【作者】 尹虹；

【导师】 马俊莲； 张子德；

【作者基本信息】 河北农业大学 ， 农产品加工及贮藏工程， 2007， 硕士

【摘要】 本项研究在已经建立的富有柿叶片的高频再生体系的基础上，对影响根癌农杆菌（Agrobacterium tumefaciens）介导富有柿叶片遗传转化的条件进行了探索。通过优化转化条件，建立了富有柿叶片遗传转化体系；PCR检测结果初步表明，已获得了ACC合成酶的RNAi的转基因植株。主要研究结果如下：1．研究了选择抗生素壮观霉素和抑菌剂头孢霉素对叶片直接分化不定芽的影响。结果表明，叶片外植体对壮观霉素非常敏感，在分化培养基中附加30mg／L的壮观霉素即可完全抑制不定芽的分化；在生根试验中，确定Sp生根压力为20mg／L。头孢霉素叶片外植体的分化没有严重影响，300mg／L的头孢霉素就能较好的抑制农杆菌的生长，且不定芽诱导率可达到46.3％。2．建立了以富有柿叶片为转化受体的根癌农杆菌遗传转化体系，详细探讨了预培养时间、共培养时间、侵染菌液浓度、侵染时间、AS浓度等因素对于T-DNA转化的影响，并优化了转化条件。结果表明，选择预培养3d的叶片，用OD₆₀₀为0.4的菌液侵染7min后，转到1／2MS＋ZT 1.0mg／L＋TDZ 0.5mg／L+AS150μmol／L的培养基上共培养4d，再通过延迟筛选4d，可获得较好的转化效果，抗性芽率最高的可达13.64％。3．通过建立的遗传转化体系，获得246株抗性苗，其中4株经PCR检测呈阳性。4．研究了暗培养时间、蔗糖浓度、光照强度、闭瓶锻炼时间对培育富有柿健壮生根苗的效果。结果表明，在蔗糖浓度为20g／L的培养基中生长的试管苗，暗培养8d，在30 000lx的光照下，闭瓶锻炼21d，对富有柿培育健壮生根苗有很大的促进作用。此条件下，培育的生根苗较好：生根率高，根系发达，试管苗生长健壮，芽点饱满。5．初步探讨了温度、湿度和移栽基质对移栽成活率的影响，生根试管苗移栽到以蛭石：珍珠岩=1∶1的基质中，进行过渡移栽。保持小拱棚温度在25～30℃。移栽前10d，保持棚内湿度在90％～100％，能获得较好的移栽成活率。更多还原

【Abstract】 In this study, an efficient transformation system had been established by Agrobacterium tumefaciens-mediated transformation technology on the base of high-frequency regeneration systems. Factors that affect the efficiency of genetic transformation of Fuyu persimmon were studied and optimized. It was originally proved by the PCR examination that the ACS gene had been transferred into Fuyu persimmon. The major results as following:1. Effects of Sp and Cef on shoot differentiation of leaf explants and rooting of in vitro shoots were investigated. The results indicated that Fuyu persimmon was highly sensitive to Sp. As its concentration increased to 30 mg/L in shoot differentiation medium, adventitious buds differentiation was completely inhibited. Selection pressures were determined that: 20 mg/L Sp for rooting of shoots. It was suitable that 300 mg/L Cef can controlled the propagation of Agrobacterium tumefaciens, and the frequency of adventitious shoot induction can reach 46.3%.2. Conditions for efficient T-DNA transformation were studied through selecting the best of pre-cultured time, the concentration of Agrobacterium tumefaciens the infection time, the co-cultured time and so on. The results indicated that Leaves pre-cultured in darkness for three days were dipped into Agrobacterium suspension for seven minutes, then co-cultured in darkness for four day on the medium of 1/2MS + ZT 1.0mg/L + TDZ 0.5 mg/L + AS 150μmol/L, the best delayed selection time was 4d, could increase the frequency of transformation. The Frequency of resistant shoots could reach 13.64%.3. In this study, we obtained 246 resistant shoots, only 4 resistant shoots were Fuyu persimmon transgenetic plants carrying ACS gene which identified by PCR.4. Effects of concentration of sucrose, dakness treatment, buttle-closed exercise and light intensity on Fuyu persimmon were studied for cultivating vigorous and healthy rooted plants. The results showed that 1/2MS medium with 20 g/L sucrose was the best basal medium, darkness cultured 8d, buttle-closed exercise should have been for 21 d in the light intensity of 30 000 Ix. In this way, the plantlet growed very well, which had many thick and long roots.5. In order to enhance survival rate of transplanting, factors of temperature, humidity, substratum was studied on trandplants environment of Fuyu persimmon. Result shows that, the Pearlite combined with Vermiculite by1:1 was the best material. The best temperature is between 25～30℃, containing the humidity with 90%～100%, could have better survival rate of-transplanting.更多还原