Study On The Gene Transferring Of Resistance-related Genes Into Wheat By Bombardment

Drought, salinity, low temperature, all these abiotic stresses have adverse effects on agricultural production. Wheat is one of the most important food crops in China, and their resistance needs to be improved. Using genetic engineering for crop improvement is one of the effective way, The tolerance to drought, salinity, low temperature of plants does not only depend on single factor but multiple genes which controll the quantitative traits. Although, using single gene is able to improve drought resistance or salt tolerance to a certain extent, but it can’t make comprehensive improvement resistance of plants .Therefore, improving or enhancing a key transcription factors is the effective ways for the comprehensive improvement of plant to biotic stresses. This study is on the transferring of resistance-related genes into wheat by bombardment, and the establishment of a transformation system for wheat regeneration system and bombardment.In this study, we have the following results:1. The optimization of the transformation system for wheat regeneration system. By comparing the nine winter wheat genotypes of different explants (immature embryos and immature tassel) in the different induction medium, differentiation and rooting medium, obsever the differentiation and seedling differences, we established an efficient wheat tissue culture regeneration system. Efficient regeneration system is the basic guarantee for genetic transformation.The immature tassels’callus induction rate is close to 100%,which is similar to immature embryo , but the differentiation rate of the two receptors is different ,the immature tassels is less than immature embryo, so the immature tassels are not suitable for conversion of the receptor, immature embryo can be used as the conversion of the receptor. Henong 827 had the best tissue cultural performance, the differentiation rate is 77.48%, the worst differentiation rate is 31.20% for the liangxin 99. 2 mg/L 2,4-D, 0.5 mg/L ABA, 10 mg/L VB1 are necessity for the immature embryo callus induction, while glutamine has little effect on it .In the process of callus differentiation, ZT is better than KT. 2 mg/L ZT is the proper concentration. NAA is essential to rooting medium, while VB2 is not the must. Kanamycin screening concentration should be at 15 mg/L and differentiation at the same time. 2. The establishment of bombardment transformation system In this study, we use different powder dosage (50μg, 100μg, 250μg, 500μg), bombardment pressure (1100 psi, 1350 psi) bombardment distance (6 cm, 9 cm, 12 cm), the times of bombardment, resumed culturing, dry culturing, etc. All these processing conditions used with Bombardment (PDS-1000/He) for gene-OsDREB2.2 transferred into immature embryo callus of different genotypes of wheat, According to the rate of green dot after the screening we established and optimized the bombardment transformation system. The results are as follows: 250μg/gun, 1100 psi, 9 cm, bombarding for 1time, a restored culture for two weeks, selected varieties of dried for 12 h.3. Obtained the transgenic plants and the genetic analysis of the OsDREB2.2 Using the system we have built, we obtained the OsDREB2.2 and EeAP2-1.1 transgenic plants.The number of the T0 period plants of OsDREB2.2 is 19, T1 period plants is 35 , lines of the T2 period is 20; the number of the T0 period plants of EeAP2-1.1 is 16. The OsDREB2.2 could transmit to their offsprings, the separation ratio of offspring does not meet the Mendel’s principles, the separation ratio is (0-1.31):1, less than the Mendel’s segregation ratio of 3:1.