Interspecific hybridization between the cultivated potato Solanum tuberosum subspecies tuberosum L. and the wild species S. circaeifolium subsp. circaeifolium Bitter exhibiting resistance to Phytophthora infestans (Mont.) de Bary and Globodera pallida (Stone) Behrens

Summary Somatic fusions between the cultivated potato Solanum tuberosum and the wild species S. circaeifolium subsp. circaeifolium Bitter were produced in order to incorporate desirable traits into the potato gene pool. Selection of the putative hybrids was based on a difference in callus morphology between the hybrids and their parents, with the hybrids showing typical purple-colored cells in otherwise green calli. In all, 17 individual calli regenerated to plants. Of the nine plants that could be transferred to the greenhouse, eight showed a hybrid and one a parental morphology. Restriction fragment length polymorphism (RFLP) analysis confirmed the hybrid character in the former group. Chloroplast counts in stomatal guard cells and flow cytometric determination of nuclear DNA content showed that four hybrid plants were tetraploid (4x), one was mixoploid (5x–8x), and the others were polyploid (6x; 8x). Three out of four tetraploid hybrids were found to be fully resistant to Phytophthora infestans, and all four hybrids were resistant to Globodera pallida pathotypes Pa2 and Pa3. It was further observed that the type and amount of steroidal glycoalkaloids varied among the tubers of the parents and the hybrids. Using the hybrids as female parents in crosses with S. tuberosum, viable seeds could be obtained. This demonstrates the potential of these hybrids in practical plant breeding.     

Resistance to potato virus Y in somatic hybrids between Solanum etuberosum and S. tuberosum x S. berthaultii hybrid

Somatic hybrids between a potato virus Y (PVY) resistant Solanum etuberosum clone and a susceptible diploid potato clone derived from a cross between S. tuberosum Gp. Tuberosum haploid US-W 730 and S. berthaultii were evaluated for resistance to PVY. All but one of the tested somatic hybrids were significantly more resistant than cultivars Atlantic and Katahdin. However, none was as resistant as the S. etuberosum parent. One hexaploid somatic hybrid, possibly the product of a triple-cell fusion involving one S. etuberosum protoplast and two haploid x S. berthaultii protoplasts, was as susceptible to PVY infection as the cultivars. Tetraploid progeny of the somatic hybrids, obtained from crosses with Gp. Tuberosum cultivars, were neither as resistant as the maternal somatic hybrid parent, nor as susceptible as the paternal cultivar parent. It appears that the introgression of PVY resistance from (1EBN) S. etuberosum into (4EBN) S. tuberosum (EBN-endosperm balance number) will be successful through the use of somatic hybridization and subsequent crosses of the somatic hybrids back to S. tuberosum.     

Unilateral and bilateral hybridization barriers in inter-series crosses of 4x 2EBN Solanum stoloniferum, S. pinnatisectum, S. cardiophyllum, and 2x 2EBN S. tuberosum haploids and haploid-species hybrids

Wild Mexican potato species are an important untapped source of useful variation for potato improvement. Introgression methods such as 2n gametes, chromosome doubling, and crossing with disomic 4x 2 endosperm balance number (EBN) bridge species have been used to overcome post-zygotic endosperm failure according to the EBN hypothesis. Stylar barriers can prevent zygote formation, bilaterally when zygote formation is blocked in both directions of the cross or unilaterally when zygote formation is blocked in self incompatible (SI) × self compatible (SC) crosses. In several Solanaceae species, the S-locus for SI has been implicated in interspecific incompatibility. The objectives of this research were to determine if: (1) disomic 4x 2EBN Solanum stoloniferum can be used as a bridge species for introgression of the Mexican 2x 1EBN species Solanum cardiophyllum and Solanum pinnatisectum, (2) pre- and/or post-zygotic barriers limit hybridization among EBN compatible Solanum inter-series crosses, and (3) reproductive barriers act unilaterally or bilaterally. Fruit formation and seed set was recorded for inter-pollinations of S. stoloniferum, 4x 2EBN chromosome doubled S. cardiophyllum and S. pinnatisectum, and 2x 2EBN S. tuberosum haploids (HAP) or haploid-species hybrids (H-S). In vivo pollen tube growth was analyzed for each cross combination with fluorescence microscopy. Attempts to create bridge hybrids between S. stoloniferum, and S. cardiophyllum or S. pinnatisectum were not successful. Pre- and post-zygotic barriers prevented seed formation in crosses involving S. cardiophyllum and S. pinnatisectum. Self compatibility in S. stoloniferum and S. pinnatisectum suggests that the S-locus does not contribute to the stylar barriers observed with these species. Alternatively, the presence of functional and nonfunctional (SC) S-alleles may explain interspecific incompatibility in intra- and inter-ploidy crosses. A non-stylar unilateral incongruity was discovered in H-S/HAP × S. stoloniferum crosses, indicating either a post-zygotic barrier, or a pre-zygotic barrier acting at or within the ovary. Furthermore, lack of S. stoloniferum pollen rejection may occur through absence of S. stoloniferum pollen-active genes needed to initiate pollen rejection, or through competitive interaction in S-locus heterozygous S. stoloniferum pollen. Introgression strategies using these species would benefit potato breeding by introducing genetic diversity for several traits simultaneously through co-current introgression.     

