| Wheat is one of the earliest plants in the world and also one of which has the widest planting area in our country. It is difficult to meet the requirement of human beings in the modern society on wheat yield and quality by the traditional breeding methods. Using transgenic technology for orientation improvement of wheat has attracted great attention and it will gradually become an effective assistance ways for developing new varieties. Studies have shown that, selecting the appropriate explants、genotypes and the composition of plant hormone had great influence on vitro regeneration and tissue culture. Wheat mature embryo regeneration and genetic transformation efficiency will be enhanced. One purpose of this experiment is to optimize the system of the wheat mature embryo regeneration with Xinong 529. The second is to selected wheat genotypes with high regeneration frequency from the 20 varieties investigated. The third purpose of the study is to explore the influencing factors by microprojectile-bombardment transformation of wheat mature embryos, the results were as follows:1. Callus induction frequency of mature embryo with cutting embryo was obviously greater than scraping pieces with a significant difference. Moreover, the speed and quality of callus formation from mature embryo with cutting was best. The frequencies of calli induction with Ma、Mb、Mc induction medium from Xinong 529 shared a similar level of above 50%. The selected Ma induction medium could obtain the highest callus rate、differentiation rate and the best quality of callus. When the KT concentration of 1.0 mg·L-1, the differentiation efficiency was the highest(34.61%). The differentiation media supplemented with 1.0 mg·L-1 KT、0.2 mg·L-1 NAA and 1 mg·L-16-BA was the optimum media for plantlet regeneration.2. 20 varieties or strains were examined by the mentioned regeneration system, obtaining 14 varieties or strains, whose callus rates above 80% and had a significant difference with the others(P<0.01). Zhengmai 366 and Z50 holding higher callus rates than control. The differentiation rate of 20 varieties varies between 16.9% to 44.2%. Otherwise, Zhengmai 366 、Z50、Chuanmai 42、Jimai 61 also possess higher differentiation rate than Bobwhite, with the highest strains “Zhengmai 366” reaching 44.2%.3. The frequencies of calli induction from Z50, Zhengmai 366 shared a similar level of above 85%. Xinong 529 got the lowest Callus induction rate(77.97%)and Embryonic callus rate(58.26%), meanwhile had a significant difference with the others. The differences among genotypes had significant effects on the regeneration rate of wheat callus of mature embryos. Zhengmai 366 had the highest regeneration rates with 39.26%.The microprojectile-bombardment transformation results showed us osmotic treatment inhibited growth of the callus in regard to fresh weight reduction. Mannitol as osmosis agent had more inactive effect on the growth and regeneration of callus than sucrose after bombardment. With dosage of gold powder 100 μg per gun plant, had no significant difference in regard to the level of GUS transient expression with gold powder 60 μg.The optimal distance between DNA-gold pellet and the target cells was 9 cm. The parameter combination of hyperosmosis by sucrose, 60 μg gold powder and 9 cm target distance exhibited good regeneration and the highest level GUS transient expression. Under these optimal conditions, the frequency of transformation for mature embryos of wheat was up to 0.67%.The transformation system of mature wheat embryos using particle bombardment has been optimized preliminarily in this study. |
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