BASIC RESARCH FOR IMPROVEMENT OF COLD AND DESEASE RESISTANCE OF PERRENIAL WEED PLANTS BY GENETIC ENGENEERING

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The goal of this research was to generate weeds that has increased tolerance to freezing and disease by genetic engineering. Experiments were focused on (1) isolation of genes that can confer plants with tolerance to freezing and disease, and (2) production of transgenic alfalfa plants by using the isolated genes.As a candidate gene for production of transgenics, WAS-3 gene, which has similarity to antifreeze proteins and antifungal proteins was isolated from winter wheat that has high degree of winter-stress resistance. The WAS-3 protein produced in transgenic wheat cells efficiently blocked growth of hyphae of the snow mold, Microdochium nivale. In addition, transgenic plants expressing the WAS-3 protein exhibited a better resistance to Fusarium oxysporum than control non-transgenic plants. We also isolated genes encoding dehydrins and group III LEA from mulberry, which exhibits extremely high freezing tolerance in winter, and several ABA and cold-responsive genes from the moss Physcomitrella patens.For production of transgenic alfalfa plants, we used Arabidopsis CBF3, a gene encoding a transcription factor that plays a critical role in the up-regulation of a number of cold-inducible genes. Agrobacterium tumefaciens carrying a construct for overexpression of the CBF3 gene was used for inoculation of leaf discs of alfalfa (Medicago sativa var. Regen-SY). Kanamycin-resistant calli were selected and shoots were regenerated by further cultivation. Nine transgenic plants were successfully regenerated. These transgenic plants showed normal growth indistinguishable from non-transgenics, suggesting they have less somatic mutations. These transgenic plants were being used for the freezing tests.