【作者】 郝曜山；

【导师】 孙毅； 李贵全；

【作者基本信息】 山西农业大学 ， 作物遗传育种， 2005， 硕士

【摘要】 本研究以玉米自交系478为受体,以质粒pBI101-Chi-Bmk为载体,在15%的蔗糖等渗溶液中,将花粉与质粒DNA混合,应用JY92-Ⅱ型超声波细胞粉碎仪进行超声波处理,然后辅以人工授粉的方法将外源基因导入到受体中,经PCR,Km涂抹检测初步证明,外源基因已整合到玉米基因组中。转化率为1.4%。 另外,本试验对影响玉米花粉介导转化的一些因素进行了研究,从等渗溶液浓度、超声波处理强度及花粉萌发辅助物的影响等方面进行了探索,认为适合玉米花粉介导的等渗溶液浓度为15%,超声波处理强度以100～200W。通过对超声波处理花粉及花粉萌发后花粉管的组织化学染色和细胞学观察,进一步证实了花粉介导植物转基因方法的可行性。 用花粉作为载体介导外源基因的转化,避免了传统的农杆菌介导法和基因枪法转化所要求的组织培养技术,转化方法简单、易操作,是一种经济、有效、能广泛应用的技术。更多还原

【Abstract】 Plasmid DNA of pB1101- Chi- Bmk was mixed with fresh pollen of maize inbred line, 478, in 15% sucrose solution . The pollen suspension was treated with ultrasonication and treated pollen were collected and pollinated on maize silks. Transformants were screened by Km selection and verified by PCR amplification. The transformation rate was 1.4%.Certain factors affecting pollen-mediated transformation in maize were also studied and discussed in the thesis, which include sucrose concentration for pollen suspesion, ultrasonication intensity, and pollen germination stimulator, etc. The optimum transformation conditions in present study were 15% sucrose solution for pollen suspension and 100~200W for ultrasonication intensity.The feasibility of polled-mediated approach in maize transformation was also confirmed by histochemical and cytological observations.Comparing with other transformation methods, the pollen-mediated transformation approach could circumvent the tedious tissues culture procedures like in Agrobacterium infection and particle bombardment, etc. This approach is simple, easy to adopt, and could be widely used in breeding practice.更多还原