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The species of the Solanum nigrum-complex are wild relatives of the cultivated potato and potentially interesting sources of genetic variation. The traditional method of introgressing a specific trait from a related species is sexual hybridisation followed by recurrent backcrossing but often one or more reproductive barriers have to be overcome in this process. The discovery of the fusion of protoplasts opened new prospects for further broadening of the genetic base of potato because this technique offered the possibility to use distantly related species.Fusion experiments were performed between diploid (2 n =2 x =24) or tetraploid (2 n =4 x =48) potato genotypes and four species belonging to the S. nigrum complex, namely Solanum nigrum (2 n =6 x =72), S. villosum (2 n =4 x =48), S. chenopodioides (2 n =2 x =24) or S. americanum (2 n =2 x =24 and 2 n =6 x =72). All five accessions of the four species of the S. nigrum -complex were able to form fusion hybrids with at least one of the potato genotypes but some combinations were more successful than others. It was shown that the ploidy level as well as the genotype were factors that influenced the somatic combining abilities. In some combinations the cell-selectable markers kanamycin or hygromycin resistance were used but they did not influence the somatic combining abilities considerably. However, such markers can be useful to improve efficient selection of somatic hybrids in sufficient numbers. Almost half (373) of the 761 somatic hybrid plants performed well in vitro , which was in striking contrast with their performance in vivo . Only 60 genotypes out of 761 somatic hybrids were vigorous in the greenhouse and were able to flower.Vigorous somatic hybrids of the fusion combinations 2 xS. americanum (+) Désirée, S. chenopodioides (+) 2 x potato and S. nigrum (+) 2 x potato were used in backross experiments. Only the somatic hybrids of S. nigrum (+) 2 x potato were successfully backcrossed to both potato and S. nigrum . First and second generation backcross progeny with S. nigrum could easily be obtained. Self-fertility was already restored in one of the BC1 genotypes. Backcrosses with potato had a much lower success rate. Only pollinations with tetraploid potato resulted in seed containing berries. Two BC1 genotypes were obtained after 5000 pollinations from which 505 ovules were cultured. The first genotype, BC1-6738, was a vigorously growing genotype, both in vitro and in the greenhouse, and flowered abundantly. The second genotype, BC1-9001, showed many abnormalities and dropped its flowers before anthesis. BC1-6738 was again crossed with tetraploid potato and also in this generation the success rate was low. Over 5000 pollinations resulted in 1750 berries from which over 3000 ovules were obtained. Twelve plants germinated from these ovules, which were not as vigorous in vitro and in vivo as the BC1 parent. Some of the BC2 genotypes were used for further backcrosses but so far no BC3 plants could be obtained.The application of genomic in situ hybridisation (GISH) made it possible to distinguish clearly between S. nigrum and potato chromosomes in mitotic and meiotic chromosome spreads. It was used to determine the genomic constitution of the somatic hybrid F21-26 and its backross progeny. F21-26 was found to be an octaploid with six genomes of S. nigrum and two of potato. BC1-6738, the result of a cross between F21-26 and 4x potato, appeared to be a hexaploid with 36 chromosomes of each of both species. Unfortunately it was impossible to determine with GISH whether the 36 chromosomes of S. nigrum represented three complete genomes. However, AFLP-data showed that none of the AFLP-specific markers that were amplified in the S. nigrum fusion parent and in the somatic hybrid was missing in BC1-6738, which is an indication that no major chromosome elimination has taken place. Most of the eleven BC2 genotypes that were analysed had a near-pentaploid genomic constitution with 14-20 chromosomes of S. nigrum and between 33 and 43 chromosomes of potato.The meiotic behaviour of BC1-6738 and BC2-9019 was also studied with GISH. Chromosome counts in nuclei of BC2 tetrad cells showed that transmission of alien S. nigrum chromosomes to BC3 progeny is likely. An average of 9.1 S. nigrum chromosomes per microspore in BC2-9019 was observed. Meiotic analysis of metaphase I in BC1-6738 and in BC2-9019 indicated clearly that allosyndetic pairing in both bivalent and trivalent formation occurs in these genotypes, which is of importance for homoeologous recombination. So far in none of the backcross derivatives recombination could be shown with GISH.BC1-6738 and eight BC2 genotypes that resulted from the backcross program with potato were tested for their resistance to Phytophthora infestans. The BC1 genotype was as resistant as the S. nigrum fusion parent but among the eight BC2 genotypes that were scored, six were resistant whereas two genotypes showed lesions on the inoculated leaves, indicating they were susceptible.Since no progeny was obtained from the BC2 genotypes, alternative approaches were sought to overcome this sexual crossing barrier. With the aim to improve the crossability of the backcross derivatives, it was attempted to alter the genomic composition of BC1-6738 and BC2-9017 by adding two haploid potato genomes by somatic hybridisation. Five diploid potato genotypes were used in these fusion experiments, of which one contained the hygromycin resistance gene. All vigorous regenerants that resulted from the fusion experiments were used for the estimation of nuclear DNA content through flow cytometry. Plants with a DNA content higher than that of the BC1 or BC2 genotypes were considered potential somatic hybrids. A total of forty-nine potential somatic hybrids resulted from fusion experiments with BC1-6738, from which 20 grew vigorously in the greenhouse and did flower. Eight genotypes produced seeded berries and five genotypes gave seedless berries after pollination with several potato cultivars. Only five potential somatic hybrids were detected among the 79 flow-cytometrically analysed regenerants from BC2-9017 (+) 2x potato fusion experiments. Two of these hybrids were rather vigorous and did flower, but pollinations with potato did not yet give any berry set.Eleven potential somatic hybrids of BC1-6738 (+) 2x potato were selected for GISH-analysis to determine their genomic composition. Theoretically the somatic hybrids were expected to contain 96 chromosomes: 36 of S. nigrum and 60 of S. tuberosum. Six of the selected genotypes had this expected genomic constitution and five genotypes deviated from this expectation. Two of these five, F108-7 and F109-6, were most likely missing one or two potato chromosomes but contained 36 chromosomes of S. nigrum. One genotype, F104-2, had 60 chromosomes of potato origin but between one and eight additional chromosomes or chromosome fragments of S. nigrum per cell were found. The fourth genotype, F101-2, had a higher number of S. nigrum chromosomes than expected (42-45) but the number of potato chromosomes was only slightly higher (41-42) than in the BC1-fusion parent which makes it doubtful that this genotype is a true somatic hybrid. The fifth genotype with a deviating chromosome number was F109-12 which had only 22 S. nigrum chromosomes left but did contain 58 to 60 chromosomes of potato.Crossing experiments were continued with selected BC1-somatic hybrids, which resulted in four backcross progeny plants. Two of these plants were even obtained without ovule culture, directly from mature seeds. Unfortunately these backcross plants were less vigorous than the somatic hybrids from which they were derived and berry set seemed to be low.It can be concluded that traits from S. nigrum have become available for the cultivated potato with the aid of protoplast fusion. However, introgression of these traits by repetitive backcrossing with potato is much more complicated then initially expected although it seems to be possible. Large numbers of starting material and a lot of work and time seem to be necessary to accomplish the introgression of S. nigrum -traits into the cultivated potato. |
