Study On The Plant Cell Fusion Of Medicago Sativa L. And Lotus Corniculatus Linn

It is reported that 40% of cultivable land and 70% of uncultivable land in the world are acid soil, and the acid soil in the south of China accounts for 21% of total Chinese land today. Alfalfa (Medicago sativa L.) is recognized as one of the most important forage legume with high quality and high production in the world, and it is also praised as "the queen of forage". With the development of stockbreeding and forage industry, the acid soil in the south of China will potentially be used for growing alfalfa. But the currently used alfalfa varieties grow poorly in the acid soil, while birdsfoot trefoil (Lotus comiculatus Linn) can grow normally in acid soil and it is praised as "the alfalfa of infertile regions". The development of biotechnology has made it possible to breed a new variety of alfalfa which can grow in acid land by protoplast fusion of alfalfa and birdsfoot trefoil. In this study, the whole tissue cultivating system of Alfalfa and birdsfoot trefoil was established, and the condition of protoplasts fusion was primarily analyzed. The results of our experiment were shown as follows:1. MS and UM were used as the basic cultivating medium. The optimal cultivating conditions of Alfalfa and birdsfoot trefoil were found through adjusting the varieties and concentration of hormones according to the orthogonal design. The most appropriate cultivating medium of Alfalfa was UM + 2,4-D 5mg·L^-1' + KT 3mgL^-1' for inducing callus, and UM + 2,4-D 5 mg·L^-1+6-BA 1mg·L^-1+KT1 mg·L^-1 for inducing embryoid callus. The most appropriate cultivating medium of birdsfoot trefoil was UM + 2,4-D 2mgL^-1 + KT 1mg·L^-1 for inducing callus, and MS + NAA0.1 mg·L^-1+6-BA 1 mg·L^-1 for differentiation, and MS + NAA 0.05 mg·L^-1 for generating roots.2. The best combination of isolating protoplasts of alfalfa was the enzyme mixture containing 2% Cellulase R-10, 0.2% Pectinase and 0.5mol/L Macerozyme R-10, and the highest yield achieved to 15.3× 10⁷ /g; The best combination of isolating protoplasts of birdsfoot trefoil was the enzyme mixture containing 2% Cellulase R-10. 0.2% Pectinase and lmol/L Macerozyme R-10, and the highest yield achieved to 7.3× 10⁷ /g3. The protoplasts of alfalfa and birdsfoot trefoil were treated by IOA and R-6G with different concentrations, The best results of this experiment were shown as follows: 7mmol / ml IOA was used to kill completely the protoplasts of Alfalfa by treating in ten minutes, and 60μg / ml R-6G to kill completely the protoplasts of birdsfoot trefoil by treating in fifteen minutes.4. Hybrid cells of alfalfa and birdsfoot trefoil were obtained using protoplast fusion by PEG method. The best fusion condition was 30% PEG (6000) with 0.3 molL^-1 Ca²⁺ and pH 9.5. The hybrid cells were selected efficiently by using IOA pretreatment of alfalfa protoplasts and R-6G pretreatment of birdsfoot trefoil protoplasts. Although the parents of hybrid cells lost the...