| The in vitro culture and regeneration of wheat compared with rice and maize are more difficult. Although the immature embryo is the most ideal explant material, however the suitable period of material selection is very short and difficult to grasp. So we can make use of the more convenient mature embryo and the potential large immature embryo to induct multiple shoot and establish multiple shoot induction system of wheat embryo. Then we can lay a foundation for the genetic transformation research of wheat. In the methods of exogenous gene transformation of wheat, the method of Agrobacterium-mediated transformation is a natural system. There are many advantages such as simplicity, cheap and single copy integration compared with biolistic bombardment. But there are many factors affecting the efficiency of Agrobacterium-mediated transformation. It is still a problem in the transformation of wheat.This experiment uses the mature embryo and the large immature embryo of different wheat varieties as the explant for culture. Through studying the influence of TDZ and 6-BA to multiple shoot induction, we can establish the system of multiple shoot induction. In addition, this experiment uses the immature embryo callus of 13 wheat varieties as receptor materials. By comparing the GUS gene transient expression rate, we intend to obtain the wheat genotypes that are sensitive to Agrobacterium. At the same time, we study the bacteria concentration and infecting time in the process of transformation to provide reference for establishing Agrobacterium-mediated wheat transformation system. This experiment mainly got the following conclusions:1.This experiment used the mature embryo of wheat and the large immature embryo which was about 18 days after pollination through 10 weeks induction training on the culture medium with TDZ and 6-BA and established the system of multiple shoot induction of wheat embryo. Firstly, we vaccinated embryo on the culture medium with TDZ and 6-BA under the condition of dark for three weeks. It was necessary to culture in low light intensity for one week and cultured in high light intensity for three weeks. Then the material could culture in high light intensity for another three weeks after successive transfer. After ten weeks of training, the embryos of wheat would induct 6.7-17.6 multiple shoot.2.We compared the influence of TDZ and 6-BA to multiple shoot induction based on different wheat varieties. The results showed that there were differences between multiple shoot induction and the optimal concentration of plant hormone in wheat varieties and the mature embryo induction effect was better than the large immature embryo: Under the condition of 2mg/L TDZ and 1mg/L 6-BA, Xinchun 9 and Zhengmai 7698 obtained the maximum number of shoot, reaching 17.6 and 16.5 respectively. It indicated that the induction effect was the best for Xinchun 9 and Zhengmai 7698 in the treatment. The condition of 2mg/L TDZ and 2mg/L 6-BA was more conducive to the multiple shoot induction of mature embryo from Changhe 25. The average induced shoot was 10.7. In large immature embryo aspects, under the treatment of 2mg/L TDZ, the induction effect was the best for Zhoumai 27. The maximum number of shoot was 6.7. The treatment of best induction effect for Zhengmai 7698 was 2mg/L TDZ and 1mg/L 6-BA. The average shoot was 9.5.3.This experiment used Agrobacterium strains with GUS gene to infect immature embryo callus from 13 wheat varieties. Through the comparison of GUS gene transient expression rate, we found that Xinchun 9, Xiaoyan 328 and Jimai 19 were the most sensitive to Agrobacterium. The transient expression of GUS was above 90%. They were appropriate as Agrobacterium-mediated transformation material. The experiment used the immature embryo callus of Xinchun 9 as the infecting material under the conditions of different bacteria concentration and infecting time. The results showed that there were no significant differences among this treatments. It was more appropriate to transform gene through Agrobacterium under the conditions of OD600=1.0 for bacteria concentration and 15 min for infecting time. |
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