). All the control calli of three rice cultivars did not show
any band (lane 5,6 and 7 of Figure 4.22).
In the case of cv.ll-llo, the hygromycin resistant rice calli obtained by co-cultivation of AGL1 (pPCV707+Gus) on nurse cells did not show any band (lane 11 of Figure 4.22). This particular calli had not shown any GUS expression (Figure 4.14). The hygromycin
resistant calli obtained by co-cultivation with LB^4404 (pIG12lHm) on tobacco nurse cells
showed the expected sized band (lane 13, 14 and 15 of Figure 4.22) and also exhibited the GUS expression (Figure 4.14). Likewise, the GUS positive transformants (Figure 4.14) obtained using LBA4404 (pIGI2lHm) co-cultivated on NB medium with acetosyringone
(lane 10 of Figure 4.22) and NB medium (lane 9 of Figure 4.22) also showed the expected
sized band. These results confirm the reliability of the histochemical analysis.
In the case of cv. Nipponbare, the hygromycin resistant rice calli obtained by co-cultivation of AGL1 (pIG121Hm) on tobacco nurse cells also gave the expected sized band (\ane22,23 and24 of Figure 4.22) and all these calli exhibited the GUS expression during histochemical analysis (Figure 4.13). The hygromycin resistant and GUS positive calli (Figure 4.13) obtained after co-cultivation of AGL1 (pIG121Hm) on NB medium with
acetosyringone (lane 19 and 20 of Figure 4.22) and on NB medium (lane 18 of Figure 4.22)
also gave the expected sized bands. These results confirmed the positive interaction of AGL1 (pIG121Hm) with the cultivar Nipponbare. The hygromycin resistant and GUS positive calli (Figure 4.L3) obtained after co-cultivation with LBA4a04 @IGI21Hm) also gave the expected sized band (lane 21 of Figure 4.22). However, this Agrobacterium
strain-plasmid combination has only produced a low transformation frequency (Table 4.6). Chapter 4. Results 64
The hygromycin resistant calli obtained by co-cultivation of AGLI (pPCV707+Gus) on NB medium gave the expected sized band (lane 16 of Figure 4.22). However, this particular hygromycin resistant calli had shown any GUS expression during histochemical analysis (Figure 4.I3).
In the case of cv. T-309, the hygromycin resistant rice calli obtained by co-cultivation of AGL1 (pIG121Hm) on tobacco nurse cells (lane 30, 31 and32 of Figure 4.22) and on NB medium with acetosyringone (lane 29 of Figure 4.22) gave the expected sized band. These calli had shown the GUS expression during the histochemical analysis (Figure 4.15). However, the hygromycin resistant calli obtained by the co-cultivation of AGL1 (pIG121Hm) on NB medium showed neither the appropriate band nor the GUS expression (Figure 4.I5). The hygromycin resistant rice calli obtained after co-cultivation of AGL1 (pPCV707+Gus) on NB medium with acetosyringone (lane 28 of Figure 4.22) and on NB medium (lane 25 of Figure 4.22) agun showed neither the expected band nor
any GUS expression (Figure 4.15). The hygromycin resistant and GUS positive rice calli obtained after co-cultivation with LBA4404 (pIG121Hm) on NB medium showed the expected sized band (lane 27 of Figure 4.22). However, this particular Agrobacterium
strain-plasmid combination has only produced a low transformation frequency (Table 4.6).
The results of histochemical analysis were confirmed after conducting the molecular analysis. The hygromycin resistant rice calli that had shown the GUS expression gave the
expected 517 bp sized band. The hygromycin resistant calli that had not shown the GUS
expression during histochemical analysis, did not give any band. However, one exception to this was hygromycin resistant rice calli (cv. Nipponbare) obtained by co-cultivation of AGL1 (pPCV707+Gus) on NB medium. This particular calli had not shown any GUS
expression during the histochemical analysis (Figure 4.13) but gave a clear expected band in
the PCR analysis (lane 16 of Figure 4.22). Lane No. Treatment Cultivar GUS PCR 1 DNA Marker 2 Plasmid pIGl21Hm + 3 Plasmid pPCV707+Gus + 4 5 control calli 7t-t70 6 control calli Nippon 7 control calli T-309 8 AGL1 (pIGl2IHw)-NB 11-nO + +
9 LB 44404 (p I G I 2 I H M ) -l\IB 7t-fl0 + + 10 LB A4404 (p I G I 2 I H M )-NB+As 7l-fl0 + + 11. AGL1 (pP CV707 + Gus)- NB+NTI 71-170 t2 AGL1 (pIG 12 I Hw)-NB+NT1 77-170 13 LB 1'4404 (p I G I 2 I H M )-NB+NTI t7-fl0 + + L4 LB A4 404 (p I G I 2 I H M )-NB+NTI 71-r10 + + 15 LB 44404 (ptG I 2 I HM )-NB+NTI 71-170 + + T6 AGL1 (pP CV707+G¿zs)- NB Nippon + l7 DNA Marker 18 AGLl (pIGl2tHw)-NB Nippon + + 19 AGLI (pIG12LHw)-NB+As Nippon + + 20 AGL1 (pIG12LHw)-NB+As Nippon + + 2t LB A4404 (p I G I 2 I HM )-NB+As Nippon + + J) AGLI (pIG 12 I Hw)-NB+NT1 Nippon + + 23 AGL1 (pIG 12 I Hw)-NB+NT1 Nippon + + 24 AGL1 (pIG 12 I Hw)-NB+NT1 Nippon + + 25 AGL1 (pPCV707+Gus)- NB T-309 26 AGL1 (pIG12LHw)-NB T-309 27 LBA44o4 (pIGI2lHM)-NB T-309 + + 28 AGL1 (pP CV707 + Gus )- NB+As T-309 29 AGL1 (pIGl2lHm)-NB+As T-309 + + 30 AGL1 (pIG 12 I Hw)-NB+NT1 T-309 + + 31 AGL1 (pIG 12 I Hw)-NB+NT1 T-309 + + 32 AGLI (pIG 12 I Hw)-NB+NT1 T-309 + + 66
