Genetic Transformation Of Wheat And Wheat Leaf Base Embryogenic Callus Induction And Plant Regeneration

Immature embryos of three varieties of wheat (Triticum aestivum L.) were transformed using Agrobacterium tumefaciens strains: EHA105/pC3301 and/or AGL-1/pD805. Factors that influence Agrobacterium- mediated transformation of wheat were investigated. Media added with 250 mg/L Cef and 100 mg/L Cb were efficient to kill bacterium during selection as well as had no negative effects to the cultures. Concentration of the strain, time of inoculation, temperature and time of co-culture had co-effects on successful transformation. Lower temperature (23-24℃) and shorter time of co-culture (3-4 d) were helpful for the differentiation of resistant calli. Immature embryos pre-cultured 1-4 d was the most adaptive explants to transformation. Adding of AS in co-culture medium enhanced the transfer of T-DNA. Washing after co-culture with liquid medium containing antibiotics was better than with either sterile water or 0.1% mannitol. Comparing three selection methods, we found that shortening the time of callus inducing selection as well as elongating the time of shoot regeneration selection under middle-strength L-PPT (3-4 mg/L) were helpful for survival of transformed plantlets. Two transgenic wheat plants were obtained from Yangmai158 transformed with EHA105/pC3301 and the integration of transgene was confirmed by PCR and southern blot analysis.Immature embryos 3-5 d pre-cultured of G8901, a wheat line weakly tolerant to Agrobacterium damage, were transformed by particle bombardment using the plasmid pC3301. Two transgenic wheat plants were obtained and the transformation event was verified by PCR and PCR-southern blot analysis. While they survived selection and transplanting, they were abnormal and sterile. A short-term regeneration system from leaf-base-derived callus of wheat(Triticum asetivum L.) was developed. Embryogenic callus formation and shoot regeneration were achieved from the first basal segments of three to four-day-old seedlings. Callus formation frequency as well as plantlet regeneration frequency was dependent on the composition of basal medium and the concentration of 2,4-D. MS medium with 4.5 μmol/L to 9.0μmol/L 2,4-D was optimal for the culture of wheat leaf base. Effects of different combinations of plant growth regulators, which were added in either callus induction medium or shoot regeneration medium, were tested. Adding of BAP in callus induction medium shortened the time of shoot emergence but could not improve the producing of embryogenic calli and green plantlets. Optimal ratio of 2,4-D, BAP and NAA gave similar regeneration frequency to control. Existence of cytokinins in regeneration medium had no effect on increasing the regeneration frequency. The regenerants could grow to normal, fertile plants after they were transferred into soil.