Chapter 1. Introduction

Transcription

1 Chapter 1 Introduction 1.1 Rice & Canola Rice (Oryza sativa) is one of the world s most important food crops, forming part of the staple diet of approximately half the world s population (Upadhyaya et al., 2000b). As the world s population increases global shortages in rice are predicted, such that a 60% increase in rice production is needed by the year 2025 simply to sustain demand (Upadhyaya et al., 2000b). Australia produces some of the highest rice yield in the world. O. sativa has a number of pests, which cause an estimated loss of 10 million tonnes of rice a year (Cheng et al., 1998). Genetic engineering offers a way of improving rice yields by introducing novel agronomic traits such as cold tolerance, resistance to pathogens and herbicides (Cheng et al., 1997). Another crop, which is becoming increasingly important to the Australian economy, is canola (Brassica napus L). B. napus is cultivated chiefly for the production of oil. Brassica sp., supply more than 13 % of the world s edible oils and rank third behind soybean and oil palm (Stewart et al., 1996). The term canola has come to describe Brassica cultivars that produce oils with less than 2% erucic acid and defatted seed meal containing less than 30mmol/g of aliphatic glucosinolates (Stewart et al., 1996; De Block et al., 1989). Glucosinolates act as non-specific antiherbivorants in plants (Benrey et al., 1998), and because of the toxicological effects of their breakdown products they are undesirable for human consumption. The reduction of these compounds in B. napus through traditional breeding has led to an increase in susceptibility to insect pathogens (Stewart et al., 1996; Benrey et al., 1998). Methods for genetically introducing traits such as pest resistance in B. napus and O. sativa are immensely valuable to industry. A number of methods for introducing genes into plants already exist. These protocols include; particle bombardment (Valdez et al., 1998), Agrobacterium mediated transformation (De la Riva et al., 1998), direct DNA 1

2 uptake (Yoo and Jung, 1995), microinjection (Escudero et al., 1996), electroporation and PEG transformation of protoplasts (Upadhyaya et al., 2000b). Most methods require transformed cells to be regenerated into whole plants, using tissue culture. Differences in regenerative ability between cultivars and even tissue types mean tissue culture procedures may not have been developed for a particular plant (Azriah and Ballah, 2000). It can take many months to regenerate cells into whole plants using tissue culture. This procedure can introduce unwanted mutations into plants through a process called somaclonal variation (Bent, 2000). O. sativa genotypes vary enormously in their regenerative response (Azria and Bhalla, 2000). B. napus has greater regenerative potential, however, it is still genotype dependent (Zhang et al., 1999; Kazan et al., 1997; Valdez et al., 1998). Transformation methods not requiring tissue culture are appealing as they are relatively inexpensive and require a low level of expertise (Bent, 2000). This is important for researchers with limited resources such as those in third world countries. This dissertation describes attempts to improve a transformation method that avoids the use of tissue culture. The method is based upon the Agrobacterium tumefaciens (A. tumefaciens) mediated seed transformation protocol, first developed by Feldmann and Marks (1987). The feasibility of Agrobacterium mediated seed transformation of B. napus and O. sativa was evaluated and attempts were made to improve the efficiency of this technique. 1.2 Plant Transformation methods Plant transformation the act of transferring foreign DNA into the genome of plants through genetic engineering techniques (Azria and Bhalla, 2000). Transformation is currently used for genetic manipulation of more than 120 species of plants, including many major economic crops (De la Riva et al., 1998; Birch 1997). Plant transformation can be divided into 2 categories, based on the method of DNA transfer. 2

3 1.2.1 Direct transformation methods Direct methods for transformation are the most commonly used techniques for the improvement and manipulation of crop species. There are a number of direct methods, which have been used to transform both B. napus and O. sativa. These include electroporation or PEG transformation of protoplasts, microinjection and microprojectile bombardment (biolistics) (Upadhyaya et al., 2000b; Upadhyaya et al., 2000a; Valdez et al., 1998; Yoo and Jung, 1995). Most forms of direct transformation rely on tissue culture methods for the production of whole transgenic plants. This is both a time consuming and expensive process, requiring both sterile conditions and controlled environments for plant growth (Birch, 1997). Transformation of O. sativa was first achieved using PEG/ electroporation mediated protoplast transformation (Brisibe et al., 2000). Protoplast transformation is inefficient and is limited by the number of tissue types that are capable of regeneration from protoplasts (Khanna and Raina, 1999; Yoo and Jung, 1995). Variations amongst plant cultivars require regeneration techniques to be refined for each plant type. Regeneration from protoplasts can also lead to tissue culture problems such as somaclonal variation (discussed Section 1.4). Despite this, 27 cultivars of O. sativa have been transformed via this method and it has paved the way for optimisation of many important parameters affecting plant transformation and transgene expression such as promoter sequences, selectable markers, reporter genes and selective agents (Upadhyaya et al., 2000b; Birch,1997). Microprojectile mediated gene delivery (biolistics) has largely overcome problems inherent in other direct methods such as protoplast methods. More than 40 cultivars of O. sativa have been transformed using biolistic transformation methods. (Upadhyaya et al., 2000b; Luthra et al.,1997). A number of regenerable tissue types can be used for transformation using the biolistic method such as immature embryos, scutellar tissue and mature embryos (Khanna and Raina et al., 1999; Valdez et al., 1998). Unfortunately, gene delivery by particle bombardment often leads to multiple copies of transgenes at one locus and these transgenes are often fragmented and rearranged (Hansen and Chilton, 1996; Hansen et al., 1997; Kohli et al., 1998). Multiple copies of transgenes can lead to their suppression due to epigenetic mechanisms such as DNA methylation (Bent, 2000; 3

4 Hansen et al., 1997; Kohli et al., 1998). Homologous recombination may also cause problems creating genetic instability of multiple copies (Hansen and Chilton, 1996; Bent, 2000). The bombardment of plant cells with particles is destructive. It causes a lot of tissue damage and is relatively inefficient. There is a common misconception that A. tumefaciens mediated transformation is not possible with monocotyledonous plants. However, Zhang (1999) and Khanna and Raina (1999) reported A. tumefaciens mediated transformation of O. sativa, eliminating the need for direct transformation methods Agrobacterium tumefaciens mediated transformation A. tumefaciens is a soil phytopathogen that naturally infects the wound sites of dicotyledonous plants (De la Riva et al., 1998; Sheng and Citovsky,1996). It has the ability to transfer DNA from itself to plant cells. Crown gall disease is caused by A. tumefaciens. In the case of crown gall disease, the T-DNA contains oncogenes, which on integration into the plant genome cause tumour growth and proliferation. The transferred T-DNA also contains opine synthesis genes, which cause transformed cells to secrete opines which A. tumefaciens can use as an energy source (De la Riva et al., 1998). The T-DNA is derived from a plasmid within the A. tumefaciens cell. These tumour inducting (Ti) plasmids are circular molecules of DNA that replicate independently of the chromosome. Only a specific portion of the plasmid (the T-DNA) is transferred to the plant. Manipulation of the T-DNA to remove oncogenes has enabled transformation of plant cells without tumour induction (Jones, 1995). Any foreign DNA placed between the T-DNA borders can be transferred to create new traits in plants (Cheng et al., 1998; Azriah and Bhalla, 2000). A. tumefaciens is grown together with the target cells (cocultivated) to begin the transformation process. The A. tumefaciens transformation process can be split up into 5 stages. They are 1) Agrobacterium adherence to the plant cell; 2) Induction of the Agrobacterium virulence region; 3) Generation of the T-DNA; 4) Transfer of the T-DNA to the plant cell and 5) Integration of the T-DNA into the plant genome (De la Riva et al., 4

