【摘要】 本研究以MS为基本培养基,在不同浓度的2,4-D与6-BA联合使用的诱导培养基上,以苜蓿子叶为外植体,诱导愈伤组织,诱导率为55%～90.6%,其中2mg/L2,4-D+1mg/L6-BA诱导率最高,达90.6%;浅黄色至淡绿色的愈伤组织在MS+2mg/LKT+0.15mg/L6-BA+0.3mg/LNAA分化培养基的分化率最高并且植株颜色深绿。丛生芽在1/2MS+2mg/L酵母提取物的生根培养基上再生成完整的苜蓿植株。以建立的苜蓿高频再生体系为基础,愈伤组织为转化的受体,用根癌农杆菌介导苜蓿,利用GUS组织化学染色法,研究影响遗传转化的若干因素,获得了100株转基因植株。乙酰丁香酮的浓度为100!mol/L,菌液浓度OD600为0.3～0.5,最佳的侵染时间为15min,共培养时间为4d,卡那霉素浓度为50mg/L时转化频率最合适。最高转化频率可达80%,Kana抗性植株Northernblotting检测表明,目的基因已整合进苜蓿基因组中。建立了苜蓿快速有效的遗传转化体系,为获得其它基因转化苜蓿,奠定基础。更多还原

【Abstract】 The explant was cotylender of alfalfa (Medicago sativa), and MS was used as the basic medium, the different combination of 2,4-D and 6-BA were added into the medium to induce the callus. The rate of inducement ranged from 55% to 90.6%, among of them 2mg/L 2,4-D +1mg/L 6-BA medium is the best (90.6%). The callus whose color was yellow and green had highest differentiation rate on the medium of MS+2mg/L KT+0.15mg/L 6-BA+0.3mg/L NAA, and the color of plants was deep green. The regenerated plants were obtained in the rooting medium (1/2MS+2mg/L yeast extract). The establishment of plant regeneration system has made the foundamentation for the studies on genetic transformation in plant breeding. Establishment of genetic transformation system of Medicago sativa callus mediated by Agrobacterium tumefaciens was studied by GUS colinration analysis and an effective protocol condition for genetic transformation of alfalfa was developed on the basis of high frequency tissue culture regeneration system and the GUS transgenic plants were achieved. Several factors affecting genetic transformation system were studied. 100 transgenic plants were achieved, the concentrations of acetosyringone were 100?滋mol/L, bacterial concentrations were OD600=0.3~0.5, the time of infection was 15min, the time of co-cultivation were 4 days, concentration of kanamycin were 50mg/L. The highest transformation frequency was 80%, Northern blotting analysis showed that the gene was integrated into genome of the plants.更多还原