【作者】 蔡兴奎；

【导师】 谢从华；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2004， 博士

【摘要】 马铃薯四倍体栽培种(Solanum tuberosum L.)的遗传基础狭窄，常规的育种手段难以突破其种质范围，扩大马铃薯的基因库是加速马铃薯育种进程的关键。马铃薯野生资源具有对病虫害、霜冻、干旱和病毒等广泛的抗性，但由于倍性水平或胚乳平衡数(EBN)的差异，大多数野生种与栽培种杂交困难，限制了这些优良基因的利用。体细胞杂交技术为利用这些野生资源开辟了一条新途径，不仅可以克服远缘有性杂交不亲和的障碍，而且可以实现胞质基因的重组和转移。 青枯病(Ralstonia solanacearum)是一种重大的细菌性病害，在热带、亚热带、部分温带、甚至一些冷凉地区普遍发生，寄主范围涉及50多个科的200多种植物，尤其易侵染马铃薯和番茄。目前，生产上对马铃薯青枯病还没有药物进行防治，综合防治措施虽可以起到一定的效果，但难以大面积推广，因此，选育抗青枯病品种无疑是最经济、有效的途径。但马铃薯栽培种没有抗源，少数具有抗性或耐性的野生种和原始栽培种又与栽培种存在杂交障碍，抗病种质资源缺乏成为马铃薯抗青枯病育种的最大障碍。本实验旨在利用体细胞杂交技术转移青枯病抗性性状，创造抗青枯病的马铃薯新种质，为马铃薯的抗病育种和倍性育种奠定基础。主要的研究结果如下： 1．建立了高效的马铃薯叶肉原生质体融合技术体系。以马铃薯栽培品种“中薯二号”经授粉诱导产生的无性系3~#、8~#(2n=4x=48)和野生种S.chacoense(2n=2x=24)的无菌苗为原生质体来源，系统研究了电融合和化学融合因素，优化了马铃薯高效融合的参数。研究表明，电融合法在交变电场(AC)100 V／cm、AC作用时间10s、直流脉冲电压(DC)1100 V／cm、DC脉冲时间60 μs、脉冲个数1次时双核融合率为4500％左右；化学融合法(PEG法)在融合诱导液(FA)作用时间15 min和固定液(FB)作用10 min条件下，其双核融合率最高为42.7％。同时比较了PEG融合和电融合两种方式对马铃薯原生质体融合效果的影响，结果显示，两种融合方式的细胞融合率和再生植株的杂种比率没有显著差异，但电融合细胞的植板率和愈伤组织分化能力均显著高于PEG融合。在本实验条件下，从四个不同的融合处理中共筛选了1212个生长旺盛的愈伤组织进行分化培养，结果有128个愈伤组织分化出芽，并发育形成完整植株，愈伤组织的平均分化频率为10.6％，不同融合处理间的愈伤组织分化频率在9.2％～16.8％之间。 2．获得了两个组合共154个体细胞杂种株系，其中略低于一半的株系为双亲的倍性之和，近90.0％的株系为单亲叶绿体类型。将首先分化形成完整植株的160个株系，经RAPD标记鉴定，表明有154个株系为真正的体细胞杂种，杂种植株的比例为96.3%，不同融合组合的杂种率在94.4%一100%之间。进一步用流式细)Jkl仪分析细胞杂种植株的倍性水平，结果表明，47.1%为六倍体、1 0.7%为八倍体、17.1%为非整倍体和25.0%为混倍体。同时将叶绿体SSR引物NTCP一9用于杂种植株的叶绿体类型检测，结果显示，大多数杂种的叶绿体并不同时具有双亲类型，89.0%的杂种植株只含有单一亲本的叶绿体类型，其中46.1%的株系为栽培种亲本类型，42.9%的株系为野生种亲本类型。具有双亲叶绿体类型的株系只占11.0%。实验还观察到由同一个愈伤组织分化形成的不同植株，其倍性水平和叶绿体类型亦存在差异。 3.具有双亲叶绿体类型的体细胞杂种，在继代培养中其叶绿体类型的传递存在分离。对两亲本的叶绿体扩增差异片段进行克隆、测序和序列比较分析，结果表明，野生种5 chacoense的特异片段大小为242bp，与Nicotianazabacum叶绿体DNA序列(登录号:200044.1)的10 220一10 408区段有92.4%的同源性。栽培种亲本特异片段大小为303 bp，与Atropa beZladonna叶绿体DNA序列(登录号:AJ316582.l)的大片段比较，有91.5%的相似性。并且与本实验克隆的差异片段具有较高同源性的序列，均为叶绿体基因组序列，由此确定扩增片段确为马铃薯叶绿体DNA。进一步跟踪分析具有双亲叶绿体类型的杂种植株，其叶绿体在继代培养中的稳定性，结果表明，其叶绿体类型在继代培养过程中存在分离。 4.病原菌接种鉴定证明，约50%的体细胞杂种重组了野生种的青枯病抗性基因。利用青枯病生理小种1号对体细胞杂种及其亲本的试管苗进行伤根接种，试验表明，体细胞杂种株系具有从高度感病到高度抗病等一系列不同程度的抗性水平。33个体细胞杂种株系的抗病性方差分析表明，有12.1%的株系比野生种S:hacoens。更抗或更耐青枯菌生理小种1号菌株，39.4%的株系其抗性水平与野生种及chacoens。没有显著差异，21 .2%的株系其抗性水平介于两融合亲本之间。另有27 .3%的杂种株系其抗性水平与栽培种亲本没有显著差异‘进一步分析植株的抗病性水平与其倍性水平和叶绿体类型之间的关系，结果表明，体细胞杂种植株的抗病性水平与其倍性和叶绿体类型之间均无直接关系。 5.马铃薯体细胞杂种的遗传变异普遍存在，其变异来源于外植体的变异程度及融合和培养过程的影响。对体细胞杂种株系的RAPD标记分析表明，相同组合的体细胞杂种间其遗传变异普遍存在，以不同的外植体为原生质体分离材料，其杂种的遗传?更多还原

