Production and characterization of fertile somatic hybrids of eggplant (Solanum melongena L.) with Solanum aethiopicum L.

Summary In order to produce fertile somatic hybrids, mesophyll protoplasts from eggplant were electrofused with those from one of its close related species, Solanum aethiopicum L. Aculeatum group. On the basis of differences in the cultural behavior of the parental and hybrid protoplasts, 35 somatic hybrid plants were recovered from 85 selected calli. When taken to maturity either in the greenhouse or in the field, the hybrid plants were vigorous, all rapidly overtopping parental individuals. The putative hybrids were intermediate with respect to morphological traits, and all of their organs were larger, particularly the leaves and stems. DNA analysis of the hybrids using flow cytometry in combination with cytological analysis showed that 32 were tetraploids, 1 hexaploid and 2 mixoploids. The hybrid nature of the 35 selected plants was confirmed by a comparison of the isoenzyme patterns of isocitrate dehydrogenase (Idh), 6-phosphogluconate dehydrogenase (6-Pgd) and phosphoglucomutase (Pgm). Chloroplast DNA (ctDNA) restriction analysis using Bam HI revealed that among the 27 hybrid plants analyzed, 10 had S. aethiopicum patterns and the 17 remaining hybrids exhibited bands identical with those of eggplant without any changes. All of the somatic hybrid plants flowered. Both parental plants had 94% stainable pollen, while the hybrids varied widely in pollen viability ranging from 30% to 85%. The somatic hybrids showed high significant variation in fruit production. Nevertheless, there was a tendency for low fertility to be associated often with S. aethiopicum chloroplast type and/or with an abnormal ploidy level, while good fertility was mostly associated with the tetraploid level and eggplant chloroplasts. Interestingly, 2 tetraploid somatic hybrid clones were among the most productive, yielding up to 9 kg/plant. As far as the fertility of the F₁ sexual counterpart was concerned, only 2 fruits of 50 g were obtained. Hybrid fertility in relation to phylogenetic affinities of the fusion partners is discussed.     

Current status in production and utilization of dihaploids from somatic hybrids between eggplant (Solanum melongena L.) and its wild relatives

The major constrains for practical exploitation of the somatic hybrids between eggplant and its wild relatives have been their sterility and tetraploidy which prevented their incorporation into breeding programs. Here we demonstrate that anther culture was successfully utilized to bring back the ploidy level to the diploid status in tetraploid interspecific hybrids between eggplant and the allied species S. integrifolium and S. aethiopicum gr. gilo. Both the relative species are resistant to Fusarium oxysporum f. sp. melongenae and to some strains of bacterial wilt (Ralstonia solanacearum) which are very destructive diseases of eggplant. Dihaploid androgenetic plants were obtained from the somatic hybrids, from the “double somatic hybrid” obtained by sexual cross of the two somatic hybrids [(eggplant + S. aethiopicum) × (eggplant + S. integrifolium)], and from tetraploid backcrossed plants between the somatic hybrid with S. aethiopicum and eggplant. Phenotypical, molecular, biological and biochemical characterization, and also artificial inoculation with Fusarium oxysporum are consistent with a recombination between the genomes of the species involved in the hybridizations. Dihaploids resistant to Fusarium were successfully backcrossed with eggplant. Besides their utility as potential valuable breeding materials, the introgressed lines obtained may be utilized in genetic and molecular studies about the resistance to Fusarium from S. integrifolium and S. aethiopicum gr. gilo.     

Production and characterization of somatic hybrids between Solanum melongena L. and S. sisymbriifolium Lam.

Summary Protoplasts of 6-azauracil (AU) resistant cell lines of Solanum melongena L. were fused with protoplasts of S. sisymbriifolium Lam. to create somatic hybrids between these sexually-incompatible species. Following fusion, colonies were selected which were capable of growth in medium containing 1mM AU. These colonies were placed on medium containing zeatin which had been shown to stimulate anthocyanin production during shoot organogenesis in tissue explants of S. sisymbriifolium but not in S. melongena. A total of 37 anthocyanin-producing colonies were identified from which 26 hybrid plants were regenerated. The morphological traits intermediate to those of the parents included: flower colour, leaf shape, and trichome density. Cytogenetic analysis revealed that all hybrids were aneuploids but their chromosome numbers were close to the expected number of 48. Isozyme analysis revealed that nuclear genes of both parents were expressed in the hybrids. In addition, isoelectric focussing of the large subunit of ribulose 1,5-bisphosphate carboxylase (Rubisco) provided evidence that each hybrid expressed only the S. sisymbriifolium chloroplast genome. All hybrids regenerated thus far have been sterile.Contribution No. 787 Ottawa Research Station     

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.     

