| Maize (Zea mays l.) as an important feed, food and industrial raw materials is sensitive to moderate salinity-alkalinity crops, and only can grow on lower salinity-alkalinity content soil,which limits its planting in the salinas.Due to the salinity resistance is quantitative trait, it is difficult to improve the salt resistance of maize by conventional breeding methods .With the rapid development of transgenic technology, biotechnology has become an effective way to improve the resistance to salts of maize. BcBCP1 gene cloned from Boea crassifolia were transformed to twenty six excellent maize inbred lines,such as Ji815,Nongda178,Zheng58 and others by Agrobacterium-mediated, Gene Bombardment, Ovary Injection and Pollen tube pathway methods,and the effect factors of genetic transformation systems were optimized at the same time .The genetic plants were tested by molecular method and the alkali tolerance resistance. This sudy is aimed at creating new stress resistance genetically modified maize germplasm, while screening and establish effective, stable, widely adaptive genetic and suitable to the cold northeast maize transformation system to breeding genetically modified corn varieties, and accelerate genetically modified maize research and industrialization process, narrowing the gap with the international advanced level and provide technical support and material support. The main research results are as follows:(1)Fifty five good maize inbred lines such as Nongda178,Xiongzhang,K10 and others were used to optimize maize immature embryos genetic transformation system by Agrobacterium-mediated partly. The system of common immature embryos was as follows: infection time was 15min, co-culture time was 3d, the concentration of PPT selection was 2mg/L; the system of immature embryos after cuting root and bud was as follows: size of cutting-up was 1-2mm, bacterial concentration was OD600=0.8; the system of embryo callus was as follows: incubating time 20min, concentrations of AS 100μmol/L; the system of maize multiple shoot was as follows: vacuum dryer 0.05Mpa infection 10 min,concentrations of AS 100μmol/L; the system of germinating embryo was as follows: oscillation incubating time 24h, concentrations of AS 100μmol/L, the concentration of PPT selection was 8mg/L .(2)Six good maize inbred lines such as Ji815,Longxi53,Luyuan92,Nongda178,Sui432 and B73 were used in some optimization of maize immature embryos genetic transformation system mediated by Gene Bombardment. He pressure 1350psi,DNA solution 3u(lcontains 300ug powder and 0.6ugDNA)can significantly improve the transformation rate.(3)Three good maize inbred lines such as He344,Nongda178,Zheng58 were used in some optimization of maize immature embryos genetic transformation system mediated by Ovary Injection.The system of it was as follows: Injection time 18h.(4)Three good maize inbred lines such as He344,Nongda178,Zheng58 were used in some optimization of maize immature embryos genetic transformation system mediated by pollen tube pathway. The system of DNA stigma mediated introduction was as follows: Suitable DNA concentration 100μg/ml, the highest transformation rate were obtained 18h after pollination;the system of pollen-mediated introduction was as follows: Treatment time10min;The system of drop DNA after opening the bract was as follows: the highest transformation rate were obtained 18h after pollination ,Suitable DNA concentration 100μg/ml.(5)36 new T2 generation of BcBCP1 transformed maize germplasm were created from 19 receptorinbred lines tested by PCR, Southern Dot blot,PCR-Southern and RT-PCR.(6)Transform germinating embryo and multiple shoot by Agrobacterium is better than others methods on transformation frequency, positive rate, operating difficulty and survival rate.(7)Nine of T2 transgenic progenies with sufficiency seed quantity were verification alkaline tolerance, among which 5 increased alkali resistance significantly compared with the control, 3 were slightly improved alkali resistance, 1 were unchanged. |
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