栽培草莓高效遗传转化体系建立及优化研究

发布时间：2018-05-06 20:33

本文选题：八倍体 + 栽培草莓　；参考：《沈阳农业大学》2017年硕士论文

【摘要】：草莓(Fragaria×ananassa Duch.)是重要的浆果类作物。随着转基因技术的应用,草莓基因的功能验证成为草莓分子生物学研究的重要内容。以往草莓的遗传转化研究多以二倍体森林草莓(Fragaria vescaa)为材料,但是森林草莓与八倍体栽培草莓(F×ananassa)在性状表现和经济价值上有所不同,因此以栽培草莓为材料进行基因功能验证更具意义。本文以不同组合的八倍体栽培草莓的种子为试材,筛选出具有高离体再生能力且对农杆菌敏感的基因型,同时以不同激素、不同侵染方式对栽培草莓的离体再生体系和遗传转化体系进行优化。主要结果如下:1.比较了 6个不同组合后代的再生能力,组合'全明星' × '石莓7号'的杂交后代和'永丽'的自交后代具有较高的再生能力,在'全明星' × '石莓7号'的杂交后代中,具有高再生能力单株的占比为57.2%,平均再生率为51.2%。'永丽'的自交后代中具有高再生能力单株的占比为46.7%,平均再生率为36.7%。2.以栽培草莓不同组合的实生种子为试材,建立试管苗无性系,筛选出具有高再生能力的草莓基因型A-19、D-10、D-34。A-19、D-10的不定芽再生率均在90%以上,平均再生芽个数在9.2-10.7之间。D-34的不定芽再生率均在85%以上,平均再生芽个数在7.6-8.9之间,均显著高于品种'全明星'。3.以优系A-19、D-10、D-34、'全明星'的叶片为外植体,利用GUS组织化学染色分析比较他们对农杆菌的敏感性。结果表明,A-19、D-10、D-34对农杆菌的敏感性均明显高于'全明星',而A-19、D-10、D-34三者对农杆菌的敏感性无显著性差别。4.以优系A-19、D-10、D-34、'全明星'的叶片为外植体进行稳定遗传转化。A-19、D-10的转化效率分别为4.3%、3.3%,均高于品种'全明星'的转化效率(1.7%),D-34侵染后无抗性芽产生。表明A-19、D-10为栽培草莓遗传转化的适宜试材。5.对栽培草莓的离体再生体系和遗传转化体系进行了优化。附加0.2 mg·L~(-1)IBA +2.0 mg·L~(-1) TDZ/6-BA的培养基可促进A-19叶片的离体再生,含有2.0 mg.L~(-1) TDZ +0.1～0.2 mg·L~(-1) IBA的培养基可促进D-10叶片的离体再生;在农杆菌介导的遗传转化中,草莓叶片接种在附加抗生素的再生培养基中会减少外植体不定芽的产生,200 mg.L~(-1)特美汀(Timentin,Tim)、200 mg·L~(-1) 头孢噻肟钠(Cefotaxime,Cef)、200 mg·L~(-1) Tim+ 200 mg·L~(-1) Cef对外植体的毒害作用无显著性差别。同时,外植体在侵染菌液之前进行预培养可促进抗性芽产生,预培养6 d最有利于栽培草莓的遗传转化。6.A-19对赤霉素具有较强的敏感性,在叶面喷施GA3和培养基附加不同浓度GA3的试验中,被处理的植株在株高和叶面积上明显增加。培养基中附加0.5 mg.L~(-1)GA3最有利于A-19的生长发育。
[Abstract]:Fragaria 脳 ananassa Duch.) Is an important berry crop. With the application of transgenic technology, functional verification of strawberry gene has become an important part of strawberry molecular biology. In the past, the genetic transformation of strawberry was studied by using Fragaria vescaa, a diploid forest, but the characters and economic value of forest strawberry and octoploid cultivated strawberry F 脳 ananassault were different. Therefore, it is more important to test the gene function of strawberry. In this paper, the seeds of different combinations of octaploid strawberry were used as materials to screen out genotypes with high regeneration ability and sensitivity to Agrobacterium tumefaciens, and different hormones were used. The in vitro regeneration system and genetic transformation system of cultivated strawberry were optimized by different infection methods. The main results are as follows: 1. The regeneration ability of the progenies of six different combinations was compared. The hybrid progenies of 'all star' 脳 'stone berry 7' and 'Yongli' self-bred progenies had higher regeneration ability, and in the cross progenies of 'all star' 脳 'stone berry 7', the progenies of 'all star' 脳 'stone berry 7' had higher regeneration ability. The percentage per plant with high regeneration ability was 57.2, and the average regeneration rate was 51.2. The percentage of single plant with high regeneration ability was 46.7in the self-bred progenies of Yongli', and the average regeneration rate was 36.70.2. With the seed of different combinations of cultivated strawberry as the test material, the clone of test-tube seedling was established, and the adventitious bud regeneration rate of strawberry genotype A-19 D-10 D-34.A-19 D-10 with high regeneration ability was all above 90%. The regeneration rate of adventitious buds with the average number of regenerated buds between 9.2-10.7. D-34 was more than 85%, and the average number of regenerated buds was between 7.6-8.9, which was significantly higher than that of 'all-star'. The leaves of A-19C D-10 D 34N 'all star' were used as explants and their sensitivity to Agrobacterium tumefaciens was analyzed by GUS histochemical staining. The results showed that the sensitivity of A-19 D-10 D-34 to Agrobacterium tumefaciens was significantly higher than that of 'all star', but there was no significant difference among the three groups in the sensitivity to Agrobacterium tumefaciens. 4. The transformation efficiency of stable genetic transformation of the leaves of A-19C D-10 D 34N 'all star' was 4. 33% and 3. 3% respectively, which was higher than that of the variety 'all star' (1. 7%) and no resistant buds were produced after infection. The results showed that A-19 D-10 was a suitable material for genetic transformation of strawberry. The in vitro regeneration system and genetic transformation system of cultivated strawberry were optimized. The culture medium supplemented with 0.2 mg L~(-1)IBA 2.0 mg L ~ (-1) TDZ/6-BA could promote the regeneration of A-19 leaves in vitro, and the medium containing 2.0 mg 路L ~ (-1) TDZ 0.1 ~ 0.2 mg / L ~ (-1) IBA could promote the regeneration of D-10 leaves in vitro, and in Agrobacterium tumefaciens mediated genetic transformation, Inoculation of strawberry leaves with antibiotics reduced the production of adventitious buds in explants (200mg 路L ~ (-1). The toxicity of Cef to explants was not significantly different from that of cefotaxime (200mg / L) Tim (200mg / L ~ (-1). At the same time, the preculture of explants before infecting bacteria could promote the production of resistant buds. The genetic transformation of strawberry was most favorable to the genetic transformation of cultivated strawberry for 6 days. 6.A-19 had a strong sensitivity to gibberellin (gibberellin). The plant height and leaf area increased obviously in the experiment of spraying GA3 on the leaf surface and adding different concentrations of GA3 to the medium. The addition of 0. 5 mg.L~(-1)GA3 to the medium was most favorable for the growth and development of A-19.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S668.4

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