Yuko Mizukami*, Isao Houmura* and Yoshimi Okada**

* Aichi-pref. Agri. Res. Center, Aichi 480-1193, Japan

**Teikyo Univ. Utsunomiya 320-0003, Japan

Viruses often damage alfalfa cultivation and seed production. In order to control of plant virus diseases, transformations of virus coat protein (CP) genes have been tried for several years. However, CP mediated protection seem to be effective against viral strains closely related to the transgene. The RNase genes derived from yeast, Pac I, are specific to double-stranded RNA. It would be expected to attack replication intermediates of RNA viruses. Transgenic tobacco with pac I showed decreases and delay in appearance of symptoms with some viruses and viroid (Watanabe, et al., 1995 and Sano, et al.,1997).

Transgenic alfalfa were produced by inoculation of cotyldon and embryogenic calli with Agrobacterium tumefaciens LBA4404, harboring the vecter PBI121 containing pac I. The explants were co-cultivated with Agrobacteria for three days on SH medium added with 50mg/l acetocyringon. After disinfecting by 100mg/l cefotaxim, alfalfa calli were proliferated and selected to be resistant to 50-100mg/l kanamycin until regeneration to plantlets. Polymerase chain reaction amplification of genomic DNA isolated from regenerants confirmed the presence of the selectable marker gene and pac I. Currently, the integration and expression of pac I and its effects of resistant levels to Alfalfa mosaic virus (AMV) in trangenic alfalfa plantlets are under examinations.

Watanabe, Y. et al. 1995. Resistance against multiple plant viruses in plants mediated by double stranded-RNA specific ribonuclease. FEBS Letters 372:165-168