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菊花高频再生体系建立及农杆菌介导FT基因转化的研究

标题: 菊花高频再生体系建立及农杆菌介导FT基因转化的研究
英文标题: Studies on Establishment of High Frequence Regeneration System and Agrobacterium-mediated Transformation of Chrysanthemum with FT
作者: 刘冰
出版时间: 2011-01-01
所在大学: 河北农业大学
关键词: 菊花,根癌农杆菌,遗传转化,FT基因
英文关键词: chrysanthemum,Agrobacterium tumefaciens,genetic transformation,FT genes
论文级别: 硕士
学位: 学位论文
导师: 肖建忠%杨际双
专业: 园林植物与观赏园艺
提交时间: 2011
摘要: 菊花是原产于我国的十大名花之一,但是绝大多数品种的菊花自然花期集中在11月左右,能够在夏季或早秋开花的只有少数花色单调、花型较小的品种.因此菊花的生产发展因为其花期问题受到了严重制约.FT基因是一个激酶抑制蛋白,可以通过对CO对FT的调节促进开花.本试验旨在通过对菊花高效再生体系及根癌农杆菌介导的转化体系的研究,将FT基因导入到菊花品种'神马'中,以期获得花期提前的菊花新品种,并为广泛运用转基因技术改良菊花品种打下基础.本试验以菊花品种'神马'为试验材料.研究了不同激素浓度配比对菊花直接诱导不定芽再生以及对生根诱导的影响,对菊花的再生条件进行了优化,从而获得了菊花叶片高速快效的再生体系.试验结果表明,将菊花'神马'叶盘接种在MS+NAA 1mg/L+6-BA 1mg/L培养基上,经过20天的组织培养后,可直接再生出不定芽,而且再生率高达95.5%,再生芽在1/2MS+NAA 0.7mg/L培养基上经过10天左右可诱导出根,生根率可达100%.在菊花品种'神马'高效再生体系建立的基础上,利用根癌农杆菌介导的遗传转化法对其遗传转化进行了研究.研究结果表明,叶盘预培养时间、侵染时间、共培养时间以及延迟培养时间均对菊花的遗传转化效率有较大影响.同时得出经过预培养12h的叶盘置于菌液侵染5min,黑暗共培养3d;延迟筛选3d时菊花遗传转化率最高.共培养后将叶盘接种到MS+NAA 1mg/L+6-BA 1mg/L+Km 20mg/L培养基上进行抗性芽的筛选培养.25天后可直接分化出不定芽,当不定芽长到1-2cm左右时转到1/2MS+NAA 0.7mg/L+Km 20mg/L培养基上进行生根筛选培养.在加km的培养基中连续筛选8周以上,将期间产生的抗性芽切下接种在含有20mg/L km的生根培养基中进行二次筛选,提取能够正常生根的抗性苗的DNA,进行PCR检测,共获得PCR阳性植株7株,转化率大约0.9%.说明目的基因FT整合到菊花的基因组中.
英文摘要: Chrysanthemum is one of the ten famous flower which originated in our country. but most varieties of them bloom concentrated in November or so,at the same time only a few a little kinds of colors and smaller type of flowers can bloom at summer or early autumn. Therefore, the development of chrysanthemum production is severely constrained because of its flowering time. FT gene is a kind of protein kinase inhibitors. The flowering time can be promoted by the regulation of CO to the FT genes. The objective of this study is to transfer the FT gene to'Jinba'through the research of the efficient regeneration system and transformation system mediated by Agrobacterium tumefaciens of chrysanthemum to obtain new varieties of flowering time earlier chrysanthemums and established a foundation of wide application of improving chrysanthemum cultivars applying transgenic method.Chrysanthemum species 'Jinba' is used in this experiment as test material. The result showed that plant growth regulators can affect shoot regeneration and root inducement of chrysanthemum. A efficient and rapid regeneration system of chrysanthemum was established. In the study we found that shoots of cultivar'Jinba'can regenerated directly from the leaf disc on the media MS+NAA(1mg/L)+6-BA(1mg/L), after 20 days of tissue culture.At the same time the shoot regeneration rate can reach up to 95.5%.Transferred shoots onto the medium 1/2MS+NAA(0.7mg/L),we can foud that roots were obtained 10 days later, and the rooting percentage was 100%.Based on the present optimal efficient regeneration system which has been obtained of'Jinba', genetic transformation was studied by using leaf discs by co-cultivation with agrobacterium. The results showed that the efficiency of genetic transformation can be greatly affected by several factors, such as the time of preculturation、infecting、co-cultivation and delay-cultivation. We found that when infected leaf disc which has been preculturaion for 12 hours in bacterium liquid for 5min, then co-cultured for 3 days in dark and delay-cultivated for 3 days, we would obtain the highest genetic transformation rate. Transferred the leaf discs which has been co-cultured for 3 days in dark onto the media MS+NAA(1mg/L)+6-BA(1mg/L)+Km(20mg/L)for selection of the regeneration resistant buds. Shoots were tured on the selection medium 25 days later, and then transferred the shoots which were at around 1-2cm to 1/2MS+NAA(0.7mg/L) +Km 20mg/L for rooting selectin.After continuous selected on the medium with Km for more than 8 weeks, cut the resistant buds down and transferred them into the appropriate rooting medium with 10mg / L Km for a second screening. Extract DNA of the resistant buds which can generate roots normally, and monitored them by PCR analysis. The PCR results suggested that transgenie has 7 positive plantlets and the transformation rate is about 0.9%.It showed that the target gene of FT has been translated into genome of chrysanthemum.