| Wheat transformation via microprojectile bombardment with immature embryo culture was strongly depended on the genotypes. This problem restricted the wide application of transgenic wheat to a great extent. Optimization of regeneration system of wheat to overcome the genotype obstacle has great significance in promoting the application of transgenic wheat to a large scale.This study employed 3 winter wheat genotypes H6756, H311 and SP8581 to compare the effect of sampling time, callus induction media, differentiation media and rooting media on wheat young spike culture. The results showed that high frequencies of green plantlet differentiation were obtained when young spikes were cultured between the formation stage of protective glume primordium and formation stage of pistil and stamen primordium. The optimum media for callus induction was MS+2,4-D 2mg/L,for green plantlet differentiation MS, and for rooting l/2MS+0.2mg/L IAA+80g/L sucrose. An efficient regeneration system for young spike culture of wheat was successfully set up.By use of the established wheat regeneration system, young spikes and immature embryos of 17 genotypes of wheat were in vi/ro-cultured to compare the callus induction frequencies and green plantlet differentiation frequencies. The 2 years results showed that there was no significant difference in callus induction frequency between young spikes and immature embryos of all the involved genotypes. But the green plantlet differentiation frequency of young spikes was higher than that of immature embryos in 15 genotypes out of all the 17 genotypes, indicating that young spike was a good explant in wheat in vitro culture.DREB1A gene was transferred via microprojectile bombardment into calli from both proper staged young spikes and immature embryos of H6756 and H311, whose frequencies of green plantlet regeneration from immature embryo calli were lower than 20%. As a result, the frequencies of gene transformation of H311 and H6756 to their young spike acceptor were 2.56% and 5.88%, respectively. While no transgenic plants were obtained in both H311 and H6756 when their immature embryo calli were used as gene acceptors.The drought tolerance of T3 transgenic lines of H6756 with DREB1A gene was identified during the germination and young plant growing periods. Although some transgenic lines showed inconsistence of drought tolerance at germination and young plant growing periods, three transgenic lines, H6756-15-20, H6756-8-18 and H6756-5-15 were selected for all their drought resistant indexes higher than the control at both germination and young plant growing periods. Such results suggested that these three transgenic wheat lines of H6756 with DREB1A gene had improved drought tolerance. |
