5.3.18
TRANSFORMATION AND EXPRESSION OF ANTIFUNGAL GENES IN WHEAT, TRITICUM AESTIVUM AND BARLEY, HORDEUM VULGARE TO INCREASE THEIR FUNGAL RESISTANCE - TWO STRATEGIES

K OLDACH, D BECKER, H LÖRZ and G LECKBAND

Department of Applied Plant Molecular Biology at the Institute of General Botany and Botanic Garden, University of Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany

Background and objectives
To improve the fungal resistance of cereals, two strategies are followed: firstly, the biolistic transformation of wheat and barley with the vst1 gene, the key enzyme in phytoalexin synthesis in Vitis vinifera; and secondly, the transformation of wheat with three genes that are involved in pathogenesis. As in grapevine, the inducible expression of the vst1 gene should result in toxic accumulation of the foreign phytoalexin resveratrol in the transgenic plants. The transformation of wheat with the three genes, afp (antifungal protein) from Aspergillus giganteus, rip (ribosome inactivating protein) and chi (chitinase) derived from Hordeum vulgare, should support defence against pathogens at the protein level. The expression of the three genes is under control of the constitutive ubiquitin1 promoter of Zea mays. With the inducible production of the phytoalexin resveratrol or the over-expression of pathogenesis-related proteins in the transgenic cereal plants, an additive possibility should be given to the transgenic plants to reduce pathogen infections.

Materials and methods
For transformation of barley, the winter-type cv. Igri was used. Growth conditions of the donor plants and the procedure for the culture of microspores were performed according to a protocol of Leckband and Lörz [1]. Microspores and immature embryos were used as material for the genetic modification of barley and wheat, respectively. For wheat transformation the spring types cvs Veery #5 and Combi, and the winter type cv. Florida were used. Growth conditions of the donor plants, immature embryos, isolation, culture and selection were carried out as described by Becker et al. [2].

Results and conclusions
The first step in the vst1 project was the analysis of the vst1 promoter in barley by means of the uidA reporter gene. An induction of the uidA gene expression by stress in stably transformed suspension cells could be observed. Next followed the stable transformation of barley microspores and immature wheat embryos with the vst1 gene under control of the vst1 promoter. As selection marker, the gene coding for phosphinothricin acetyltransferase (PAT) was used. In all plants analysed, vst1-specific mRNA and the activity of stilbene synthase (STS) could be detected. A first hint for an improved resistance against fungal infection of the vst1 barley was given by reduced leaflet infection of the transgenic line in comparison to control plants of cv. Igri with the fungus Botrytis cinerea [1].

In transformation experiments with wheat and the afp, rip and chi genes, more than a dozen wheat lines were produced. The independence of the lines and the inheritance of the transgenes were analysed by genomic Southern experiments. The expression of the antifungal genes was shown by Northern analysis in the transgenic parental and the following T1 generation. By these two genetic approaches, transgenic barley and wheat plants show additive possibilities, a foreign phytoalexin or overexpressed PR-proteins, to prevent or to reduce fungal infections.

Further experiments in planta and especially with other fungi are necessary in longer scale experiments to support the first results with B. cinerea.

References
1. Leckband G, Lörz H, 1997. Theoretical and Applied Genetics, in press.

2. Becker D, Brettschneider R, Lörz H, 1994. Plant Journal 5, 583-592.