ßigure 4.22 The PCR analysis of hygromycin resistant rice calli for the presence of uidA gene fragment. 67
(A) Figure 4.1 culture of rice suspension cells (cv. 7147Ð on AA medium without or (B) with tobacco nurse cells. 68
rnedíunr (-a) lFfrgn¡ne 4.2 Regenreraûñopr of'riae caH[Å (cv" 77-lt70] cuåtuned om '4.,4. wf,thou¡t or (ts) wåtË¡ tohacco nurse celås'
rnediurn Fågure 4.3 Regeneration of nice calli (cv. Nipponbare) aultured on ,A.A (A) without or (B) with tohacco nurse cells" 69
Figure 4.4 Plantlet regeneration from rice callus (cv.77-170)' (A) Single shoot formation in rice callus cultured without nurse cetls, (B) Multiple shoot formation in rice callus cultured with nurse cêlls. 7A
tiT 1l - ./ì;.,fr ,.,"'
Figure 4.5 Muttiple shoot formation on rice calli (cv.77-170) cultured on tobacco nurse cells. 71
Figure 4.9 Transient expression of GUS in rice calli (cv. Nipponbare) after one week of co-cultivation with LB^4404 (pIG121Hm) on tobacco nurse cells. E
gi
ii a ¿ìû It t Ò t t" ib Ç. * *'* t ,¡ 1; ¡b
Figure 4.10 Rice callus culture (A) Control calli grown on NB medium (B) Control calli grown on NB-H50 medium, (C) Putatively transformed rice calli {cv.77-170, AGL1 (pIG121Hm)) on NB-H50 medium. 72
Figure 4.11 (A) Complete expression of GUS in the putatively transformed trygromycin resistant rice calti {cv.77-170, AGL1 (pIGl2lIIm)} stained in X-gluc along with (B) control calli.
Figure 4.12 Chimeric expression of GUS activity showing different levels of blue coloration in different sectors of the rice calli. 73
,] !
calli of Fñgure 4.13 GUS expression in putativety transformed hygromycin resistant three Agrobøctevíum sttain- rice 4cv. Nipponhare) obtained aiter co-cultivation with plasmid comhinations on three different media'
Strain Treatment Well Number None Negative control Al l-412 A9-A10 AGL 1 Gl21 -cv.77 -170 Positive control NB A1-44 CI-C4 AGLI þPCV7O7+Gus) NB+As NB+ Nurse Cells F1-F6 NB B1-812 El-El2 AGL1 þIG121ÉIm) ¡g+As Dl-D12, NB+ Nurse Cells Gl-Gl2, Hl-H12 NB A5-47 LBA4404 (pIGl2lHm) NB+As c5-cl2 NB+ Nurse Cells F7-FT2 v4
Ðs
!
!
I I I I I
i I
t
ìFågure 4.14 G{.IS expression in puÉatively tnansforrned hygromycin resistant calli of nice (cv" 77-17t) obtained af,ter co-cuttåvation with three Agrobacteríwm strain- plasrnid comhinations on three different media"
'Well Strain Tneatrnent Numher None Negative control Al l-412 AGL I (pIGl2 lHm)-cv.77-170 Positive control A9-410 NB A1-44 AGL1 þPCV7O7+Gus) NB+As cl-clz NB+ Nurse Cells NB A5-A.7 AGL1 (pIG121Hm) NB+As D1-D6 NB+ Nurse Cells NB B1-87 LBA44A4 þIG121Hm) NB+As El-El1 NB+ Nurse Cells F1-F12, G1-G6 75
€i
^\ r3, t¿ ^
Li
a-
':
of Figure 4.15 GUS expression in putatively Éransformed hygromycin resistant calli Agrobacterium strain-plasmid rice 4cv. T-3t9) obtained after co-cultivation with three combinations on three different media.
Strain Tneatment Well Number None Negative control Al l-412 A9-410 AGL 1 Gl21Hm v.71-170 Positive control NB A1-44 AGL1 (pPCV707+Gus) ¡g+As C1.C5 NB+ Nurse Cells El-E12 NB B1-86 AGL1 (pIG121Hm) NB+As Dl-D11 NB+ Nurse Cells G1-G11 NB A5 LB^4404 (pIG121Hm) NB+As C6-C] NB+ Nurse Cells F1-F4 76
NB 77-170 Nurse Cuf
! ; ? I :i? ct I , -v o a. ¿ t: ê t,+ f' I rù I .t !t I a ç a I ,l . t t t f, ñ à t '"a.- It wgl ': t ¡ ,ìt ó t3 ¡ ç 4 .a r¡ , ,'{t .t
AGt. A6t- LBA pl6 12l- Hm Pcvrþ7 pl6 l2l'llm
Figure 4.16 Selection of putatively transformed calli of rice cultivar 77-170 co-cuitivated with ûhree Agrobacteyiwm slrain-ptasmid aombinatiûns on NB rnediurn with tobacco nurse cells"
77-t7A
a l rBa . ?D Lr¡
a a 'l .o A6L .lrl ,l l2l=llm ,t a Êlr, ,,, ¡+ .,4.. t
l¡ { Ò rÞa l: rl a I .|l a ì Þ pi-\l /D7 - G ¡ t{ '.r r'C t ¡r a ,, gH ffi
Figure 4.L7 Selection of putatively transformed calli of rice cultivar 77-110 co-cultivated on three media with three Agrobacterium strain-plasmid combinations' 't'l
NB N ipPon Nurse Cul
t ( ^ 'lt t t o t l-. a I tt a o a . rD. a ¡ ,a o e., a a Þ' i. I I t. ¡ ¿1 o et 3 è t. { û o û ? ! D' F .b ,{ a a ¡t I a s ) t T aa' t -t a I a ¡t t.b' î. r{. I a 5l .l ,
AGt- A6[- LBA pCV7b7-ct pl6 l2!-llm pl6 12l- llrn
Figune 4"X.8 Selection of putatively transformed aalli of nice cultivan Nípponbare co-cultivated with thnee Agrobøcteri.wwe strain-ptasmid aomhinatitns tn NB medium witËr tobacco nurse cells. @ .Þ I ù t .¡ I ä " e .{ r,:i Þl A 1 ' @ b ?- s
t 4 f It a a , *lr rr a @ a g o¡ o T a
a t D a'1 ôt a ¡ @ 4 T g B m
Figure 4.19 Selection of putatively transformed calli of rice cultivar Nipponbare co-cultivated on three media with three Agrobacteriwm strain-plasmid combinations. 78
NB T-309 NUTSC Cul
! \. rå ¡ a. a å q rl e ú "È" É; t '¿rÒ + T. ta ,3 {l ,t-[' t t :t s I I I o tÒ 0 J t¿ *,
AGL.I A6L.I LBA pcvrÞ'l P l6 l2l-Hm pl6 12l'llm
Figure 4.20 Selection of putatively transformed calli of rice cultivar T-309 co-cultivated with three Agrobøcteríum strain-plasmid combinations on NB medium with tobacco nurse cells.
