| Alfalfa is with the world’s oldest cultivated history and is widely distributed, and is a forage crop with high quality, yield and high benefit. At the same time, alfalfa play an important role to improve soil nutrient and prevent soil and water loss. However, in order to satisfy more production and development demand, an important subject have been put forward, which means that more effort is needed to improve alfalfa genetic features and to create new forage varieties. Now a new way has been provided to improve resistance and ecological suitability of alfalfa because the plant protoplasts fusion technology is an effective measures to overcome cross-incompatibility and increase the genetic variation coefficient. Then research alfalfa intraspecific somatic cell fusion conditions could lay the foundation for interspesific somatic cell fusion of alfalfa and other grass species. So this study took with cold and drought resistance of Medicago sativa subsp. Varia and with insufficient resistance of Medicago sativa L. cv. Gannong No.4as materials, to discuss the effects of different phytohormone types and combinations on inducing callus and establishing regeneration system. Based on which the effects of enzyme combinations, mannitol concentration, enzymolysis time, phytohormone and culture density for callus protoplast isolation and culture. Meanwhile, the effects of cell inactivation treatment, electric field condition and density on protoplasts asymmetric fusion were studied. The results indicate that:1. The best induced medium with cotyledon of Russian variegated alfalfa as explant was MS+1.0mg·L-12,4-D+0.5mg·L-16-BA+0.5mg·L-1KT, the induction ratio was91.1%; the best induced medium with hypocotyl of Russian variegated alfalfa as the explant was MS+1.0mg·L-12,4-D+1.0mg·L-1-BA+1.0mg·L-1NAA, the induction ratio was93.3%. The best induced medium with cotyledon and hypocotyl of Gannong No.4alfalfa as the explant was MS+2.0mg·L-12,4-D+1.5mg·L-16-BA++1.5mg·L-1NAA, and the induction ratios were88.9%and92.2%, respectively.2. The best differentiation medium for Russian variegated alfalfa calli were MS+0.5mg·L-1KT+0.3mg·L-1NAA, the differentiation ratios of cotyledons and hypocotyls were91.1%and83.3%, respectively. The best differentiation medium for Gannong No.4alfalfa callus were MS+0.5mg·L-1KT+0.5mg·L-1NAA, respective differentiation ratios of cotyledons and hypocotyls were88.9%and82.2%.3. The optimal seedling formation medium for Russian variegated alfalfa and Gannong No.4alfalfa was1/2MS+1.0mg·L-1IBA, and the ratio were96.7%and86.7%, respectively.4. For hypocotyl calli of Russian variegated alfalfa, the optimum enzyme combinations was2%cellulase onnzuka R-10+0.5%Pectinase Y-23+0.3%Macerozyme R-10, the optimum mannitol concentration was0.55mol·L-1, the optimum enzymolysis time was12hours and the optimum callus subculture time was14days, and the optimum pre-plasmolysis were CPW-11solution for1h, the yield and viability of protoplasts were3.40×106·g-1and73.1%.5. For hypocotyl calli of Gannong No.4alfalfa, the best callus subculture time was12days, the optimum enzyme combinations was2%cellulose onnzuka R-10+0.5%Pectinase Y-23+0.3%Hemicellulase Onozuka+0.6mol·L-1mannitol, the optimum enzymolysis time was10～12hours, and the optimum pre-plasmolysis was CPW-11solution for1h, the yield and viability of protoplasts were3.34×106·g-1and82.8%.6. The protoplasts from hypocotyl calli of Russian variegated alfalfa in the solid-liquid comprehensive cultivation mode, the optimum hormone combination was1.5mg·L-12,4-D+1.0mg·L-16-BA, and the optimum culture density was3×105ml-1, follow the above appropriate condition, the cell division ration was36.3%, with less browning phenomenon, the cell clusters were with compact structure and sustainable division capability.7. For protoplasts from hypocotyl calli of Gannong No.4alfalfa in the solid-liquid comprehensive cultivation mode, the optimum hormone combination was2.0mg·L-12,4-D+1.0mg·L-16-BA, and the optimum culture density was3×105ml-1, follow the above appropriate condition, the cell division ration was37.9%, with less browning phenomenon, the cell clusters were with compact structure and sustainable division capability.8. For the protoplast electro fusion, the optimum inactivation pretreatments was: protoplast been treated with5min ultraviolet radiation for Russian variegated alfalfa and been treated with6mmol·L-1iodoacetamide for Gannong No.4alfalfa, respectively.9. The optimum electrofusion parameters of Russian variegated alfalfa protoplast and Gannong No.4alfalfa protoplast was AC electric field intensity for15V·cm-1～20V·cm-1, frequency for2000～2500kHz, DC electric field intensity for200V·cm-1～250V·cm-1, pulse width for40μs and pulse3times; and the optimum protoplast density was3×105ml-1～5×105ml-1. Following the above appropriate conditions, the highest protoplast binary fusion frequency reached13.8%. |
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