Production of somatic hybrids between frost-tolerant Solanum commersonii and S. tuberosum: characterization of hybrid plants

Somatic fusion of mesophyll protoplasts was used to produce hybrids between the frost-tolerant species Solanum commersonii (2n=2x=24) and dihaploid S. tuberosum (2n=2x=24). This is a sexually incompatible combination due to the difference in EBN (Endosperm Balance Number, Johnston et al. 1980). Species with different EBNs as a rule are sexually incompatible. Fifty-seven hybrids were analysed for variation in chromosome number, morphological traits, fertility and frost tolerance. About 70% of the hybrids were tetraploid, and 30% hexaploid. Chloroplast counts in stomatal guard cells revealed a low frequency of cytochimeras. The frequency of aneuploids was relatively higher at the hexaploid level (hypohexaploids) than at the tetraploid level (hypotetraploids). The somatic hybrids were much more vigorous than the parents, and showed an intermediate phenotype for several morphological traits and moderate to profuse flowering. Hexaploid hybrid clones were less vigorous and had a lower degree of flowering than the tetraploid hybrid clones. All of the hybrids were female fertile but male sterile except for one, which was fully fertile and self-compatible. Many seeds were produced on the latter clone by selfing and on the male-sterile clones by crossing. The somatic hybrid plants showed an introgression of genes for frost tolerance and an adaptability to cold from S. commersonii. Therefore, the use of these somatic hybrids in breeding for and in genetic esearch on frost tolerance and cold-hardening is suggested.     

Plant regeneration from in vitro culture of anthers of Solanum chacoense Bitt. and interspecific diploid hybrids S. tuberosum L. x S. chacoense Bitt.

Summary Production of plants from cultured anthers of Solanum chacoense clone IP 33, of its interspecific diploid hybrids with S. tuberosum clones IP 354 and IP 372, and of a complex Solanum hybrid containing in its genome S. ajanhuiri is reported. Genotypic differences were found to influence both the induction phase and the regeneration process. Hybrids derived from clone IP 354 of S. tuberosum were much more responsive in culture than hybrids from clone IP 372. Altogether, 507 plants were regenerated and 309 were cytologically analyzed. Of these, 52% were haploid, 47% diploid and 1% mixoploid or tetraploid. A number of diploid plants probably originated from unreduced microspores and some genetic consequences of this event are discussed.     

Fertile Solanum tuberosum+S. commersonii somatic hybrids as sources of resistance to bacterial wilt caused by Ralstonia solanacearum

 The wild potato relative Solanum commersonii is reported to carry resistance to bacterial wilt disease caused by Ralstonia solanacearum. To overcome sexual incompatibilites due to differences in ploidy and endosperm balance numbers, somatic hybrids were made that combine the S. tuberosum and S. commersonii genomes. The resulting somatic hybrid plants are vigorous, but their disease resistance level and their fertility was unknown. We therefore tested the S. commersonii and S. tuberosum source material cv Superior, potato cv Atlantic and six somatic hybrid lines for resistance to a virulent strain of R. solanacearum (race 3, biovar 2) at 28°C. As expected, S. commersonii was significantly more wilt-resistant than the cultivated potatoes. In five of the six somatic hybrid lines, disease resistance levels were similar to that of the resistant S. commersonii parent. The resistance level of the sixth somatic hybrid was intermediate, significantly different from both S. commersonii and S. tuberosum. In controlled crosses, the somatic hybrids in this study proved both to be male- and female-fertile and were self-compatible. More importantly, the somatic hybrids can be crossed with S. tuberosum to produce viable seeds. Received: 23 June 1998 / Accepted: 13 October 1998     

