A METHOD FOR ' in embryo' transformation with agrobacterium of wheat and production of transgenic fertile plans over-expressing the AISAP gene, isolated from the halophyte grass ' A littoralis', for improvement of abiotic stress tolerance

摘要

The invention relates to a plant transformation method through treating germinating cereal seeds with Agrobacterium. Seeds of wheat at the early germinating stage were used as receptors and the binary pCAMBIA-1390W plasmid, harboured in a disarmed Agrobacterium strain EHA105, carrying an inserted T-DNA fragment was used as a gene donor. The T-DNA is composed with the hygromycin phosphotransferase gene (denoted bpt, hph or aph IV confers resistance to the hygromycin antibiotic) under the control of 35S CaMV promoter and the AISAP gene (coding for a stress associated zinc finger protein), isolated from the halophyte grass Aeluropus littoralis, under the control of the Act 1 promoter. The recombinant Agrobacterium strain was injected in the embryos of imbibed seeds and during the two days of co-cultivation the T-DNA fragment was transferred to the germ line. Then the seedlings from the injected seeds were incubated in a solution containing cefotaxim to remove Agrobacterium and finally were grown in green house during a whole cycle. ln some of the produced seeds, PCR amplification verified the integration of the alien AISAP and hpt genes into the receptor plant and can inherit to the next generation.