【Abstract】 The quite narrow genetic basis of the cultivated potato {Solarium tuberosum L.) and the difficulty in breakthrough of germplasms segregation entail the enlargement of Solamum gene pool. Many wild species and primordial cultivated types of potato exhibit comprehensive resistance or tolerance to pathogens, virus, pests, frost and drought, which are highly attractive to breeders. The application of these desirable genes is confined, however, as differences in ploidy level or endosperm balance numbers that cause sexual incompatibility or low fertility of the progenies. Somatic hybridization, however, provides a new approach to make use of these wild recourses, bypassing sexual incompatibilities to widen the germplasms scope and recombine the cytoplasmic DNAs.Bacterial wilt, a severe and devastating plant disease caused by Ralstonia solanacearum, occurs widely in tropical, subtropical, warm temperate areas and even in some cool regions. It affects more than 200 plant species distributed over 50 families, particularly potatoes and tomatoes. Currently, resistant or tolerant cultivars are mainly utilized for disease management since chemicals are not always effective to diseases control and most of them have negative effects on ecological environments. Therefore, the worldwide strategy of potato bacterial wilt control has been consisted on development of new resistant varieties. Fortunately, some wild or related cultivated species are known to be resistant or highly tolerant to bacterial wilt, such as Solunum chacoense and S. phureja, and thus are potential sources for resistance. However, cross incompitibility with cultivated species provents their use in potato breeding. The purpose of the present research was to incorporate resistance against bacterial wilt from ?chacoense or 5. phureja into cultivated potato by somatic hybridization. The major results are as following:1. Establishment of a high efficient fusing system of potato mesophyll protoplasts. This study was primarily attempted to optimize the fusion parameters using mesophyll protoplasts isolated from potato clones 3# and 8# (2n = 4x = 48), which were derived from c.v. Zhongshu 2 by pollination inducing, and diploid species S. chacoense (2n = 2x = 24). It was shown that binuclear fusion frequency reached up to 45.0 % with the following electrofusion parameters: alternate current (AC) 100 V/cm, AC duration 10 s, directcurrent (DC) 1 100 V/cm, 1 DC pulse with 60 us. The frequency of PEG induced fusion reached 42.7 %, as FA solution treated for 15 min and FB 10 min respectively. The PEG and electrofusion methods were employed to look into their effects on the protoplast fusion. The results showed that no much difference existed in the fusion efficiency between these two methods, except that higher cell plating frequency and stronger plant differentiation ability of callus were accompanied with electrofusion. From four different fusion combinations we have isolated 1 212 vigorously growing calli, from 128 calli of which (10.6 %) shoots regenerated and developed into intact plants. The regeneration frequency ranged between 9.2 %~16.8 % depending on the fusion combination.2. There were 154 somatic hybrids derived from the two fusion combinations were obtained, of which nearly half had a ploidy level summed to the parental materials and about 90 % performed either one of the parents’ chloroplast DNA type. Total 160 plants differentiated and regenerated earlier were subjected to RAPD analysis. The results revealed that 154 plants (96.3 %) were true somatic hybrids with a combination range between 94.4 -100 %. Further analysis of ploidy level using flow cytometry demonstrated that 47.1 % were hexaploids, 10.7 % octoploids, 17.1 % amphiploids and 25.0 % mixoploids. The identification of chloroplast types of the somatic hybrids was conducted with chloroplast SSR marker, NTCP-9. The results indicated that only 11.0 % of the hybrids combined the two parental chloroplasts while the majority contained the chloroplast from either of the fusion parents. It w更多还原