Preliminary Study On Transgenic Maize Resistant To Viruses

New transgenic plants expressing various enzymes were conducted to initiate studies to evaluate broad spectrum resistance to viruses. The Ribonuclease L (RNase L) and 2',5'-synthetase (2-5A) genes were co-transformed into immature embryos of the inbred maize lines Z3 and Z31 by Agrobacterium tumefaciens LBA4404. In addition an RNase III gene and its mutation (Glu 117 Lys) were used to transform additional maize lines as above. After 11 weeks of selection by G418 on D medium, resistant calli were transferred to a high sugar medium for development. At the same time, a partial cDNA clone of the rice black streak dwarf virus (RBSDV) segment SI in pAct-Sl and the bar gene in pAHC25 were co-transformed into maize calli through bombardment with a gene gun.Ninety-four plants were regenerated with the 2-5A system and PCR-Southern blot analyses revealed that seven lines positive for both 2-5A and RNase III (Double Positive) were obtained from Z3, and 12 double positive lines were obtained from Z31. The RNaseIII transformations yielded 124 regenerated plants of which 51 contained wild type RNase III and 73 had the mutant RNase III. The highest efficiency of transformation was 8.3% by PCR-Southern assay. Thirty-six plants were regenerated from resistant calli transformed with pAct-Sl and in this case, the highest positive transformation efficiency was 1.54% and the lowest was 0.42%. PCR-Southern analysis indicated that 7 positive plants were obtained, but 4 of these were sterile. PCR and Southern analyses of the T] generation plants showed the pAct-Sl gene was stable in these transgenic plants.A prokaryotic expression vector containing a fusion of the 464 N-terminal amino acids of the P2 protein of RBSDV-Hbm was constructed and expressed as a fusion protein (P2-N, 66kDa) in E. coli. The purified protein was used to immunize rabbits for preparation of antiserum to characterize P2. The resulting antibodies are in the preliminary stages of testing.