Study On Agrobacterium-mediated Genetic Transformation Of Cotyledonary Node In Medicago Sativa L.

Alfalfa (Medicago sativa L.) is one of the most widely cultivated perennial forage legume, which has high yield and protein. With the development of animal husbandry, it is essential to breed new alfalfa varieties with high quality and high yield. Alfalfa has complex genomic information because of autotetraploid, and it is difficult to cultivate new varieties by traditional breeding methods such as hybrization and backcrossing, due to cross-pollination and self-incompatibility.Recently, molecular breeding which is based on plant genetic transformation has been received widespreadly. In addition, research of plant functional genomics must used genetic transformation technology. In this study, two methods by Agrobacterium-mediated genetic transformation using alfalfa cotyledonary node were explored and transgenic alfalfa plants were gotten eventually, which provided two effective ways to introduce genes for alfalfa and stimulate the development of genetic transformation technique in legume plants. The main results were as follows:1. Agrobacterium-mediated direct differentiation of adventitous buds from alfalfa cotyledonary node was optimized. The cotyledonary node was cut from 8-days old aseptic seedlings and these explants were infected with Agrobacterium solution for 15 min, and then were transferred onto the co-cultivation medium (MS+1.0 mg/L 6-BA+100 mg/L AS) for 3-4 d in the dark. The optimum medium for shoot induction was MS+1.0 mg/L 6-BA+0.1 mg/L NAA+100 mg/L Kan+400 mg/L Carb, and the optimum medium for root regeneration was 1/2MS+0.2 g/L YE+100 mg/L Kan+400 mg/L Carb, respectively. When there were several thick and stout roots, the plantlets could be trained for 3-5 d and then transplanted. Some of the regeneration alfalfa were identified to be transgenic plants with PCR amplification.2. Another genetic transformation method was established by the way of non tissue-culture. One of the cotyledons of 4-days old aspectic alfalfa seedling was cut and infected with Agrobacteium for 60 min, then was co-cultured in the dark. After 2 days they were transferred to the vermiculite in humid environments for 10 d. Then, those survived alfalfa were transplanted into pots for continuing growth. At last, the positive transgenic plants were identified by PCR.3 transgenic alfalfa were chosen and moved to open space for blooming, pollinating and seeding. No abnormal phenomena were observed form these transgenic plants and their seeds were harvested ultimately.