Studies On The Optimization Of Wheat Genotypes And Culture System And Transferring Anti-TrxS Gene Into Wheat

The objective for this study was to transfer Anti-TrxS into wheat cultivars. Three aspects was studied in this paper. Firstly , selecting the optimal genotypes as receptors for wheat genetic transformation and optimize the culture system of immature embryo; Secondly, transferring Anti-TrxS into wheat by methods of microprojectile bombardment, pull en tube pathway and ovary injection;Thirdly, analyzing Anti-TrxS Gene of the transformed plants by PCR. The main results were as follows:1. In order to select the optimal wheat genotype for immature embryo culture and gene transformation. 19 wheat genotypes were studied. The result showed that the difference among the genotypes was significant. Yumai70 had high rate of callus induction (95.5%), but the frequency of green primordial differentiation was only 65.4%; Yumai66 had the highest rate of green primordial differentiation, but the frequency of plant regeneratation was only 21.7%;Guomai No.l had the lowest frequency of green primordial differentiation(39.1%), but half of the green primordials could be differentiated into plantlets; 89 (117) had high frequency of callus induction (93.8%), green primordial differentiation's.3%) and plant regeneratation(32.2%) respectively. So it was the best wheat genotype for genetic transformation. Yumai 18-64 also had the higher frequency of plant regeneratation(2 7.3 %).2. The factors influencing culture ability of immature embryo,such as genotype, medium and physical treatment were studied in the process of the immature embryo culture.The result showed that immature embryos of different genotypes required different embryo age, medium and culture condition. Media with different ratios of exogenous hormones effect the differntiation ability of immature embryossignificantly. In the inducation culture,there was difference among different genotypes,but they had a same trend. There was significant interaction between genotype and medium in the differentiation culture. The treatments of desiccation, refrigeration before the differentiation culture can significantly affect the plant regeneration of the calli.The optimal culture system for immature embryo from 89(117), Yumai18-64 was 0.7-1.2 mm(dimeter)immature embryo in the size-on the inducation medium IV (MS + 2,4-D 2 mg/L + ABA 0.5 mg/L) and dark culture for 20 days -on subculture medium?(MS + 2,4-D 1 mg/L + ABA 0.5 mg/L + KT 1 mg/L) for 15 days -refrigeration for 10 days-on the differentiation medium E or I(E:MS+ZT4/NAA/1.5mg/L; LMS+6-BA2/IAA 0.5mg/L) for 15 days-on rootage medium-translpant into pots.3. Based on the optimization of genotype and culture system, the Anti-TrxS and bar genes were co-transferred to the immature embryos (calli) from Yumail8-64 as receptor by bombardment. 3~5 mg/L Bialaphos was added into the medium to select resistant callus during callus induction and regeneration culture. 32 regenerated plantlets were obtained and 11 of them grown normally.4. Anti-TrxS. and Bar genes were co-transferred to 2036 flowers of receptors, Yumai18, Yumai34,Yumai47, Yumai49,Yumai70,Zhengmai9023,OOzhong13, Jimai No.2, YangaoNo.1, 95519, Zhengxin99K Zhengxin992 and Luomai No.4 through pullen tube pathway. 1616 seeds from transferred receptors with a percentage of 79.4 % and 1424 plants from1616 seeds with a percentage of 88.1% were obtained. Leaf genome DNA from T1 plant of different genotypes and treataments were analyzed by PCR and 16 positive lines were obtained among 31 tested lines.5. Two genes were also co-transferred to 1206 flowers of Yumail8,Yumai49 Yumai70,Zhengxin991, 95519 by methods of ovary injection. 89 seeds (7.4% of the flowers) and 41 plants (46.1% of seeds)were achieved. Leaf genome DNA of Zhengxin991 TI plants strain was identified by PCR analysis.