Research On Multiple Shoots From Wheat Immature Embryo And Mature Embryo

Wheat is one of the most widely grown crops in the world, Improving quality, enhancing resistance and increasing production has became very important. Genetic transformation is an important means of improving wheat varieties, while establishing the efficient system of in vitro tissue culture regeneration is a basis for genetic transformation of wheat. At present, the main recipient of wheat genetic transformation is Callus of wheat immature embryo. However, Promotion and application of Wheat Callus used as the receptor of genetic transformation is constrained by several factors, such as drawning by seasonal restrictions, chimeric phenomenon of transformed plants and depending on the gene gun. Shoot tips as a receptor for genetic transformation in rice, corn and other crops have been applied, but it has not successfully reported because of the poor culture characteristics of the wheat stem tip in vitro. While shoot tips as a receptor will provide new ways and methods for establishing Agrobacterium-mediated genetic transformation system and solving the chimeric phenomenon of genetic transformation. Wheat immature embryo and mature embryo used as the experimental material to study the differentiation about multiple shoots through optimiating impact factor of pretreatment, germination and induction. In order to exploring the conditions and factors of effect on inducting multiple shoots from wheat immature embryos and mature embryos, setting up the efficient system of multiple shoots from wheat immature embryo and mature embryo and providing better receptor of genetic transformation. Achieving the following researches:1. In order to solve the phenomenon of chimera in vitro shoots translating directly into seeding, wheat immature embryo of five common varieties were used as the experimental material to study the effect of TDZ, 6-BA, 4-PU, TDZ combined IBA and TDZ combined 6-BA on formation of multiple shoots from stem apex of wheat immature embryo. The results showed that, added different concentration of TDZ in the germination medium impacted on germination rate of wheat immature embryo: with the concentration of TDZ increasing within the scope of the design gradient, the germination rate of Xinong183 was increasing, while the germination rate of Qianjinzao, Xiaoyan22, Yangmai12 and Yangmai18 were first increased and then decreased, when the concentration of TDZ was 4mg·L-1, the germination rate of Qianjinzao was the best with 92.2%, 20.4% significant higher than that of control. Added TDZ in the germination medium also was to the benefit of inducting multiple shoots. Different hormone specie, concentration and combination had a significant impact on the differentiation of multiple shoots in the program of induction. Under the premise of the best hormone concentration and combination, when 3mg·L-1 TDZ and 0.5mg·L-1 IBA was combined, the regeneration rate of multiple shoots was the highest, the 3mg·L-1 TDZ and 1mg·L-1 6-BA was combined, it was better, then followed by 3mg·L-1 TDZ, 1mg·L-1 6-BA and 2mg·L-1 4-PU in the five induction medium. The best method of formation on the multiple shoots from wheat immature embryo was 4mg·L-1 TDZ to germinate, 3 mg·L-1 TDZ and 0.5mg·L-1 IBA to induct. Differentiation efficiency of the five wheat materials was different, which the regeneration rate of multiple shoots of Yangmai18 was the highest, then followed by Yangmai12, Qianjinzao, Xinong183 and Xiaoyan22. This result could provide some references in order to set up efficient system of multiple shoots from wheat immature embryo and select better receptor of genetic transformation.2. In order to provide better receptor of genetic transformation and to decrease the phenomenon of chimera in vitro shoots translating directly into seedling. Four common wheat varieties were used as the experimental material to study the effect on TDZ hormone when the wheat was in preprocessing. The results showed that, when the wheat was steeped in TDZ preprocessing, bud germination rate was decreased, but regeneration rate of multiple shoots and budding number of each shoots were increased observably. When the concentration of TDZ was 5mg·L-1, the rate of multiple shoots and budding number come to a head and Yangmai 18 is the best reaching 50.9% and 2.82; While the concentration of TDZ was 4mg·L-1, in medium for embryo germination, regeneration rate of multiple shoots and budding number of each shoots of wheat reached the top. When both concentration of TDZ and 6-BA were 3mg·L-1, regeneration rate of multiple shoots of wheat was the best and the effect of TDZ was better than 6-BA. Combination cytokinin with auxin had an effect on regeneration rate of multiple shoots of wheat. When 3mg·L-1 TDZ and 1mg·L-1 6-BA was combined and 3mg·L-1 6-BA and 0.05mg·L-1 2,4-D(2,4-dichlorophrnoxyacetic acid) was combined the result of induction was better, but was lower than cytokinin added independently. The higher the ratio was, the lower the induction effort was. Added different species of organic additives and inducted days had a important impact on the differentiation rate of multiple shoots from wheat mature embryo. The results showed that, when added 500mg·L-1 AHC, 250mg·L-1 L-Asp and 200mg·L-1 L-Glu on the induction medium and inducted days were 30 days, regeneration of multiple shoots and budding number of mature embryo is the highest, which the highest regeneration of multiple shoots and budding number of yangmai18 respectively reached 81.4% and 7.99, 7.1% and 1.28 significant higher than that of control. Genotype had great effect to induction of multiple shoots of wheat, of all the four materials of wheat, the regeneration rate of multiple shoots of Yangmai 18 was the highest, Yangmai 12 second place, next Qianjinzao, Xinong 183 and Xiaoyan 22 were the lowest.3. In considering, within the scope of the design gradient, the best method of formation on the multiple shoots from wheat immature embryo was 4mg·L-1 TDZ to germinate, 3mg·L-1 TDZ and 0.5mg·L-1 IBA combined to induct. The best method of formation on the multiple shoots from wheat mature embryo was 5mg·L-1 TDZ to pretreat, 4mg·L-1 TDZ to germinate, 3mg·L-1 TDZ and 500mg·L-1 AHC, 250mg·L-1 L-Asp and 200mg·L-1 L-Glu to induct. Inducted days were 30 days.