Field evaluation of tetraploid somatic potato hybrids

Tetraploid somatic hybrids, derived from ten different fusion combinations, were grown in the field to study the inheritance of quantitatively-controlled agronomic traits. The data from two seasons showed that the heterosis in yield varied between 70% and 230% compared to the mid-parent value of the dihaploid fusion clones. The relative yield of the hybrids compared to two standard varieties (= 100%) ranged from 47% to 106%. Despite large differences in yield between the single hybrids of the same fusion combination, no significant differences could be detected between the hybrids. The analysis of the yield components revealed that the heterosis was mainly due to a highly increased tuber weight, whereas the tuber number was in general intermediate to that of the parent clones. In three combinations, the starch content of the hybrids was 15–20% higher than the midparent value and in the others it was intermediate. Fusion of clones with long oval tubers and with round or round-oval ones showed the round/round-oval form was dominant. Fusion combinations of tuber flesh colour indicated that in most cases yellow was dominant to white, while red skin colour was dominant over yellow.     

Expression of reducing sugar accumulation in interspecific somatic hybrids of potato

Summary A somatic hybridisation programme was undertaken to evaluate the expression of reducing sugar accumulation in potato. Interspecific hybrids created between the Solanum tuberosum cultivar Record and the diploid species Solanum phureja were evaluated at the morphological and molecular levels. These analyses indicated that the protoplast regenerants were partial (asymmetric) hybrids which had undergone elimination of S. phureja chromosomes. Tubers of the parents exhibited significant differences for reducing sugar accumulation during cold storage with S. phureja having lower levels of glucose and fructose than Record. The somatic hybrids resembled the S. phureja parent in terms of reducing sugar accumulation demonstrating that low reducing sugar accumulation is dominant to high reducing sugar accumulation in these particular genotypes. These results are discussed in relation to the exploitation of asymmetric hybridisation for the production of potato genotypes for the potato processing industry.Abbreviations BSA (Bovine serum albumin) - GA₃ (Gibberellic acid) - MS (Murashige and Skoog) - NAA (-Naphthaleneacetic acid) - RAPD (Randomly Amplified Polymorphic DNA)     

Molecular characterisation of inter and intra-specific somatic hybrids of potato using randomly amplified polymorphic DNA (RAPD) markers

Summary Protoplast fusion allows the transfer of both mono- and polygenic traits between species that are sexually incompatible. This approach has particular relevance for potato, and somatic hybridisation has been used to introduce a range of disease resistance genes from sexually incompatible wild species into the cultivated potato gene pool. In addition, protoplast fusion allows the resynthesis of tetraploid genotypes from pre-selected diploid or dihaploid donor parents. A limiting factor for the efficient exploitation of this technology in potato breeding is the difficulty of unequivocally identifying nuclear hybrids (heterokaryons). In order to facilitate the identification of hybrids at an early stage following fusion, Randomly Amplified Polymorphic DNA markers (RAPDs) have been used to characterise molecularly both inter- and intra-specific somatic hybrids of potato. RAPD markers detect naturally occurring polymorphism in the donor genotypes and utilise short oligonucleotide primers of arbitrary nucleotide sequence in combination with the polymerase chain reaction (PCR). The exploitation of RAPDs in the characterisation of both somatic and sexual hybrids is discussed.     

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.     

Cytological analysis of tetraploid hybrids between sweet potato and diploid Ipomoea trifida (H. B. K.) Don.

Summary Tetraploid F₁ hybrids between Ipomoea batatas, sweet potato (2n = 6x = ca. 90), and diploid (2n = 2x = 30) I. trifida (H. B. K.) Don. showed various degrees of fertility reduction. The present study aimed to clarify its causes by cytological analysis of meiotic chromosome behavior in the diploid and sweet potato parents and their tetraploid hybrids. The diploid parents showed exclusively 15 bivalents, and the sweet potato parents exhibited almost perfect chromosome pairing along with predominant multivalent formation. Their hybrids (2n = 4x= 57–63) formed 2.6–5.0 quadrivalents per cell, supporting the autotetraploid nature. The meiotic aberratios of the hybrids were characterized by the formation of univalents, micronuclei, and abnormal sporads (monad, dyad, triad, and polyad). The causes underlying these aberrations were attributed in part to the multivalent formation, and in part to a disturbance in the spindle function. Three hybrids showing serious meiotic aberrations were very low in fertility. The utilization of the sweet potato-diploid I. trifida hybrids for sweet potato improvement is described and, further, the role of interploidy hybridization in the study of the sweet potato evolution is discussed.     

