| In this study, some transgenic cotton plants with a phytase gene were obtained successfully via partical bombardment of apical meristems. A large amount of kanamycin-resistant embryogenic callus was obtained via Agrobactetium-mediated method. PCR analysis showed that some plants which transformed by pollen tube-path way contained PhyA gene. This research aim to enhancing germplasm for modern cotton molecular breeding and providing an economic way for the crops utilizing organic phosphorus in the soil.The regenerated plants of cotton from the shoot apical meristem are more easily obtained compared to from other explants. Some naked shoot apical meristems of NDKB18 were bombarded with gold particles coated with the plasmid PC-KSA2300 containing phytase gene (PhyA) and neomycin phosphotransferase (NPTII) gene. Thereafter, a more efficient regeneration system was established by comprehensive contrasts. Apices were cultured on MSB medium supplemented with 30% sucrose. The bombarded parameters were as follows, Helium pressure at 1350psi, the vacuum pressure at 28 inche Hg, target distance at 9cm, Single shot. Subsequently the bombarded meristems were cultured in MSB medium with 1g·L-1 of activated charcoal for 4-7 days, then transferred to the selective medium MSB+Kan (70, 80, 100, 110, 130,140mg·L-1). A total of 15 kanamycin (Kan) resistant plants were obtained through six times of selection. PCR analysis showed that 8 resistant plants contained PhyA gene. PCR-Southern blotting confirmed the integration of PhyA gene in the genome of transgenic cotton. Transformed plantlets could be obtained in about 3 months after particle bombardment. The transformation ratio was 2.97% in this experiment.In this study, the susceptive Agrobacterium EHA105 were introduced the plasmid PC-KSA2300 containing phytase gene (PhyA) , and the Agrobacterium which can using transformation directly were obtained successfully. in order to establish an Agrobacterium-mediated transformation system of cotton, We use the obtained Agrobacterium EHA105 infected embryogenic callus and investigated factors affecting transformation efficiency. The results showed that, Kanamycin has different effect to the different varieties of embryogenic callus and those varieties have different growth trends. The EC were infected within 5-10min and were co-cultured under 18-21℃ for 1-2d in darkness has got better culture effect, after kanamycin selection culture, some kan-resistant embryogenic callus were obtained. The PCR analysis showed that the target gene had been integrated into cotton genome.About of 1400 cotton bolls were transformed by pollen tube-path way, 638 of grown bolls were obtained and the frequence of grown bolls is 45.57%. Cotton seeds from the treated bolls were planted in the growth chamber. When seedlings attained one fully opened leave, 2000mg-L"' of kanamycin was painted on and plants were evaluated for resistance. Therefore, A total of 56 Kanamycin (Kan) resistant plants were obtained, PCR analysis showed that 7 resistant plants contained PhyA gene. |
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