r'3Õi'
a I a t 4 I Þ ù t t rt 3 ¡rl & a F a t I 'a a ta a
+1 ¡o ta¡ ¡ t ..Ð @ I rJ a
c .Þ I .a \ a à? t tr .l + tiå '. @ t'rtt a .r- 't I t
NE Ar. ffi
Figure 4.21 Selection of putatively transformed calli of rice cultivar T-309 co-cultivated on three media with three Agrobacteríum strain-plasmid combinations. 19
Figure 4.23 Transient expression of GUS in the immature embryos of rice {cv" 77-170) after bombarding with plasmid constructpl cT-Ð.
-l ,l,.l
I
ligure 4.24 Transient expression of GUS in suspension cells of rice (cv.17-170) after bombarding with plasmid construct pACT-D.
þ Chapter 5
Discussion
5.1 MICROPROJECTILE BOMBARDMENT OF RICE
The microprojectile bombardment of rice was done using PDS-1000/He particle gun.
Suspension cells, immature embryos and scutellum derived calli of three cultivars of rice
were bombarded with three different plasmid constructs. The osmoticum treatment of the expression. The |d explant 4 hours before bombardment enhanced the transient GUS j plasmolysis of the cells due to osmoticum treatment is knc'.r,n to reduce the tissue damage by preventing the leakage of protoplasm after the bombardment (Vain et al., 1993). The calli were shot twice, once on each side and the immature embryos were shot four times, twice on each side. There are previous reports that indicate repeated bombardments increase the transformation frequency. There was an increase in the transformation frequency after three successive bombardments of ma\ze suspension cells (Klein et aI', 1988). In case of wheat embryos, double bombardment increased the transient GUS expression, however subsequent bombardments resulted in greater tissue damage with no increase in the transformation frequency (Lonsdale et aL, I99O). In contrast, double bombardments of barley immature embryos and suspension cells was shown to be deleterious (Kartha et a1.,19S9). In this experiment, there was an increase in the transient expression of GUS following multiple bombardments in both rice calli and immature Ì embryos. However, levels of transient gene expression frequently far exceed those of stable expression (Southgate et aI., 1995). In this study levels of transient expression was considered as scale for effîciency of gene delivery. The amount of cell damage after
bombardment of rice calli or immature embryos was not observed. t 80 Chapter 5. Discussion 81
The selection of calli was carried out on NB-H50 medium containing hygromycin (50 mgtL). The hygromycin resistant calli of cv. 11-110 continued normal growth on NB-H50 medium, while contrul calli turned brown, when both were grown on same selection plate. The proliferating rice calli on NBH-50 medium were histochemically analysed for GUS expression. There was no GUS expression recorded in any of the hygromycin resistant rice calli. The plasmid vectors pACT-D and PGli-G'rs were co-transformed with p35S-Hygro. It is therefore, assumed that only p35S- Hygro might be integrated in the rice calli or immature embryos. The frequency of co-transformation of two genes located on different plasmids is less than when both located on the same plasmid (Southgate et a1.,1995). Due to repeated bombardments the cells might have been severely damaged leading to death of the cells. This might have further reduced the frequency of co-transformation. Since no molecular analysis was conducted, the specific reasons for the lack of GUS expression is not known. Further. the hygromycin resistant calli failed to regenerate into plants when transferred to NBRH-30 medium. The reasons for lack of regeneration of calli are not known.
