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玉米再生体系的建立及SsBHMT基因遗传转化的初步研究

标题: 玉米再生体系的建立及SsBHMT基因遗传转化的初步研究
英文标题: The Establishment of Plant Regeneration System on Maize and Studies on Transformation of SsBHMT
作者: 张慧杰
出版时间: 2010-01-01
所在大学: 河北农业大学
关键词: 玉米,植株再生体系,农杆菌,遗传转化,SsBHMT
英文关键词: Regeneration system,Agrobacterium tumefacien,Genetic transformation,SsBHMT
论文级别: 硕士
学位: 学位论文
导师: 余爱丽%刘国振
专业: 植物学
提交时间: 2010
摘要: 玉米是重要的粮食作物和饲料作物,建立高效、稳定的玉米遗传转化体系无疑具有重要的理论意义和应用价值,而良好的受体及植株再生体系是转化的重要前提.本试验对自交系综3、综31、连87、齐319、H04-36、旱21、Mo17以及连87与综3、综31、H04-36和齐319的杂交F1的幼胚的愈伤组织诱导和植株再生体系进行了研究,结果表明玉米愈伤组织培养受基因型的限制,也受培养基和诱导激素的影响;综31*连87的F1、综31、综3和齐319的Ⅱ型胚性愈伤组织诱导率高、植株再生能力较强,适宜作转化受体;Y6较适宜作综31*连87的F1、综31、综3和齐319的诱导培养基.同时摸索了玉米自交系连87、H04-36、综3、综31、旱21和齐319的成熟胚愈伤组织诱导和植株再生的条件,结果表明继代培养基中添加10mg/L的AgNO3、100mg/L的DTT和150mg/L的L-半胱氨酸,玉米成熟胚的胚性愈伤组织诱导率高;获得自交系综3、综31和H04-36的再生植株共6株,初步建立了玉米成熟胚再生体系.另外对玉米幼胚和成熟胚组织培养反应进行比较,表明幼胚愈伤组织诱导率、愈伤组织性状和幼苗分化率优于成熟胚的;幼胚愈伤组织诱导率和分化率高的基因型其成熟胚的也相应较高,存在一定相关性,据此可利用成熟胚为外植体初步筛选玉米基因型,从而克服幼胚取材时间不便的障碍.农杆菌介导玉米萌动胚基因转化方法,试验操作相对简单,不依赖组织培养技术,可以快速得到转基因植株,是非常方便可行的遗传转化方法.本试验采用此方法将SsBHMT基因转化玉米萌动胚,首先对划胚方法进行了改良,将玉米种皮剥开后划胚,有效地控制了划胚的力度和深度,提高了转化率效率;然后筛选出最适农杆菌浸染时间为20min、浸染液中添加AS的最佳浓度为200μmol/L、抗性筛选剂PPT最适筛选浓度200mg/L;最后经PCR检测,初步确定获得了4株转基因植株.本研究初步建立了玉米遗传转化体系,为探讨SsBHMT基因对玉米生长发育、抗逆等方面的作用奠定了基础.
英文摘要: Maize (Zea Mays L.) is a widely grown cereal crop and the most important fodder crop in the world today.It is significant in theory and application to establish an efficient and stable genetic transformation system of maize. Good receptor and plant regeneration system is an important prerequisite for transformation. Immature embryos from Zong 3, Zong 31, Lian 87, Qi 319, H04-36, Han 21, Mo 17 and Zong 31*Lian 87, Zong 3*Lian 87, Qi 319xLian 87, H04-36*Lian 87 were used for the study on the callus induction and regeneration. The resules show that callus culture of maize was significant affected by maize genotypes, also by medium and hormones.The Zong 31*Lian 87, Zong 31, Zong 3 and Qi 319 was suitable as the receptor for their higher performance both in induction of TYPEⅡembryonic callus and plant regeneration. Y6 was more suitable for Zong 31 x Lian 87, Zong 31,3 and Qi 319 as induction medium. Immature embryo regeneration system was established preliminarily. The mature embryos callus induction and plant regeneration conditions were also explored of lian 87, H04-36, Zong 3, Zong 31, Han 21 and Qi 319. The results show that the maize mature embryo callus induction was higher when subculture medium supplemented with 10 mg/L of AgNO3,100 mg/L of DTT, and 150 mg/L of L-cysteine; 6 regenerated plants were obtained from the inbred lines Zong 3, Zong 31 and H04-36. Mature embryo regeneration system was established initially. And compared the response of immature embryo and mature embryo in tissue culture, the results showed that the callus induction and plant regeneration was better than those of the mature embryo. The immature embryo callus induction of a maize genotype was higher and its mature embryo callus induction was higher too, there was correlation. Accodingly it can solve the inconvenience of immature embryo sampling time by screening maize genotypes preliminarily useing mature embryos as explants.Agrobacterium tumefaciens-mediated genetic transformation in germinating embryo of maize was very convenient and a practical method. It was relatively simple in operation and did not rely on tissue culture. Transgenic plants can be gotten rapidly. The method of cutting embryo was improved by peeling corn seed coat before cuting embryos. The strength and depth of cutting embryos was controled effectively to improve the transformation efficiency. Through optimizing the transformation conditions, the optimal inoculation time was 20 min, the optimal concentration of AS in co-culture medium was 200μmol/L, PPT concentration was 200 mg/L in the selecting solution. Four transgenic plants were obtained by PCR testing. The study established a system of genetic transformation of maize, which provides a basis for further researching the role of animal's SsBHMT gene on growth development and resistance of maize.