【作者】 马伶俐；

【导师】 柳小妮； 刘晓静；

【作者基本信息】 甘肃农业大学 ， 草业科学， 2008， 硕士

【摘要】 紫花苜蓿(Medicago sativa)为生长面积最大,利用价值最高的优质牧草。如何进一步提高其耐盐碱抗旱特性,扩大其种植范围,是当前苜蓿生产面临的重要研究课题,基因工程的发展和应用为苜蓿抗盐育种开辟了一条新的途径。本研究探讨了和田、阿尔冈金、甘农1号、甘农3号、甘农4号、陇中、德福、牧歌、润不勒、三得利10个苜蓿品种组织培养程序,确定出再生能力最强的品种及外植体。利用农杆菌介导法转化BADH(甜菜碱醛脱氢酶)基因,建立了一套农杆菌介导苜蓿基因转化的优化体系,主要研究结果如下:1.再生体系的建立试验表明,苜蓿不同品种间再生能力差异较大,在试验中所采用的10个品种中,再生能力的强弱依次为:甘农4号﹥牧歌﹥陇中﹥三得利﹥阿尔冈金﹥德福﹥润不勒﹥甘农3号﹥甘农1号﹥和田。胚轴是紫花苜蓿最佳的愈伤诱导外植体材料,最佳诱导培养基是MS+2mg/L2,4-D+0.25mg/LKT+30g/L蔗糖,愈伤组织诱导的最佳品种是甘农4号。10个品种最适的分化培养基为MS+0.5mg/LKT+0.1mg/LNAA+20g/L蔗糖。最佳诱导生根的培养基为1/2MS+0.5mg/LNAA+15g/L蔗糖。综合考虑各品种再生能力的强弱,最后确定以甘农4号苜蓿作为基因转化受体。2. BADH基因转化体系的建立以侵染后甘农4号号外植体的抗性愈伤组织出愈率、抗性芽形成率以及抗性苗的获得为指标建立了转化体系。以培养7天的甘农4号的胚轴为受体材料,预培养3天,用于转化的侵染时间为10 min,脱菌中使用500mg/L的羧苄青霉素,菌液浓度为OD6000.3～0.5,共培养3天,50mg/L Kan的选择压下筛选90多天。通过上述转化体系获得了5株抗卡那霉素的再生植株,经PCR检测,均表现为阳性。更多还原

【Abstract】 Abstract: Alfalfa (Medicago sativa) is the biggest for the growth area and the highest quality forage grass. Development and application of genetic engineering opened up a new way for the alfalfa in resisting salt breeding. Tissue culture technique of ten alfalfa breeds(Hetian,A’ergangjin,Gannong NO.1,Gannong NO.3,Gannong NO.4,Longzhong,Muge,Defu,Runbule,Sandeli) was researched and the BADH gene was transferred to alfalfa by Agrobacterium mediated transformation in this paper. The main achievements of our rearch are following:1) The establishment of regeneration systems. The test indecated that to different breeds,the regeneration ability isn’t same.From stronge to low,the order is:GannongNO.4>Muge>Longzhong>Sandeli>A’ergangjin>Defu>Runbule>Gannong NO.3>Gannong NO.1>Hetian.To different explant,hypocotyl has the best ability to induce callus. The effective media for inducing callus was on MS supplemented with 2,4-D 2.0 mg·L-1 and KT 0.25mg·L-1 and sucrose 30g·L-1, the best breeds for inducing callus was Gannong NO.4 . The effective media for bud inducing was MS containing KT 0.5mg·L-1and NAA 0.1mg/L and sucrose 20g·L-1.The effective root inducing meida was on 1/2MS supplementedwith NAA0.5 mg·L-1 and sucrose 15 g·L-1.Because of the high or low of the regeneration ability,the test conform Gannong NO.4 Alfalfa as the gene transform ingredients.2) Transformation system of BADH gene establishment: 500 mg·L-1 Carb could restrained growing of agrobacterium effectively.According to we established transformation system:Cultivated 7 days hypocotyls as explants as mediator, preculture 2 days, 10 minutes infection time and OD6000.3～0.5, 3 days coculture. After more than 90 days selection 3 regenerated Kan-resistant plantlets were abtained. The result of PCR analysis stated that the detected Kanamycin resistant plants transgenic BADH gene were all positive.更多还原