5 1998; Kohli et al., 1998). These steps are essential to the A. tumefaciens mediated transformation process (De la Riva et al., 1998; Escudero et al., 1996). Figure 1.1. A. tumefaciens transferring T-DNA to a plant cell. Adherence is the first step in the transformation process. Mutant A. tumefaciens incapable of attaching to plant cells were shown to be incapable of transformation (De la Riva et al., 1998), by increasing A. tumefaciens ability to adhere to plant tissue transformation can be enhanced. This may be achieved using such surfactants as silwet L-77 (Clough and Bent, 1998). 2) The virulence genes ( vir genes) in A. tumefaciens are essential for initiation of the T- DNA production process. The vir genes are located both on the chromosomal DNA of A. tumefaciens and on some plasmids. They are responsible for the processing, transfer and integration of the T-DNA (Nan et al., 1997; Chateau et al., 2000; Raineri et al., 1993). 5

6 Vir genes monitor the environment for signs of cell damage, signs are often components of the plant cell wall such as phenolic compounds. On contacting such stimuli the vir genes facilitate transfer of DNA. Induction of the vir region can be achieved artificially using tobacco extract, phenolic compounds such as acetosyringone or some sugars common in dicotyledonous cell (Raineri et al 1990; Orlikowska et al., 1995; Winans et al, 1988). The last 3 steps in the A. tumefaciens transformation process are difficult to manipulate and are unaltered during transformation. Success in obtaining transgenic plants depends on the susceptibility of target cells to A. tumefaciens (Chateau et al., 2000). O. sativa like many other important crop species such as wheat, corn and barley is monocotyledonous and as such is not a natural host for Agrobacterium tumefaciens (Khanna and Raina, 1999; Upadhyaya et al., 2000a). A. tumefaciens mediated gene transfer to rice was thought not to be possible due to the initial difficulty in transforming this species. The use of appropriate target tissue and conditions have contributed to successful transformation (Upadhyaya et al., 2000a; Escudero et al., 1996). A. tumefaciens transformation has been the most widely used method of gene transfer in B. napus (Zhang et al., 1999). The efficiency of Agrobacterium tumefaciens-mediated transformation technique in B. napus is influenced by cultivar specificity, donor plant age and explant type (Zhang et al., 1999). One problem of A. tumefaciens transformation is that the T-DNA is randomly inserted into the plant genome. This can disrupt genes and may be detrimental to the plant (Bent, 2000; McNevin et al., 1993). Benefits of the A. tumefaciens transformation system include discrete, low copy number, unrearranged DNA insertions (Upadhyaya et al., 2000a; Upadhyaya et al., 2000b; Bent, 2000). The A. tumefaciens transformation method is efficient and can transfer relatively large segments of DNA into the plant genome compared to direct transformation methods (Elliot et al., 1998). In general transgene expression is greater with A. tumefaciens mediated transformation then with direct methods (Elliot et al., 1998). A. tumefaciens mediated transformation results in fewer mosaic plants than are observed through direct transformation systems (De la Riva et al., 1998; Hansen et al., 1997). A. tumefaciens generally has a higher transformation frequency than direct transformation methods, does not require expensive transformation 6

7 equipment and has a more predictable pattern of foreign DNA integration than other methods (Rainer et al., 1990). The lower copy number leads to fewer problems since multiple copies of transgenes can lead to instability of their expression. Epigenetic mechanisms such as gene silencing, tend to inactivate multiple copies of transgenes and homologous recombination may cause genetic instability of multiple copies (Hansen and Chilton, 1996). 1.3 Factors affecting A. tumefaciens mediated transformation. As mentioned previously the ability of A. tumefaciens to adhere to plant cells is vital to the transformation process. Gaining access to cells is important so that A. tumefaciens can adhere to cells and transfer T-DNA. Induction of the vir region is also important, beginning the T-DNA production process. Agrobacterium strain, plasmid used, tissue culture medium, cell density and plant genotype have also been found to influence transformation by A. tumefaciens (Amoah et al., 2000) Agrobacterium strain Many Agrobacterium strains differ in their ability to transform plant cells (Klee et al., 1987). This is in part because of the different virulence regions present on the Agrobacterium chromosome. Genes for cell adherence are also important in controlling the virulence of Agrobacterium strains. Alterations of the genes such as the vir genes have helped develop super virulent Agrobacterium strains. Many strains of Agrobacterium have been used as delivery systems Virulence induction Improvement of the A. tumefaciens system involves the stimulation of the vir genes. Phenolic compounds that are released by wounded dicotyledonous plants stimulate the vir region (Khanna and Raina, 1999). A. tumefaciens recognises such compounds and starts T-DNA synthesis in response to them. Wounded monocotyledonous plants produce 7

8 different compounds to dicotyledonous plants. In most monocotyledonous plants the chemicals released on injury are not recognised by A. tumefaciens, therefore artificial induction of the vir region is required. One phenolic compound, commonly used for this purpose, is acetosyringone (Nan et al., 1997; Winans et al., 1988; Pavingerova and Ond_ej, 1995). Induction of the vir genes prior to cocultivation increases the transformation efficiency of many A. tumefaciens strains in monocotyledonous and dicotyledonous plants (Khanna and Raina, 1999) Vectors There are 2 types of vectors used in A. tumefaciens mediated transformation systems, cointegrate vectors and binary vectors. Binary vectors are plasmids that contain origins of replication from a broad host-range plasmid (Warden, 1988). These replication origins allow autonomous replication of the vector in A. tumefaciens. Binary vectors can be used in almost all A. tumefaciens species as long as there are vir helper genes provided to aid the generation and transfer of the T-DNA (Klee et al., 1987; Walden, 1988; Potrykus, 1991). Often helper plasmids are used to induce the binary plasmid to generate and transfer T-DNA. Helper plasmids have no T-DNA, but contain virulence genes to induce T-DNA generation and transfer in the binary plasmid. Larger DNA segments can be transferred using binary plasmids as compared to cointegrative vectors. Cointegrate vectors contain the T-DNA on the same plasmid as the vir genes. These vectors are constructed by recombining a disarmed Agrobacterium and a small vector plasmid, which is engineered to carry a gene of interest between a right and a left T-DNA borders of the T-DNA region (Babaoglu et al, 2000). Recombination takes place through a single crossover event in a homologous region present in both plasmids. A problem encountered with cointegrate vectors is their large size, which makes their manipulation in the laboratory difficult (Babaoglu et al, 2000). Consequently, the binary vector system is the system of choice, as it allows the small plasmid to be introduced and amplified in Escherichia coli and then later transferred into Agrobacterium for plant transformation (Babaoglu et al, 2000). 8