A comparison of performance of tetraploid progenies produced by diploid and their vegetatively doubled (tetraploid) counterpart parents

 Two diploid potato parents, W5295.7 and W5337.3, and their colchicine-doubled tetraploids were used as male to cross with three cultivars: ‘Raritan’, ‘Shepody’ and ‘Superior’. Both diploids produced 2n gametes via first division restitution (FDR) during meiosis. Field experiments were carried out on seedlings of the 12 hybrid progenies in 1991 and 1992. Six of the progenies represented the tetraploid-diploid (4x-2x) hybrids and the other six tetraploid-tetraploid (4x-4x) progenies. The genetic consequence of FDR gametes was examined by comparing the mean and genotypic variance of six tuber traits of 4x-2x and 4x-4x progenies. Tuber appearance, eye depth, specific gravity and tuber size showed significant differences between the means of 4x-2x and 4x-4x progenies from either both or one of the diploid parents. The genotypic variances of these traits in 4x-2x progenies were reduced in various degrees from those observed in 4x-4x progenies. Major genes responsible for the inheritance of these traits are probably located close to the centromere. The two diploid parents may also carry alleles of different size and direction of non-additive genic effects in their 2n gametes. The 4x-2x progenies also outperformed the 4x-4x progenies with respect to the means of total and marketable yield. There were thus heterotic gene loci for high yield located close to the centromere. No marked reduction of genotypic variance, however, was observed. The results suggest that genes for yield would be scattered between the centromere and the site for maximum recombination. Received: 24 April 1996 / Accepted: 26 July 1996     

Chondriome-type characterization of potato: mt α, β, γδɛ and novel plastid-mitochondrial configurations in somatic hybrids

 One hundred and eighty dihaploid clones used for protoplast fusions, and 144 tetraploid German potato cultivars were analysed for their cytoplasms using 11 homologous mt DNA-probes, and were classified as mitochondrial (mt) types α, β, γ, δ, and ɛ according to their RFLP patterns. From the 4x cultivars, 79 had the typical mt-type β of Solanum tuberosum being different from the 46 cvs which had the mt-α type and 19 others with mt-γ. A dendrogram shows their relationships to other Solanum species. The distantly related mt-ɛ was only found in di-haploids, and particularly in clones deriving from Solanum phureja and Solanum andigena. Accessory mt types will be actualized on website (http://www.edv.agrar.tu-muenchen.de/pbpz/ mm/mt/al1.htm). In order to evaluate the genetic potential of novel plastid-mitochondrial configurations we have analyzed four representative populations, which derive from different fusion-combination classes: [α (+) β], [α (+) γ], [α (+) δ] and [α (+) ɛ]. On the mitochondrial expression level, hybrids from an [α (+) ɛ] fusion could be distinguished by in-organello translation from [α (+) β] hybrids, and other di-haploids, by an additional translation product of 15 kDa. In fusion parents with mt-α and -γ an additional atp6 reading frame is detectable in sub-stoichiometric amounts by the use of specific PCR primers. The gene differs from the original 211 bp 3′ from the stop codon. Novel RFLP-patterns in 10% of the somatic hybrids were due to a high-rate replication of this pre-existing parental genome region. A second characteristic for somatic hybrids was the partial addition of parental mt sub-genomes. The major part of them revealed a new organization in their mt genomes at the mt-type characteristic loci rpl5, rps14, cob, rps10, coxI and rpl2, which contain recombination-specific repeats homologous to Petunia spp. and Nicotiana. A schematic model for the formation of novel mitochondrial genomes in potato somatic hybrids is provided. Received: 7 November 1998 / Accepted: 30 November 1998     

Analysis of fertility in somatic hybrids of Nicotiana rustica and N. tabacum and progeny over two sexual generations

Summary Somatic hybrid plants, produced between Nicotiana rustica and N. tabacum by heterokaryon isolation and culture and also by mutant complementation, were examined regarding their ability to set seed. From a total of seventeen independent somatic hybrids, three were found to be partially self-fertile while the others did not set seed. Differences regarding the methods of hybrid selection, parental varieties and chloroplast composition of hybrids did not appear to be significant regarding the ability of plants to set seed. Much variation in fertility was observed in subsequent generations and by recurrent selection of the most fertile, over two generations, it was possible to increase the level of self-fertility in some of the progeny. One R2 derivative possessed approximately a tenfold higher level of self-fertility than it's somatic hybrid parent. The presence of genetic markers from both parents were observed in all progeny indicating their hybrid nature.     

