Study On Agrobacterium Tumefaciens-mediated Transformation Of Bt And PTA Genes In Medicago Sativa L. Cv. Zhongmu No.1

Alfalfa (Medicago sativa L.) is the most widely cultivated forage legume, with approximately 3,500,000-hectare planting area in the world. However, large area of alfalfa creates favorable ecological condition for the occurrence and prevalence of alfalfa insect pest. In recent years the insect pest problem has been becoming increasingly severe and has become one of the primary restrictive factors for the alfalfa industrialized development. Along with the biological technology development and its extensive application, transgenic techniques can be taken to improve the alfalfa trait.The alfalfa cv."Zhongmu NO.1", which was cultivated through the conventional big field breeding technology by our laboratory, was selected as experimental material. Firstly, we used the hypocotyl and the cotyledon as explants for the tissue culture systematic research to find out the explant with high differentiation rate and its suitable basic culture medium. Secondly, we carried out antibiotics hypersensitivity experiments as to the alfalfa, and experiments relating to factors affecting transformation rate, including the state of the Agrobacterium tumefaciens, the time of invading, co-cultural time etc. In this way, we established the transformation system. We used the cotyledon as the acceptor and UM as the basic culture medium. The explants were immersed in the bacterial suspension with OD600 of 0.6 for 10 minutes, then transferred to the culture medium which was covered with a sterile filter per dish for 3-day co-culture, and finally transferred to the selected culture medium with ppt 8mg/l and Carb 400mg/l. Finally, we induced the Bt and PTA genes into the"Zhongmu NO.1"mediated by Agrobacterium tumefaciens EHA105 and got the ppt resistant plants. Preliminary PCR and RT-PCR experiments were carried out for verification. We will do the further hybrid analysis and the insect resistance experiments.