5.2 EFFECTS OF TOBACCO NURSE CULTURE
5.2.1Regeneration of Rice Suspension Cells
Rice suspension cells are not only the best source of protoplasts, but also serve as an excellent explant for optimisation of parameters involved in cereal transformation experiments. In temperate conditions the growth and seed setting in rice is poor. In such situations it is difhcult to have continuous supply of immature embryos for transformation experiments. The initiation and maintenance of embryogenic suspension cells is one of the
better solutions for such problems. However, the regenerability of suspension cells reduces over a time period. The regeneration of plants from explant is an important step in
production of transgenic plants. Low seed set in rice plants and low regeneration frequency in rice suspension cells were the main obstacles at the coÍlmencement of the current transformation experiments. The fîrst experiment was designed to enhance the regeneration
frequency of rice suspension cells prior to using them for transformation experiments. Chapter 5. Discussicn 82
Suspension cells of two cultivars of rice, cv. l7-Il0 and cv. Nipponbare, maintained in AA medium for more than two years, were used in this experiment. The AA medium supports better growth of the rice suspension cells as the main source of nitrogen is amino acids (Toriyama and Hinata, 1985). The suspension cells were plated on the solid AA medium for two weeks before using them for the experiment. This helped in maintenance of uniformity during the selection of calli clusters. The calli clusters were cultured on tobacco nurse cells covered with Whatman no. 1 filter paper, which was pre- wetted with liquid AA medium. Therefore, the calli were not in direct contact with the medium for 14 days.' It appears that this factor might have helped the calli to lose water resulting in partial desiccation. There are reports that regeneration of rice calli (Rance et aI., 1994; Jaln et at., 1996) or suspension cells (Tsukahara and Hirosawa, 1992) is enhanced by partial desiccation. Up to a 50Vo reduction in the water content of the calli has been shown to be beneficial for plant regeneration in rice (Tsukahara and Hirosawa,1992). However, in this experiment, liquid AA medium was
added after 7 days to the calli on the nurse cells. Therefore, the total loss in water content of the calli could not have been substantial. This supports the view that reduction in the water content alone is not enough for enhancement of regeneration frequency
(Tsukahara and Hirosawa, 1992).
Among the two regeneration media tested, the calli on NBR medium regenerated into plants while calli on MS9 medium dried and completely died. The NBR medium has BAP (3 mglL) and NAA (0.5 mg/L), while MS9 has BAP (1 mgll-) and NAA (1 mg/L). The growth regulator BAP is required for the regeneration of plants in rice. Therefore NBR medium with higher BAP concentration might have supported the regeneration of
shoots from the rice calli.
Initial beneficial effects of tobacco nurse cells were observed once the chlorophyll
synthesis had started in the rice calli under the light. Nurse cell treated rice calli, on NBR medium, started turning green within two days after transferring to the light. This was an early indication that rice calli cultured on tobacco nurse cells were more embryogenic than the control calli. The tobacco nurse cells increased the regeneration frequency of these two cultivars of rice by 8-9 times more than the control calli. This increase in regeneration Chapter 5. Discusston 83 frequency could have been much higher if all the green calli had given rise to the shoots. Multiple shoot formation was also observed in the calli cultured on the nurse cells. In both the cultivars, nurse cell treatment gave rise to multiple shoots while control calli mainly formed single shoot.
There are previous reports of use of nurse cells for protoplast culture in rice. In Japonica rice, nurse culture of fast growing rice cells were necessary for induction of protoplast division (Kyozuka et al., 1987). In case of Indica rice, the fast growing Japonica rice cell
lines were used as nurse cells for the protoplast culture (Lee et aI., 1989). It is reported by Kyozuka et al., 1981, that the colonies derived from the rice protoplasts, cultured on the nurse cells, had the typical appearance of embryogenic calli, and that might have had correlation with the high' frequency of plant regeneration. The protoplasts of wheat (Triticum aestivum) formed the colonies with use of nurse cells (Kyozuka et aI., 1987). In this experiment, the nurse culture of fast growing tobacco cells were used, as the fast growing Japonica rice cell lines were involved in the regeneration.
It is well known fact that the tobacco plant is easily manipulated under in vitro conditions
and plants can be regenerated even from the leaf discs. Tobacco cells grow very rapidly in
suspension as well as on solid media. There could be unknown factors released by the fast growing tobacco nurse cells which might have enhanced the plant regeneration in
suspension cells of rice. It is also possible that these factors released by the tobacco nurse
cells might be in the right proportion to enhance the regeneration. These unknown factors
might have played an important role in restoring the embryogenic properties of the rice calli cultured on tobacco nurse cells. It has now been shown that tobacco nurse cells have
benef,rcial effects on the regeneration rice suspension cells and this enables the use of rice
suspension cells in aciual transformation experiments.
5.2.2 Agrobacteríum Mediated Transformation of Rice
In this experiment, the effects of tobacco nurse cells on Agrobacterium mediated transformation of rice were evaluated. Three Agrobacterium stra\n-plasmid combinations were co-cultivated with the calli of three rice cultiva¡s on three co-cultivation media. The Agrobacterium cultures were grown in the AAM medium and incubated at 25o C for Chapter 5. Discussion 84
12-16 hours. During this 12-16 hour period, the bacteria seemed to be acclimatised to the new medium. The bacterial cultures at ODaoo = 0.3 were used for co-cultivation with the rice calli. This procedure of culturing of bacteria in the induction medium fot 12- 16 hours might have a positive effect in enhancing the Agrobacterium infection. The induction medium has acetosyringone (100 pM) which might have induced the vir genes of
Agrobacteriumto the optimum stage in that 12-16 hour period. The results discussed in the following paragraphs would indicate the possible effects of pre-induction of Agrobacterium.
