Study On Cell Fusion Technology Of Medicago Sativa And Melilotus Suaveolen

It is a shot-term and effective biotechnology that make use of the protoplast fusion to transfer the good gene.now this technology was researched on tomatoes,tobacco,rice and other plants at home and abroad. Now many species between species and others or even a subfamily of somatic cell hybrid access to hybrid cell lines or hybrid plants.At present cell fusion technology has been widely used for forage in many areas of research and alfalfa and other forage resistance breeding,reduce swelling of hazards increase dry matter digestibility, biodegradable materials development,biological soil improvement, vaccines and biological agents such as the activity has made progress and breakthroughs But the protoplast fusion research has not been reported at home and abroad.In this study,the whole tissue cultivating systerm of Alfalfa and Melilotus suaveolen was established, and the condition of protoplasts fusion was primarily analyzed. The results of our experiment were shown as follows:1. MS was used as the basic cultivating medium. The optimal cultivating conditions of Alfalfa and Melilotus suaveolen was found through adjusting the varieties and concentration of hormones according to the orthogonal design. The most appropriate cultivating medium of Alfalfa was MS + 2,4-D 3.0mg/L + 6-BA 0.3 mg/L for callus. The most appropriate cultivating medium of Melilotus suaveolen was MS + 2,4-D 1.5mg/L + 6-BA 0.3 mg/L.2. The best combination of isolating protoplast of alfalfa was enzyme mixture containing 2% Cellulase R-10, 0.2% Pectinase and 0.5% Macerozyme R-10,and highest yield achieved to 1.9×107/g, hydrolysis time is 10h, the protoplast vitality achieved 60%; The best combination of isolating protoplast Melilotus suaveolen was enzyme mixture containing 2% Cellulase R-10, 0.3% Pectinase and 1% Macerozyme R-10 hydrolysis time is 15h, the protoplast vitality achieved 62.5%.3. The protoplast of Melilotus suaveolen was treaed by IOA with different concentrations, the best of this experiment was shown as follows: 3mmol/ml IOA was used to kill the protoplast of Melilotus suaveolen by treating ten minutes. 4. The protoplast of alfalfa was treaed by UV, the best of this experiment was shown as follows: RadiationOf UV is 375 uw/cm2 was used to kill the protoplast of alfalfa by treating one minutes.5 Hybrid cells of alfalfa and Melilotus suaveolen were obtained using protoplast usion by PEG method, The best fusion condition 40% PEG(6000) can obtain the hybrid cell ,the Fusion rate is 12.5%.