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.     

Nuclear genomic composition of asymmetric fusion products between irradiated transgenic Solanum brevidens and S. tuberosum: limited elimination of donor chromosomes and polyploidization of the recipient genome

Summary The production of asymmetric somatic hybrid calli after fusion between gamma-irradiated protoplasts from transgenic Solanum brevidens and protoplasts from S. tuberosum are reported. Transgenic (kanamycin-resistant, GUS-positive) S. brevidens plants and hairy root clones were obtained after transformation with Agrobacterium tumefaciens LBA 1060 (pRi1855) (pBI121) and LBA 4404 (pRAL4404) (pBI121), and A. rhizogenes LBA 9402 (pRi1855) (pBI121), respectively. Leaf protoplasts isolated from the transgenic plants or root protoplasts from the hairy root clones were fused with S. tuberosum leaf protoplasts, and several calli were selected on kanamycin-containing medium. The relative nuclear DNA content of the hybrid calli was measured by flow cytometry (FCM), and the percentages of DNA of the S. brevidens and S. tuberosum genomes in the calli were determined by dot blot analysis using species-specific DNA probes. Chromosome-specific restriction fragment length polymorphism (RFLP) markers were used to investigate the elimination of specific S. brevidens chromosomes in the hybrids. The combined data on FCM, dot blot and RFLP analysis revealed that 18–62% of the S. brevidens DNA was eliminated in the hybrid calli and that the RFLP marker for chromosome 7 was absent in seven out of ten calli. The absence of RFLP markers for chromosomes 5 and 11 hardly ever occurred. In most of the hybrids the ploidy level of the S. tuberosum genome had increased considerably.     

QTL analysis of potato tuberization

Quantitative trait loci (QTLs) affecting tuberization were detected in reciprocal backcrosses between Solanum tuberosum and S. berthaultii. Linkage analyses were performed between traits and RFLP alleles segregating from both the hybrid and the recurrent parent using a set of framework markers from the potato map. Eleven distinct loci on seven chromosomes were associated with variation in tuberization. Most of the loci had small effects, but a QTL explaining 27% of the variance was found on chromosome 5. More QTLs were detected while following alleles segregating from the recurrent S. tuberosum parent used to make the backcross than were detected by following alleles segregating from the hybrid parent. More than half of the alleles favoring tuberization were at least partly dominant. Tuberization was favored by an allele from S. berthaultii at 3 of the 5 QTLs detected by segregation from the hybrid parent. The additive effects of the QTLs for tuberization explained up to 53% of the phenotypic variance, and inclusion of epistatic effects increased this figure to 60%. The most common form of epistasis was that in which presence of an allele at each of 2 loci favoring tuberization was no more effective than the presence of a favorable allele at 1 of the 2 loci. The QTLs detected for tuberization traits are discussed in relationship to those previously detected for trichome-mediated insect resistance derived from the unadapted wild species.Paper number 54 of the Department of Fruit and Vegetable Science, Cornell University     

Karyotypic variability in plants of Solanum melongena regenerated from callus grown in presence of culture filtrate of Verticillium dahliae

Summary Plantlets were regenerated from calli derived from leaf expiants of three genotypes of Solanum melongena (two parental genotypes and their hybrid). The cytological analysis showed that a) plants regenerated were all mixoploid, b) toxic medium (basal medium added with filtrate culture of Verticillium dahliae) was able to evidence karyotypic differences between genotypes not displayed by plants regenerated from callus grown on control medium, c) chromosomal mosaicism persists up to plant maturity and also in the selfed progeny. The results are discussed in terms of a selective process involving genes controlling chromosome number and/or a direct effect of toxic medium on the activity of the same genes.This research is supported by a grant from ERSO (Ente per la Ricerca e Sperimentazione in Ortoflorifrutticoltura e Sementi) — Regione Emilia Romagna     

Field assessment of dihaploid Solatium tuberosum and S. brevidens somatic hybrids