9 The natural virulence of A. tumefaciens varies from strain to strain. The virulence of some strains can be increased by the introduction of a supervirulent plasmid, such as ptok47, which carries extra copies of some of the virulence genes (Babaoglu et al, 2000). Super binary vectors, in which extra copies of virulence genes are on the binary vector itself, have proven useful in the transformation of cereals, as in the case of rice (Hiei et al., 1997). Certain disarmed strains of A. tumefaciens have been used extensively for several years to carry binary and super binary vectors, an example being LBA4404 (Hoekema et al., 1983) Promoters Promoters control the initiation of transcription, influencing both the level of gene expression (Upadhyaya et al., 2000b). The use of appropriate promoters for transgene expression is important for successful transformation and transgene expression (Upadhyaya et al., 2000b; Upadhyaya et al., 2000a). In this study the cauliflower mosaic virus 35S is the only promoter used (CaMV35S). The promoter is most active in the genus Brassica (Walden, 1988), but it has been shown to be active in a wide range of tissues and in many crop species including O. sativa (Wilkinson et al., 1997). Various versions of the CaMV35S promoter display great variation in activity between independently transformed lines and between different plant species (Wilkinson et al., 1997) Selective markers There are a number of genes, which confer resistance to a range of antibiotics and herbicides. These genes have been used in plant transformation protocols to select for transformed cells. Genes such as the neomycin phosphotransferase (nptii) and hygromycin phosphotransferase (hptii), confer resistance to kanamycin and hygromycin respectively. Once incorporated into the plant cell genome, transformed plant cells are capable of growing on selective media. This is extremely useful in separating 9

10 untransformed plants (which would otherwise perish or show severe growth retardation on selective media) from transformed ones Reporter genes Reporter genes give transformed cells unique abilities, which allow them to be distinguished from non-transformed cells. The b-glucuronidase (GUS) gene has been used extensively as a reporter for gene expression in plants (Haseloff et al., 1997; Jefferson, 1987). The GUS system is sensitive and shows low background endogenous activity. However biochemical tests for its presence are generally destructive, due to the cytotoxic substrate used to detect its expression. The substrate used for its detection is 5- bromo-4-chloro-3-indolyl glucuronide (X-gluc). The product produced by glucuronidase action on X-Gluc is not coloured. Instead, the compound must undergo an oxidative dimerization to form the insoluble and highly coloured indigo dye. This dimerization is stimulated by atmospheric oxygen, and can be enhanced by using an oxidation catalyst such as a potassium ferricyanide/ferrocyanide mixture (Jefferson, 1987). The green fluorescent protein (GFP) is an alternative reporter gene to GUS. It is derived from the jellyfish Aequorea victoria and can be visualised with the use of a hand held UV lamp. It has been used extensively as a non-destructive scorable marker gene for plant transformation and can be detected without the need for invasive techniques or the addition of cofactors (Haseloff et al., 1997). The continual improvements to this gene ensure it will gain widespread use throughout the scientific community Attachment To transfer T-DNA, A. tumefaciens must first adhere to the plant cell. In some transformation systems adding a surfactant to transformation solution enhances adherence of A. tumefaciens to plant cells. Surfactants work by lowering the surface tension. A popular surfactant is Silwett L-77, which is an organosilicane compound of low phytotoxicity (Richardson et al., 1998; Bechtold et al., 2000). 10

11 1.3.8 Wounding of plant tissue The wounding of plant tissue is a critical step in A. tumefaciens mediated transformation as it allows the bacteria greater access to host cells. Vacuum infiltration is one way of achieving this (Bent, 2000). Vacuum infiltration involves immersing plants in A. tumefaciens suspension and applying a vacuum, causing air pockets within the tissue to be sucked out. On release of the vacuum the negative pressure forces the bacteria into the cells thus increasing the susceptibility of the host. The vacuum infiltration also stimulates the plants wound response, which may increase the virulence of the Agrobacterium (Clough and Bent, 1998; Bent, 2000). Liquid nitrogen treatment of tissue can cause small fissures and channels in cells due to damage caused by intracellular and extracellular ice formation. In theory this should allow Agrobacterium greater access to the cells within the seed. Extracellular masses of ice can damage the structure of organs therefore freezing with liquid nitrogen could decrease the seed viability (Gonzàlez-Benito et al., 1998). 1.4 Plant Tissue Culture Almost all transformation methods involve the use of plant tissue culture. Plant tissue culture involves the propagation of plant cells in vitro, with the purpose of regenerating individual cells into mature plants. Plants that are regenerated from single cells are homogeneous for the engineered trait ie. the gene of interest is present in every single cell within the resulting plant (Bent, 2000). Plant regeneration is influenced by many factors, including culture environment, culture medium composition, explant source, and genotype (Azria and Bhalla, 2000). There are a number of problems associated with plant tissue culture. Many agronomically valuable genotypes are not easy to manipulate in vitro because of their poor regenerative ability (Azria and Bhalla, 2000). O. sativa varies enormously in its regenerative response even amongst individual genotypes (Azria and Bhalla, 2000, Upadhyaya et al., 2000a, 11

12 Valdez et al., 1998). Plant tissue culture is labour-intensive involving continual cultural maintenance and strict adherence to aseptic protocols. It is also time consuming to regenerate a whole plant from a single cell (Ye et al., 1999; Bent, 2000). During plant tissue culture a number of mutations may occur ranging from single base changes, small rearrangements or even the loss of entire chromosomes, causing reduced fertility amongst generated plants, this phenomenon is called somaclonal variation (Ye et al., 1999). Unwanted epigenetic changes may also occur such as gene silencing (Upadhyaya et al., 2000b). Plants derived from tissue culture often need to be screened to ensure they have suffered minimal genetic damage (Bent, 2000). 1.5 In planta transformation In planta transformation refers to recombinant DNA techniques which do not use tissue culture to obtain transformed plants. In planta transformation can provide a high throughput method for obtaining transformants whilst minimising labour, expense, and required expertise (Bent, 2000; Clough and Bent, 1998; Bechtold et al., 2000). Only a few in planta transformation experiments have been achieved using direct transformation methods. These include; DNA uptake by whole embryos of rice by imbibition (Junhi and Guhung, 1995), particle bombardment of cells around apical meristems (Bent,2000; Birch, 1997), injection of naked DNA into ovaries (Bent, 2000; Bent,2000), and electroporation into intact meristems in planta (Chowrira et al., 1995; Touraev et al., 1997). As these methods have been difficult to reproduce they have not been widely adopted. The direct in planta methods require expensive equipment to transform cells or specialised training to perform the experiments successfully. The exception to this is DNA uptake by rice embryos, however, this technique is not particularly reproducible. More reliable in planta methods of transformation have been achieved using A. tumefaciens. These include; transformation of cauliflower seeds by injection of A. tumefaciens (Eimert et al., 1992), co-cultivation of whole or split sunflower embryo apices with A. tumefaciens (Schoneberg et al., 1994), A. tumefaciens treatment of germinating corn (Graves and Goldman, 1986), A. tumefaciens inoculation of excised primary and secondary inflorescence shoots of A. thaliana (Katavic et al., 12

