| Wheat is one of the most important food crop which is planted widely in the world. However, wheat is the last one of the gramineous crop which was transformed successfully. The main reason is that the transformation mediated by Agrobacterium tumefaciens or microprojectile bombardment depend on tissue culture and regeneration, which is influenced greatly by genotype. Only a few highly regenerated varieties can be transformed, while a large quantity of elite cultivars can not be transformed successfully due to their poor regeneration property. This result in the transformation of wheat develops slowly and can hardly be applied in breeding practice. The development of foodstuffs processing and improvement of living standard have created a great demand for high quality bread-making wheat. The high molecular weight glutenin subunit (HMW-GS) are closely associated with the bread-making quality of wheat flour. Therefore, It is theoretically and practically significant to use genetic transformation of high quality HMW-GS gene to improve wheat quality.This investigation, based on the clone of HMW-GS 1Bx14 gene, the plant regeneration system of elite wheat cultivars was studied, and the immature embryo was transformed by Agrobacterium tumefaciens and microprojectile bombardment with the high quality HMW-GS gene 1Bx14. The method of pollen tube pathway was also explored. The major results are as follows:1. The callus induction and plant regeneration of immature embryo, immature inflorescences and mature embryo from different common wheat cultivars was investigated. The result showed that the frequency of callus induction was higher than 90 percent and there was no significant difference between explants and varieties. While the frequency of callus differentiation and plantlet regeneration of different wheat Varieties and explants are quite different. Callus induction, differentiation and regeneration were independent, and there was no significant correlation among them. Generally, the callus differentiation and plantlet regeneration frequency of immature embryo was highest, immature inflorescence take second place, and mature embryo is lowest. The frequency of immature inflorescence callus differentiation rank downward in the order Mianyang 19, Luoyang 8716, Xiaoyan107, Shaan354, Xiaoyan22, Shaan150, Zhengmai 9023, Xinong 88. The frequency of immature embryo callus differentiation, from high to low, was Mianyang 19, Xiaoyan107, Xiaoyan22, Zhengmai 9023, Luoyang 8716, Shaan354. The frequency of mature embryo callus differentiation was Shaan354 > Mianyang 19 > Luoyang 8716 > Xiaoyan107 > Xiaoyan22 > Zhengmai 9023. Most of the differentiated callus of Mianyang19 formed multiple shoot clumps and the frequency of regeneration was highest among all the varieties, therefore the immature embryo of Mianyang 19 can be the most ideal explants for transformation.2. In order to improve bread-making quality of flour and produce transgenic plants free of selectable markers, a linear DNA construct consisting of a minimal expression cassette with the HMW-GS 1Bx14 gene was transformed into wheat cultivar Mianyang19 by microprojectile bombardment. The transformants were selected by PCR instead of markers gene. 2480 callus of Mianyang19 was transformed and seven transgenic plants were identified from a total of 1,219 transformants, yielding a transformation frequency of 0.28%. Four of the seven transformants have the normal phenotype, and the other three(A1-14, B2-8 and D5-10)demonstrate phenotypic variation among which two transformants (B2-8 and D5-10) could not form seeds and A1-14 had only 3 seeds. An SDS-PAGE analysis of 82 T1 seeds confirmed that the 1Bx14 gene was expressed in the progenies of three transgenic T1 seeds (L2, L4 and L5), while the expression was weaker than other endogenous subunits. The three T1 progenies of A1-14 were male sterility. it is feasible to obtain marker-free transformants using the linear expression cassette transformation approach coupled with PCR selection.3.The plant expression vector of HMW-GS 1Bx14 gene was constructed, and a total of 652 callus of Mianyang 19 immature embryo was transformed by microprojectile bombardment. After selected with hygromycin, 11 callus was differentiated to plantlet and 8 of which survived in greenhouse. The result of PCR and PCR-southern showed that 1Bx14 have been transformed into seven plantlet and the frequency of transformation was 1.07%. Three transgenic plants demonstrated the expression of transgene and the expression of other subunits was suppressed in varying degrees.4. The genetic transformation systems of wheat Mediated by Agrobacterium was investigated. The result showed that Luoyang 8716 was more sensitive to Agrobacterium strain LBA4404 than Mianyang 19. For the two varieties, the infection frequency of Agrobacterium strain EHA105 was higher than LBA4404. The optimal procedure to Mianyang was OD600 0.8×30 min and OD600 1.0×30 min for strain EHA105 and LBA4404, respectively. To Luoyang 8716, the Optimum procedure was OD600 0.8×30 min and OD600 0.8×60 min, respectively. Selected by hygromycin and analyzed by PCR, two positive plant was gained from 483 callus of Miangyang 19, and one was got from 321 callus of Luoyang 8716 using Agrobacterium strain LBA4404. Using Agrobacterium strain EHA105,five positive plant was regenerated from 452 callus of Miangyang 19, and two from 315 callus of Luoyang 8716.5. The HMW-GS 1Bx14 gene was transformed into five wheat varieties by pollen tube pathway, and transformants were identified by PCR. The result showed that the differences between varieties and treatments were significant, and the average frequency of transformation was 0.56 %. The transformation frequency of Shaan354 by treatment 2 was the highest (1.01%), follow by treatment 4 (0.93%). The frequency of Shaan 893 by treatment 1, treatment 3 and Xiaoyan 107 by treatment 1 was over 0.8%. Therefore it is a potential way to transform wheat by pollen tube pathway.
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