Study On The Breeding Of Disease-resistant Germplasmas Materials In Maize By Using Modern Biotechnology Methods

Immature embryo calli of four maize genetypes (87-1 (R), 87-1 ×Zong3 (G), Zheng22 × 87-1 (H), Zheng22 ×HI (A ×B) (T) )were used as receptor of PAP target gene,which was isolated from pokweed (phytolacca Americana) leaves .The PAP gene and the Kanamycin resistant gene were cotransformed into embrogenetic calli by high velocity micro-projectile bombardment. Before and after bombardment the calli were cultured at a highly osmotic medium (containing 0.4M mannitol for 4h and 16h respectively). Resistant calli were obtained after three generations of selection with 25~40mg.L-1 G418, the concentration range were defined according the result of concentration gradient experiment. On the differentiation media, 18 plants were regenerated from G418-resistant calli. They were identified by PCR, dot-blot and Southern blot. The results showed that 5 seedlings were positive. It indicated that PAP gene had been transferred and integrated into the genomes of maize's regenerated plants, and the transformation-efficiency was up to 27.8%. From this experiment, it could be concluded that co-transformation is an effective method even though target gene and resistant gene were constructed into two different plasmids.Six maize genetypes' embryogenic calli were used as start material for selection of resistant-calli stalk rot mutant by somatic variation. The crude toxin filtrate was extracted from Fusarium graminearum. Pathogenicity test showed that the toxin could inhibit the germination of corn seeds, and growth of corn plumules and radicles. It means that the toxin was effective. Two different screening procedures were used in the experiment, one was from low to high, the toxin concentration was 10%-20%-30%; the other used high concentration directly by 30%-30%-30%. During the process of screening, the effection of inhibitation of toxin extraction on the calli was evident at the first two times and not evident at the third time. Toxin-tolerant calli were obtained after three generations of selection. A lot of seedlings were generated on the differentiation media. However, only few of them could produce seeds after transferring them into fields. Disease-resistant identification of R1 was made by inoculating pathogen, some progeny from regenerated plants showed significantly stronger resistance topathogen than that of control plants, but the others hadn't evident difference in comparison with control plant, such as Hh4-1, Nh1-2, Nh1-3, It could infer that some resistant calli were physiological adaptability, while others were the actual mutants. These results also showed that the resistance at the cell level were not always as same as whole plants.