13 1994), co-cultivation of germinating A. thaliana seeds with A. tumefaciens (Feldmann and Marks, 1987) and A. tumefaciens infection of the cotyledonary node of germinating sunflower and peanut seeds by excision of one of the cotyledons (Sankara Rao and Rohini, 1999a; Sankara and Rao Rohini, 1999b). Experiments have shown that a number of different tissues can be successfully transformed. Incision experiments with peanut have shown that embryo axes subjected to wounding and inoculation with A. tumefaciens are capable of transformation without the need for tissue culture. Inoculated embryos germinate into plants ex vitro to produce transgenic seed. Experiments of this kind have also been performed using soybean (Chee, 1989). Graves and Goldman (1986) report that A. tumefaciens appears to infect scutellar and mesocotyl cells of germinating corn (Zea mays) seeds and that the resulting plants are transformed, although these plants are chimeric. Many non-tissue culture approaches transform the cells in or around the apical meristems. This meristematic tissue is left to develop into a mature plant, some of which should produce seed. Seed derived from the transformed meristematic tissue will be transgenic (Bent, 2000). The frequency of transformation varies not only with plant genotype but also with the transformation system used. This shows that A. tumefaciens mediated transformation can be achieved without the use of any tissue culture. Further proof of this is provided by Feldman and Marks (1987) who achieved transformation of mesocotyl cells of germinating seeds using A. tumefaciens. In an extension of these experiments, A. tumefaciens suspension is applied to inflorescences of A. thaliana, which are then allowed to set seed (Bouchez and Bechtold, 1995; Richardson et al.,1998). In this method (termed floral transformation) A. tumefaciens appears to transform the ovule of flowering A. thaliana plants (Desfeux, et al., 2000; Bechtold et al., 2000; Ye G et al., 1998). Advantages of in planta transformation methods include lower rates somaclonal variation, simpler means for transformation thus facilitating transformation intensive procedures such as positional cloning and insertional mutagenesis (Forsthoefel et al., 13

14 1992; Franzmann et al 1995). Due to a reduction in regeneration time, these methods allow for rapid testing of DNA constructs (Bent, 2000; Feldmann, 1991; Feldmann, 1992). In planta transformation methods are particularly important for O. sativa as attempts to regenerate plants from tissue such as protoplasts have proven particularly difficult and vary greatly amongst genotypes. In planta methods eliminate the need for immature embryos, callus or suspensions in transformation experiments (Valdez et al., 1998; Feldmann et al., 1995). The most popular in planta transformation method used today is the floral dipping method (Bent, 2000). Floral dipping protocols developed for A. thaliana so far are impractical for larger plants such as B. napus due to the large volumes of A. tumefaciens suspension required to treat plants. Modifications such as spraying suspension onto floral areas are an improvement, however they pose problems such as A. tumefaciens contamination and worker safety issues. Seed transformation eliminates these problems. Seed provide a large number of transformation targets that require only minimal amounts of A. tumefaciens suspension to treat, are relatively easy to treat and have a large potential for transgenic plant production. Seed transformation was first developed by Feldmann and Marks (1987), using Arabidopsis thaliana seed. This process involved the cultivation of seed in the presence of A. tumefaciens suspension. Seeds were allowed to develop into plants and to set seed. Not all the cells within the plant were transformed by this method, so the seed produced was germinated on selective media to determine if it was transformed. 1.6 Research aims The aim of this study is to develop an easy and inexpensive A. tumefaciens in planta transformation method for seed of B. napus and O. sativa. The regulating factors involved in A. tumefaciens seed mediated transformation will be investigated and the feasibility of the method assessed. 14

15 Chapter 2 Materials and Methods 2.1 Plant Material Seeds of Brassica napus cv. Oscar and Oryza sativa cv. Amaroo were obtained from the Farrer Centre, Charles Sturt University, Wagga Wagga. Seed of Arabidopsis thaliana of geographic race, Columbia (Col-O) were used for floral transformation. 2.2 Seed Surface Sterilisation Seeds were surface sterilised for 15 minutes using 1.25% sodium hypochlorite (bleach) and 0.15% Triton X-100 (v/v), followed by 5 rinses with sterile water. 2.3 Plasmids pcambia1301 and pcambia1304 Both pcambia1301 and pcambia1304 binary vectors were obtained from the Centre for Application of Molecular Biology to International Agriculture (CAMBIA). pcambia1301 and pcambia1304 containing a hygromycin resistance gene (hpt) and Figure 2.1: T-DNA of pcambia1301 and pcambia

16 gusa gene on the T-DNA. Both are driven by the CaMV35S promoter, and are used to select for transformed plant tissue. pcambia1301 contains an intron near the N- terminus of the gusa-coding region, to prevent its expression in A. tumefaciens. pcambia1304 T-DNA is comprised of a gfp/gusa fusion gene, and has no intron, therefore it can be expressed in both plant tissue and A. tumefaciens. Both plasmids carry the nptii gene outside the T-DNA region, for antibiotic selection of transformed bacteria. 2.4 Bacterial strains Escherichia coli (E. coli) E. coli strain XL-1 blue was obtained from Stratagene. The genotype of XL-1- Blue is reca1 enda1 gyra96 thi-1 hsdr17 supe44 rela1 lac [F proab lacl q Z6M15 Tn10 (Tet r )] (Bullock et al., 1987) A. tumefaciens A. tumefaciens strains AGL-1 and EHA105 were obtained from CAMBIA. AGL-1 genotype is AGL0 (C58 ptibo542) reca::bla, T-region deleted Mop(+) Cb(R) [AGL0 is an EHA101 with the T-region deleted, which also deletes the aph gene] (Lazo et al., 1991). EHA105 is a Km(S) derivative of EHA101 (Hood et al., 1993). EHA101 genotype is C58 ptibo542; T-region::aph, Km(R); A281 derivative harboring peha101, T-DNA replaced with nptii, elimination of T-DNA boundaries unconfirmed, super-virulent (Hood et al., 1986). 16

