| New Chinese spring wheat varieties (lines) with desirable agronomic and yield characters were used as explants to investigate the tissue culture and genetic transformation parameters in this study. The plasmid pDM803, carrying the bar gene, conferring resistance to the herbicide, and the screenable uidA gene, was transferred into wheat by particle bombardment. The main results showed as following:1. This is the first report about the effects of 2, 4, 5-T and NAA on wheat tissue culture, particularly in comparison with 2, 4-D in detail. The frequency of embryogenic callus and increment of callus fresh weight on 2, 4, 5-T media were higher than those on 2, 4-D media, and in the presence of 2, 4, 5-T the precocious germination of explants for all genotypes were significantly suppressed. For immature embryos cultured on MS medium, 2 mg/L of 2, 4-D was optimal, and the highest frequency of embryogenic callus (33.50%) was observed. For the mature embryos on N6 medium, 4 mg/L of 2, 4-D was optimal.2. The results indicated that the differentiation frequency (62-76.39%) of callus from immature was higher than that(38.57-47.76%) of callus from mature embryo. Proper partial desiccation of callus before transferred to regeneration medium could improve the frequency of plant differentiation. Different hormones combinations (KT , IAA , NAA , TDZ , 6-BA and ZT ,etc. ) significant affected the bud differentiation frequency , among which , 0.5mg/L TDZ increased the frequency of shoot formation. Supplementing with 0.5mg/L IBA and 0.25mg/L NAA in rooting medium was optimal. Choline Chloride and Paclobutrazol could facilitate root formation.3. We have first reported that the effects of AgNCh on tissue culture of common wheat. Addition of AgNO3 could significantly improve embryogenic callus frequency and callus growth, whereas it could reduce the callus necrosis and callus induction frequency. Strong genotypic effects were observed on callus induction frequency, embryogenic callus frequency and callus necrosis frequency among the four genotypes. It also was found that there were significant genotype >3 concentration in the different stage of wheat immature embryos culture.4. Growth inhibition of Agrobacterium by Carbenicillin and Cefotaxitin was valuated. Cefotaxitin was significantly more toxic than carbenicillin in induction of callus in wheat. lOOmg/L Cefotaxtin almost inhibited Agrobacterium growth, while Carbenicillin was 250mg/L. The induction frequency of callus was 84.7% supplementing with 250mg/L Carbenicillin in medium. Carbenicillin of low concentration showed auxin effects in tissue culture and disturbed hormone proportion. Thus, the ideal concentration of carbenicillin was 500mg/L when used in genetic transformation of wheat mediated by Agrobacterium-mediated. Wheat callus was more sensitive to hygromycin and G418 than to kanamycin. Hygromycin and G418 as the selection marker in genetic transformation of wheat was optimal, and the ideal selecting pressure was 25mg/L.5. The objective of this experiment was to optimize the major parameters controlling DNA delivery by particle bombardment to immature embryos of elite Chinese spring wheat {Triticum aestivum L.) cultivars. Efficiency of DNA (uidA gene) delivery was assessed by determining transient GUS (p-glucuronidase) expression in bombarded calli. Among the estimated parameters, acceleration pressure, bombardment distance, chamber vacuum pressure, bombardment times and osmotic conditioning of culture had significant effects on the transient gene expression. A bombardment procedure suitable for elite Chinese spring wheat cultivars was developed, allowing a highly efficiency DNA delivery with reduced damage of target calli. A total of 429 resistant calli from cultivar Chuannongl6 were obtained and 59 of them regenerated plants. PAT activity and the bar gene in transformed plants were detected by leaf painting with Basta solutions and PCRamplification, respectively. A total of 13 herbicide-resistant and partially resistant plants were screened out of 59 regenerated plants, among which 6 had the expected fragment corresponding to the bar gene PCR amplification. Thus, the transformation frequency of Chuannongl6 was 1.40% by PCR result.6. The mature seeds were implanted on Petri dish, in which concentration of Basta was 0, 3, 6, 9 and 12mg/L respectively. The sensitive responsibility of 4 genotypes with Basta were estimated. The results showed that seedling could be refrained and the rate of etiolation rate increased with concentration improving of Basta. Effects on seedling height and root length of Basta were significant difference. Etiolated seedlings appeared after sowing 4-5 days when concentration was 9mg/L. The selection effects to wheat with this treatment was appropriate. Spraying the offspring of 6 transformation lines with 0.25% Basta in field, the segregation Basta resistance in Ti was investigated. The results showed the bar was inherited as Mendelian single gene in offspring of all lines contained the bar gene. The segragation of resistance : sensitivity to Basta was 3:1 by X2-test. |
