Studies On Transformating Pepc Of Maize Into Wheat Via Particle Bombardment

Immature inflorescences and immature embrys of 16 wheat genotypes obtaining the valuable application in Huang Huai District, were cultured to select the optimum genotype for transgenic acceptor by comparing the callus induction rate, propagation rate, regeneration rate. Construction of wheat expression vectors carrying gus and bar.This experiment was conducted to study sensibility of immature embryos to hygromycin and L-PP,and the optimum selection pressure to hygromycin and L-PPT had been ascertained. Influencing factors of particle bombardment transformating external source genes into immature embryos were studied. Finally, resistant plants obtained were detected.The main results showed as follows:1. Acceptor genotypes screen of genetic transformation from wheatThe results showed that the time of callus induction had no difference among immature inflorescences of 16 wheat genotypes.But the quality of immature inflorescences callus had much difference.The difference among callus induction frequency was very obvious. The difference of propagation rate among genotypes was also existed. The propagation of K35 always kept the highest in four times of subculture. The callus of zhou19,01H186-20-24,2000H841-15-1-3,2000 H 1012-2-1-5,zhengmai9023 grew comparatively more quickly, and the propagation was relatively higher.When the callus of immature inflorescences were transferred into differentiation medium, significant difference of frequency of green spot and green plantlet of immature inflorescences callus among 16 wheat genotypes was existed. Regeneration rate of 16 wheat genotypes came down equally, but the decrease is different among genotypes.Thereby, differentiation ability keeping showed certain difference. The frequency of green spot of zhou19,2000H863-13-4-4,2000H841-15-l-3,2000H1012-2-1,K35 always kept relatively higher in four times of subculture.The frequency of green plantlet of zhou19,2000H863-13-4-4,2000 H841-15-1-3,2000H1012-2-1-5 always kept relatively higher in four times of subculture.The time of callus induction had no difference among immature embryos of 16 wheat genotypes. The quality of immature embryos callus had much difference.The difference among callus induction frequency was not existed. The immature embryos callus of guan35,zhou19,01H186-20-24,2000H841-15-1-3,2000H 1012-2-1-5 grew comparatively quickly, and the propagation was relatively higher. The frequency of green spot of zhou19,01H186-20-24,2000H863-13-4-4,2000H841-15-1-3,2000H1012-2-1-5,K35 always kept relatively higher in four times of subculture. The frequency of green plantlet of zhou19,2000H863-13-4-4,2000H1012-2-1-5 always kept relatively higher in four times of subculture, and they grew exuberantly.Combined with the experimental results on immature inflorescences and immature embryos, zhou19和2000H1012-2-1-5 could be used as optimum acceptor genotypes. The frequency of green plantlet of immature embryos callus was significant higher than that of immature inflorescences callus and materials quantity of immature inflorescences were restricted.Thereby, Compared with immature inflorescences, immature embryos were more suitable to be explants for tissue culture.2.Construction of wheat expression vectors carrying high efficiency photosynthesis genesHigh efficiency photosynthesis gencs(pepc) from the C4 crops (maize) was inserted into expression plasmid and expression vector pPC46 carried the gus gene and the bar gene was obtained. By enzyme restriction and PCR, the vector pPC46 was constructed successfully and efficiently.3.The experiment about sensitivity of immature embryos callus to hygromycin and L-PPTThe sensitivity of zhou19和2000H1012-2-1-5 to hygromycin was different.The resistance of zhoul9 to hygromycin was higher than that of 2000H1012-2-1-5.The concentration of hygromycin 110 mg/L was optimun for immature embryos callus of zhoul9.The concentration of hygromycin 80 mg/L was optimun for immature embryos callus of 2000H1012-2-l-5.The immature embryos callus of zhou19 and 2000H1012-2-1-5 were cultured in subculture medium added L-PPT.But there were no significant difference on immature embryos callus among the groups.Then they were transferred into differentiation medium, the results of which showed that the sensitivity of immature embryos callus of the different groups was different.The differentiation selection concentration of L-PPT 6 mg/L was optimum for immature embryos callus of zhou19 and 2000H1012-2-1-5.4. Study on influncing factors of bombardment transformation system in wheat.By comparing GUS transient expression and green buds differentiation, amount of gold particle, bombardment distance, bombardment pressure and bombardment times were studied.The results showed that the optimum parameters about bombardment transformati- on system were 900psi, 9cm, 250μg/gun and 1 time, respectively.5.Detection of resistant plants.Expression vectors pPC46 was transformated into immature embryos, referencing the bombardment parameters had been optimized, then callus differentiated green buds.Immature embryos had differentiated buds and roots and immature leaves were detected by GUS staining analysis, which showed that expression vectors carrying target gene had been transformated into wheat.