Rescuing abortive inter-EBN potato hybrids through double pollination and embryo culture

Summary Regeneration of inter-EBN hybrids among potato species was achieved using embryo rescue techniques. Tetraploid hybrids between 4x(2EBN) Solanum stoloniferum x 4x(4EBN) S. tuberosum Gp. Andigena as well as diploid hybrids between 2x(1EBN) S. chancayense x 2x(2EBN) S. chacoense were obtained by culturing immature hybrid embryos in nutrient medium. Identification of appropriate embryo developmental stages was critical in developing a suitable protocol for rescuing viable hybrid embryos. The use of IVP clones as the second pollinator in 4x(2EBN) x 4x(4EBN) crosses reduced premature fruit drop and helped to identify triploid hybrids. Morphological and cytological examination confirmed true hybridity for a few of the regenerated plants. Male sterility and meiotic abnormalities were characteristic of the hybrids. Several S. stoloniferum-Andigena hybrids were successfully backcrossed to Gp. Andigena.Cooperative investigation of Vegetable Crops Research Unit, USDA, Agricultural Research Service, and the Wisconsin Agricultural Experiment Station Note: Reference to a specific brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of similar nature not mentioned     

Somatic hybrids produced by protoplast fusion between S. tuberosum and S. brevidens: phenotypic variation under field conditions

Summary Phenotypic and flowering characteristics of hybrid plants generated by protoplast fusion between a tetraploid S. tuberosum line and diploid S. brevidens were assessed under field conditions. Hybrids were compared to both clonal parental material and protoplast-derived plants of each parent. Almost all of the hybrids were hexaploid. A wide range of variation in morphological characters was observed for hybrids and protoclones. Flowering was markedly reduced in protoclones. The majority of hybrids flowered, had viable pollen and set tubers. Tuber and pollen characteristics of hybrids produced from individual fusion calli also varied. The potential usefulness of fusion hybrids in potato improvement is discussed.     

Somatic hybridization in the genus Solanum: S. tuberosum and S. brevidens

Somatic hybrid plants were regenerated from fused mesophyll protoplasts of an albino potato (Solanum tuberosum spp. tuberosum) variant and Solanum brevidens, a non-tuber bearing species which is sexually incompatible with S. tuberosum. These somatic hybrid plants represent the first example of direct hybridization between potato and members of the taxonomic group Etuberosa, and offer the potential for introgressing valuable germplasm from Solanum species outside the sexually compatible range into a worldwide crop species.     

A cytogenetic and phenotypic characterization of somatic hybrid plants obtained after fusion of two different dihaploid clones of potato (Solatium tuberosum L.)

Summary Somatic hybrid plants of various ploidy levels obtained after chemical fusion between two dihaploid clones of potato Solanum tuberosum L. have been analysed by cytological, morphological and molecular methods. The hybrid nature of tetraploid and hexaploid plants and the genome dosage in hexaploid hybrids were confirmed by Giemsa C-banding. Tetraploid and hexaploid hybrids showed numerical as well as structural chromosome mutations. The latter occurred mainly in the nuclear organizing chromosome. The tetraploid hybrids were more vigorous than the dihaploid parents as demonstrated by an increase in height, enlargement of leaves, increase in the number of internodes, restored potential for flowering and increased tuber yield. The grouping of tetraploid somatic hybrids into various classes on the basis of leaf morphology revealed that plants with a full chromosome complement were more uniform than aneuploids. Many hexaploid somatic hybrids were also more vigorous than the dihaploid parents and could be grouped into two different classes on the basis of floral colour and tuber characteristics, the differences being due to their different dosage of parental genomes. Most of the tetraploid somatic hybrids showed pollen development halted at the tetrad stage as one of the parental clones contained a S. Stoloniferum cytoplasm. However, one tetraploid plant produced pollen grains with high viability. The chloroplast genome in the hybrid plants was determined by RFLP analysis. All of the hybrids had a cpDNA pattern identical to one parent, which contained either S. Tuberosum or S. Stoloniferum cpDNA. A slight preference for S. Tuberosum plastids were observed in hybrid plants. No correlation between pollen development and plastid type could be detected.     

