草莓离体再生体系的建立及遗传转化

发布时间：2018-03-24 02:22

本文选题：草莓　切入点：再生体系　出处：《河北大学》2017年硕士论文

【摘要】：草莓(Fragaria×ananassa Duch)属蔷薇科草莓属,是人们日常生活中重要的水果产品之一。栽培草莓品种多为八倍体,是高度的杂合体,使用常规育种方法费时费力,且育种较难。而且,草莓在生长过程中,随着其无性繁殖世代的延续,草莓病毒病会逐渐积累,严重影响草莓的产量和果实的品质,因此,草莓苗一般需要2~3年进行一次换苗。本研究为了快速高效的获得草莓实生苗和提高草莓试管苗的分化率和增殖系数,并缩短其再生周期,分别以草莓种子、叶片、叶柄和试管苗基部作为外植体,通过改变其培养过程中激素配比及浓度,建立了以试管苗基部为外植体的高效再生体系。为了进一步提高其分化效率,将试管苗基部划分为草莓试管苗叶柄托叶结合部位和试管苗基部两部分作为外植体,建立了更加快速高效的再生体系。采用SRAP分子标记技术检测通过不同途径获得的草莓植株的遗传稳定性,探索检测子代与母本的相似程度,为离体快速繁殖保存草莓种质资源提供了技术依据。草莓在生长过程中同样会受到外界环境的影响。盐碱地是我国分布十分广泛的土壤类型,盐胁迫会抑制植株的组织和器官的成长及发育。据统计,盐害可导致全球农作物每年减产20%以上。如何提高草莓的抗盐性,是目前亟待解决的问题。本试验室吴科瀛研究结果表明导入Cjhppd基因的烟草抗盐性显著提高。本研究建立农杆菌介导的草莓试管苗基部为受体的遗传转化体系,将Cjhppd基因导入草莓植株从而获得转基因植株,研究其转基因性状,为利用基因工程技术进行草莓品质改良奠定基础。主要研究结果包括:1.建立了高效的草莓再生体系:以草莓试管苗叶柄托叶结合部位和试管苗基部作为外植体,分化培养基为MS+IBA0.2mg·L-1+6-BA1.5mg·L-1。相较于以叶片、叶柄作为外植体进行离体快繁,草莓试管苗叶柄托叶结合部位和试管苗基部分化均无明显的“愈伤组织”产生,可直接分化不定芽,且再生周期短。分化率可达到100%,增殖系数平均分别为6个和6.73个芽体,最高为13个,分割分化芽转移至诱导生根培养基,可形成正常植株,生根率达90%以上。2.采用CTAB法提取草莓基因组DNA,并建立了草莓适宜的SRAP-PCR反应体系。17对随机引物组合可以扩增出清晰、稳定的条带,共扩增出199条带,多态性条带占比为58.79%,其置信概率为99.99999%,可以用于鉴别任何一种样品,同时证明草莓基部分化苗的遗传稳定性较高,为离体快速繁殖保存草莓种质资源提供了一定的依据。3.以gfp基因作为标记基因,建立了以草莓试管苗基部为转化受体的农杆菌介导的草莓的遗传转化体系,最高转化率为22.73%。4.利用上述体系将Cjhppd基因导入草莓获得转化植株,试验得到转基因草莓植株。
[Abstract]:Strawberry Fragaria 脳 ananassa Duchbelongs to the genus Rosaceae, which is one of the most important fruit products in people's daily life. The cultivated strawberry varieties are octaploid and highly heterozygous. It takes time and effort to use conventional breeding methods, and breeding is difficult. During the growth of strawberry, with the continuation of its asexual reproduction generation, strawberry virus disease will gradually accumulate, which seriously affects the yield and fruit quality of strawberry. Strawberry seedlings usually need to be changed once for 2 ~ 3 years. In order to obtain strawberry seedlings quickly and efficiently and to improve the differentiation rate and multiplication coefficient of strawberry plantlets in vitro, and to shorten their regeneration cycle, strawberry seeds and leaves were used in this study. As explants, petiole and base of test-tube plantlets were used as explants. By changing the proportion and concentration of hormones in the culture process, an efficient regeneration system was established, which took the base of test-tube seedlings as explants, in order to further improve its differentiation efficiency. The base of the test-tube seedling was divided into two parts: the base of the leaf petiole and the base of the test-tube seedling as explants. A more rapid and efficient regeneration system was established. The genetic stability of strawberry plants obtained from different ways was detected by SRAP molecular marker technique. It provides a technical basis for rapid propagation and preservation of strawberry germplasm resources in vitro. Strawberry will also be affected by the external environment during its growth. Saline-alkali land is a widely distributed soil type in China. Salt stress inhibits the growth and development of plant tissues and organs. According to statistics, salt damage can lead to a reduction of more than 20 percent a year in global crops. The results of Wu Keying study in our laboratory showed that the salt tolerance of tobacco introduced into Cjhppd gene was significantly improved. A genetic transformation system was established in which the base of strawberry tube seedlings mediated by Agrobacterium tubuloides was a receptor. The transgenic plants were obtained by introducing Cjhppd gene into strawberry plants, and the transgenic characters were studied. The main results were as follows: 1. An efficient strawberry regeneration system was established. The explants were based on the binding site of leaf petiole and the base of test-tube seedling of strawberry test-tube seedling. The differentiation medium was MS IBA0.2mg L-1 6-BA1.5mg L-1.Compared with leaves, petiole was used as explant for rapid propagation in vitro. There was no obvious "callus" in the base differentiation of petiole and basal part of petiole of strawberry plantlets, and adventitious buds could be directly differentiated. The regeneration period is short, the differentiation rate can reach 100%, the average multiplication coefficient is 6 and 6.73 buds respectively, the highest is 13. The differentiation bud is transferred to the induction rooting medium, and the normal plants can be formed. The rooting rate was over 90% .2.The genomic DNA of strawberry was extracted by CTAB method, and the suitable SRAP-PCR reaction system. 17 pairs of random primer combinations could amplify clear and stable bands and amplify a total of 199 bands. The percentage of polymorphic bands is 58.79 and its confidence probability is 99.999999.It can be used to identify any kind of samples and to prove that the basal differentiation seedlings of strawberry have high genetic stability. The results showed that the genetic transformation system of strawberry mediated by Agrobacterium tumefaciens was established by using gfp gene as marker gene and in vitro rapid propagation and preservation of strawberry germplasm resources. The highest conversion rate was 22.73. 4. The transgenic strawberry plants were obtained by introducing Cjhppd gene into strawberry.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S668.4

【参考文献】