Optimization Of Regeneration System Of Tissue Culture And Transformation Of KN2 Gene In Wheat

Wheat tissue culture and regeneration system is not completely established as in rice, the efficiency of genetic transformation is much lower than that in rice. Optimization of regeneration system of wheat will be significant in promoting the application of transgenic wheat in large scale. In recent years, low-temperature is a common stress that always has adverse effect on the growth and productivity of wheat. In this study, the KN2 gene encoding superoxide dismutase cloned from Antarctic fish was transferred into wheat to improve their capacity of cold-resistance.Ten wheat genotypes were used to compare the effect of different media on callus induction and differentiation and effect of different ways of inculation, subculture and culture period before transformation on wheat immature and mature embryos culture and genetic transformation efficiency. The result showed that the callus induction media supplemented with 0.2mg/L ABA,1.25mg/L CUSO4 was the optimum media for callus induction and differentiation, the differentiation media supplemented with 5mg/L KT, 0.25mg/L CUSO4 was the optimum media for plantlet regeneration. High frequencies of plantlet regeneration were obtained when whole callus was used to subculture. Pre-incubation of wheat immature embryos for 4 days before transformation seemd to be the optimum pre-incubation period. Half-matured embryos inoculation and callus induction media supplemented with CH, VB1, Pro and Gln were both benefit for culturing mature embryos.Expression vectors carrying KN2 gene and Bar marker gene were transformed into immature embryos from L23, K199, Bobwhite to improve their capacity of cold-resistance using particle bombardment. Two hundred and sixty-five resistant plantlets from immature embryos of L23 were obtained. PCR analysis identified 92 Bar-positive transgenic plants,98 KN2- positive transgenic plants and 37 double positive transgenic plants. The transformation frequencies were 14.91%,15.88% and 6.00%, respectively. The KN2 gene and Bar marker gene were also transferred into the callus derived from ten-week induction of immature embryos of L23, K199, Bobwhite. Forty two resistant plantlets were obtained. PCR analysis identidfied 17 Bar-positive transgenic plants,19 KN2- positive transgenic plants and 6 double positive transgenic plants. The frequencies of gene transformation were 1.58%,1.77% and 0.56%, respectively. In addition, we also obtained transgenic seeds from 6 L23,4 K199 and 7 Bobwhite regenerated plants.