Developing New Germplasma Of Salt-Tolerant Medicago Sativa L. Via Transgenic Technology

Limitation of growth, yield and quality of crop by soil salinization has become a common problem in the world. With the characteristics of high content of protein, abundance of vitamins, minerals and amino acids, agreeable taste, alfalfa(Medicago Sativa L.) is one of the most important leguminous forages, and plays a very important role in the agriculture, animal husbandry and ecological construction in areas of the northwest China. However, most alfalfa cultivars are unsuitable to grow in saline-alkali soil because of their low salt-tolerant abilities. Therefore growing high-yield, salt-tolerant alfalfa strains to use the salt soil efficiently and improve the forage yield is an effective way for development of the agriculture, animal husbandry and maintenance of the ecological environment. Rapid development of modern biotechnology in breeding has provided an efficient tool for breeding stress-tolerant alfalfa.In this study, to improve its salt-tolerance, we applied genetic improvement to the alfalfa cultivar Algonquin, and obtained the salt-tolerant germplasma. The main results of this study are as follow: 1. The primary assessment of the salt-tolerance of Algonquin seeds show that effective salt selecting concentration was 225 mmol/L and 300 mmol/L NaCl for Algonquin in sprout stage and seedling stage, respectively.2. The total DNA of mangrove (Rhizophora apiculata L.) was transformed into the alfalfa (Medicago sativa L.) by the pollen-tube pathway.12 To seedlings with improved salt-tolerant trait were selected. The RAPD analysis was performed in these 12 To seedlings and their parental recipients and donors, and the band diversities were newcomer, specific to donor and absent in recipient. Sequences were analyzed for the specific band in donor of the primer S178 and the newcomer of the primer S198 in Ms-ra3 plantlet, respectively. The results confirmed that the exogenous DNA had been integrated into the recipient's genome, and improved salt resistance of To seedlings may result from the introduction of exogenous DNA. Then the T1 seeds were harvested when To plants were transplanted into the experimental plot and self-pollinated with manual assistance. The salt-tolerance related physiological and biochemical indices of the T1 seedlings were measured when the T1 seedlings were treated with 300 mmol/L NaCl, and these results confirmed that the salt-tolerance of T1 seedlings was significantly improved. The T2 salt-tolerance strains were obtained when T2 seedlings were treated with 0.6% NaCl for 21 days.3,The effect of different components of the culture media and hormone proportion on regeneration of hypocotyl, cotyledons and cotyledonary node of Algonquin was analyzed comparatively, and these results indicated that the cotyledonary nodes growing in the SH medium containing 3.0 mg/L 6-BA are suitable for genetic transformation, and the favorable concentration of IBA for root-inducing culture is 0.5 mg/L. Then the Na+/H+ antiporter gene SeNHX1 of Salicornia europaea L. was transferred into Algonquin by Agrobacteria-mediated method.36 transgenic plants were proven to have integrate the SeNHX1 gene by PCR detection, and 6 of them developed healthily in greenhouse. The expression of SeNHX1 gene was confirmed in 5 transgenic plants by RT-PCR analysis, and their salt-tolerance related physiological and biochemical indices were measured by treatment with 0.6% NaCl. The results showed that most of salt-tolerance related physiological and biochemical indices of 5 transgenic plants are superior to the controls, and one of transgenic plants, MsNHX75, superior to the else. The T1 transgenic straints were obtained when T1 seedlings were selected by PCR.4,The promoter of the salt-inducible gene MsPRP2 was cloned from Algonquin. It is 1521 bp, containing two large AT-rich regions and a single G-box, and sharing 93.7% identity with reported sequence (AF028841) in GenBank. The expression vector pPZP221 (MsPNN), which contains the SeNHX1 ORF driven by the promoter of the salt-inducible gene MsPRP2, was constructed successfully. Then the regenerated Algonquin plantlets, which transformed with pPZP221 (MsPNN) by Agrobacteria-mediated method, were got.