Potato germ plasm enhancement with disomic tetraploid Solanum acaule. II. Assessment of breeding value of tetraploid F1 hybrids between tetrasomic tetraploid S. tuberosum and S. acaule

The breeding value of tetraploid F₁ hybrids between tetrasomic tetraploid S. tuberosum and the disomic tetraploid wild species S. acaule was examined. The F₁ hybrids showed a tuber yield and appearance comparable to those of their cultivated parent, indicating a potential as acceptable breeding stocks despite the 50% contribution to their pedigree from wild S. acaule. The cytological behavior of the tetraploid F₁ hybrids was examined to determine the probability of recombination for the introgression of S. acaule genes. The majority of the meiotic configurations at metaphase I was bivalents and univalents with mean frequencies of 17.6 and 9.9, respectively. Further, a low frequency of trivalents and quadrivalents was observed. An acceptable low level of meiotic irregularities were observed at the later stages of microsporogenesis, and a reasonable level of pollen stainability was obtained. Therefore, these hybrids could likely be employed for further introgression. From the cytological observations, the following speculations were drawn: (1) some genomic differentiation exists between the S. acaule genomes, (2) at least one of the S. acaule genomes may be homoeologous to the S. tuberosum genomes, (3) intergenomic recombination would likely occur due to the nature of the genomic constitution of the hybrids, and (4) the nature of sesquiploidy of the hybrids may facilitate efficient introgression and establishment of unique aneuploid and euploid recombinant genetic stocks.     

Application of somatic hybrids between dihaploids of potato Solanum tuberosum L. and wild diploid species from Mexico in breeding: Generation and backcrossing of dihaploids of somatic hybrids

The efficiency of an original approach to involvement of the valuable genetic pool of wild diploid potato species from Mexico is estimated. The essence of this method is in generation of dihaploids (2n = 2x = 24) of tetraploid somatic hybrids (2n = 4x = 48) followed by backcrossing with dihaploids of Solanum tuberosum. A haploid producer, S. phureja IvP35, was used to generate ten dihaploids of S. tuberosum + S. pinnatisectum, all of which crossed with fertile S. tuberosum dihaploids and developed plump viable seeds. This gives the possibility of an efficient introgression of the genes valuable for breeding from wild species to the bred plants at a diploid level, which has several advantages compared with the corresponding procedure at a tetraploid level. A part of the dihaploids produced was compatible (the pollen tubes reached the ovary) with diploid and tetraploid forms of S. pinnatisectum; however, no viable seeds were developed. The attempt to generate the dihaploids of S. tuberosum + S. bulbocastanum somatic hydrides using the haploid producer S. phureja IvP35 was unsuccessful.     

Somatic hybridization between the diploids of <Emphasis Type="Italic">S.</Emphasis> × <Emphasis Type="Italic">michoacanum</Emphasis> and <Emphasis Type="Italic">S. tuberosum</Emphasis>

Interspecific somatic hybrids between a diploid potato clone DG 81-68 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuber-bearing species Solanum × michoacanum were generated and analyzed. About 30 regenerants displaying an intermediate morphology were obtained as a result of three separate PEG-mediated fusion experiments. The RAPD analysis confirmed the hybridity of all the regenerants. About 50% of the hybrid plants exhibited vigorous growth and were stable in culture, while the rest of them rooted poorly and grew slowly in vitro. Most of the hybrid clones were at the tetraploid level (70%), while 30% of the clones examined were at the hexaploid level. The S. × michoacanum (+) DG 81-68 hybrids with growth anomalies were aneuploid. The variation in late blight resistance of the hybrid clones was found in detached leaflet tests, with enhanced resistance characteristic for three tetraploid hybrids.     

