Establishment Of Plant Regeneration System Of Alfalfa

Alfalfa is widely known because of high content of crude protein,strong adaptability,and contains a variety of vitamins and minerals,is a kind of high quality forage widely cultivated in the world,so it has the name of "the king of grass",it is the material basis for the development of animal husbandry.Alfalfa 806 has many advantages,such as cold resistance,drought resistance,salt tolerance and high forage yield,but there are also serious diseases and insect pests,Therefore,we need to apply transgenic technology to improve and obtain new transgenic varieties with target characters.In this experiment we used alfalfa Longmu 806 sterile Seedling Cotyledon,hypocotyl,leaf and root as explants,through the study of four kinds of materials in vitro callus induction and differentiation,obtain Alfalfa Regeneration,discuss the culture medium of different hormone concentration,the regeneration effect on Alfalfa in in different stages,the results of this research are as follows:Induction callus:Among the four explants,the highest inoculation efficiency was hypocotyl,and the explants were significantly higher in the medium A6MS with 2.0 mg/L 2,4-D and 0.25 mg/L KT And the callus ability of four explants was:hypocotyl>root>cotyledon>leaves.The results of the induction showed that the medium used in combination with K-2 and KT was superior to the medium used in combination with 2,4-D and 6-BA,from the callus state and the induction rate.Effects of dark culture on callus:dark culture treatment for a period of time on cotyledons and leaves callus to promote a positive effect,after training two kinds of explants induction rate increased by 20%to 30%.Bud differentiation:Four explants were adventitious bud differentiation,and the explants were the highest in the culture medium B5:MS with 0.4 mg/L KT,The results of four explants were as follows:hypocotyl>root>cotyledon>leaves,The differentiation rates were 62.4%,59.4%,54.6%and 50.5%respectively.The results showed that the differentiation rate of hypocotyls was the highest under the same culture conditions,and the effect of kinetin alone was the best.Callus induction time has a certain effect on adventitious bud differentiation,hypocotyl explants induced by 10,20,30,40 days later,the differentiation rate were 30.5%,50.7%,62%,59%;embryogenic callus rate reached the highest at 30 d,and the best differentiation effect,namely 62%.In 5 different gradient concentrations of sucrose,hypocotyl and cotyledon explants were 2,20g/L concentration is the highest differentiation rate were 67%and 53.5%,when the sucrose concentration is more than 20g/L,browning increased significantly,and the differentiation rate decreased with the increase of sucrose concentration.Finally,the best explants were hypocotyl explants,The culture conditions were callus induction medium A6:MS,additional 2 mg/L 2,4-D and 0.25 mg/L,The induction time of callus was 30 days.The differentiation medium B5:MS added 0.4 mg/LKT,and the differentiation sucrose concentration was 20 g/L.The regeneration system cycle is 60?70d.