Establishment Of Immature Embryo Transgenic Accepter System And Transformation Of CYP735A Gene By Agrobacterium Tumefaciens In Three Maize Inbred-lines

Maize(Zea Mays L.) is the most important cereal crop and fodder crop in the world. Transformed good gene into fine maize inbred lines by genetic engineering is one of the important approaches for maize breeders currently. In maize transformation study, good receptor and plant regeneration system is not only an important prerequisite for transformation, but also helpful to promote transformed cells differentiation and transformed plants regeneration, and it has important significance for maize improvement and application by genetic engineering. In this dissertation, in this study, three fine maize inbred lines, 18-599, Qi319, A188, were used to establish immature embryo transgenic acceptors and to study the factors affecting transformation process, and CYP735A gene was transformed into embryogenic callus by Agrobacterium-mediated transformation. Maize genotypes and other factors on rates of transformation was studyed. The efficient immature embryo transgenic acceptor provides the basis for transformation of target gene to fine maize inbred lines in the future. Here, the main results were showed as following:1. The immature embryos vaccination time(physiological status) and plant tissue culture conditions plays an important role in callus induction and plant regeneration conditions. The results showed that, the callus from the immature embryos of 10¹5 days after pollinization with length of 1.0².0 mm, and incubated at 25℃in the darkness with scutellum upward grew fasest and formed most typeâ…¡embryonic callus in three maize inbred lines. Higher concentration of 2,4-D in the induction medium was critical factor for producing primary calli. Supplement with 3.0 mg L^-1 of 2,4-D and 0.2 mg L^-1 of NAA in the subculture medium significantly promoted the formation of embryogenic callus for 18-599 and Qi319, while 3.0 mg L^-1 of 2,4-D and 0.2 mg L^-1 of NAA for A188. In process of differentiation, 0.5mg L^-1 KT could promote the differentiation of embryogenic calli. 0.5 mg L^-1 ABT could promote the roots regeneration and numuber.2. The research results of the factors which affected transformation efficiency showed that the transformation conditions for different genotypes were different. The transformation efficiencies of Qi319 and 18-599 were good when Agrobacterium OD₆₀₀ < 0.4, and so were that of A188 when Agrobacterium OD₆₀₀ < 0.2. Optimized cocultivation was performed with filter paper at 20℃for 3 days which was controled easily and beneficial to improve T-DNA transformation efficiency; in the three round of screening culture, the appropriate screening concentration of paromomycin for Qi319 and A188 was 80, 100 and 80 mg L^-1, while 100, 120 and 100 mg L^-1 for 18-599.3. CYP735A gene was transformed into embryogenic callus from three maize inbred lines by Agrobacterium-mediated transformation with the optimized transgenic acceptor system. About 300 seedlings were obtained after differentiation culture and about 30% transplanted seedlings were survilal of which 8 transgenic plants of Qi319 and 4 transgenic plants of 18-599 were PCR-positive. Two of these Qi319 PCR-positive transgenic plants are RT-PCR-positive. A total number of 3 RT-PCR-positive plants of 18-599 were obtained. Preliminary results have confirmed that CYP735A and NPTâ…¡gene have been integrated into the corn genome.4. Embryogenic callus induction of maize was affected by genotypes and callus culture conditions, The callus induction rate of different genotypes was different, and were respectively 94.5%, 93.1% and 85.2% for 18-599, Qi319 and A188. The callus induction rate of 18-599 and Qi319 is higher than A188, and callus growth is good. They are the good materials for transgenic research. But the callus of A188 is white and not suitable for several times of successive transfers of culture.