【作者】 喻艳；

【导师】 柳俊；

【作者基本信息】 华中农业大学 ， 细胞生物学， 2013， 博士

【摘要】 体细胞杂交技术不仅能够克服有性生殖障碍,而且可以有效地同时转移核基因与胞质基因,促使优良性状在种内、种问、属间得到整合,从而获得有性杂交无法得到的优良种质资源,丰富现有栽培种资源的遗传背景。青枯病是茄科植物的重要的细菌性病害,其危害在马铃薯上仅次于晚疫病。目前我国马铃薯栽培品种缺少青枯病抗源,而存在于相关种中的优良抗性资源由于有性杂交不亲和又难以利用。因此,本研究旨在利用对称及非对称融合技术,将抗青枯病茄子优良抗性基因染色体片段整合到马铃薯栽培种中,创制抗青枯病马铃薯新资源。同时期望利用非对称融合技术转移马铃薯染色体片段到茄子中,建立马铃薯染色体片段导入系,为马铃薯遗传基础研究搭建新平台。取得的主要研究结果如下：1.亲本原生质体培养体系建立研究在对亲本倍性确认的基础上,采用本实验室建立的马铃薯原生质体培养方法,选用7个马铃薯基因型进行原生质体培养研究。结果表明,不同基因型间培养反应差异较大。前期液体浅层培养反应较好的只有3个基因型,包括8#、AC142、AC239.后续培养中,分化培养只有AC142能再生植株。此外,马铃薯二倍体野生种S.chacoence(C9701)虽然前期培养生长缓慢,但在继续培养中可以分化植株。茄子E508来源于抗青枯病基因型“本地红茄"的种子,组织培养下无性繁殖用于原生质体培养体系建立。使用不同的预处理方法,培养基和激素组合,研究显示,培养4周的试管苗,采用CM培养液黑暗预处理24h,适宜于茄子原生质体分离；CM.-I为适于茄子原生质体前期培养的培养基；后续培养的适宜激素组合为1.0mg,L NAA+0.1mg/L2,4.D+0.25mg/L ZT+0.25mg/LBA。2.马铃薯-茄子对称融合及其体细胞杂种鉴定研究以二倍体马铃薯AC142和茄子E508为融合亲本,进行原生质体对称融合,共获得了n7个再生植株。使用15对马铃薯SSR标记引物和6对茄子SSR标记引物,鉴定确认了34个株系为体细胞杂种。流式细胞仪分析了17个体细胞杂种的倍性水平,结果显示,17个被测杂种株系中,3个系峰值与四倍体马铃薯种相似；8个系含有两个二倍体马铃薯基因组和1个二倍体茄子基因组(六倍体)；另有6个非整倍体。对一个与马铃薯四倍体峰值相似的系PE29-1和一个按峰值推测含有四倍体马铃薯基因组和二倍体茄子基因组的杂种PE3-4,进行染色体计数,结果证明PE3-4染色体数为72,PE29-1染色体数为48,与流式细胞仪测定结果相符。3.对称融合体细胞杂种染色体组成分别选取7个体细胞杂种(4个六倍体,1个四倍体和2个非整倍体),采用基因组原位杂交(GISH)分析了其染色体组成。结果显示,4个六倍体杂种均含有48条马铃薯染色体和24条茄子染色体。非整倍体的体细胞杂种PE60-10含54条马铃薯染色体,16条茄子染色体和7条重排染色体。对标记检测为杂种、倍性分析为四倍体的PE29-1进行GISH分析,结果未检测到茄子的杂交信号。上述结果表明,在马铃薯-茄子的融合中,即使是对称融合,也很难获得具有二倍体马铃薯和二倍体茄子染色体的杂种,二倍体马铃薯在体细胞杂种中更容易加倍为为四倍体,这种加倍是两个马铃薯细胞和一个茄子细胞融合产生、还是在培养过程中马铃薯染色体的加倍目前还不清楚。在非整倍体杂种中更容易发生染色体重排。4.对称杂种的胞质组成研究采用在双亲间具有多态性的线粒体引物P4(pumD)分析了34个体细胞杂种的线粒体组成。结果显示,除了PE29-4和PE57-4两个株系只含有马铃薯亲本带以外,其它32个杂种株系线粒体均为双亲带型,且在所有含有双亲带型的杂种均出现了新带型,表明双亲线粒体基因组在融合后进行了整合。选用两个亲本间具有多态性的叶绿体引物NTCP9和NTCP12,对体细胞杂种叶绿体基因组组成进行了分析。结果显示34个体细胞杂种叶绿体扩增带型均与马铃薯亲本一致。上述结果说明,在体细胞杂种中,线粒体更容易发生重组,而叶绿体则只能保留一个亲本的基因组,且可能偏向远缘融合中提供核背景较多的亲本一方。5.对称杂种的青枯病抗性评价本研究对11个生根正常的体细胞杂种初步进行了青枯病抗性评价。使用青枯菌1号生理小种对杂种株系试管茵进行伤根接种,9个株系表现为抗青枯病,其中PE4-1的抗性显著高于抗病茄子亲本,其余8个系与之相当。选择试管苗接种后呈现抗性且钵栽生长正常的3个株系进行室外接种,3个株系抗性水平与茄子亲本相当,表明马铃薯-茄子原生质体融合成功地将茄子的青枯病抗性转移到马铃薯中。6.马铃薯-茄子非对称融合及其融合子鉴定研究采用紫外线照射原生质体破坏供体亲本的染色体结构,以利于融合中的染色体片段插入。以茄子E508为供体、马铃薯四倍体栽培种8拌为受体的非对称融合中,共获得73个再生株系。使用流式细胞仪对47个株系进行倍性分析,结果表明,36个株系为四倍体,6个为混倍体,2个为非整倍体,1个为八倍体,还有2个株系在测定过程分别出现主峰数量减少和峰值改变的现象,说明在继代过程中植株的染色体可能发生丢失。对16个非对称融合再生株系形态特征进行了观察评价,它们均能结薯,但在薯形、叶片形态、长势等性状上具有不同程度的变异。这些变异是否因为茄子的染色体片段插入而引起,还有待进一步研究。对马铃薯栽培种DH讷16为供体、茄子E508为受体的非对称融合中,共获得335个愈伤组织,到论文撰写时获得12个再生植株。由于大部分再生株系生根困难,生长缓慢,本研究只能对再生较早、能够生根的3个株系进行了鉴定分析。选用STI024、STI046和STM10883对在亲本间具有有多态性的引物进行分析,3个再生株系均含有马铃薯和茄子特异带,表明它们为体细胞杂种。胞质分析表明,杂种株系的线粒体为双亲融合型,叶绿体为偏马铃薯亲本类型。更多还原

【Abstract】 Somatic hybridization can not only get rid of sexual incompatibility, but also promote transfer of desire traits intra-/inter-species or inter-genus by fusion simultaneously the nuclear and cytoplasmic genes. This strategy has been considered irreplaceable by conventional crossing in enhancement of plant germplasms to enrich the genetic resources of modern cultivars. Bacterial wilt caused by Ralstonia solanacereaum is an important disease of Solanaceae plants, its impacts on potato is just after late blight. It is lack of resistance resources in solanum tuberosum, and difficulties exist in efficient use of wild species-derived resistance due to cross incompatibility. Therefore, for improving potato resistance, present research aimed to integrate