| Genetic transformation is an important tool for studying genes function and breedingnew varieties. Wheat (Triticurn aestivum L) is one of the cereal crops recalcitrant totransformation, in literatures, the highest transformation efficiency obtained throughAgrobacterium tumefaciens- mediated was just over 10％, which is far below than that ofrice. One of the main factors limiting transformation is the callus regeneration ability,which is dependent of genotype. Immature embryos are currently the main source forgenetic transformation but mature embryos and anthers have also been used. Matureembryos are arguably one of the best explants for genetic transformation because of itsunlimitedness in sources and no growth season restriction. However, efficient regenerationsystem using the mature embryos as explants in wheat is still not available. This studyaimed at clarifying the genetic basis of wheat tissue culture response and to establish moreefficient Agrobacteriurn tumefaciens-mediated transformation system in wheat.We started with studying the genetic analysis of tissue culture response (TCR) of wheatmature embryos. We found that callus derived from mature embryos of wheat cultivar'Nanda2419' has good regeneration ability. Using a RIL population derived from cross of'Wangshuibai' with 'Nanda2419', QTLs for callus induction from mature embryos andcallus regeneration were mapped. Through whole genome scanning, we identified five, fourand four chromosome regions conditioning, respectively, percent embryos forming callus(PEFC), percent calli regenerating plantlets (PCRP), and number of plantlets perregenerated callus (NPRC). The major QTLs QPefc.nau-2A and QPcrp.nau-2A weremapped to the long arm of chromosome 2A, explaining up to 22.8％and 17.7％of therespective phenotypic variation. Moreover, two major QTLs for NPRC were detected onchromosomes 2D and 5D, which together explained 51.6％of the phenotypic variation.To study the genetic mechanism underlying the TCR of immature embryos of wheat, callus induction and regeneration using the above-mentioned population were conductedtwice. In the first trial, the immature embryos were sampled from plants grown in thegreenhouse in the winter of 2005; in the second trial, the immature embryos were sampledfrom plants grown in the field in the summer of 2006. Through whole genome screening,eleven chromosome regions conditioning percent embryos forming embryogenic callus(PEFEC) and two conditioning the percent caUus-let regenerating plant (PCRP) wereidentified. The main loci QPefec.nau-3B.2,QPefec.nau-5B and QPcrp.nau-3A weredetected in both experiments.Based on this study and other reports, group 2, 3 and 5 chromosomes of wheat areimportant for tissue culture, and the corresponding genes were conserved among cereals.TCR of immature embryos, mature embryos and anthers of wheat might be under control ofsome common or tightly linked genes. The PCR-markers linked to TCR QTLs wouldfacilitate germplasm identification and genetic improvement of wheat tissue culture.To improve the efficiency of Agrobacterium-mediated genetic transformation in wheat,pre-culture times, Agrobacteria strains and cell densities were studied. As a result, 3dpre-culture on the regeneration medium we used is beneficial to transformation of lineB31697. For line B31394, OD600=0.6～0.8 was the optimal cell density of theAgrobacterium strain used in transformation. Among the four Agrobacterium strain usedin transformation, including AGL I, EHA101, EHA105 and LBA4404, LBA4404produced the lowest transformation efficiency. Based on these results, 69 genotypes weretransformed and genotypes with transformation efficiency higher than that of controlcultivars Yangmai158 and Bobwhite were obtained.In addition, we constructed the RNAi vectors of wheat ubiquitin fusion degradationprotein gene (UFD), Betalain Glucosyl transferases gene (BoGT), calreticulin (Crt) and lateembriogenesis abundant protein gene (LEA). We also constructed an over-expression vectorfor the LEA gene.
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