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

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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.

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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;

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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.

1.2.2 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.,

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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).

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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

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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).

1.3.1 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.

1.3.2 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

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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).

1.3.3 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).

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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).

1.3.4 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).

1.3.5 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

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untransformed plants (which would otherwise perish or show severe growth retardation

on selective media) from transformed ones.

1.3.6 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.

1.3.7 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).

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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,

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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.,

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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.,

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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.

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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 pCAMBIA1304.

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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

2.4.1 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 laclqZ6M15 Tn10 (Tetr)]

(Bullock et al., 1987).

2.4.2 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).

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2.5 Culture of bacterial strains

2.5.1 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.

2.5.2 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).

18

2.6 Bacterial Transformation

2.6.1 E. coli

2.6.1.1 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.

2.6.1.2 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.

2.6.1.3 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.

2.6.1.4 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.

19

2.6.2 A. tumefaciens transformation

2.6.2.1 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.

2.5.6.2 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

2.7 .1 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.

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 OD600 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.

2.7.2 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 25-28˚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

2.8.1 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/M2/sec for 2 weeks, after which

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

2.6.

2.8.2 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, 1991. 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.

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.

2.10.1 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.

2.10.2 Vacuum infiltration of seed with A. tumefaciens

2.10.2.1

A. tumefaciens strains were cultured, resuspended and diluted as per Section 2.9.1. A

minimum of 20 O. sativa seed and 100 B. napus seed were placed into separate 50ml

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.

2.10.2.2 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.

2.10.3 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 OD600nm 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/m2/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.

24

2.10.4 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 OD600nm of

0.8 in sterile distilled water with the following concentrations of Silwet L-77- 0.00, 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 2.7.2 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).

2.10.5 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.

25

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

Right side is O. sativa.

26

Chapter 3

Results

3.1 Bacterial transformation

3.1.1 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:7638068 ). 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.

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:7638083).

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.

3.1.2 Transformation of A. tumefaciens with pCAMBIA 1301 and pCAMBIA 1304.

DNA extracted from transformed E. coli was used to transform Agrobacterium strains

AGL-1 and EHA105 by electroporation. Transformation efficiency was greater than 1.4

¥ 105 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.

29

3.2 Assessment of plasmid function in A. tumefaciens.

3.2.1 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.

30

3.2.2. 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.

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

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-1 1 756

AGL-1 2 579

AGL-1 3 159

EHA105 1 691

EHA105 2 85

EHA105 3 80

water+silwet 1 1206

Water 1 1785

Table 3.2: The treatments used on A. thaliana with the floral transformation method and

the number of seed collected for each treatment.

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.

34

3.3.1. 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., 2000.

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.

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).

3.3.2 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-1 1 0

AGL-1 2 0

AGL-1 3 0

EHA105 1 0.290

EHA105 2 0

EHA105 3 0

water+silwet 1 -

water 1 -

Table 3.3: Transformation efficiency obtained using the floral transformation method of

A. thaliana.

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.

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.

3.4.1 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.

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-1 71 7.52 X 1011

EHA105 66 1.54 X 1011

O. sativa Treatment Percentage Germinated Cells per ml

Water 96 0

AGL-1 96 7.52 X 1011

EHA105 96 1.54 X 1011

Table 3.4: Effect of A. tumefaciens concentration on seed germination.

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.

40

3.4.2.1 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.

3.4.3 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.

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.

42

O.

sativa

Agrobacterium Silwet L-77 concentration Percentage Germination

AGL-1 0.01 98

AGL-1 0.05 98

AGL-1 0.1 100

AGL-1 0.5 95

AGL-1 1.0 97

EHA105 0.01 97

EHA105 0.05 97

EHA105 0.1 100

EHA105 0.5 97

EHA105 1.0 97

none 0 98

none 1.0 92

B.

napus

Agrobacterium SilwetL-77 concentration Percentage Germination

AGL-1 0.01 95

AGL-1 0.05 95

AGL-1 0.1 94

AGL-1 0.5 94

AGL-1 1 94

EHA105 0.01 96

EHA105 0.05 96

EHA105 0.1 94

EHA105 0.5 95

EHA105 1.0 85

None 0 100

None 1.0 95

Table 3.6: The effect of various silwet concentrations on germination of B. napus and O.

sativa.

43

3.4.4 The effect of liquid nitrogen treatment on transformation and germination of B.

napus and O. sativa seed treated with A. tumefaciens.

B. napus and O. sativa seed were immersed in liquid nitrogen for various lengths of time.

After immersion, A. tumefaciens strains AGL-1 or EHA105, containing pCAMBIA1301

O. sativa Time

(minutes)

Treatment Percentage germinated

0 No Agrobacterium 95

1 No Agrobacterium 95

2 No Agrobacterium 85

0 AGL-1 92

1 AGL-1 91

2 AGL-1 89

0 EHA105 93

1 EHA105 89

2 EHA105 86

B. napus Time

(minutes)

Treatment Percentage germinated

0 No Agrobacterium 92

1 No Agrobacterium 91

2 No Agrobacterium 86

0 AGL-1 100

1 AGL-1 98

2 AGL-1 98

0 EHA105 85

1 EHA105 91

2 EHA105 94

Table 3.7: Germination efficiency of B. napus and O. sativa after immersion in liquid

nitrogen and co-cultivation with A. tumefaciens.