eggplant chromosome fragment carrying bacterial wilt resistance genes into potato by symmetric and asymmetric protoplast fusion. Meanwhile, the eggplant introgression lines by incorporating potato chromosome fragments could be obtained through asymmetric fusion strategy to establish a novel platform for potato genetics. Main results gained are as bellow.1. Establishment of protoplast culture system for fusion parents. Based on ploidy confirmation of potatoes, with previously developed method, seven potato genotypes were chosen for the protoplast culture. The results showed that response to the culture conditions was genotype-dependent indicated by difference in callus formation during the shallow-liquid culture phase at the beginning of protoplast culture. Three genotypes,8#, AC142and AC239, performed better than others. Only AC142regenerated plants in the following culture procedure. In addition, C9701, a succession of S. chacoense, could form plants although it grew slowly at early stage of the culture.The eggplant parent E508with bacterial wilt resistance was derived from seed of a cultivar "BendiHongqie". The in vitro grown plantlets were used for the protoplast culture. After test of the method for priming and combination of media and hormones, the protocol was optimized that a24h dark priming in CM medium of the leaves taken from 4week-old plants was available for protoplast isolation, CM-I medium was desire for early stage of the protoplast culture, and the combination of1.0mg/1NAA+0.1mg/12,4-D+0.25mg/l ZT+0.25mg/1BA was favorite for late stage culture.2. Fusion and somatic hybrids identification of potato-eggplant. The symmetric protoplast fusion was conducted between a diploid potato AC142and eggplant E508. Thirty-four of117regenerated plants were identified somatic hybrids with15potato specific SSR markers and6eggplant SSR specific markers. Seventeen hybrids first obtained were subjected to ploidy test by flow cytometer. The results, showed that, by aligning to the absorbent peaks of the parents,3hybrids were similar to potato tetraploid,8(hexaploid) were combination of2diploid potato and1eggplant genomes, and the rest6denoted aneuploid. The chromosome counting of hybrid PE29-1(showed peak value of potato tetraploid) and PE3-4(showed peak value of tetraploid potato+diploid eggplant) conformed the flow cytometer results that PE29-1has48chromosomes while PE3-4has72.3. Chromosome composition of symmetric hybrids. The genome in situ hybridization (GISH) was applied to7somatic hybrids (4hexaploids,1tetraploid,2aneuploid) to elucidate their chromosome composition. The results demonstrated that the hexaploids consisted of48potato chromosomes and24eggplant chromosomes, while the aneupolid PE60-10contained54potato chromosomes,16eggplant chromosomes and7rearranged chromosomes. However, the tetraploid hybrid PE29-1identified by SSR markers failed to observe the signal of eggplant chromosomes by GISH. The above findings indicated rare tetraploids derived from diploid potato and diploid eggplant even for symmetric fusion. The diploid potato is likely to be doubled in the process of protoplast fusion, for example in hexaploid hybrids, but it remains unknown whether this doubling comes from a fusion of two diploid potato cells and a diploid eggplant cell, or the diploid potato cell is doubled during the process of protoplast culture. It seems that the chromosome rearrangement is in preference to occur in aneuploids. 