Study On Dissociation And Culture Of Protoplasts Of Medicago Sativa L.

Alfalfa (Medicago sativa) cultivation is the world's earliest and most widely distributed, nutrient-rich, arid and semi-arid regions indispensable for improving soil nitrogen-fixing perennial legume forage quality, known as "the king of grass" reputation. But its yield, quality and resistance and so on to be further improved, and the existing pasture can no longer meet the growing demand for animal husbandry, cultivating high-yield high-quality new varieties of alfalfa with strong resistance has become a study of a number of breeders.The primary objectives.Because apomixis characteristics of alfalfa, using conventional breeding methods to cultivate new varieties has been greatly restricted. Plant cell engineering and genetic engineering technology to enhance the resistance of alfalfa provides a new way. In this study, four alfalfa varieties (Qingshui alfalfa, Algonquin, Gannon1, Euroke) for the materials, the establishment of alfalfa callus regeneration system as a basis for the establishment of alfalfa protoplast isolation and culture.1. In order to establish the basis for protoplast culture and cell fusion of Medicago sativa L.,the plant regeneration was studied using hypocotyls of Qingshui alfalfa( varieties of wild alfalfa), Algonquin, Gannon 1 as explants. Results indicate that:(1) 4 alfalfa cultivars hypocotyl callus formation followed by Algonquin 93.7 %>Gannon1,92.6 %>Qingshui alfalfa92.2 %>Euroke87.7 %,The additional 1 % sucrose.(2) The best callus inducement medium of 4 alfalfa varieties in was MS + 2.0 mg/L2,4-D + 1.0 mg/LNAA + 2.5 % sucrose + 0.6 % agar, the highest induction rates as follows: 91.7 %Algonquin, 89.6 %Euroke, 88.3 %Qingshui alfalfa, Gannon1, 87.3 %.(3) The optimal medium of differentiation of 4 alfalfa varieties was MS + 0.5 mg/L KT + 0.3 mg/LNAA +2.5 % sucrose + 0.6 % agar and the highest callus differentiation rate were : Gannon1 93.0 %, 89.7 % Qingshui alfalfa, Euroke 86.5 %, 84.5 % Algonquin.(4) The best rooting medium of 4 alfalfa varieties was 1/2MS + 0.1mg/LNAA + 1 % sucrose + 0.6 % agar and the best rooting rate were as follows: Gannon1, 96.0 %, Algonquin.94.6 %, 93.3 % Euroke , 92.5 % Qingshui alfalfa .2. To alfalfa varieties Algonquin soft-induced embryogenic callus and cotyledons were used for protoplast preparation. The protoplasts were isolated through enzyme digestion. The effects of different composition of enzyme, time of enzyme digestion, calli age and osmoticum on protoplast isolation were studied and discussed the callus protoplasts of the KM8P split of medium. The results showed that:(1) Highter yields and viabilities of protoplasts were obtained from embryogenic calli 12 d with 2 % cellulase + 0.5 % PectolaseY-23 + 1 % hemicellulase conditions, the enzyme 12 h, enzyme-osmotic pressure of 0.55 mol/L. Protoplasts in the KM8P + 2.0 mg/L2 ,4-D +1.0 mg/LNAA culture medium 4 d after the first cell division, splitting the frequency of up to 50.2 %and small cell clusters could be seen after 2 to 3 weeks in the culture. At this moment, the addition of the protoplast culture medium with 0.1mol/ L～0.2mol/L mannitol 2 or 3 time was needed for the continuous protoplasts division to form calli.(2) Highter yields and viabilities of protoplasts were obtained from Algonquin alfalfa cotyledons 2 % cellulase + 0.5 % PectolaseY-23 + 1 % hemicellulase conditions, the enzyme 8 h, enzyme-osmotic pressure of 0.55 mol/L .