The Expression Of A Inducible Male Sterility Gene And A Fertility Restore Gene In Tobaccos And The Transformation To Wheats

Genetically engineered male sterility is one of the important methods to better use heterosis in crops. In our laboratory a chemically regulatable male sterile (RMS) expression system had been constructed based on the antisense actin strategy and putatively transgenic tobacco plants with Kan-resistance had been obtained. In the meantime, an expression system of chimeric male sterility restore gene with barstar had been constructed and Kan-resistant tobacco plants also regenerated. In order to define whether the system can work or not in transgenic plants, and if working, what is their efficiency, this study identified true transgenic plants from Kan-resistant ones on the molecular level first, and then observed their pollen fertility after copper induction by pollen germination in vitro. Meanwhile, two expression vectors mentioned above were also introduced into two weaker winter-type wheat varieties, by Agrobacterium-mediation and/or biolistic bombardment, and the transformants were identified by PCR. Here are the main results:1.Among putative RMS-transformants of tobacco 42 were PCR-positive;2.Among putative barstar-transformants of tobacco 16 were PCR-positive;3.With Agrobacterium-mediation, a total of 53 lines of G418-resistant wheat plant (37 from variety "YM158" and 16 from "YM10") were regenerated from the immature embryo-derived calli transformed with RMS, and 2 RMS-transformants from "YM158" were PCR-positive.4.With biolistic delivery of RMS, 12 lines of G418-resistant wheat plants were obtained from the calli of "YM10", and PCR-positive plant was not observed.5.From barstar-transformed calli of "YM158" mediated by Agrobacterium, 18 plants with G418-resistance were obtained, and 2 of them were PCR-positive.6.From calli of "YM10" bombarded with barstar gene, 85 plants with G418-resistance were regenerated, and 6 of them were PCR-confirmed.7.RMS-transgenic tobacco plants and their untransformed control grew and developed normally before copper-induction. After foliar spraying 0.5-1.5 uM CuSO4, once every 5 days, the flower-buds of some RMS-transformed plants appeared withered, brown or even dropped, although the leaves remained unattached. Upon copper induction, 2 flowers from 1 of 6 transgenic plants in 5 lines were observed phenotype-male sterile, and 1 flower from each 6 plants to have lower rate of pollen germination than that of the untransformed control, with a decreasing range from 9.7% to 81%.8.If sprayed 0.5-1.5 uM CuSO4 every day, the leaves of transgenic tobacco plants turned to wither too, and the damage aggravated with the increase in the copper concentration used and the spraying times.The preliminary results showed the RMS-transgenic tobacco plants were insensitive to copper induction and the RMS system used failed to produce male sterility in transgenic tobacco.