Summary Following both chemical and electrical fusion of protoplasts of a dihaploid line of potato (Solanum tuberosum), (PDH40), with those of the wild species, Solanum brevidens, 11 and 40 somatic hybrid plants, respectively were obtained. Fifteen of these somatic hybrid genotypes and the two parents were studied further in a small field trial to assess field performance and phenotypic variability. In the UK, somatic hybrid plants are classified as genetically engineered organisms, and the UK Advisory Committee on Genetic Manipulation have imposed various restrictions on field experiments. Examination of the somatic hybrids in the field showed extensive phenotypic variability, and no two genotypes were identical. Some of the variation reflected changes in chromosome numbers, but other factors were also involved. Half the somatic hybrid genotypes produced tubers in the field, although the tubers were smaller and differed morphologically from those of PDH40. The results of the study suggest that the extent of somaclonal variation manifested in somatic hybrids is greater than that found in protoplast-derived plants of potato. The implications of this observation and the current regulations concerning field experiments of somatic hybrid plants in the UK are discussed.     

Plant regeneration from the protoplasts of Solanum tuberosum, S. nigrum and S. bulbocastanum

The mesophyll protoplasts were isolated from the Solanum tuberosum (S. tbr) clones of different ploidy level (4x Bzura cv., 2x H-8105, and 2x ZEL-1136) as well as from the wild species: S. bulbocastanum (S. blb, 2x) and two accessions of S. nigrum (S. ngr, 6x). Additionally, the protoplasts were isolated from the cell suspensions of Bzura cv. and H-8105 clone. The conditions of protoplast isolation as well as the media for their culturing and regeneration, were selected and optimized for the studied genotypes. For mesophyll protoplasts, the shooting calli were produced by all the cultured protoclones except that of S. bulbocastanum. The shoots excised from the protoplast-derived calli developed into whole plants in all the studied potato clones but only in one accession of S. nigrum, i.e. S. ngr var. gigantea. As for suspension-cell-derived protoplasts, only H-8105 clone produced the regenerative type of calli, though normal shoots could not be obtained. The regenerative capacity of the protoplasts isolated from leaves and cell suspensions is compared and discussed. We regret to report the death of M. Sc. Maria Borkowska after the completion of this work.     

QTL analysis of potato tuber dormancy

The potential loss of chemical sprout inhibitors because of public concern over the use of pesticides underscores the desirability of breeding for long dormancy of potato (Solanum tuberosum L.) tubers. Quantitative trait locus (QTL) analyses were performed in reciprocal backcrosses between S. tuberosum and S. berthaultii toward defining the complexity of dormancy. S. berthaultii is a wild Bolivian species characterized by a short-day requirement for tuberization, long tuber dormancy, and resistance to several insect pests. RFLP alleles segregating from the recurrent parents as well as from the interspecific hybrid were monitored in two segregating progenies. We detected QTLs on nine chromosomes that affected tuber dormancy, either alone or through epistatic interactions. Alleles from the wild parent promoted dormancy, with the largest effect at a QTL on chromosome 2. Long dormancy appeared to be recessive in the backcross to S. berthaultii (BCB). In BCB the additive effects of dormancy QTLs accounted for 48% of the measured phenotypic variance, and adding epistatic effects to the model explained only 4% more. In contrast, additive effects explained only 16% of the variance in the backcross to S. tuberosum (BCT), and an additional 24% was explained by the inclusion of epistatic effects. In BCB variation at all QTLs detected was associated with RFLP alleles segregating from the hybrid parent; in BCT all QTLs except for two found through epistasis were detected through RFLP alleles segregating from the recurrent parent. At least three dormancy QTLs mapped to markers previously found to be associated with tuberization in these crosses.Paper number 55 of the Department of Fruit and Vegetable Science, Cornell University     

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.     

Fertile somatic hybrids of Solanum species: RFLP analysis of a hybrid and its sexual progeny from crosses with potato

Summary Restriction fragment length polymorphism (RFLP) markers were used to distinguish the chromosomes of Solanum brevidens from those of potato (S. tuberosum) in a fertile somatic hybrid. The hybrid had markers that account for all 24 chromosome arms from each parent, indicating that the hybrid contained at least one copy of each chromosome from each parent. The markers were then used to follow segregation of chromosomes in sexual progeny that resulted from a cross of the somatic hybrid with the potato cultivar Katahdin. Approximately 10% of the sexual progeny lacked one or more of the markers specific to S. brevidens. No one chromosome or marker appeared to be lost preferentially. This infrequent absence of a chromosome marker derived from the wild parent could be explained by intergenomic pairing and recombination. The loss of a marker band for chromosome 8, coupled with the retention of two flanking markers, suggested that a small region of DNA was deleted during regeneration of the somatic hybrid. These results show the value of RFLP analysis when applied to somatic hybrids and their progeny. Clearly, RFLPs will be useful for following the DNA from wild species during its introgression into potato cultivars.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

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.     