44

was added to seed and incubated at room temperature in the dark for 7 days. Seed were

stained for b-glucuronidase activity and germination (Table 3.7) and transformation

efficiency was determined. No transformation was evident for this experiment.

3.4.5 Inoculation of B. napus and O. sativa with Agrobacterium via seed incision.

Experiments with Arachis hypogaea L. and Brassica oleracea (Rohini and Rao, 2000;

Eimert et al.,1992) have shown that seed transformation can be achieved by inoculating

A. tumefaciens into seed incisions. The effect of seed incision on different seed tissues

was investigated to determine the influence it had on germination and transformation.

Incisions on seed of B. napus and O. sativa were created using a syringe inoculated with

either A. tumefaciens strain AGL-1 or EHA105. Both strains carried pCAMBIA1301.

Seed incision allows A. tumefaciens to bypass the seed coat, providing greater access to

cells. Incisions made on O. sativa targeted either the embryo or the endosperm depending

on the treatment. Incisions made into the embryo were expected to be detrimental to seed

development as vital structures within the embryo may be damaged during incision.

Therefore another treatment, specifically targeting the endosperm was trialed. The

embryo utilises the endosperm as a food source whilst developing into a self-supporting

seedling. By targeting the endosperm for incision, it may be possible to transform it

without adversely affecting germination. Due to the size of B. napus and the difficulty in

its manipulation, one seed incision strategy consisted of targeting the hypocotyl. The

hypocotyl causes a visible lump in the seed coat. This is where the cotyledons first

emerge during germination. By targeting this region it was hoped that tissue within the

embryo would be transformed. The other strategy consisted of pricking opposite ends of

the B. napus seed in an attempt to transform embryonic tissue.

Figure 4.1 shows the sites of incision for the various treatments. No transformation was

observed for this experiment, indicating that the treatment does not dramatically increase

the efficiency of transformation. The effects of wounding and inoculation on germination

of B. napus and O. sativa seed are outlined in Table 3.8.

45

Table 3.8: The effect of inoculating A. tumefaciens strains AGL-1 and EHA105 into

incisions targeting tissue of endosperm and embryo of B. napus and O. sativa.

46

Chapter 4

DISCUSSION

In order to evaluate the feasibility of the transformation method, construct function of

pCAMBIA1301 and pCAMBIA1304 was assessed using restriction endonuclease

analysis, the potato disk assay and floral transformation of A. thaliana. After establishing

plasmid function, pCAMBIA1301 was chosen to investigate a number of modifications

to the seed transformation method. The methods that were investigated involved

supplementing cocultivation medium with Silwet L-77, vacuum infiltrating seed with A.

tumefaciens suspension, direct inoculation of seed through seed incision and immersion

of seed in liquid nitrogen prior to cocultivation. The effect of A tumefaciens on seed

germination and transformation was assessed.

Restriction endonuclease analysis of the plasmids isolated from E. coli XL-1 blue

transformed with pCAMBIA1301 and pCAMBIA1304, revealed an extra Nhe I site in

pCAMBIA1301, indicating the presence of a small insertion. Restriction mapping

showed the insertion to be situated outside T-DNA region therefore it was anticipated

that it would not interfere with expression in transformed cells.

The function of pCAMBIA1301 was demonstrated in plants in two ways. The first

method employed an assay in which potato disks were inoculated for several days in the

presence of A. tumefaciens strains containing pCAMBIA1301. _-glucuronidase activity

was detected by histochemical staining, suggesting successful transformation of potato

tissue using pCAMBIA1301. A. tumefaciens strain EHA105 showed a slightly stronger

expression than AGL-1 in this assay. The potato disk assay might be useful for rapid

testing of new constructs prior to transformation of plants.

Plasmid function was further assessed using a common method of transforming A.

thaliana called floral transformation, described by Clough and Bent (1998). This method

achieved transformation in 2 seeds as evidenced by their resistance to hygromycin and

47

the presence of _-glucuronidase activity. This data suggests A. tumefaciens strains AGL-1

and EHA105 are capable of transforming plant tissue and the gusA gene is likely to be

expressed. Treatment of budding A. thaliana using one application of EHA105 indicated

0.29% of seed collected from treated plants as being transgenic. This was similar to the

reported transformation frequency of 0.23% obtained by Clough and Bent (1998).

Increasing the number of A. tumefaciens applications caused the seed yields from A.

thaliana to decrease. It was noted that in the controls, plants sprayed with water had a

48% greater yield than those plants treated with silwet L-77 and water. Clough and Bent

(1998) reported that reapplication of A. tumefaciens within a space of 4 days had a

detrimental effect on plant growth. In this experiment, repeated applications of A.

tumefaciens were performed every 5 days. This may explain why there was such a

dramatic effect on seed yields.