After co-cultivation for 2 days on three different media, the growth of the bacteria was observed. The lower density of bacterial culture seemed to be important in the recovery of embryogenic calli after co-cultivation. The growth of ba.cteria is also dependent on the period of co-cultivation; longer the period, higher the growth of bacteria. The overgrowth of the bacteria during co-cultivation reduces the chances of recovering the embryogenic calli. The calli co-cultivated on the tobacco nurse cells had minimum growth of bacteria, while the calli co-cultivated on NB medium and NB+As medium had higher bacterial growth on them. In some cases, the entire callus was covered with the bacteria. The tobacco nurse cells were covered with the sterile Whatman No. I paper, on which the rice
calli were co-cultivated with the Agrobacterium. The presence of this filter paper might be one of the reasons for less growth of bacteria. In contrast, in the other two media, the co-cultivation was carried out on the medium which gave scope for overgrowth of the Agrobacterium. After co-cultivation of 2 days on different media, the calli were washed with timentin (150 mg/L) in lX MS macro salts solution. Extensive washing was required where the overgrowth of bacteria was observed. The calli were placed on the sterile fìlter paper to drain all the water after washing procedures. It was observed that draining all the water from the calli after washing enhanced the growth and recovery of calli after co- cultivation (visual observations). The calli were then placed on the recovery medium NB-T150 for 2-3 weeks without any selective agent'
The calli placed on the recovery medium NB-T150 (appendix 1) seemed to have undergone shock from the co-cultivation with Agrobacterium and the washing procedures. The calli
appeared dull, shrivelled and soft. However, after one week, the calli co-cultivated on the tobacco nurse cells hid already started showing the signs lf recovering their embryogenic
properties. These calli were compact, yellowish and resuming the growth. The recovery of Chapter 5. Discussion 85 embryogenic calli after co-cultivation wrth Agrobacterium was enhanced by tobacco nurse cells. There was a clear cultivar difference observed in their ability to recover the embryogenic properties of calli after co-cultivation. The cv.'s 17-170 and Nipponbare had higher recovery ability compared to cv. T-309. The nurse cells helped the calli to regain their embryogenic properties after co-cultivation in all the three cultivars of rice tested. It
has been shown in the previous experiment that tobacco nurse cells restore the embryogenic properties of the rice calli. Here in this experiment, the highest recovery of rice calli after co-cultivation with Agrobacterium on tobacco nurse cells supports the results of the previous experiment. The less growth of bacteria on the rice calli might also have been
additional cause for the higher recovery of embryogenic calli. The rice calli were cultured for 2-3 weeks on NB-T150 medium without any selection pressure. This factor might have
helped the rice calli to recover from the adverse effects posed during co-cultivation. This recovery period of 2-3 weeks might also have provided enough time for the T-DNA for stable integration and expression. During this period the transformed cells might have produced enough quantity of enzymes to counteract the selective agents. After 2-3 weeks, the embryogenic calli were transferred to the selection medium NB-TH50 (appendix 1), which contains timenlin (150 mg/L) and hygromycin (50 m!,L).
After 8 weeks of selection on the NB-TH5O medium, the hygromycin resistant calli were tested for the expression of GUS. The GUS expression in hygromycin resistant calli is reliable indicator of gene expression in plant cells because the uidA gene in plasmid pIGt2IHm does not express in the Agrobacterium (Ohta et al., 1991). Therefore, the
number of GUS expressing calli was considered to determine the transformation frequency in this experiment. The GUS expression in the rice calli varied from complete with uniform
expression throughout the calli to chimeric expression. In some calli the expression was
complete and the entire calli turned blue upon addition of X-gluc stain. In case of chimeric
expression, the intensity of blue coloration varied in different segments of the same callus.
However, the presence of uidA gene in GUS expressing calli was also confirmed by PCR
analysis. Based on the number of GUS expressing rice calli, it has been observed that there is an interaction between the plant genotype and the particular Agrobacterium sttain- plasmid combination used. The AGL1 (pIG121Hm) was found to be effective in transforming cv. Nipponbare and cv. T-309, whereas the LBA4aOa (pIG121Hm) was effective on cv. 17-L10. Similarly, differences in the interaction of plant genotype and Chapter 5. Discussion 86
Agrobacterium have been observed with pea (Puonti-Kaerlas et aI., 1989), soybean (Owens and Smigocki, 1988), willow (Vahala et al., 1989) and rice (Raineri et aI., 1991) transfornation. The Agrobacterium strain and host specificity have also been reported for beet (Krens et al., 1988), tomato (Komari, i9S9) and chrysanthemum (van Wordragen
et al., 199 1 ) transformation.
After 8 weeks of selection the hygromycin resistant calli were transferred from NB-THSO
medium solidified with 8Vo agar medium to NB-TH5O medium solidihed w\th 4Vo agarose.
The escapees that survived during the first round of selection turned brown and died, while
the potential transformants continued the normal growth. The uptake of hygromycin by rice calli from nutritional medium might have been enhanced by the use of agarose as solidiffing agent. The number of calli surviving after 16 weeks of selection was counted and used for statistical analysis. Among three rice cultivars, after 16 weeks of selection, the
cv. Nipponbare recorded highest transformation frequency of 94Vo after co-cultivation with AGL1 (pIG121Hm) on tobacco nurse cells followed by treatments co-cultivated on acetosyringone (70Vo) and a low transformation frequency on NB medium (51Vo). In contrast, the cultivar T-309 showed poor transformation frequency of 3OVo on tobacco
nurse cells and !\Vo onNB medium with acetosyringone. The cultivar 77-I70 showed high transformation frequency on tobacco nurse cells (507o) followed by treatments co-cultivated on acetosyringone (44Vo) and a low transformation frequency on NB medium (24Vo)' These results show the difference in response of these three cultivars of rice to Agrobacterium mediated transformation. Similarly, the differences in response of various Indica rice cultivars to Agrobacterium mediated transfbrmation has been observed
(Rashid et a\.,1996). Therefore, it might suggest that cultivar screening for Agrobacterium mediated transformation is crucial before commencing the actual transformation of crop
plants.