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.     

Evidence for tetrasomic inheritance in a tetraploid Solanum commersonii (+) S. tuberosum somatic hybrid through the use of molecular markers

In order to assess the potential for interspecific recombination between the cultivated Solanum tuberosum (tbr) and the sexually isolated wild species Solanum commersonii (cmm), genetic analysis of a F₂ progeny obtained by selfing one tetraploid cmm (+) tbr somatic hybrid was performed through molecular markers. For this purpose, the extent of disomic and/or tetrasomic inheritance of species-specific RAPD and AFLP markers was determined by following their segregation in a 90-genotype progeny, and testing all the possible segregation ratios in a selfed tetraploid progeny. The RAPD analysis performed using 16 primers revealed that the cmm-specific RAPDs were mainly (93.7%) duplex markers and were equally distributed between loci with a disomic (46.7%) and tetrasomic (53.3%) inheritance. The AFLP analysis led to the identification of 272 (58%) informative AFLPs, which were either cmm- or tbr-specific markers. About 63% of cmm-specific AFLPs were duplex loci, most of which (92.6%) were inherited as tetrasomic loci. As regards the tbr-specific AFLPs, the percentage of simplex loci (52.9%) was higher than that of duplex loci (32.6%), and among the latter most (88.5%) were inherited as tetrasomic loci. Overall, 130 duplex markers were found, of which 53.1% were cmm-specific and 46.9% were tbr-specific. Out of 130 markers, 18 (13.8%) were inherited as disomic, and 112 (86.2%) as tetrasomic, loci. This implies that the majority of duplex markers were located on chromosomes which at meiosis tend to randomly pair as bivalents or to form tetravalents. The total number of simplex loci was 119, and most of them (82.3%) were tbr-specific loci. In some cases the observed segregation ratios even allowed us to clearly determine whether a random chromosome or chromatid segregation was detected. This was the case of three cmm-specific RAPDs, 19 cmm- and 25 tbr-specific AFLPs, which fit a 20.8:1 or 2.5:1 ratio, both cases for which a clear random chromatid segregation can be assumed, since they represent the limit of segregation expected when the distance between the locus and the centromere always leads to a cross-over event. The percentage of ascertained crossing-over events was around 37% out of the tetrasomically inherited loci clearly identified (128 loci), a value indicating that the flow of genes from the sexually isolated S. commersonii to the cultivated potato is possible, for at least a large proportion of genes. Received: 23 July 2001 / Accepted: 9 August 2001     

Chloroplast and mitochondrial DNA composition of triploid and tetraploid somatic hybrids between Lycopersicon esculentum and Solanum tuberosum

The chloroplast (cp) DNA type and mitochondrial (mt) DNA composition of 17 somatic hybrids between a cytoplasmic albino tomato and monoploid potato (A7-hybrids) and 18 somatic hybrids between a nitrate reductase-deficient tomato and monoploid potato (C7-hybrids) were analyzed. Thirteen A7-hybrids and 9 C7-hybrids were triploids (with one potato genome); the other hybrids were tetraploid. As expected, all A7-hybrids contained potato cpDNA. Of the C7-hybrids 7 had tomato cpDNA, 10 had potato cpDNA and 1 hybrid contained both tomato and potato cpDNA. The mtDNA composition of the hybrids was analyzed by hybridization of Southern blots with four mtDNA-specific probes. The mtDNAs in the hybrids had segregated independently from the cpDNAs. Nuclear DNA composition (i.e. one or two potato genomes) did not influence the chloroplast type in the C7-hybrids, nor the mtDNA composition of A7- or C7-hybrids. From the cosegregation of specific mtDNA fragments we inferred that both tomato and potato mtDNAs probably have a coxII gene closely linked to 18S+5S rRNA genes. In tomato, atpA, and in potato, atp6 seems to be linked to these mtDNA genes.     

Cytogenetic analysis of Lycopersicon esculentum (+) Solanum etuberosum somatic hybrids and their androgenetic regenerants