4. Cytoplasm composition of symmetric hybrids. The polymorphic mitochondria primer P4(pumD) was employed to dissect the mitochondrion components of34somatic hybrid. Except for PE29-4and PE57-4that contained only potato bands, all hybrids showed integration of the bands from two fusion parents. Furthermore, there were novel bands detected, indicating recombination of parental mitochondria genomes after fusion. Two polymorphic chloroplast markers, NTCP9and NTCP12, were used for chloroplast genome analysis of the hybrids. Interestingly, all34hybrids showed only potato chloroplast. Present results suggested that, in somatic hybrids obtained, mitochondria are often to recombine, whereas the chloroplast of one parent is more capable to retain than the other, this may occur preferentially to the parent contributing dominant DNA dosage in the hybrids.5. Bacterial wilt resistance of symmetric hybrids. The disease assessment was carried out with11somatic hybrids rooted normally. The plantlets grown in vitro were inoculated with cell suspension of R. solanacearum race1. There9hybrids scored resistance, of which PE4-1showed a higher resistance level than the resistant eggplant parent and other8hybrids had no difference compared to the resistant control. Three hybrids which could grow normally in pots and showed resistance in vitro were tested for resistance to bacterial wilt. Three inoculated somatic hybrids showed high resistance similar to the eggplant fusion parent. These results demonstrate that the protoplast fusion is a feasible approach to successfully transfer bacterial wilt resistance from eggplant to potato.6. Asymmetric fusion of potato+eggplant and identification of somatic hybrids. Eggplant E508was exposed to UV light treatment to breakdown the chromosomes as the donor to fuse with potato protoplast of8#as recipient. Forty-seven of73regenerated plants were assayed with the flow cytometer and the results showed36tetraploids similar to potato parent8#,6mixoploids,2aneuploids and1octaploid, as well as2with varied peak values that may be resulted from chromosome elimination during the subcultures. The variations in plant morphology of16regenerated plants were observed in tuber shape, leaf shape and growth vigour. However, all the16regenerated plants can produce tubers normally. It worth further clarifying if the morphology change comes from the insertion of eggplant chromosome fragments.In the fusion with eggplant E508as recipient and UV-treated dihaploid potato Ne16as doner,12plants were generated from335calli. Since most of them were difficult in rooting and slow in growth, only3rooted plants were taken for further analysis. Amplification with3polymorphic markers, STI024, STI046and STM1088, confirmed the rooted plants were somatic hybrids. Furthermore, the cytoplasmic analysis revealed that these hybrids exhibited an integrated mitochondrion pattern of the parents and a potato chloroplast pattern.更多还原