The Comparison Of Different Regeneration Systems And Agrobacterium Mediated ANHX1 Gene Transformation Of Alfalfa

Soil salinity and alkalinity is a cosmopolitan problem confronting humankind. In China, there are large areas of saline and alkaline land with the tendency of increasing year after year. Alfalfa (Medicago sativa), known as"the king of forage", with its medium tolerant to salinity, cannot grow well in high saline soil. So it is significant to breed salt-tolerant alfalfa varieties both for the further improvement of saline land and development of alfalfa production. The development of biotechnology provides advanced techniques for plant genetic improvement which traditional breeding lacks. The purpose of this research was to improve the salt-tolerance of alfalfa by means of biotechnology, providing basis for its application in saline soil improvement.Sodium is one of the major toxic cations in plants. Plant cells deal with the excessive sodium by pumping Na~+ from the cytoplasm into the vacuole by Na~+/H~+ antiporter, which can protects the cytoplasm from excessive Na~+ damage and decrease the osmotic potential to attract water into the cells. The Na~+/H~+ antiporter is responsible for this process. The first Na~+/H~+ antiporter gene (AtNHX1) in higher plants was cloned from Arabidopsis in California University. In this research, we planed to establish an efficient regeneration system of alfalfa and transfer AtNHX1 into alfalfa by agrobacterium mediation to achieve high salt-tolerance.After comparison of different regeneration systems in 5 alfalfa varieties including Xinjiangdaye, Longdong, Zhongmu No.1, Xinmu No.1 and Algonquin, an efficient alfalfa regeneration system was established based on the organogenesis; according to which, AtNHX1 was transferred into alfalfa mediated by Agrobacterium, and Kan-resistance plants were obtained. The main results are as follows:1 The comparison and optimization of regeneration systems. Compared with embryogenesis, the organogenesis approach showed the advantages of shorter regeneration period, higher regeneration frequency, wider suitability and without vitrification. Xinjiangdaye had the highest regeneration frequency (376.3%) and short regeneration period (about 60 days). Longdong gave the lowest regeneration frequency (120.3%) and a similar regeneration period of about 60 days. Zhongmu No.1, Xinjiangdaye, Longdong, Xinmu No.1 and Algonquin showed a much shorter (40d) regeneration period in organogenesis system than in embryogenesis approach. Meanwhile, their regeneration frequencies were increased by 2.36, 4.68, 16.9, 1.99 and 1.56 times, respectively. In organogenesis system, the best shoot differentiation...