Inclusion of acetosyringone in the co-cultivation medium was shown to be essential in the previous studies of rice transformation (Hiei, et al., 1994; Rashid et al., 1996). Transient
expression of GUS was obtained when co-cultivation was done on tobacco feeder cells, while acetosyringone had.no effect in transformation of Moricandia arvensis (Rashid et al., 1996). However, in this current work, cultivars Nipponbare and 71-l7O produced GUS expressing calli even when tobacco nurse cells and acetosyrinSone were omitted from the Chapter 5. Discussion 87 co-cultivation media albeit at a lower frequency. The new procedure of culturing the
Agrobacterium in the induction medium might have been the possible reason for the success of transformation on co-cultivation medium without any phenolic compounds. Previous research on the optimisation of parameters for higher vir gene induction have identihed several important factors. The conditions required for vlr induction are; pH of < 5.1
(Stachel et al., 1985), temperature below 30" C (Stachel et aI., 1985), a carbon source such
as sucrose (Alt-Moerbe et aI., 1988) and addition of phenolic compounds such as acetosyringone. These optimum conditions were provided through AAM induction
medium (Hiei et al., 1994) during the pre-induction. Therefore, with these growing conditions, the vir genes of Agrobacterium might have been higttly activated, thus
eliminating the requirement of vir gene inducers in the co-cultivation medium. However, by the use of acetosyringone or tobacco nurse cells in the co-cultivation medium, the
transformation frequency was increased.
In the current work, co-cultivation on tobacco nurse cells resulted in highest transformation frequency with all the three rice cultivars tested. This enhancement in transformation
frequency was particularly noticeable when there was a positive interaction between the rice
cultivar and the Agrobacterium strain-plasmid combination. The effect of tobacco nurse cells or addition of acetosyringone was not effective when the Agrobacterium strain-
plasmid combination was not appropriate to that rice cultivar. In the case of cv.77-I70, the strain AGLl (pPCV707+Gus) resulted in higher transformation frequency when co-cultivated on NB medium with acetosyringone than on tobacco nurse cells. However, the data showed that LBA4404 (pIGIZlHm) was much more effective in cv.77-l7O and the use of tobacco nurse cells was also beneficial. Therefore, the positive interaction between Agrobacterium strain-plasmid combination and rice cultivar appears to be important for optimising the beneficial effects of tobacco nurse cells. This effect was also observed with interaction of other two rice cultivars, Nipponbare with AGLI (pIG121Hm)
and T-309 with AGL1 (pIG12lHm).
In a study of effect of age of the callus (3 week old and more than 3 months old), Rashid et aI., 1996, reported that former showed the GUS activity, while no expression was detectable in the latter, after co-cultivation with the Agrobacterium. However, in the current experiments, all the calli used were more than 4 months old at the time of co- Chapter 5. Discussion 88 cultivation. Both transient GUS activity one week after co-cultivation and stable GUS expression after 8 weeks on selection medium, were obtained using these calli. However, the age of the calli might have important role in determining the final transformation frequency which is measured by the number of transgenic plants regenerated.
The PCR analysis of the hygromycin resistant calli was conducted to confirm the presence of uidA gene in the GUS expressing rice calli. The randomly picked hygromycin resistant calli for PCR analysis included those that showed GUS expression and also those that did not. The expected sized band of 517 bp was obtained in all the calli that showed the GUS expression and the control plasmids, while no bands were observed in the control calli and the calli that did not show GUS expression. However, the hygromycin resistant calli of cv. Nipponbare obtained by AGL1 (pPCV707+Gus) showed the expected sized band while it did not show any detectable GUS expression. This indicates that tbe uidA gene might be present in the rice calli but not expressed. In this particular case, the reasons for lack of expression of uidA gene in rice calli are not known.
It has been reported that inclusion of acetosyringone in co-cultivation media enhanced gene transfer in Antirrhinum maju.s, soybean (Godwin et aI., 1991), mustard (Hadfi and Bastschauer, 1994), orange (Kaneyoshi et aI., 1994), apple (James et aI., 1993), pea
(Davies et al., 1993) and it was necessary.in rice transformation (Hiei et aI., 1994 ; Rashid et al., 1996). Different amounts of acetosyringone have been used, for instance 200 pM acetosyringone was necessary in Antirrhinum majus and soybean (Godwin et al., 1991),
100 pM in Japonica rice (Hiei et al., 1994) and 50 pM in Indica rice (Rashid et al., 1996). Although addition of acetosyringone in the co-cultivation medium was not essential in the current work, it did increase transformation frequency when it was added. In the current work 100 ¡tly'r acetosyringone was used for both the bacterial pre-induction medium and co-cultivation medium. Among different phenolics tested, acetosyringone and syringaldehyde have been reported to enhance virulence of C58 and 4281 in transformation of Antirrhinum majus (Holford et al., 1992).
Apart from phenolic compounds, the extracts or inducing factors released from dicotyledonous plants or cell cultures have been used for higher vir gene induction. In
number of cases, the suspension cells of tobacco have been used as feeder cells during co- Chapter 5. Discussion 89 cultivation of explant with the Agrobacterium. In the transformation of pea, Lulsdorf et aI., 1991, obtained highest transformation frequency (777o) with the use of tobacco nurse cells during co-cultivation. In the transformation of peanuts, addition of tobacco leaf extract to the bacterial culture enhanced transient GUS expression, while inclusion of acetosyringone did not have any significant effect (Cheng et aI., 1996). Similarly, transient expression of GUS was obtained when co-cultivation was done on tobacco feeder cells, while acetosyringone had no effect in transformation of Moricandia arvensis (Rashid et al., 1996). The tobacco nurse cells enhanced the transformation of Anthurium (Chen and Kuehnle, 1996). The tomato explants pre-cultured on tobacco feeder cells before co-cultivation resulted in increased transformation frequency (Hamzaand Chupeau, 1993). In rice transformation, 3-4 days old seedlings were co-cultivated in petri dishes containing filtrate from potato suspension cultures (Chan et al., L992). The results of this experiment confirm the previous findings about the positive effect of tobacco nurse cells in the Agrobacterium mediated transformation.
These previous reports and the results of the current work indicate that induction of virulence genes is a complex procedure which requires more than acetosyringone alone.