17 2.5 Culture of bacterial strains E. coli E. coli were streaked on LB agar- consisting of 10g of Bacto tryptone, 5g of Bacto yeast extract, 10g of NaCl and 15g of agar made to 1litre with distilled water (ph 7.0). Plates are incubated at 37 C for 24hrs. Flasks containing LB were inoculated with single colonies and cultured using an orbital shaker at 37 C at 200rpm for 24hrs. E. coli containing pcambia1301 or pcambia1304 were streaked out on LB agar containing kanamycin (50µg/ml). Liquid cultures were inoculated with single colonies into flasks containing LB with kanamycin (50µg/ml). Cultures were grown using an orbital shaker at 37 C with shaking at 200rpm for 24hrs A. tumefaciens A. tumefaciens strain AGL-1 was streaked out on LB agar containing carbenicillin at 100µg/ml. Liquid cultures were inoculated with single colonies into LB nutrient solution with Carbenicillin (100 µg/ml). Cultures were grown for 3 days at 25 C with shaking at 150rpm. A. tumefaciens strain EHA105 was streaked out on LB agar containing rifampicin (10µg/ml). Liquid cultures were inoculated with single colonies into flasks containing LB and rifampicin (10 µg/ml). Cultures were grown for 3 days at 25 C with shaking at 150rpm. A. tumefaciens strains containing pcambia vectors were cultured as above, except that kanamycin was added at a concentration of 50µg/ml. Cultures grown for plant transformation were supplemented with 400µM acetosyringone to preinduce the virulence genes (Khanna and Raina, 1999). 17

18 2.6 Bacterial Transformation E. coli E. coli transformation E. coli strain XL-1 blue was transformed with pcambia1301 and pcambia1304 according to Stratagene instructions for Epicurian Coli XL1-Blue Competent Cells Extraction of plasmid DNA from E. coli. Plasmid DNA was extracted for E. coli cultures using the Wizard Plus Minipreps DNA Purification System (Promega) according to the manufacturers instructions. The concentration of extracted DNA was determined after electrophoresis on a 1% agarose gel by comparison with a known concentration of lambda DNA digested Restriction endonuclease digestion of plasmid DNA Plasmid DNA extracted from E. coli was digested according to manufacturer recommendations (Promega) using 5 units of restriction enzyme per µg of DNA and 2µl of the buffer supplied, in a total reaction volume of 20µ. Digests were performed for 2 hours at recommended temperature Agarose gel electrophoresis DNA digests were analysed on a 1% agarose gel by electrophoresis. A 100ml gel was made by dissolving 1g of agarose in 100ml of 1 X TAE buffer and ethidium bromide 0.5µg/ml. TAE buffer was comprised of 0.04M Tris-acetate and 0.001M EDTA as described by Maniatis (1989). The 1X TAE buffer was also used as electrophoresis buffer. Electrophoresis was performed at a constant voltage of 95V for 4 hours. 18

19 2.6.2 A. tumefaciens transformation Preparation of electrocompetent A. tumefaciens cells A. tumefaciens cells were made electrocompetent by placing overnight liquid cultures on ice for 20 minutes in 50ml Falcon tubes then centrifuging at 5000 rpm for 15 minutes. Supernatant is poured off and cells were resuspended in 50 ml of cold distilled water. Cells were washed as above 4 times. Cold sterile 10% glycerol (10 ml) was added. Cells were resuspended and then centrifuged at 5000 rpm for 10 minutes. The supernatant was removed and the pellet was resuspended in 3 ml of ice cold 10% glycerol Transformation of A. tumefaciens. Plasmid DNA extracted from E. coli was used to transform A. tumefaciens via electroporation. The electroporation was performed using the IBI genezapper 450/2500 using a capacitance of 21µF, a voltage of 2500V and a resistance of 200_. Aliquots of 80µl of A. tumefaciens cells were electroporated using 5µl of plasmid DNA at 0.05µg/µl was used. 2.7 Floral transformation A. thaliana growth and floral transformation. Seed of Arabidopsis thaliana cv. Columbia Col-O were placed in 4 C fridge for 2 days and then planted into presoaked pots containing one third Yates Seed Raising mix, one third sand and one third perlite. The pots were supplemented with Scotts Osmocote. Plastic wrap was applied to each pot and several pinholes made in the plastic. Pots were transferred to the growth room. Growth conditions consisted of continuous light at 22 C. Plants were watered gently by spraying every second day. A. thaliana were transplanted into 15 cm diameter pots. Plants were placed 2 per pot and were sub-irrigated every second day. After approximately 8 weeks plants began to bud. The primary bolts of A. 19

20 thaliana were removed. When the secondary bolts were 2-10 cm long they were sprayed with A. tumefaciens suspension. This suspension is the same as the floral dip suspension used by Clough and Bent (1998). It comprised of A. tumefaciens strain AGL-1 or EHA105 resuspended to 0.8 at OD 600 in 5% sucrose and 0.05% Silwet L-77 (Silwet l-77 kindly donated by Dr Julie Glover of Groupe Limagrain Pacific, Australian National University). Either 1, 2 or 3 applications of A. tumefaciens suspension were spaced 5 days apart. The 3 treatments were repeated for both A. tumefaciens strains AGL-1 and EHA105. Two controls were used, one consisted of applying a solution of Silwet L-77 and water to floral tissue, and the other consisted of spraying water only. Plants were kept within plastic bags overnight to maintain humidity, since plants had to be kept out of direct sunlight after treatment A. tumefaciens was applied at night B. napus growth and floral transformation Seed of B. napus cv. Oscar were placed in a 4 C fridge for 2 days and then planted 2 seeds per pot. Pots were presoaked before sowing and consisted of Yates Seed Raising mix supplemented with Scotts Osmocote. B. napus was placed in the PC2 glasshouse at 25 C under natural lighting. A temperature of C was maintained. Seedlings were later transplanted one plant per pot. Approximately 6 months after germination B. napus were ready for floral transformation. Due to the late budding not all plants were budding for the first A. tumefaciens treatment. Treatment of B. napus was the same as for A. thaliana except applications were repeated every seven days. 2.8 Selection of transformed plants Determining concentration of hygromycin suitable for selecting transformed plants. Seed of A. thaliana and B. napus were germinated on selective plates consisting of 0.8% water agar and hygromycin concentrations of 0, 25, 50 and 75µg/ml. Plates were incubated at 25 C under continuous white light of 25µE/M 2 /sec for 2 weeks, after which 20

