Establishment Of An Efficient Agrobacteriu-mediaed Transformation System For Genetic Improvement Of Medicago Sativa

Alfalfa (Medicago sativa) is one of most important legume forage. Because of the high forage yield and comprehensive nutrition, it is called the "the king among forages" and acts a very important role in animal husbandry all over the world, as well as in China. However, in current, China increasingly serious soil salinization, desertification, drought and the frequent pests and diseases have greatly affected the yield and quality of alfalfa, the acid soils in South china also limit alfalfa introduction and cultivation, at the same time, many nutritional quality problems of alfalfa such as low dry matter digestibility and animal tympanites yet not be improved. Therefore, to cultivate well resistance and high quality new varieties of alfalfa has an important significance.The traditional breeding methods need long period, and are lack of breeding resources. In recent years, tissue culture and genetic transformation technology have provided a convenient method of cultivating new varieties of alfalfa. Since the first success of genetically modified alfalfa in 1986, many alfalfa genetic transformations have been reported. However, the existing system of genetic transformation of alfalfa showed lower transformation efficiency, limiting the quality of foreign gene in the transformation of alfalfa, thus limiting access to superior new varieties of alfalfa.In this study, an alfalfa cultivar "Derby" with important agronomic traits is used as the recipient for transformation, the selectable marker and target genes both dual function bar gene, using the Agrobacterium-mediated methods which is commonly used in genetic transformation of alfalfa. The explant selection, antibiotic screening, culture time, the concentration of Agrobacterium infection solution and other factors were tested. Finally, an efficient alfalfa regeneration and transformation system was established. The major findings are as follows:1). Cotyledon was the optimal transformation explant, which has the highest callus induction rate, up to 97%. The best bialaphos screening concentration is 2.5mg/L, the best pre-culture time is 3 days, the optimal Agrobacterium strain OD6oo value of liquid infection is 0.5-0.7,8-10 min is the best time for Agrobacterium infection to cotyledon, and reasonable co-culture time is 2-3 days. From above system, the highest rate of resistant callus is up to 30.5%. 2). After repeated screening in the medium containing bialaphos, about 100 lines of To resistant plants was obtained.3). The resistant plants were detected by PCR, the result suggests that all of the resistant plants were positive transgenic plants.4). The regeneration plants and wild-type plants leaves were sprayed with herbicide Basta 5%o solution, all of the PCR positive plants showed resistance to herbicide, further indicating our alfalfa transformation system is efficient and reliable. This study laid a solid foundation for alfalfa materials innovation.