Production of potato intraspecific somatic hybrids with improved tolerance to PVY and Pythium aphanidermatum

Somatic hybridization can be an interesting alternative for the selection of heterozygous and vigorous potato plants through combination of dihaploid genomes. The resulting hybrids can harbour interesting characters and thus can be used in agriculture if they are in agreement with agronomic criteria.

In this report, we used an intraspecific somatic hybridization technique for the production of tetraploid potato lines. Two parental combinations were used in protoplast electrofusion procedure: Aminca-Cardinal and Cardinal-Nicola. The selection of somatic hybrids was based on in vitro plant vigour. Therefore, among the 75 regenerated plants obtained from Aminca-Cardinal fusion, 3 putative hybrids were retained and 2 plant lines were selected among the 54 regenerated from the Cardinal-Nicola fusion. Heterosis was observed in the larger hybrid tuber size compared to the parents’. Our results also showed a precocity in the in vitro tuberization for the hybrids. Moreover, all of the regenerated putative hybrids were tetraploid (2n=4x=48 chromosomes). Isocitrate dehydrogenase and malate dehydrogenase isoenzyme analyses confirmed the hybrid nature of these lines. A molecular characterization performed by PCR amplification of simple sequence repeats and inter-simple sequence repeats confirmed that all these lines were somatic hybrids.

The effect of potato virus Y infection on these hybrid lines was tested by mechanical inoculation of plants cultivated in a greenhouse. The majority displayed a reduction of infection rate associated with a delayed appearance of symptoms compared to the parents. Moreover, complete resistance was noted for one hybrid line (CN2). All hybrids also showed improved tolerance to Pythium aphanidermatum infection during tuber storage or after plant inoculation.     

Expression of freezing tolerance in the interspecific F1 and somatic hybrids of potatoes

 The expression of freezing tolerance was examined in interspecific F₁ and somatic hybrids of potatoes using 20 species and 34 different combinations between hardy and sensitive species. In the field, the frost tolerance of hybrids resembled either that of the hardy parent, the sensitive parent, or the parental mean, depending on the species combination and the genomic ratio (ratio of the number of sets of chromosomes contributed from each parent). Similar phenomena were observed when the non-acclimated freezing tolerance (NA) and the acclimation capacity (ACC) (two independent genetic components of freezing tolerance) were evaluated separately under controlled environments. In general, the expression level of freezing tolerance was higher in hybrids with more genomes contributed from the hardy parent than from the sensitive parent. In addition, the effectiveness or combining ability of genes conferring freezing tolerance from the hardy species also showed some influence on the expression of freezing tolerance. All three parameters, namely NA, ACC and acclimated freezing tolerance (AA) (NA plus ACC), were significantly correlated to the frost tolerance exhibited in the field. This indicates that the controlled freezing test used in this study could provide a good estimate of field performance. The implications of these results in breeding for freezing tolerance in potatoes are discussed. Received: 21 July 1998 / Accepted: 29 September 1998     

Mitochondrial DNA patterns are similar in gametosomatic and somatic hybrids of two Nicotiana species

Summary The segregation and recombination patterns of mitochondrial genome in the somatic hybrids of Nicotiana tabacum and N. rustica were studied by RFLP analysis using four heterologous mitochondrial DNA probes, namely cytochrome oxidase subunit I (COI), cytochrome oxidase subunit II (COII), 26s rDNA and 5s-18s rDNA. These RFLP patterns were compared with those of the gametosomatic hybrids of these two species. A preponderance of N. rustica type patterns was observed in the somatic hybrids. One of the somatic hybrids had N. rustica type pattern with COI probe, novel pattern with COII, and 26s rDNA probe and N. tabacum type pattern with 5s-18s rDNA probe. These patterns are identical to those of some of the gametosomatic hybrids and could only be due to the recombination of mitochondrial genomes of the two parents. The extent and the nature of recombination of mitochondrial genomes is similar in gametosomatic and somatic hybrids.     

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     

Selection of somatic hybrids between diploid clones of potato (Solanum tuberosum L.) transformed by direct gene transfer

Summary Five diploid potato clones have been transformed by electroporation of protoplasts with different selectable markers. The resulting diploid regenerated plants have been used in somatic hybridization. It has been shown that hybrid cell selection on the basis of antibiotic or herbicide resistances brought by the two parents of fusion is an efficient method for the recovery of tetraploid somatic hybrids.     