The aim of the study was to characterize genomic relationships among cultivated tomato (Lycopersicon esculentum Mill.) (2n=2x=24) and diploid (2n=2x=24) non-tuberous wild Solanum species (S. etuberosum Lindl.). Using genomic in situ hybridization (GISH) of mitotic and meiotic chromosomes, we analyzed intergeneric somatic hybrids between tomato and S. etuberosum. Of the five somatic hybrids, two plants were amphidiploids (2n=4x=48) mostly forming intragenomic bivalents in their microsporocytes, with a very low frequency of multivalents involving the chromosomes of tomato and S. etuberosum (less than 0.2 per meiocyte). Tomato chromosomes showed preferential elimination during subsequent meiotic divisions of the amphidiploids. Transmission of the parental chromosomes into microspores was also evaluated by GISH analysis of androgenic plants produced by direct embryogenesis from the amphidiploid somatic hybrids. Of the four androgenic regenerants, three were diploids (2n=2x=24 or 2n=2x+1=25) derived from reduced male gametes of the somatic hybrids, and one plant was a hypertetraploid (2n=4x+4=52). GISH revealed that each anther-derived plant had a unique chromosome composition. The prospects for introgression of desirable traits from S. etuberosum into the gene pool of cultivated tomato are discussed. Received: 2 August 2000 / Accepted: 4 December 2000     

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.     

Asymmetric somatic hybrid plants between an interspecific Lycopersicon hybrid and Solanum melongena

Summary Asymmetric somatic hybrid plants were obtained by a modified PEG/DMSO fusion procedure between protoplasts derived from suspension cells of an interspecific tomato hybrid, Lycopersicon esculentum x L. pennellii, and mesophyll protoplasts of Solanum melongena, eggplant. The tomato hybrid was previously transformed with Agrobacterium tumefaciens and contained the kanamycin-resistance marker gene. Prior to fusion, the donor protoplasts of the tomato hybrid were gamma irradiated at 9.0 krad. Thus, non-division of irradiated tomato hybrid protoplasts coupled with kanamycin sensitivity of eggplant enabled selection of somatic cell hybrids. Forty-nine calli selected post-fusion regenerated leaf-like structures in the presence of 50 mg/l kanamycin. However, only four of the 49 calli regenerated intact shoots which rooted in the presence of 50 mg/l kanamycin and were later transferred to the greenhouse. Analysis of phosphoglucoisomerase and peroxidase isozymes, and Southern hybridization with a nuclear-specific pea 45 S ribosomal RNA gene confirmed somatic hybrid status. Cytology revealed that the four hybrid plants had chromosome numbers of 45, 60, 42 and 57, respectively; they were all sterile.     

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     

Induction of atrazine resistance and somatic embryogenesis in Solanum melongena

 The effects of atrazine on cotyledon cultures of Solanum melongena were investigated with a view to establishing a system for in vitro selection of resistant mutants. At herbicide levels producing little growth inhibition some chlorophyll loss occurred associated with the production of albino shoots. At 15 mg/l bleaching was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated with severe suppression of growth. Mutagenesis with EMS resulted in herbicide-resistant mutants based on the embryogenic ability of mutagenised explants placed on medium containing selective levels of sucrose (0.2%) and atrazine (15 mg/l). Different morphogenetic responses were observed when the levels of sucrose (0.2–5%) were altered. Somatic embryogenesis was observed at low sucrose concentrations (0.2–0.5%). Both embryogenesis and shoot regeneration occurred in 1% sucrose. Shoot regeneration was maximum in 2% sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3–5%). However, lowering of sucrose concentration from 2% to 0.2% caused complete bleaching, permitting the selection of herbicide-resistant mutants. Received: 26 November 1996 / Accepted: 20 December 1996     

GISH confirmation of somatic hybrids between Solanum melongena and S. torvum: assessment of resistance to both fungal and bacterial wilts

Interspecific somatic hybrids between Solanum melongena L. (2n = 2x = 24) and two accessions of Solanum torvum Sw. (2n = 2x = 24) were produced in view of transferring resistance to two soil-born pathogens, Ralstonia solanacearum and Verticillium dahliae, from the wild species into the cultivated eggplant. All somatic hybrids were phenotypically homogenous and intermediate between the parents. Their hybrid nature was confirmed by analysis of isozymes and RAPDs. They showed reduced pollen viability, and all but one possessed the chloroplasts from either one or the other parent. As S. melongena and S. torvum chromosomes were morphologically indistinguishable, genomic in situ hybridisation (GISH) was applied to recognise the chromosomes from each parent in the hybrids. As expected, the selected tetraploid plants contained one complete set of chromosomes from each fusion partner. On spread preparations, the two parental genomes were not spatially separated at any time of the cell cycle. Translocation or recombinant chromosomes could not be demonstrated in the mitotic metaphase. Tests for resistance performed in vitro by using suspensions of two strains of R. solanacearum (race 1 and 3) and filtrate of culture medium of one strain of V. dahliae, revealed that S. melongena was susceptible, whereas both accessions of S. torvum had high levels of resistance. Except for two hybrid clones, which were found susceptible to race 3, as was S. melongena, all somatic hybrids tested showed good levels of bacterial and fungal resistance, either intermediate or as high as that of the wild parent.     