Tobacco is a dicotyledonous crop plant which is readily transformed by the Agrobacterium. Number of different factors required to induce virulence genes of Agrobacterium ate identified including phenolic compounds .released by wounded plant cells. The higher number of GUS expressing rice calli and hygromycin resistant calli obtained using tobacco nurse culture suggests that some factors other than acetosyringone aÍe released by the fast growing tobacco cells that are important for higher induction of Agrobacterium. The unknown factors released by the fast growing tobacco nurse cells may have enhanced the recovery of embryogenic properties and also may have resulted in higher induction of vir genes of Agrobacterium, and these combined effects may have resulted in the observed higher transformation frequency. Chapter 5. Discussion 90
5.3 SUMMARY
Both Agrobacterium mediated transformation and the biolistic approach have been used in this project. The results obtained indicate that the Agrobacterium mediated transformation was effîcient in this study. In this project, the main features of the Agrobacterium mediated transformation were, the ease of handling, low cost equipment and high transformation frequency; while on other hand, involvement of high cost equipment, complex handling procedure and lack of clear results were associated with the biolistic approach. Previous reports and the present studies of successful transformation of rice indicate that the Agrobacterium mediated technique might have the potential to transform the crop plants even outside its natural host range. However, it might be essential to provide special conditions to mimic the natural T-DNA transfer environment.
5.4 FUTURE DIRECTIONS
Various approaches to improve regeneration of rice calli and suspension cells, ie., desiccation of calli, water stress and osmotic stress, have been reported in the past. In this project the regeneration of long term rice suspension cells has been improved using tobacco nurse culture. The elfects of the treatment le., improvement in the regeneration frequency and multiple shoot formation, are known, however the reasons for these effects remain unknown. Future experiments should be conducted to try and characterise these unknown factors released by tobacco nurse cells, and to compare the utility of the nurse culture method with the previously reported water dehydration methods. This technique can be easily tested on crop plants wherever the regeneration frequency has been found to be low.
In the case of Agrobacterium mediated transformation technique, it has been shown that tobacco nurse cells are beneficial in two ways; fîrstly, by enhancing the recovery of embryogenic properties of calü after co-cultivation, and secondly, improving the transformation frequency due to higher vir gene induction of Agrobacterium. In the current
work, the transformation frequency was based on the number of hygromycin resistant calli which showed the GUS expression. However, the experiments should be conducted to Chapter 5. Discussion 9T verify the actual transt'ormation trequency by regenerating the transgenic plants. In principle, the tobacco nurse culture technique can be applied to other crop plants, especially the cereals, which are usually considered to be recalcitrant to Agrobacterium mediated transformation.
The microprojectile bombardment technique has frequently been used in transformation of crop plants where Agrobacterium med\ated approach is still not possible. However, the transformation frequency in most reports is low and was nonexistent in the current project.
The effects of repeated bombardments on frequency of transformation should be thoroughly studied. In the future experiments it is suggested that a single plasmid containing the required genes be used for transformation. The tobacco nurse culture technique can be used for regeneration of potentially transformed calli. It seems that further refinements in the methodology will be required before application of this technique to cereals is routine.
The results and procedures described in this project have mainly been concerned to the transformation of rice (Oryza sativa). It is to be hoped that the results reported in this thesis will be of immediate benefit to future workers in the field. 92
Appendix 1.
Nutritional Media for Plant Tissue Culture
AA ANE NB NBPR NBR MS MSS CSV (mg/L) (múL) (me/L) (me/L) (me/L) (mg/L) (mg/L) MACROSALTS NH¿N0¡ I 650 1650 1240 KNO¡ 2830 2830 2830 2830 1900 1900 255 (NH¿)zSO+ 463 463 463 463 (NFI¿)zPO¿ 460 KHzPO¿ t70 400 400 400 400 t70 170 MgSO¿.7HzO 370 185 185 185 r85 370 370 400 CaClz.2HzO 440 166 166 r66 166 440 MO 200 KCI 2940 MICROSALTS MnSO¿.4HzO 22.3 l0 l0 l0 10 16.9 16.9 l3 H¡BOs 6.2 10 3 3 3 6.2 6.2 5 ZnSO¿.7HzO 8.6 10 2 2 2 8.6 8.6 I KI 0.83 0.8 0.7 0.7 0.7 0.8 0.8 I Na2MoO4.2H2O 0.25 0.025 0.25 0.25 0.25 0.025 0.025 0.1 CuSO¿.5HzO 0.025 0.025 0.025 0.025 0.025 0.025 0.025 o.2 CoClz.6HzO 0.025 0.025 0.025 0.025 0.1 IRON (mg/L) 0.025 28 28 28 NA2EDTA 3'7.3 5t.3 37.3 3'7.3 5 t.) 37.3 J /.J 37.3 FeSO¿.7HzO 27.8 27.8 27.8 27.8 27.8 27.8 27.8 27.8 VITAMINS Ascorbic acid Biotin Ca-pantothenate Choline chloride Folic acid 0.4 myo-lnositol 100 100 100 100 100 100 100 1000 Nicotinic acid 0.5 5 0.5 0.5 5 p-Aminobenzoic acid Pyridoxine-HCl 0.1 l 0.5 0.5 05 Riboflavin Thiamine-HCl 0.5 5 10 l0 10 0.1 0.1 5 Glycine 75 2 2 AMINO ACIDS Glutamine 87'l 500 500 500 Argenine 228 Proline 500 500 500 Aspartic acid 266 Asparagine OTHERS Casein hydrolysate r00 400 300 300 300 Sucrose 20,000 20,000 30,000 30,000 30,000 30,000 30,000 30,000 Coconut water 20 ml 20 ml 20 ml 20 ml Maltose Glucose 5,000 Mannitol 5,000 2,4-D 2 I 2 2 2 BAP 0.5 z 3 ABA 5 NAA 0.5 I 05 tAA 0.5 Kinetin 0.2 0.5 93
SELECTION MEDIUM
Selection Medium Basal Medium Antibiotics (mg/L)
NB-H5O NBz Hygromycin 50 NB-T150 NBz Timentin 150 NB-TH5O NBz Hygromycin 50 Timentin 150
NBPR-H5O NBPR Hygromycin 50
NBPR-THsO NBPR Hygromycin 50 Timentin 150
NBR-H3O NBR Hygromycin 30
NBR-TH3O NBR Hygromycin 30 Timentin 150
MSO-H5O MSO Hygromycin 50
MSO-TH5O MSO Hygromycin 50 Timentin 150 94
Appendix 2
Bacterial Medium, Reagents and Solutions.
