【作者】 樊超红；

【导师】 王跃进； 张剑侠；

【作者基本信息】 西北农林科技大学 ， 果树学， 2006， 硕士

【摘要】 本研究以爱莫无核叶片、叶柄和无核白花药、子房等外植体为试材,通过器官发生途径和胚状体发生途径建立了葡萄再生体系;并将携带有中国野生葡萄芪合酶基因的植物表达载体pWR306,通过农杆菌介导法对爱莫无核叶片叶柄、长穗无核白叶柄、无核白胚状体及胚性愈伤进行遗传转化,研究了潮霉素(Hyg)筛选浓度、头孢霉素(Cef)脱菌浓度、农杆菌侵染浓度、预培养时间等遗传转化因素;并通过Hyg筛选、PCR检测、Southern杂交等方法对外源基因的整合进行了检测。取得的主要结果如下:1.葡萄再生体系的建立(1)器官发生途径再生体系的建立以爱莫无核葡萄的叶片、叶柄为材料,通过研究不同激素浓度组合对外植体愈伤诱导及不定芽分化的影响,建立了葡萄器官发生途径的再生体系。叶片再生适宜培养基为MS+TDZ 2.5mg·L-1+NAA 0.1mg·L-1,再生率为21.57%;叶柄再生适宜培养基为MS+TDZ 1.0mg·L-1,再生率为2.08%;不定芽伸长培养基为MS+TDZ 0.25mg·L-1+NAA 0.01mg·L-1;生根成苗培养基为1/2MS+IBA 0.1mg·L-1。(2)胚状体发生途径的建立以无核白葡萄花药、子房为材料,研究葡萄胚状体发生途径的再生。结果表明:无核白的花药和子房胚性愈伤组织诱导最佳培养基分别为MS+BA 4.0mg·L-1+2,4-D 0.5mg·L-1+3%蔗糖和MS+BA 4.0 mg·L-1+2,4-D 1.0mg·L-1+蔗糖30g·L-1;胚状体诱导培养基为MS+BA 4.0mg·L-1+蔗糖30g·L-1;在无激素的MS培养基中胚状体很快萌发;成苗培养基为WPM+蔗糖15g·L-1。2.遗传转化体系的优化(1)Hyg选择压的确定以爱莫无核叶片和扩繁单芽茎段为试材,研究Hyg对葡萄叶片不定芽再生及植株生长的影响,确定了Hyg筛选浓度和筛选方式。葡萄遗传转化后进行延迟筛选,并逐步增加潮霉素的浓度,起始浓度为3mg·L-1再逐步增加至12mg·L-1可减少对再生的抑制。(2)脱菌素Cef浓度的确定以爱莫无核叶片为试材,研究Cef对不定芽再生的影响,确定了Cef的最佳脱菌浓度为400mg·L-1。(3)农杆菌侵染浓度的确定分别以叶片、叶柄、胚状体和胚性愈伤为试材,研究在不同浓度农杆菌下脱菌的难更多还原

【Abstract】 The regeneration systems of Emerald Seedless and Thompson Seedless were established by organogenesis and somatic embryogenesis, respectively. The novel stilbene synthase gene which was carried in the pWR306 binary vector was introduced into leaves and petioles of Emerald Seedless, into petioles of Long Thompson Seedless, into somatic embryos and embryogenic callus by Agrobacterium-mediated. Concentration of hygromycin for selection, concentration of cefotaxime for wiping off bacterium, concentration of Agrobacterium and time of pre-culture were studied. The foreign genes were confirmed by selection of hygromycin, PCR analysis and Southern blotting. The mainly results were:1 Producing of regeneration system(1)Organogenesis system Regeneration system from leaf and petiole of Emerald seedless grape using thidiazuron was studied. The results were: Regeneration from leaf was easier than from petiole. The combination 2.5mg·L-1 TDZ with 0.1mg·L-1 NAA resulted in the highest frequency of adventitious bud production from leaf. The adventitious bud regenerating rate is 21.57%. The medium MS+TDZ 1.0mg·L-1 was the best for bud regeneration from petiole with regeneration rate of 2.08%. The optimum medium for bud elongation is MS+TDZ 0.25mg·L-1 + NAA 0.01mg·L-1.The medium 1/2MS+IBA 0.1mg·L-1 was better for roots inducing.(2)Somatic embryogenesis system Somatic embryogenesis and plant regeneration from anthers and ovaries of Thompson Seedless (Vitis vinifera L) were studied. The results were: The best combination of hormones for embryogenic callus inducement of Thompson Seedless from anther and ovary are BAP 4.0 mg·L-1、2,4-D 0.5 mg·L-1 and BAP 4.0mg·L-1、2,4-D 1.0mg·L-1, respectively. Somatic embryos are induced on embryonic callus inducing medium but no growth regulator. The plantlets and new embryoids were produced on free-hormone MS medium. The medium WP+sugar 15g·L-1 was the best for complete plant forming.2 Improvement of genetic transformation system更多还原