21 time a qualitative assessment of growth was made. This method was used to identify transformed seed obtained from the floral transformation methods described in section Histological staining for _-glucuronidase (GUS) enzyme activity. To score transformation, histochemical staining was performed to reveal _-glucuronidase activity in plants. _-Glucuronidase enzyme activity was detected using the substrate 5- bromo-4-chloro-3-indolyl glucuronide (X-GLUC, Progen) (as described by Jefferson, 1987). Histological staining gives a blue colouration in the presence of the enzyme. Tissues were vacuum infiltrated with the substrate (200µg/ml X-gluc in 50mM phosphate buffer, ph 7.0, supplemented with 0.01%(v/v) Triton X-100) for 3 minutes then incubated at 37 C for 16. A positive control (transgenic tobacco tissue expressing GUS driven by the CaMV35S promoter) and a negative control (consisting of tissue not treated with A. tumefaciens) were also assayed. 2.9 Potato disk assay This assay was based on the antitumourigenic potato disk assay used by Galsky et al, 1981 and Lazo et al, Red potatoes (Solanum tuberosum L.) were sterilised for 20 minutes with 1.25% sodium hypochlorite (bleach) and 0.15% (v/v) Tween 20. The ends of the potatoes were removed and then sterilised for a further 10 minutes. Potatoes were rinsed in sterile water then cut into disks of 1.2cm diameter by 0.5cm width. Discs were placed in petri dishes containing 1.5% water agar. Five disks were used per petri dish and 3 petri dishes were used per treatment. A drop of A. tumefaciens suspension (0.05ml) was applied to each disk and spread over the surface of the disk. Untransformed A. tumefaciens strains AGL-1 and EHA105 were inoculated onto potato as controls. A water control was also used. Petri dishes were sealed with parafilm and stored in the dark at 22 C for 7 days. After 7 days, disks were placed into 50ml falcon tubes and stained for _- glucuronidase activity. 21

22 2.10 Seed transformation Seed transformation was based on the A. tumefaciens mediated seed transformation protocol described by Feldmann and Marks (1987). Modifications included resuspending A. tumefaciens in sterile water to form the suspension used for cocultivation. Cocultivation was performed in petri dishes instead of flasks and the resulting seedlings were stained after 7 days. Feldmann and Marks allowed seedlings to develop into plants and then tested the resulting seed for transformants using selective media. In this study the likelihood of transgenic seed developing was determined from the localisation of stained tissue Effect of A. tumefaciens concentration on germination and transformation A. tumefaciens cultures transformed with pcambia1301 of strains AGL-1 and EHA105 were centrifuged at 5000 rpm for 15 minutes, cells were resuspended in an equal volume of water. Serial dilutions of A. tumefaciens suspensions were prepared in sterile distilled water. 50µl of each dilution were spread onto LB plates and incubated at 25 C for 3 days. Bacterial colonies were counted for each plate and the A. tumefaciens concentration determined. A minimum of 20 seeds per dish was used for O. sativa and 100 seeds for B. napus. Seeds were placed in petri dishes containing 2 filter papers, then 7mls of the appropriate A. tumefaciens solution were added to each petri dish. Water was used in as a control in place of A. tumefaciens. Plates were sealed with parafilm and incubated in the dark for 7 days at 22 C. Seedlings were placed in 50ml falcon tubes and stained for _- glucuronidase activity Vacuum infiltration of seed with A. tumefaciens A. tumefaciens strains were cultured, resuspended and diluted as per Section A minimum of 20 O. sativa seed and 100 B. napus seed were placed into separate 50ml 22

23 falcon tubes to which 7mls of appropriate A. tumefaciens suspension was added. Tubes were placed under a vacuum for 4 minutes then 50µl of A. tumefaciens suspension was spread onto LB agar plates and incubated for 3 days at 25 C. Vacuum infiltrated seed and solution were placed into sterile petri dishes containing 2 filter papers per dish. Sterile water was used in place of A. tumefaciens suspension as a control. Petri dishes were sealed with parafilm and incubated in the dark at 22 C for 7 days. Seedlings were placed in 50ml falcon tubes and stained for _-glucuronidase activity Viability of vacuum infiltrated A. tumefaciens suspensions AGL-1 and EHA105. A. tumefaciens cultures AGL-1 and EHA105 were placed into 50 ml falcon tubes and were placed under vacuum for 0, 4, 8 and 12 minutes. Serial dilutions were made in sterile distilled water. 50 µl of each dilution was spread onto LB agar plates, which were incubated at 25 C for 3 days. Bacterial colonies were counted Liquid Nitrogen Treatment B. napus and O. sativa seeds were immersed for 0, 1 or 2 minutes in liquid nitrogen using a tea strainer and then placed in a petri dish with 2 filter papers. A minimum of 20 seeds were used per treatment for both B. napus and O. sativa. Cultures of A. tumefaciens were centrifuged at 5000 rpm for 15 minutes. The supernatant was poured off then the pellet resuspended to an OD 600nm of 0.8 using sterile distilled water. Then 7 mls of the appropriate A. tumefaciens strain was added to the seed. Plates were incubated under continuous light (25µE/m 2 /sec) at 22 C for 7 days. The control consisted of applying 7 mls of water after the various immersion times in liquid nitrogen. After 7 days the seed were stained for _-glucuronidase activity. 23

24 Effect of Silwet on transformation and germination of B. napus and O. sativa seed. Liquid cultures of A. tumefaciens strains were grown then resuspended to an OD 600nm of 0.8 in sterile distilled water with the following concentrations of Silwet L , 0.01, 0.05, 0.10, 0.50 and 1.00% (v/v). A minimum of 90 B. napus seeds or 30 O. sativa seeds were used per treatment. Surface sterilised seed was placed in petri dishes containing 2 filter papers. To each dish, 7 mls of A. tumefaciens/ Silwet L-77 solution was added. Seed and A. tumefaciens were cocultivated for 7 days at 22 C in the dark. Staining for _- glucuronidase was performed as per section except that the catalysts potassium ferricyanide and potassium ferrocyanide were added to the staining solution to a final concentration of 0.5mM each (Jones, 1995) Seed injection This experiment was based on the protocol of Eimert et al (1992). Seed of B. napus and O. sativa were incised with a Terumo needle of size 0.45 X 13mm. Prior to incision the needle was dipped in an A. tumefaciens solution of either AGL-1 or EHA105 containing pcambia1301. Treatments were split according to A. tumefaciens strain and site of incision. The incisions for B. napus consisted of targeting the hypocotyl region of the canola seed, as indicated by a raised region on the testa. The raised region is the point at which the cotyledons first emerge. The other treatment consisted of pricking opposite ends of B. napus seed. In both cases the needle tip penetrated the seedling no more than 1-2mm deep. Sites of incision for O. sativa consisted of targeting the embryo as one treatment, and the endosperm as another. The needle tip was allowed to penetrate approximately 3mm deep into the seed. 24

25 Figure 2.2: Diagram of incision sites on B. napus and O. sativa. Left side is B. napus. Right side is O. sativa. 25

26 Chapter 3 Results 3.1 Bacterial transformation Restriction enzyme analysis of pcambia1301 and pcambia1304. Plasmid DNA was isolated from E. coli was digested with restriction endonucleases. The sizes of the DNA fragments were determined after electrophoresis on a 1% agarose gel (figure 3.1). Sizes of fragments are presented in Table 3.1. The number of fragments and sizes observed for pcambia1301 were in-consistent with the predicted sizes based on the published sequences (GENBANK accession number GI: ). The restriction endonuclease Nhe I was expected to produce 2 fragments of sizes 3444 and 8393 bp s. The observed values were 2399, 3467 and 6918 bp s. This result suggests that an extra Nhe I site is located outside the T-DNA, at the 5 end of the npt II gene (Figure 3.2). Figure 3.1: Restriction analysis of plasmid DNA extracted from E. coli strain XL-1 blue transformed with pcambia1301 and pcambia1304. Lane 1- lambda DNA digested with Hind III, Lanes 2-7, pcambia1301 DNA. Lanes 8-13, pcambia1304 DNA. 26