Early tapetal degeneration and meiotic defects are involved in the male sterility of Solanum commersonii (+) S. tuberosum somatic hybrids

 Somatic hybridization between Solanum commersonii and S. tuberosum resulted in the production of male-sterile hybrid plants, except for one fully male-fertile hybrid. The male-sterile hybrids exhibited a“pollen-less” phenotype, with rare pollen grains which were abnormal in shape and exine sculpture. Microsporogenesis and tapetal development were investigated both in male-sterile and male-fertile somatic hybrids to assess the cytological events that were involved in male sterility. The pattern of male sterility was complex, arising through mechanisms expressed at both sporophytic and gametophytic levels. Various abnormalities occurred first in the tapetum, and later during meiosis-II and cytokinesis. These caused the degeneration of the sporads and of the microspores when they were released. In the male-fertile hybrid, normal development of the tapetum and pollen mother cells was restored. The hypothesis that tapetal breakdown, meiosis-II and cytokinesis defects are related to each other, and depend on nuclear-mitochondrial interactions, is discussed. Because of the formation of multivalent chromosome configurations, it is likely that gene exchange between S. commersonii and S. tuberosum can occur in somatic hybrids, offering potential perspectives for the introgression of useful traits from S. commersonii into S. tuberosum. Received: 10 December 1996/Accepted: 21 March 1997     

Analysis of intra-specific somatic hybrids of potato (Solanum tuberosum) using simple sequence repeats

We have utilised simple sequence repeat (SSR) polymorphism to analyse two sets of potential intra-specific hybrids of potato. Two primer pairs were used and both showed that one set of fusion products could not be true heterokaryons. In the other set, one of the primer pairs showed that unique bands in each of the parents were present in all of the hybrids, unambiguously demonstrating hybridity. This simple and robust, high-resolution assay can be used at the callus level and is amenable to automation, making it possible to reduce greatly the time required to screen a large number of potential somatic hybrids.     

Comparison of the organization of the mitochondrial genome in tomato somatic hybrids and cybrids

Summary The organization of the mitochondrial genome in somatic hybrids and cybrids regenerated following fusion of protoplasts from cultivated tomato, Lycopersicon esculentum, and the wild species, L. Pennellii, was compared to assess the role of the nuclear genotype on the inheritance of organellar genomes. No organellar-encoded traits were required for the recorvery of either somatic hybrids or cybrids. The organization of the mitochondrial genome was characterized using Southern hybridization of restriction digestions of total DNA isolated from ten cybrids and ten somatic hybrids. A bank of cosmid clones carrying tomato mitochondrial DNA was used as probes, as well as a putative repeated sequence from L. pennellii mitchondrial DNA. The seven cosmids used to characterize the mitochondrial genomes are predicted to encompass at least 60% of the genome. The frequency of nonparental organizations of the mitochondrial genome was highest with a probe derived from a putative repeat element from the L. pennellii mitochondrial DNA. There was no difference in the average frequency of rearranged mitochondrial sequences in somatic hybrids (12%) versus cybrids (10%), although there were individual cybrids with a very high frequency of novel fragments (30%). The frequency of tomato-specific mtDNA sequences was higher in cybrids (25%) versus somatic hybrids (12%), suggesting a nuclear-cytoplasmic interaction on the inheritance of tomato mitochondrial sequences.     

Analysis of chloroplast and mitochondrial segregation in three different combinations of somatic hybrids produced within Brassicaceae

Summary Mitochondrial and chloroplast DNA were characterized in three different combinations of somatic hybrids produced between different species within Brassicaceae. The fusions were made between B. campestris and B. oleracea, B. napus and B. nigra and between B. napus and Eruca sativa. The combinations represent interspecific hybridizations, but the phylogenetic distance between the species used in each instance is different. Whereas the B. campestris (+) B. oleracea and the B. napus (+)B. nigra hybrids are both examples of intrageneric hybrids, B. campestris is more closely related to B. oleracea than B. napus is to B. nigra. The fusion of B. napus and E. sativa represents an intergeneric hybridization. Since hybrids were produced with reproducible and uniform fusion and culture methods, a comparison of chloroplast and mitochondrial segregation and mitochondrial DNA (mt-DNA) rearrangements could be made between the combinations. The segregation of both chloroplasts and mitochondria was biased in the B. napus (+)B. nigra and the B. napus (+)E. sativa combination. The nonrandom segregation of chloroplasts and mitochondria could be due to the different ploidy levels of the fusion partners and/or reflect differences in organelle replication rate. Furthermore, segregation of mitochondria was correlated to the differences in phylogenetic distance between the species used in the fusions. However, mitochondrial segregation, in contrast to chloroplast segregation, could in all combinations also have been affected by the cell type used as protoplast source in the fusions. All different chloroplast types could be established within each combination. Hybrids containing chloroplast from one parent together with mitochondria from the other parent were found in two of the combinations, although the majority of the hybrids had mt-DNA that was altered compared to the parental species. The rearranged mt-DNA found in most hybrids was an effect of the heteroplasmic state following protoplast fusion rather than of the tissue culture methods, since no mt-DNA rearrangements were found in B. napus plants regenerated from protoplast culture. The mtDNA restriction patterns of the hybrids with rearranged mt-DNA indicated that specific regions of the mt-DNA were involved in the rearrangements following protoplast fusion.     