Microsporogenesis in three tetraploid somatic hybrids of potato and their di(ha)ploid fusion partners

Summary The microsporogenesis of three somatic hybrids of potato, i.e. one tetraploid Solanum tuberosum (+) S. phureja, one tetraploid and one hypertetraploid S. tuberosum (+) desynaptic mutant, has been examined and compared with the microsporogenesis of the di(ha) ploid fusion partners. The somatic hybrids had a first meiotic division with uni-, bi-, and multivalents like that of tetraploid potatoes, illustrating introgression and dominance over desynapsis. Abnormal spindle orientations at second meiotic division, sporad types with reduced and unreduced cells and viable pollen occurred at various frequencies. Pollen fertility could not be predicted on the basis of pollen fertility of the fusion partners. Pollen sterility was partially due to abnormal chromosome numbers. Only the tetraploid S. tuberosum (+) desynaptic mutant produced normal amounts of viable seeds.     

Novel inter-series hybrids in Solanum, section Petota

Sexual hybrids between distantly related Solanum species can undergo endosperm failure, a post-zygotic barrier in inter-species hybridizations. This barrier is explained by the endosperm balance number (EBN) hypothesis, which states that parents must have corresponding EBNs for viable seed formation. Tests for inter-crossability were made involving the Mexican species Solanum pinnatisectum Dunal. (series Pinnatisecta, A^piA^pi, 1EBN), autotetraploids of this species, Solanum verrucosum Schlechtd. (series Tuberosa, AA, 2EBN), haploids (2x, 2EBN) of the South American S. tuberosum L. (series Tuberosa, A₁A₁A₂A₂, 4EBN), and F₂ haploid-species hybrids with South American species (AA, 2EBN) S. berthaultii Hawkes, S. sparsipilum (Bitter.) Juz. and Bukasov and S. chacoense Bitter. The development of hybrid endosperms was investigated for these combinations by confocal microscopy with regard to cell-division timing and tissue collapse. Novel sexual diploid (AA^pi) and triploid (AA^piA^pi) inter-series hybrids were generated from S. verrucosum × S. pinnatisectum crosses by using post-pollination applications of auxin. F₁ embryos were rescued in vitro. The hybrid status of recovered plants was verified by microsatellite marker analysis, and the ploidy was determined by chromosome counting. The application of phytohormones in inter-ploidy S. pinnatisectum × S. tuberosum crosses, however, did not delay endosperm collapse, and embryos were not formed. Other diploid, 1EBN species tested in remote hybridizations with Group Tuberosum were S. cardiophyllum Lindl., S. trifidum Correll, and S. tarnii Hawkes and Hjert., series Pinnatisecta, and S. bulbocastanum Dunal., series Bulbocastana. Based on the analysis of post-zygotic reproductive barriers among isolated species of section Petota, we propose strategies to overcome such incompatibilities.     

Protoplast-fusion-derived Solanum cybrids: application and phylogenetic limitations

Summary We established interspecific Solanum cybrids in order to study the intrageneric nuclear-organelle compatibility and the introgression of advantageous plasmone-coded breeding traits into potato. Cybridization was performed by the donor-recipient protoplast-fusion procedure. We found that the plastomes of S. chacoense, S. brevidens, and S. etuberosum could be transferred into the cybrids having S. tuberosum nuclear genomes; chondriome components were likewise transferred from the former species into these cybrids. The combination with S. chacoense as organelle donor and potato as recipient resulted in green fertile plants with potato morphology. By using S. etuberosum as an organelle donor and potato as recipient, male-sterile cybrid plants, most of them having pigmentation abnormalities, were obtained. The combination of S. brevidens with potato resulted in palegreen (almost albino) regenerants. The latter albino plantlets had both the chloroplast DNA and the mitochondrial DNA of the donor (S. brevidens) and did not survive the transfer into the greenhouse. An immediately applicative result of this study is the de novo establishment of male-sterile plants in a potato cultivar. Such plants should be useful as seed parents in the production of hybrid, true-potato seeds.     