27 Enzymes used; Lanes 2&8- Nhe I, Lanes 3&9- EcoR I, Lanes 4&10- BstE II, Lanes 5&11- Xho I, Lanes 6&12- EcoR I and Nhe I, Lanes 7&13- BstE II and Xho I. This shows that the T-DNA region of pcambia1301 is unaffected and should not introduce unknown genetic material during transformation of plants. pcambia1301 therefore contains a small insert at the location of the extra Nhe I site of up to 1.5kb. The Figure 3.2: Restriction map showing extra Nhe I site in pcambia1301. Extra site is bordered by a rectangle. Fragment lengths are observed values rather than exact sizes. fragments from pcambia1304 appear consistent in size and number with expected sizes from the published sequence in GENBANK (Accession number GI: ). 27

28 Table 3.1: Expected and observed fragment sizes of digested pcambia1301 and pcambia1304 DNA. Fragment lengths were determined by plotting the log of the molecular weight marker in bp s against the distance each fragment had migrated Transformation of A. tumefaciens with pcambia 1301 and pcambia DNA extracted from transformed E. coli was used to transform Agrobacterium strains AGL-1 and EHA105 by electroporation. Transformation efficiency was greater than cfu s per mg of plasmid DNA. Transformed strains were selected on LB agar and kanamycin (50_g/ml). DNA was extracted from transformed Agrobacterium strains and digested with restriction endonucleases. Difficulties in purifying DNA from A. tumefaciens using the methods of Maniatis (1982) and the Wizard Plus Minipreps for the isolation of Binary Plasmids from Agrobacterium tumefaciens protocol (Promega) were experienced. Problems included chromosomal DNA contamination, degradation by non-specific endonucleases and insufficient yields of plasmid DNA. As an alternative strategy, plasmid function was assessed using other methods. 28

29 3.2 Assessment of plasmid function in A. tumefaciens Assay for endogenous b-glucuronidase activity in A. tumefaciens. Agrobacterium strains AGL-1 and EHA105 transformed with pcambia1301 and pcambia1304 were able to grow in the presence of kanamycin indicating the presence of functional nptii genes. These transformed bacteria were stained for _-glucuronidase activity (Figure 3.3). Untransformed A. tumefaciens strains AGL-1 and EHA105 and AGL-1 and EHA105 transformed with pcambia1301 showed no b-glucuronidase activity. b-glucuronidase activity was observed in AGL-1 and EHA105 strains harbouring pcambia1304. Since there was no intron within the gusa gene to prevent translation in A. tumefaciens, the CaMV35S promoter appears to function to some degree. Expression of genes in Agrobacterium driven by the CaMV35S promoter have been previously reported as weak to non-existent (Elliott et al, 1998). Figure 3.3: _- glucuronidase staining of Agrobacterium tumefaciens strains. Left to right; GUS positive control, AGL-1 untransformed, EHA105 untransformed, AGL-1 + pcambia 1301, AGL-1 + pcambia1304, EHA105 + pcambia1301, EHA105 + pcambia1304. Blue staining indicates the presence of b-glucuronidase activity. 29

30 Assay for b-glucuronidase activity on potato disks transformed with A. tumefaciens strains AGL-1 and EHA105 using pcambia1301. A. tumefaciens strains AGL-1 and EHA105 carrying pcambia1301 were inoculated onto potato disks set in water agar. Disks were incubated at room temperature in the dark for 7 days then stained for GUS expression. It is assumed that some cell proliferation of transformed cells will occur allowing expression of the gusa gene to be visualised. Expression of GUS indicated that AGL-1 and EHA105 carrying pcambia1301 successfully transformed plant tissue and expressed b-glucuronidase activity (Figure 3.4). Of those treated with AGL-1 carrying pcambia1301, 4 strong blue stained regions were observed, indicating GUS expression. On potato disks inoculated with EHA105 carrying pcambia1301, 6 strong blue stained regions were observed. Each treatment in the assay consisted of 15 potato disks. 30

31 Figure 3.4: Assay of b-glucuronidase on potato disks transformed with A. tumefaciens strains AGL-1 and EHA105 using pcambia1301. Arrows indicate sites of _- glucuronidase activity. EHA105 appeared slightly more efficient at transforming cells. A faint Light blue staining was observed on all treatment groups, indicating the presence of bacterial or fungal surface contamination. No strong colourations were seen on disks inoculated with water or untransformed A. tumefaciens. The results suggest that both AGL-1 and 31

32 EHA105 containing pcambia1301 are capable of transforming plant tissue and that the transformed tissue can express the gusa gene. 3.3 Floral transformation of A. thaliana and B. napus. To further assess whether A. tumefaciens strains containing pcambia1301 were capable of transformation, A. thaliana was transformed using the standard floral transformation method of Clough and Bent (1998). Transformation of A. thaliana is well documented and has the benefit of yielding transformed seed in as little as 6 weeks. A. thaliana is closely related to oilseed crop plants, such as Brassica napus (Theien, 2000). The reliability of this method lead to its adoption for use with B. napus to check construct function of pcambia1301. Flowering A. thaliana plants were split into treatment groups consisting of 4 plants. Each group was sprayed with Agrobacterium strains AGL- 1 or EHA105, both containing pcambia1301. Plants subsequently set seed, Table 3.2 shows the number of seed collected for each treatment. Interestingly, seed yield decreased with the increased number of A. tumefaciens applications. Transformed A. thaliana seeds were selected on water agar hygromycin plates (Section 3.3.2). Treatment No. of applications Total no. of seed collected AGL AGL AGL EHA EHA EHA water+silwet Water Table 3.2: The treatments used on A. thaliana with the floral transformation method and the number of seed collected for each treatment. 32

33 B. napus was transformed according to the A. thaliana floral transformation method. Figure 3.5 shows the different floral stages present at the time of the first application of A. tumefaciens. Figure 3.5: Different stages of B. napus development during first application of A. tumefaciens according to the floral transformation experiment. At the time of writing, seed from B. napus hadn t been collected due to late flowering and seed set. 33