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.     

Mitochondrial DNA rearrangements in somatic hybrids of Solanum tuberosum and Solanum brevidens

Summary Thirty somatic hybrids between Solanum tuberosum and Solanum brevidens were analysed for mitochondrial and chloroplast genome rearrangements. In all cases, the chloroplast genomes were inherited from one of the parental protoplast populations. No chloroplast DNA alterations were evident but a range of mitochondrial DNA alterations, from zero to extensive intra- and inter-molecular recombinations, were found. Such recombinations involved specific recombination hot spots in the mitochondrial genome. Not all hybrids regenerated from a common callus possessed identical mitochondrial genomes, suggesting that sorting out of mitochondrial populations in the callus may have been incomplete at the plant regeneration stage. Sorting out of organelles in planta was not observed.     

Production of somatic hybrids by electrofusion in Solanum

Summary Conditions are described for large scale electrofusion of mesophyll protoplasts of dihaploid S. tuberosum with those of diploid S. brevidens. Overall fusion frequencies of 20%–30% were achieved, and following fusion, large numbers of protoplast-derived calli were obtained. Putative somatic hybrid plants were selected from the regenerated shoots by examining their morphological characteristics. Twenty-one somatic hybrids were confirmed by isoenzyme analysis and six somatic hybrids were further confirmed by Southern hybridization. Tetraploid hybrids were obtained, but cytogenetic studies indicated that more of the regenerated hybrids were hexaploid than had previously been found following chemical fusion of the same partners. Some advantages of electrofusion over chemical fusion are discussed.     

Transfer of fungal tolerance through interspecific somatic hybridisation between Solanum melongena and S. torvum

Tolerance to Verticillium wilt is found in a wild relative of eggplant, Solanum torvum. To transfer this tolerance to eggplant (S. melongena), protoplast fusions between eggplant and irradiated S. torvum protoplasts were performed. Putative hybrids were regenerated and evaluated for Verticillium tolerance and field value. A total of 12 plants were eventually selected to be incorporated into the breeding program, as they showed a high degree of Verticillium tolerance under field conditions combined with the morphological characters of eggplant, including normal seed set. DNA analysis showed one of these tolerant plants to have a changed pattern, with some changes corresponding to specific pattern characteristics of S. torvum. Received: 12 April 1998 / Revision received: 9 September 1998 / Accepted: 29 November 1998     

Analysis of chromatin, nuclear DNA and organelle composition in somatic hybrids between Solanum tuberosum and Solanum sanctae-rosae

A protoplast fusion strategy has been applied to advance aspects of a potato breeding programme. A sub-population of somatic hybrids, selected for agronomic potential, between tetraploid Solanum tuberosum cv. Brodick and a diploid EBN2 accession, S. sanctae-rosae was subjected to detailed molecular analysis. This study reports the use of simple sequence repeats (SSRs) to identify nuclear hybrid genomes and PCR and DNA-DNA analysis to determine organelle composition in somatic hybrids derived from these parents. SSR analysis revealed somatic hybrids containing the genetic background of S. tuberosum cv. Brodick with some specific markers from S. sanctae-rosae. One somatic hybrid contained the chloroplasts derived from S. sanctae-rosae, and several hybrids had detectable RFLP mitochrondrial DNA profiles, indicating genetic re-arrangements. We also examined the use of DNase I sensitivity to the genomic and ribosomal RNA sequences in these somatic hybrids as an indicator of changes in chromatin structure. Chromatin and DNAse I analysis showed differential sensitivity to increasing levels of nuclease; DNA from several somatic hybrids was found to be resistant to DNase I compared to the parental plants. The significance of the findings to somatic cell genetics and plant breeding studies is discussed. Received: 6 July 1999 / Accepted: 29 February 2000     

Segregation analysis of RFLP markers reveals a tetrasomic inheritance in apomictic Paspalum simplex.

Apomictic tetraploid Paspalum simplex was crossed with colchicine-doubled diploid sexual plants belonging to the same species. Homologous genomic probes were selected from a partial PstI genomic library for their capacity to detect alleles specific to the apomictic parent, and their segregation was analyzed in the F1 progeny. High levels of polymorphism between apomictic and sexual genotypes were recorded. The heterozygosity was high in both tetraploid and diploid genotypes but the differences between them were not as great as expected. In the sexual parent, some markers segregated as either a monoallelic duplex or a diallelic duplex, while several allelic configurations were observed in the apomictic parent. The segregation of double-dose monoallelic fragments demonstrated the tetrasomic inheritance of apomictic P. simplex. The correlations between apomixis, ploidy level, and tetrasomic inheritance are discussed.