Improved isolation of dihaploidsolanum tubersoum protoplasts and the production of somatic hybrids between dihaploidS. tuberosum andS. brevidens

Summary A modified protoplast isolation technique, applicable to a range of dihaploidSolanum tuberosum genotypes, has been developed. A combination of high calcium and high pH was used to fuse mesophyl protoplasts of dihaploidS. tuberosum (PDH40) and the diploid wild speciesS. brevidens. Large numbers of colonies were obtained after fusion and putative hybrids selected on the basis of phenotype from regenerated shoots. From these, 11 somatic hybrid plants have been identified by their isoenzyme patterns and morphologic characteristics. Four of these hybrids had the expected chromosome number of 48. The approach of mass culture after fusion followed by selection of hybrids from regenerated shoots and the application of somatic hybridization to potato breeding are discussed.     

Chloroplast DNA variation between the common cultivated potato (Solanum tuberosum ssp. tuberosum) and several South American relatives

Summary Chloroplast DNA (ctDNA) from the tuberbearing Solanum species tuberosum, vernei, phureja, and chacoense has been compared by restriction endonuclease analysis. Digestion by Hind III or Xba I reveal no differences, but digestion with Bam HI and Eco RI reveals minor differences in the ctDNA among these species. The ctDNA restriction patterns of the tetraploid common cultivated potato of North America and Europe, S. tuberosum ssp. tuberosum and the South American tetraploid, S. tuberosum ssp. andigena are identical for all four restriction endonucleases. These data suggest that ssp. tuberosum and ssp. andigena contain similar ctDNA and therefore may share a common ancestor, or direct lineage. The ctDNA restriction patterns of S. vernei and S. chacoense are identical for all four restriction endonucleases, and S. phureja ctDNA, can be distinguished from the other diploid ctDNAs by digestion with Bam HI. None of the diploids analyzed contain ctDNA identical to the tetraploids and therefore either did not contribute their chloroplast genomes to the evolution of the tetraploids, or the ctDNA has diverged since this evolutionary event. The ctDNAs studied did not contain restriction polymorphisms which could be correlated to cytoplasmic male sterility in Solanum. This is the first demonstration of ctDNA diversity in the tuber-bearing Solanum species.     

Characterisation of somatic hybrids between Solanum tuberosum and its frost-tolerant relative Solanum commersonil

 Somatic hybrids between three dihaploid Solanum tuberosum (2n=2x=24) genotypes and the frost-tolerant, diploid, relative Solanum commersonii (2n=2x=24) were analysed for variation in morphological traits, fertility and frost tolerance. The somatic hybrids were more vigorous than their parents and in many ways resembled a tetraploid S. tuberosum. All of the hybrids flowered profusely, although the male fertility was largely dependent on the S. tuberosum genotype used. In one hybrid combination all plants were both male- and female-fertile, while in the other two combinations the majority of plants were male-sterile but female-fertile. The somatic hybrids showed an increase in direct frost tolerance when compared with the dihaploid S. tuberosum parents, and to a varying extent had gained the capacity to cold acclimate. These somatic hybrids will be used in breeding programmes involving repeated cycles of anther culture and somatic hybridisation. Received: 20 May 1997 / Accepted: 12 June 1997     

Interspecific somatic hybrids between Solanum bulbocastanum and S. tuberosum and their haploidization for potato breeding

Protoplast fusion between incongruent Solanum bulbocastanum and S. tuberosum haploids was accomplished to produce hybrids combining elite traits from both parents. We identified 11 somatic hybrids out of 42 regenerants analyzed through ISSR markers. Some hybrids had loss or gain of fragments compared to the parents, likely due to rearrangements and deletions of chromosome segments after fusion, and/or to somaclonal variation during hybrid regeneration. Increased heterotic vigor for some traits as well as high diversity was observed as the effect of both ploidy and fusion combination. Microsporogenesis analysis indicated the occurrence of multivalent configurations and several meiotic abnormalities, such as chromosomes bridges and various spindle orientations. Since all hybrids were sterile, in vitro anther culture was employed for haploidization as a possible strategy to overcome barriers to hybridizations. Haploids were obtained from all the tetraploid S. bulbocastanum (+) S. tuberosum somatic hybrids tested, although with differences in both the number of embryos per 100 anthers cultured and the number of differentiated green plantlets. This is the first report on the successful production of haploid plants from S. bulbocastanum (+) S. tuberosum hybrids.