【作者】 凌英华；

【导师】 李加纳； 殷家明；

【作者基本信息】 西南农业大学 ， 作物遗传育种， 2005， 硕士

【摘要】 油菜是世界重要的油料作物之一,也是重要的食用油和蛋白饲料来源,因而受到国内外油菜育种工作者的高度重视。分子生物技术的发展与实践应用为油菜的研究提供一种有效的研究手段,给油菜育种赋予了新的活力。 为研究F₂无性系群体作为可重复作图群体的效率,本论文运用组织培养手段,将甘蓝型油菜F₂种子进行组织培养,以试管苗茎尖、子叶和下胚轴为材料,分别进行快繁培养,获得油菜F₂无性系群体;研究F₂无性系群体后代再生植株田间性状表现,结合SSR分子标记技术检测其变异情况;初步探讨不同外植体在相同条件下的再生频率和其后代再生植株在分子水平上的变化和差异,得到以下结论: 1.试验结果表明:甘蓝型油菜F₂代分离群体中不同材料茎尖分化频率在初次继代培养表现不一,在相同培养条件下,随着继代次数的增加,茎尖分化频率提高,几乎达到100%,表明茎尖经多次的继代培养可以消除（或缩小）油菜基因型对分化频率的影响。 2.在遗传背景和培养环境一定的条件下,不同外植体的诱导频率表现出明显的差异,子叶的芽再生频率明显比下胚芽的再生频率高;在培养环境一定、材料不同条件下,子叶芽再生频率相差不大,而下胚轴芽再生频率表现一定的差异。 3.由茎尖途径培养获得119个F₂无性系株系,其再生植株在苗期株系内生长整齐一致;蕾薹期株系内植株在植株高低、大小、株型等性状上表现基本一致,部分植株表现矮小,一株系内的一植株在叶片大小、形状和茎等性状发生变异,并表现为败育;株系内所有植株全部开花所需时间主要集中在5d到12d,有10个株系的初花总天数超过15d,表现为初花期提前;茎尖途径形成的再生植株R₁代变异系数分析发现:在一次有效枝起点上表现出的差异最大,变异频率为44.54%;而在主花序长和角果长度这2个性状上变异最小（变异频率均为0）;在株高、一次有效分枝数和主花序有效长上虽然有一定的变异,但变异的频率都很小;可能受环境和管理的双重影响,F₂无性系群体的R₁代单株在有效果数、主序有效果数和每果粒数上表现出了较大的变异。 4.以亲本DNA为模板,从100对甘蓝型油菜SSR引物中选取20对多态性高的作为标准引物,对F₂无性系群体03H109进行SSR扩增,发现F₂茎尖无性系的变异频率为0.0059%,接近自然变异频率,说明茎尖再生植株的变异频率极低,后代株系的稳定性高,可以作为构建F₂无性系群体的外植体材料;子叶和下胚轴的的变异频率分别为0.5271%和0.2095%,均高出自然变异率几百倍,说明子叶和下胚轴再生植株的变异频率较高,后代稳定性差,不易作为构建F₂无性系群体的外植体材料。 5.一般说来,F₂无性系群体的变异频率随着培养时间的增加而增加,因此应尽量减少继更多还原

【Abstract】 Rapeseed is a worldwide important oil crops, also is a key resource of main edible oil and protein feed, so rape breeding workers attach importance to it. The development and application of molecular biophysical techniques supply an efficient means to the scientific research in rape, and bring creative methods to rape breeding.In order to identify the efficiency of clonal F2 population as a repetitive colony in the genetic mapping, plantlets were regenerated by tissue culture of F2 rapeseeds （Brassica napus L.）. A clonal F2 population of rapeseed was propagated by stem tip, cotyledon and hypocotyls of the plantlets. This essay focused on the agronomic characters of the regeneration plants, identified the variation by SSR markers. Besides, the variety of regenerated plantlets between different explants in the same condition is primarily studied on the molecular level. And the main conculusions are as follows:1. The frequencies of stem tipes differed among F2 segregation populations in the first subculture. Under the same condition of culture, with the increasing of subculture, the frequencies of stem tipes increased, almost 100%, so it indicated that multi-subculture could reduce the affect -rapeseeds （Brassica napus L.） genotype affect regeneration frequencies of stem tipes.2. Under certain conditions as genetic background and cultural environment, shoot regeneration frequencies varied significantly from different explants, the rate of frequency in cotyledon was much more higher than that in hypocotyls; when the material was changed, there was almost equality in cotyledon, while difference in hypocotyls.3. The regeneration plants 119 materials of F2 clonal population from stem tipes growed uniformly in lines in seedling stage; in bud and stooling stage, size and shape of plants exhibited basic accordance, except partial dwarfish plants. However, in one line a plant’s characters—size, shape of leaf and stem, had variation. Young blooming period of all plants concentrated mainly between 5d and 12d, but individuals of ten lines exceeded I5d, advanced bloom. The result of analysis about CV （coefficient of variation） in R₁ generation suggested that variation in zero point for 1st branches was the highest, and it’s frequency of variation was 44.54%; while main inflorescence length and pod length had no variation; variation in stem heigth, No. of 1st branches and effective length of main inflorescence were low; pods per plant, No. of pods of main raceme and seeds of per pods displayed evident variation, due to affect of the environment and management.4. SSR amplified with 100 pairs of primers, which cover the whole rapeseed （Brassica napus L.） genome using the parents DNA as template. Clone 03H109 were studied using SSR by 20 pairs with polymorphism selected from those 100 pairs. 1 DNA polymorphism was found in the stem tip更多还原