Plants may require more time between treatments so as to recover from the deleterious

effects of the detergent. Silwet L-77 when combined with A. tumefaciens had an additive

effect on lowering seed yields. Future research might trial different time intervals

between applications to determine the effect on transformation.

Floral transformation is impractical for larger plants due to difficulty in treating them.

Floral spraying has overcome this problem, allowing easy treatment of floral tissue,

however it raises a number of concerns which still make it an impractical transformation

method. There is the issue of A. tumefaciens containment, A. tumefaciens is capable of

transforming human HeLa cells and as such could pose a threat to workers who inhale the

A. tumefaciens as an aerosol (Kunik et al, 2000). Silwet L-77 used in floral

transformation can affect corneal tissue therefore may pose a health risk to workers.

After determining that pCAMBIA1301 was capable of transformation and expression,

seed transformation was attempted using seed injection, Silwet L-77 treatment, seed

immersion in liquid nitrogen and vacuum infiltration. The effects on transformation and

germination efficiency are assessed below.

48

Several dilutions of A. tumefaciens suspension were used in cocultivation with B. napus

or O. sativa. The highest A. tumefaciens concentration did not appear to greatly effect

germination efficiency compared to the water control. This is in contrast to reports made

by Feldmann and Marks (1987) who found high concentration of A. tumefaciens to be

deleterious to seedling development. In previous studies the cocultivation medium

consisted of a nutrient solution, this experiment used water only. It is unlikely that high

concentrations of A. tumefaciens will survive long in water medium, limiting the chance

of seed damage.

The effect of vacuum infiltration of A. tumefaciens on transformation and germination

efficiency was assessed. Transformation was not enhanced in this study by this method

nor did it appear to adversely affect germination of seed or the survival of A. tumefaciens.

Plates used to assess contamination levels, revealed contaminants, that were likely to

originate from within treated seed. Individual colonies were stained for _-glucuronidase

activity and revealed that a yellow forming colony mildly expressed _-glucuronidase

activity. This could explain the faint staining observed in most of the staining

experiments. To avoid this, future experiments could include antibiotics in the _-

glucuronidase staining solution to prevent growth of contaminants.

A novel method for aiding penetration of A. tumefaciens into seed was attempted using

liquid nitrogen. Seed of B. napus and O. sativa were immersed in liquid nitrogen prior to

cocultivation in A. tumefaciens suspension. Damage sustained from the freezing and

thawing of seed was thought to make seed tissue more susceptible to A. tumefaciens

infiltration. No positive effect on transformation efficiency was observed, but

surprisingly, only a slight decrease in seed viability was seen.

To increase the efficiency of seed transformation Silwet L-77 was added to the

cocultivation mixture. Silwet L-77 is reported to increase transformation efficiency in

floral transformation experiments by increasing A. tumefaciens adherence to plants

49

(Clough and Bent, 1998; Richardson et al, 1998). No increased transformation was

observed in this study. At concentrations of 0.5%, the effects of Silwet L-77 were

detrimental to seed development producing poor root morphology and browning in B.

napus, which may have been necrosis. In O. sativa Silwet L-77 prevented leaf shoots

from forming. Seed germination of B. napus and O. sativa did not appear to be effected

by Silwet L-77 at any concentration, however seed germination was determined by the

emergence of a root from the seed coat. Silwet L-77 had a significant effect on seed

growth at 1.0% (v/v) Silwet L-77, where growth of B. napus and O. sativa seedlings

appeared retarded and unlikely to survive even if removed to a Silwet L-77 free medium.

Inoculation of A. tumefaciens through seed incision was adopted from in planta methods

reported for safflower and cauliflower (Eimert et al, 1992; Orlikowska et al, 1995). No

increase in transformation efficiency was observed using this technique. There was no

effect of wounding on germination for O. sativa and very little effect on B. napus.

No transformation events were observed for any of the seed transformation methods

employed in this study indicating that transformation might not be possible using this

method. It is possible that the transformation efficiency was quite low and that the

number of seeds screened in this study was not high enough to detect transformation.

Future research could include the use of alternate reporter genes that are less expensive to

test for, allowing a greater number of seed to be screened. The reporter gene gfp requires

no substrate for the detection of its expression and can be observed visually with the use

of a hand held UV light (Haselhoff et al, 1997).

Another possibility is that the transformation event involved in seed mediated

transformation may occur later in the development of the seedling. This is the case for

seed transformation of A. thaliana (Feldmann and Marks, 1987), although not for peanut

or safflower (Eimert et al, 1992; Rohini and Sankara Rao, 2000a). Transformation would

not be detected at the time of staining if the transformation event occurred later in plant

development. If transformation did occur later in seed development, this protocol would

50

still be useful since it is much easier to treat a single seed then it is to treat a whole plant.

Future research might involve growing seedlings into mature plants and testing the seed

they produce for transformation.

51

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