5.3.30
PATHOGEN-RESISTANT GRAIN LEGUMES BY GENE TRANSFER TECHNIQUES

JS MILLER, FM WALLS and G RAMSAY

Scottish Crop Research Institute (SCRI), Invergowrie, Dundee DD2 5DA, UK

Background and objectives
The European Community imports about 16 million t of protein-rich feedstuffs each year, and grain legumes provide the best source of filling this deficit. Fungal diseases have a very high economic cost for peas and faba beans, and the creation of new forms of resistance to fungal pathogens may be an ideal way to reduce crop losses and the need for prophylactic or remedial sprays. Several fungal pathogens including Ascochyta, Botrytis, Fusarium and Aphanomyces are very important across a range of grain legumes. The proposed method for introducing increased fungal resistance is to insert genes which produce proteins already identified as having antifungal properties. The project aims to improve transformation technology for the European grain legumes and to apply this technology to introduce so-called antifungal genes. Pathology of the fungal organisms will then be investigated using transgenic plants expressing the antifungal genes.

Materials and methods
At SCRI, several types of explants of Vicia faba (faba bean) and Lupinus albus (white lupin) have been tested in transformation experiments. The Ag 11 strain of Agrobacterium tumefaciens was used for inoculations. Transient expression levels were estimated using GUS assays, and cultivars or lines showing the highest levels of transient expression were transferred to media consisting of M+S salts, Gamborg B5 vitamins, 30% sucrose (w/v) and 10 mM thidiazuron. Selection and testing of these potential transformants is ongoing
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Results and conclusions
It was observed that explants arising from an embryo axis slicing method [1] produced the highest levels of transient GUS expression. Explants which were inoculated with Ag11 containing the test construct pGINI, carrying a herbicide resistance gene bar, produced buds and shoots in culture and survived on selection medium containing 5 mg/l phosphinothricin. The construct pSCPI has also been used. This contains the gene for polygalacturonase-inhibiting protein (PGIP, derived from raspberry). These explants are also being cultured on selection medium. PG1Ps are cell wall-bound, leucine-rich repeat proteins that have been shown to be involved in plant resistance to fungi [2]. Polygalacturonases (PGs) degrade the extracellular matrix providing a carbon source for fungal growth and development, and facilitating host cell penetration. PGIP inhibits this process and this causes the increased formation of oligogalacturonides which are potent elicitors of plant defence responses.

Other genes of interest include chitinase, osmotin and stilbene synthase. Chitinase is a PR protein which hydrolyses the -1,4 linkages of chitin, a component of fungal cell walls. Compounds such as these have been reported to act synergistically with membrane-affecting compounds like osmotin to inhibit pathogenic fungi. It has been suggested that osmotin-like proteins may permeabilize fungal cell membranes and together, these two enzymes have been shown to inhibit in vitro spore termination of F. oxysporum and the hyphal growth of other fungi. Stilbene synthase is the key enzyme in the biosynthesis of the phytoalexin resveratrol. This is synthesized in one step from precursors available in all plants, but the gene for stilbene synthase is absent in most of the important crop plants.

All the genes mentioned here are known to have antifungal properties and it is planned to test them, individually or in combination, for their efficiency against a range of important pathogens

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References
1. Schroeder HE, Schotz AH, Wardley-Richardson T et al., 1993. Plant Physiology 101, 751-757.
2. Jones DA, Jones JDG, 1997. Advances in Botanical Research 24, 89-167.