【作者】 高翠萍；

【导师】 云锦凤； 石凤翎；

【作者基本信息】 内蒙古农业大学 ， 草业科学， 2007， 博士

【摘要】 苜蓿雄性不育系在杂种优势利用中具有重要价值。1978年,吴永敷教授从草原一号杂花苜蓿(Medicago varia Martin.cv.‘Caoyuan No.1’)中选择出雄性不育株Ms-4,其雌蕊育性低,杂交制种结实率低,在生产中很难大面积推广应用。因此,快速有效地获得新的优良不育系是迫切需要解决的问题。许多作物利用杂交选育出了优良雄性不育系。为提高育种效率,需要对杂交后代进行早代选择。本研究采用两种杂交方式转育雄性不育株,杂交方式一:选取具有优良性状的多个父本与不育系杂交,从F1代中选取农艺性状优良的可育株与不育系回交,以不育系为轮回亲本进行有限回交,对回交早代进行选择;杂交方式二:选取具有优良性状的多个父本与不育系杂交,从F1代中选取不育株与原父本回交,以选取的具有优良性状的父本为轮回亲本进行回交。通过采用组织培养、细胞形态学、同工酶和分子标记等方法和技术,分析了原不育系雄性不育性及杂交方式一的早代雌蕊育性,初步研究结果如下:1利用多父本与不育系杂交,从F1代中选取农艺性状优良的可育株与不育系回交,以不育系为轮回亲本进行了有限回交,对回交后代进行花粉染色反应和植株活体鉴定雄性育性,回交一代共得到24个不育株。2在苜蓿雄性不育系Ms-4没有找到保持系之前,组织培养是苜蓿雄性不育系扩繁的有效途径。叶柄可作为诱导愈伤组织的良好外植体,愈伤诱导率为85.6%;附加一定比例的脯胺酸和硫酸铵的培养基有利于体细胞胚的诱导,体胚诱导率为90%,且苜蓿雄性不育系Ms-4体细胞胚生产性能高。3对回交一代24个不育株几个与雌蕊育性相关的农艺性状调查和柱头、花柱细胞形态学特征分析结果表明:回交一代不育株BC1A1-21和BC1A1-22结实率显著提高,柱头膨大较明显,即乳突细胞外露面积隆起较大,毛状体细胞也相对长且外展;花柱横切面宽且长,表皮细胞体积较大,表皮和通道细胞之间有6-8层薄壁细胞和多条维管束,通道细胞排列紧密。两者雌蕊育性提高。4采用聚丙烯酰胺凝胶垂直板电泳技术,分析了亲本与杂交后代不育株不同生育时期的叶片和小花过氧化物酶(POD)、酯酶(EST)同工酶的谱带差异,结果表明:杂种F1代POD和EST同工酶谱为双亲的互补型或有杂种特征带。BC1代POD和EST同工酶谱偏向于轮回亲本。5采用RAPD技术,分析了亲本及杂交后代基因组DNA遗传差异,获得了与苜蓿雄性不育性有一定连锁关系的分子标记一个,记为MSS126438。利用此标记结合表型鉴定法共获得BC1代雄性不育株14株,与表型育性鉴定结果基本一致,是对表型育性鉴定出的不育株的进一步筛选。更多还原

【Abstract】 The male sterile line of alfalfa is important material for heterosis .The male sterile line Ms-4 of alfalfa was selected from Medicago varia Martin.cv.‘Caoyuan No.1’by professor Wuyongfu in 1978.It can not be widely used in production because it’s pistil has lower fertility .So selecting the new better male sterile lines of alfalfa rapidly and effectively is the urgent problem to be solved.The good male sterile lines of crop were selected by backcross .So backcrossing is an effective method for improving male sterile line. It need select the early generations from backcross offspring for improving breeding efficiency. In this study, two methods of backcross were used. One is male sterile line in crossing with male parents that have better characters, then select fertile plants from F1,using it in backcrossing with male sterile line. The other is male sterile line in crossing with male parents that have better characters, then select sterile plants from F1, using it in backcrossing with male parents .The sterility of male sterile lines and pistil fertility of early generations from backcross offspring were analysed by tissue culture, cell morphology, isoenzyme and molecular marker .The results showed:1 There are 24 male sterile lines were selected with pollen staining reaction and bioassay by the method that using male sterile line in crossing with male parents that have better characters, then select fertile plants from F1,using it in backcrossing sterile line.2 Tissue culture is an effective method for propagation of male sterile line of alfalfa before maintainer line was found. Blad stems are good explants of inducing callus. Callus inducing rate is 85.6%.The medium that add Proline and Ammonium Sulfate is benefit for somatic embryos inducing. The rate of somatic embryos inducing is 90%. The productivity of somatic embryos is high.3 The results of agronomic character and cell morphology of stigma and style of 24 sterile plants selected from early generations showed: the setting percentage of BC1A1-21 and BC1A1-22 is improved significantly; the stigma enlarge significantly ,the area of mastoid cells exterior is large; hairy cells are longer and abduction.Style is larger and wide; there are 6-8 layers cells between epidermal and passage cells ; The arrangement of passage cells was tight .The fertility of BC1A1-22 and BC1A1-21 is improved significantly.4 The POD and EST isoenzyme of parents and cross generations in different grow periods and organs were analysed by using polyacrylamide gelelectrophoresis. The results showed: the isoenzyme pattern of F1 are complementary type of parents or have distinguished bands.The isoenzyme pattern of BC1 is lean to recurrent parent.5 The genome DNA of parents and cross generations were analysed by RAPD. The results showed: MSS126438 is the molecular marker that link to male fertility of alfalfa. BC1A1-1,3,6,9,21,22,27、BC1MC1-4,6,7,11,13,14,15 are confirmed male sterile plants by MSS126438. The result is approximatly consistant in above methods. It is further identifying method for male fertility.更多还原