农杆菌介导CBL基因转化菊花的研究

发布时间：2018-04-27 14:29

本文选题：菊花 + CBL基因　；参考：《沈阳农业大学》2017年硕士论文

【摘要】：菊花(Chuysanthemum morifolium)是菊科菊属(Chrysanthemum)的多年生草本花卉,因其种类繁多、色彩丰富、形态各异而深受世人喜爱。本研究首先以菊花品种'C008'的顶芽为外植体获得了该品种的无菌苗;其次,以菊花品种'C008'和'A'的无菌苗叶片为试验材料,建立了高频再生体系;然后在筛选并分析影响遗传转化因素的基础上,建立了两个菊花品种稳定的遗传转化体系,利用农杆菌介导的方法将CBL基因成功转入了菊花品种'C008'中。本研究为获得菊花转基因新品种及抗性研究奠定了基础,主要试验结果如下:1.菊花品种'C008'无菌苗的获得:用0.1%升汞消毒5 min,萌发率最高,可达92%。菊花品种'C008'的茎段增殖培养基为MS+0.3mg·L-16-BA+0.3mg·L-1NAA。2.菊花品种'C008'和'A'再生体系的建立:菊花品种'C008'和'A'不定芽分化最适培养基分别是 MS+3.0 mg·L-1 6-BA+0.1 mg·L-1NAA 和 MS+1.0 mg·L-16-BA+0.3 mg.L-1NAA;生根的最适培养基为 1/2MS+0.2 mg.L-1NAA 和 1/2MS+0.3mg.L-1NAA;炼苗移栽成活率分别为84%和80%;抑制菊花品种'A'内生菌产生的最适头孢霉素浓度为50mg·L-1。3.菊花品种'C008'和'A'遗传转化体系的建立:菊花品种'C008'潮霉素筛选不定芽浓度为3.0 mg.L-1,不定芽生根的筛选浓度为3.0 mg.L-1,头孢霉素的抑制浓度为300 mg·L-1;菊花品种'A'潮霉素不定芽筛选浓度为1.0 mg.L-1,不定芽生根的筛选浓度为3.0mg.L-1,头孢霉素的抑制浓度为300mg·L-1。菊花品种'C008'的遗传转化体系为:预培养2 d,菌液浓度OD600=0.5-0.6,稀释倍数为50倍,侵染时间8 min,共培养2d、延迟培养2d,该品种共获得抗性植株41株,PCR检测表明,阳性植株为5株,证明CBL基因已转化到菊花'C008'的基因组DNA中,转化率为12.2%。菊花品种'A'的遗传转化体系为:预培养1d,菌液浓度OD600=0.5-0.6,稀释倍数为50倍,侵染时间10 min,共培养1 d、延迟培养2 d,该品种虽有抗性芽产生,但并未获得抗性植株。
[Abstract]:Chuysanthemum morifolium is a perennial herbaceous flower of Chrysanthemum of Compositae. In this study, the apical buds of chrysanthemum cultivar C008 'were used as explants to obtain the sterile seedlings of this variety, and the leaves of aseptic plantlets of Chrysanthemum chrysanthemum cultivars C008' and A' were used as experimental materials to establish a high frequency regeneration system. On the basis of screening and analyzing the factors affecting genetic transformation, the stable genetic transformation system of two chrysanthemum varieties was established, and the CBL gene was successfully transferred into the chrysanthemum cultivar C008'by Agrobacterium tumefaciens. This study laid a foundation for the study of transgenic varieties and resistance of chrysanthemum. The main results are as follows: 1. The aseptic seedling of chrysanthemum variety C008 'was obtained: the germinating rate was the highest, up to 92%, when 0.1% mercuric chloride was sterilized for 5 min. The stem segment proliferation medium of chrysanthemum cultivar C008 'was MS 0.3mg L-16-BA 0.3mg L-1NAA.2. Establishment of Regeneration system of Chrysanthemum cultivars C008 'and A': the most suitable medium for adventitious bud differentiation of chrysanthemum cultivars C008' and A' was MS 3.0 mg L-1 6-BA 0.1 mg L-1NAA and MS 1.0 mg L-16-BA 0.3 mg 路L -1 NAA, respectively, and the optimum medium for rooting was 1/2MS 0.2 mg.L-1NAA and 1/2MS 0.3 mg 路L -1 NAA, respectively, and the optimal medium for seedling transplantation was MS 3.0 mg L -1 6-BA 0.1 mg L-1NAA and MS 1.0 mg L-16-BA 0.3 mg 路L -1 NAA, respectively. The survival rate was 84% and 80%, respectively, and the optimal concentration of ceftomycin was 50mg L-1.3. Establishment of genetic Transformation system for Chrysanthemum cultivars C008 'and A': the concentration of adventitious buds, rooting and cefomycin were 3.0 mg 路L ~ (-1), 3.0 mg 路L ~ (-1) and 300 mg 路L ~ (-1), respectively. The concentration of shoot screening was 1.0 mg 路L ~ (-1), the screening concentration of adventitious bud rooting was 3.0 mg 路L ~ (-1), and the inhibitory concentration of cefamycin was 300mg L ~ (-1). The genetic transformation system of chrysanthemum cultivar C008 'was as follows: Pre-culture for 2 days, concentration of OD600N 0.5-0.6, dilution multiple of 50 times, infection time of 8 min, co-culture for 2 days and delayed cultivation for 2 days. PCR analysis of 41 resistant plants showed that there were 5 positive plants. The results showed that CBL gene had been transformed into the genomic DNA of chrysanthemum chrysanthemum C008 'and the transformation rate was 12.2%. The genetic transformation system of chrysanthemum cultivar was as follows: 1 day of pre-culture, 0.5-0.6 of bacterial solution concentration, 50 times of dilution, 10 min of infection time, 1 day of co-culture and 2 days of delayed cultivation. Although resistant buds were produced, resistant plants were not obtained.
【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】：S682.11

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