LB Medium (Sambrooket a1.,1989) Bacto Tryptone 10 gms/L Yeast Extract 5 gmsll NaCl 10 gms/L
Adjust to pH = 7.0 with 5N NaOH
YEP Medium
Bacto Peptone 1.0 gms/L
Yeast Extract 1.0 gmsll
NaCl 0.5 gms/L pH=7.5
TCM Buffer
Tris HCI 10 mM
MgCl2 10 mM
CaCl2 10 mM
DNA Extraction Buffer (Sambrook et aI., 1989) Tris HCI 100 mM NaCl 100 mM
EDTA 10 mM
Sarcosyl 0.3%o pH = 8.5 95
TE Buffer (pH = 8.0) (Sambrook ¿f aL,1989) Tris HCI (pH 8.0) 10 mM EDTA (pH 8.0) 1mM
PCR Master Mix
10X Reaction Buffer 2.0 ¡t"l
50 mM MgCl2 0.6 pl
10 mM dNTP's 0.4 pl
PrimerMix 0.8 pl
Nanopure H2O 15.1 ¡rl Taq polymerase (SUnits/pl) 0.1 pl
X-Gluc Solution
X-gluc stock solution (5-bromo-4-chloro-3-indolyl-B-D-glucuronide)
50pg X-gluc (PROGEN)
5 Opl N,N'-Dimethylformamide (Aj ax Chemicals)
Stored at -20"C
X-gluc buffer (modified from McCabe et aI., 1988, and Mendel et aI., 198e)
50mM NaPO4 (pH 7.0)
10mM NazEDTA 0.5mM IÇFeCNe 0.5mM K:FeCNe
l%o vlv Triton X-100
X-gluc staining solution (modified from McCabe et al., 1988, and Mendel et al., 1989)
20ml X-gluc buffer
200p1X-gluc stock
Filter sterilise through 0.2pm ltlter
Stored at -20"C 96
Appendix 3 Plasmid Maps
Appendix 3 a
Smal Bgl lEcoRl Pstl Pvull Pstl Sstll 35S Pstl Pvull hptll Sphl Hindlll nos EcoRV dual35S EcoRl LB Xhol Kpnl Ncol pPCV707*Gus Sstll uidA
Xbal
35S poly A Xbal BamHl Pstl pN BstEll Sphl Hindlll Sstll Pvull Aphl Pstl EcoRV Pvull Sphl Sphl 97
Appendix 3 b
Smal EcoRl Bglll EcoRl
Pstl Pvull Pstl 3ss Sstll hptll
Pvull Hindlll nos
Pvull
pPCV707 Sstll LB pNOS Kpnl Pstl
Sstll nptll Pvull
Sphl
Xbal BstEll RB Aphl Sphl 98
Appendix 3 c
Sphl Psrl Pvull H¡ncll EcoRV
EcoRl l5s
Ncol
TL
on
pRTL2Q-GUS
GUS
Sæl
35S polyA Xb¿l BåmHl
Pstl Pvull Sphl Hnd 99
Appendix 3 d
Bam Hl LB nos Pstl EcoRl hptll
3ss Sal I Sst I nos pIGl21Hm 'I -d
,t! I intron-GUS
Sna I
Sal I Xbal Bam Hl nos nplll R Hindlll 35S Pstl phl Sphl Pstl
! 100
tx e 'il Appendix 3 I
Nael mnl Sspl Sspl scä Pvull Pvul Kpnl f1 (-)ori EcoRl
I LacZ
Amp 35S
pBS-3SS Hygro-nos
EcoRl
ColEl ori hptll
Pvull Sacl Hindlll
I i
! I !.
I'
I
t
I Appendix 3 f
pAHC25 Hindlll(,) (+) Ubl-Gus Spht Ubf-Bar Psrl(r) Sal I (60ú) +1
B0r ll (qtq) bla Promolol Eco Rr (trot)_ Eton Pstl(t 111) PUæ lrlmn Sall (æos) Xbal '(zorr) BamHr(zøt+) Smal ( zoz:J (tosc)eco nt r¡os 3' (c7re)eert 6rio) satt GUS 5r5l)Bgl ll BAR NOs s' [66sr) spnt $all (6\'t e) Exon i6ert) sPnt Promoler (6t1r) Bamttt (arts)xtat' Eco Sact (:9 ro) Eco Rl Crtcr) (ern)satt Sal (ql1)) H¡ndlll(q\?5) Pstl Bglll (*ttt) (6n3) (stzt) Sphl +l Psil (qr81)
Adclirional XbaI sitcs in plasnúd at: 45. 1015, 1390. lól 8. ¿219. 5209. 5564. 5192 Sornc a¡c subjcct to dam.methylaûon. t02
Appendix 3 g
Xmnl Sspl Nael Scal Pvul Pvull Pvul Kpnl f1c) ongln LacZ Ampicillin Nos Eco R1
ColEl pBS-Glia-2-GUS ongrn
Lacl GUS Pvull Gliadin Sacl Hind lll Xbal Xbal Spel
Ncol Sma I
Sma I Bam Hl
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