| 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. |
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