34 Determination of hygromycin concentration suitable for selection of transformed plants. Seed of untransformed A. thaliana and B. napus were germinated on 0.8% water agar containing varying concentrations of hygromycin, to determine the concentration at which untransformed plants could be differentiated from transformed ones. The effect of hygromycin on the germination of A. thaliana is shown in figure 3.6. At 0_g/ml hygromycin, both A. thaliana and B. napus displayed good root and cotyledon development. Seeds were germinated on media containing 0, 25, 50 and 75 mg/ml of hygromycin. Hygromycin 25_g/ml was the lowest concentration tested, at which all A. thaliana failed to germinate as indicated by the failed emergence of root and cotyledons from the seed. This concentration was subsequently used to select for hygromycin resistant A. thaliana plants from seed obtained via the A. thaliana floral transformation method, consistent with Bechtold et al., Figure 3.6: Untransformed seed germinated on various hygromycin concentrations. A- A. thaliana 25_g/ml hygromycin. B- A. thaliana 0_g/ml hygromycin. C- B. napus on 25_g/ml hygromycin. D- B. napus on 0_g/ml hygromycin. 34

35 B. napus seedlings displayed various degrees of growth retardation with all hygromycin concentrations. Hygromycin concentration of 25_g/ml was chosen as the selective concentration at which to select for transformants obtained from the B. napus floral transformation experiment, as root growth was severely inhibited at this concentration (figure 3.6) Selection of transformed plants. Seed collected from A. thaliana treated via the floral transformation method were germinated on water agar containing hygromycin 25_g/ml. Most A. thaliana seed displayed limited growth and development on hygromycin at 25_g/ml. Seeds germinating from water treated A. thaliana (controls) had very poor root growth (approximately 1-2mm, see figure 3.7A), and did not penetrate very deeply into the medium. Seeds from controls and seedlings showing relatively normal growth were stained for GUS activity. The number of seed collected for each treatment and the transformation efficiency obtained for each is listed in table 3.5. EHA105 transformed with pcambia1301 (single application) produced two seedlings, which displayed good root and leaf development (figure 3.7B). They both stained positive for GUS activity indicating that the GUS construct is functional and that these seedlings had been transformed. Treatment No. of applications Transformation efficiency (%) AGL AGL AGL EHA EHA EHA water+silwet 1 - water 1 - Table 3.3: Transformation efficiency obtained using the floral transformation method of A. thaliana. 35

36 Figure 3.7: A. thaliana seedlings that were obtained from the floral transformation experiment and germinated on 25_g/ml hygromycin media. A- Seedling obtained from water treated A. thaliana (control) and stained for GUS expression. B- Stained seedling obtained from A. thaliana plants treated with EHA105 (one application). Note blue staining in root, hypocotyl and cotyledons indicating constitutive GUS expression. The seed produced from treated B. napus was not tested due to time constraints. 36

37 3.4 Seed transformation experiments with B. napus and O. sativa. The effects of different factors involved in A. tumefaciens transformation were investigated in order to determine feasibility of the seed transformation method The effect of A. tumefaciens concentration on seed germination and transformation in B. napus and O. sativa. Various concentrations of A. tumefaciens were co-cultivated with B. napus and O. sativa seeds to study the effect of Agrobacterium concentration on seed germination and transformation efficiency. A. tumefaciens is a plant pathogen, hence is likely to have an adverse effect on germination. Based on this assumption experiments were designed to determine the concentration of A. tumefaciens suspension that would achieve maximum transformation whilst minimising detrimental effects on seed germination. No transformed seeds were detected indicating that not enough seeds were screened. Light blue staining occurred in treated groups as well as in the controls. Blue staining was most frequently observed in tissue near the interface between the staining solution and the air. In some treatments the staining solution had a bluish tinge and appeared turbid after incubation. This suggests that microbial contamination had occurred (figure 3.8). Table 3.4 shows that the maximum A. tumefacines suspension differed only slightly from that of the water control in both B. napus and O. sativa. Hence the overall concentration of A. tumefaciens had a minimal effect on seed germination. 37

38 Figure 3.8: B. napus co-cultivated with water (control) and stained for GUS activity. Note the staining at the interface. B. napus Treatment Percentage Germinated Cells per ml Water 72 0 AGL X EHA X O. sativa Treatment Percentage Germinated Cells per ml Water 96 0 AGL X EHA X Table 3.4: Effect of A. tumefaciens concentration on seed germination. 38

39 3.4.2 Vacuum infiltration of seed and assessment of bacterial contamination Vacuum infiltration has been used in transformation protocols to aid infiltration of A. tumefaciens into tissues of A. thaliana (Clough and Bent, 1998). Vacuum infiltration is reported to improve transformation by providing A. tumefaciens access to intercellular spaces within plant tissue. Figure 3.9: LB plate inoculated with suspension taken from vacuum infiltrated O. sativa seed. A. tumefaciens number was monitored to study survival rates after vacuum infiltration. Figure 3.9 shows plates used for this purpose covered with a number of different colony types not removed by surface sterilisation This suggests that there may be bacteria present underneath the seed testa or within the seed itself. Colonies from the contaminated plates were stained for _-glucuronidase activity, revealing GUS expression in the yellow colonies, which could account for the background staining in figure 3.8. Vacuum infiltration did not improve the transformation efficiency nor did it adversely effect germination. 39

40 Viability of A. tumefaciens after vacuum infiltration. A number of transformation methods use vacuum infiltration to facilitate transformation of seed. This experiment was designed to test the effects of vacuum infiltration on the Table 3.5: Agrobacterium viability after vacuum infiltration for 0, 4, 8 and 12 minutes. viability of A. tumefaciens strains AGL-1 and EHA105. The results (Table 3.2) show no apparent trend between length of vacuum infiltration period and Agrobacterium viability for AGL-1. EHA105 showed a gradual decrease in viability over time, however the effect was minimal The effect of Silwet L-77 on transformation and germination of B. Napus and O. sativa seed. Silwett L-77 is a surfactant with low phytotoxicity used to increase transformation efficiency in floral transformation methods. It facilitates greater access to the seed by reducing surface tension, possibly helping A. tumefaciens to penetrate seed tissue. This experiment tested the effects of various concentrations of silwet L-77 in cocultivation solution on transformation and seedling development of B. napus and O. sativa. Six treatment were of 1.0, 0.5, 0.1, 0.05, 0.01 and 0% v/v of Silwet L-77. The effect of silwet concentration on seedling development of B. napus is shown in figure 4.0. The staining solution used to detect _-glucuronidase activity was modified to include oxidation catalyst s potassium ferricyanide and potassium ferrocyanide. The catalysts were added to prevent non-specific tissue staining near the interface. Despite the addition of these catalysts, non-specific staining was apparent at the water air interface. 40

41 Figure 3.10: B. napus seed germinated in varying concentrations of Silwet L-77. A- 1.0% Silwet v/v, B- 0.5% silwet v/v, C-0.1% silwet v/v, D- 0% v/v silwet. The effects of silwet L-77 were the same for both A. tumefaciens strains. Seed were counted as germinated if the seed coat was ruptured and signs of root growth were evident. No major effects on germination were observed (Table3.3), however at 1%v/v Silwet L-77 application extremely restricted the growth of both B. napus and O. sativa such that they were not likely to survive even if transferred to another medium. Rice shoots were inhibited by increasing amounts of Silwet L-77. No transformed seeds were observed indicating that the method was not efficient enough for the number of seeds tested. 41