Establishment Of Hygromycin Selection System In Wheat And Rice Gene Ghd7 Transformed To Wheat

Wheat is one of the most important food crops in the world,developing high yielding variety is one of the most important goals of breeders.The traditional breeding mainly used hybridization to broke three factors,which are negatively related to wheat production,for the high yield of wheat,while it cost long time and greatly influenced by environmental factors;Transgenic technology cost less time and can directly transform the exogenous gene to explants,was widely applied in wheat breeding.The mature embryo induction and immature embryo calli of zheng 9023 was used for researching the influence of hygromycin on the regeneration to establish hygromycin selection system in wheat;Immature embryo calli of zheng9023,han6172,the dragon wheat 31,the dragon wheat 31 and the mature embryo induction of zheng9023 was used for genetic transformation of rice pleiotropic gene Ghd7 by particle bombardment.The main results were as follows:1.The sensitivity to hygromycin of mature embryo calli and immature embryo induction of zheng 9023 was studied.The results showed that 50 mg/L hygromycin influencing for 30d was suitable for screening after transformation;The PCR analysis for hpt showed that the positive frequency were 86.33%and 91.67%,it indicated that 50mg/L hygromycin selecting for 30d was significant for screening,therefore,the hygromycin selection system of genetic transformation in wheat was established.2.Directly screening and screening after 15d convalescence was used for selecting immature embryo calli of four varieties.Regeneration frequency of convalescence respectively increased by 38%,12.59%,153.25%and 12.59%than directly screening,indicated that convalescence could promote regeneration of transformed calli.Different degree of promotion indicated that there were differences on hygromycin sensitivity among different genotypes.3.The four varieties immature embryo calli and mature embryo induction of zheng9023 transformed by particle bombardment,and got 279?566 resistant plants respectively,which could complete their normal life cycle.4.GUS staining of resistance tissue after genetic transformation,young plant,floral and root showed GUS positive,indicated that gus integrated into the wheat genome and could be passed on to offspring.5.PCR analysis for Ghd7 of part of TO generation from immature embryo calli and mature embryo induction showed 81.96%and 70.83%positive efficiency respectively.Ghd7 PCR analysis for 12 lines of Ti generation showed 77.59%PCR-positive,and 10 of them appeared separation with 3.46:1 separation ratio,indicated that Ghd7 integrated into the wheat genome and can be passed on to offspring;12 lines of T2 generation showed 81.82%Ghd7 PCR-positive and there was still a certain degree of separation;it was a further prove of that Ghd7 had been integrated into the genome of wheat.6.The genetic transformation efficiency of immature embryo calli and mature embryo induction was compared,the transformation efficiency?regeneration frequency?GUS-positive ratio and PCR-positive ratio were increased by 8.07%?344.66%?29.57%?5.34%respectively;it indicated that mature embryo induction and immature embryo calli both were excellent receptor for genetic transformation.