Molecular,cytogenetic and morphological characterization of somatic hybrids of dihaploid Solanum tuberosum and diploid S. brevidens

Summary Fifty-eight somatic hybrid plants, produced both by chemical (11) and electrical fusion (47) of protoplasts of dihaploid Solanum tuberosum and S. brevidens, have been analysed by molecular, cytological and morphological methods. The potentially useful euploid plants constituted 34% of the total, of which 20% were tetraploid and 14% hexaploid; the remainder were aneuploid at the tetraploid, hexaploid and octoploid levels. Analysis of chloroplast DNA showed that 55% of hybrids contained chloroplasts from S. brevidens and 45% from S. tuberosum. Hexaploids, the products of three protoplasts fusing together, were analyzed with specific DNA probes, and this revealed that nuclear genome dosages could be either 21 S. tuberosumS. brevidens, or vice-versa. Chloroplast types of hexaploids were not influenced by nuclear genome dosage, and all six possible combinations of genome dosage and chloroplast types were found amongst tetraploids and hexaploids. To examine the morphology of the hybrid population and its possible relation to the chromosome number and chloroplast DNA type, 18 morphological characteristics were measured on greenhouse-grown plants and analyzed by principal component and canonical variate analyses. Both analyses showed that nuclear ploidy has the most prominent influence on the overall morphology of the hybrids. Differential parental genome expression in the morphology of the hybrids is discussed. These results provide useful data on the range of genetic combinations that can be expected to occur amongst somatic hybrid plants.     

New mitochondrial genome organization in three interspecific somatic hybrids of Medicago sativa including the parent-specific amplification of substoichiometric mitochondrial DNA units

Three somatic hybrid plants produced by protoplast fusion between Medicago sativa and each of the three species Medicago coerulea, Medicago falcata and Medicago arborea have been analysed for the composition of their mitochondrial DNA. Restriction fragment length polymorphism (RFLP) analysis of mitochondrial genes in somatic hybrids and their parental lines showed various degrees of rearrangement. The M. sativa+M. coerulea hybrid retained all of the M. coerulea-specific bands but lost all the major M. sativa- specific bands. The M. sativa+M. falcata hybrid showed only M. sativa-specific bands together with non-parental bands, and the M. sativa+M. arborea hybrid showed a partial incorporation of bands from both parents together with non-parental bands. The three different outcomes were attributed mainly to differences in the genetic distance between the parents of each hybrid. Analysis of the sexual progeny of the M. sativa+M. coerulea hybrid showed that a residual mitochondrial DNA subunit of M. sativa was retained in the hybrid cytoplasm. This subunit was amplified and inherited in a mutually exclusive, allelic-like fashion with its M. coerulea homologous counterpart in the sexual progeny of the hybrid. Possible mechanisms for the partitioning of mitochondrial DNA in the generative lineage of the somatic hybrids are discussed in relation to the creation of new nucleus-cytoplasm assortments otherwise impossible to obtain by a sexual cross in Medicago. Received: 5 January 2001 / Accepted: 23 March 2001     

Analysis of the mitochondrial DNA of the somatic hybrids of Solanum brevidens and S. tuberosum using non-radioactive digoxigenin-labelled DNA probes

Summary Mitochondrial (mt) DNAs of somatic hybrids obtained by electrical and chemical fusion of mesophyll protoplasts of S. brevidens and a dihaploid line of S. tuberosum PDH 40 were analysed by Southern hybridization using the digoxigenin-labelled mtDNA sequences nad5 or orf25. In the Southern analysis of the hybrid mtDNA probed with nad5, most of the 19 hybrids analyzed had an RFLP pattern similar, but not identical, to one of the parents, S. tuberosum, PDH40. Nineteen percent of the hybrids had most of the S. brevidens fragments. Five of the hybrids had an identical RFLP pattern to either one of the parents while another two hybrids had novel RFLP patterns. Similar results were obtained by Southern analysis with orf25. These results clearly show that mtDNA rearrangements had occurred at a high frequency in the somatic hybrids. There were no differences in the frequencies of rearrangements observed between the hybrids regenerated from chemical and electrical fusions.