Electrofusion of protoplasts from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>, <Emphasis Type="Italic">S. bulbocastanum</Emphasis> and <Emphasis Type="Italic">S. pinnatisectum</Emphasis>

This paper discusses a number of experiments performed, involving the fusion by an electric field of mesophyll protoplasts from Solanum tuberosum cv. Bintje, S. tuberosum dihaploid clones 243, 299 and the wild tuberous disease-resistant species S. bulbocastanum and S. pinnatisectum. Three fusion experiments (S. bulbocastanum + S. tuberosum dihaploid 243, S. pinnatisectum + S. tuberosum cv. Bintje and S. pinnatisectum + S. tuberosum dihaploid 299) yielded 542 calli, the 52 ones of which produced shoots. Obtained regenerants were estimated by the flow-cytometry (FC) and RAPD analysis to determine hybrid plants.The utilisation of the FC as a useful method for detecting somatic hybrids is also discussed in this paper. The combination S. bulbocastanum + S. tuberosum dihaploid 243 led to the creation of eight somatic hybrids, the combination S. pinnatisectum + S. tuberosum cv. Bintje yielded four somatic hybrids and the combination S. pinnatisectum + S. tuberosum dihaploid 299 resulted in no hybrid regenerants. Morphology in vitro, growth vigour and production of tuber-like structures were evaluated in hybrid plants. Plants were transferred in vivo for further estimation (acclimatization, habitus evaluation and tuberization ability).     

Somatic hybridization between potato and Nicotiana plumbaginifolia

Summary Electrofusion was carried out between mesophyll protoplasts from the transformed diploid S. tuberosum clone 413 (2n=2x=24) which contains various genetic markers (hormone autotrophy, opine synthesis, kanamycin resistance, -glucuronidase activity) and mesophyll protoplasts of a diploid wild-type clone of N. plumbaginifolia (2n=2x=20). Hybrid calli were obtained after continuous culture on selection medium containing kanamycin. Parental chromosome numbers, determined at 2 months after fusion, revealed hybrid-specific differences between the individual calli. On the basis of these differences three categories of hybrids were distinguished. Category I hybrids contained between 8 and 24 potato chromosomes and more than 20 N. plumbaginifolia chromosomes; category II hybrids had between 1 and 20 N. plumbaginifolia chromosomes and more than 24 potato chromosomes; category III hybrids contained diploid or subdiploid numbers of chromosomes from both parents. The hybrids were evenly distributed over the three categories. After a 1-year culture of 24 representative hybrid callus lines on selection medium the karyotype of 10 hybrids remained stable, whereas 8 hybrids showed polyploidization of the genome of one parent, together with no or minor changes of the chromosome numbers of the other parent. Six hybrids showed slight changes in the hybrid karyotype. The elimination of chromosomes of a particular parent was not correlated to their metaphase location. The processes of spontaneous biparental chromosome elimination leading to the production of asymmetric hybrids of different categories are discussed.     

Somatic hybrids between Solanum etuberosum and diploid,tuber bearing Solanum clones

Electrofusion was used to obtain somatic hybrids between Solanum etuberosum (2n=2x=24) and two diploid potato lines. These hybridizations were conducted to determine if haploidxwild species hybrids are better fusion partners than conventional S. tuberosumGp. Tuberosum haploids. Restriction fragment length polymerase (RFLP) analyses of the putative somatic hybrids confirmed that each parental genome was present. The somatic hybrids between S. etuberosum and a haploid S. tuberosum clone, US-W730, were stunted and had curled, purple leaves. In contrast, somatic hybrids between S. etuberosum and a haploidxwild species hybrid (US-W 730 haploidx S. berthaultii), were vigorous and generally tuberized under field conditions. These hybrids were designated as E+BT somatic hybrids. Analyses of 23 E+BT somatic hybrids revealed a statistically significant bias towards the retention of S. etuberosum chloroplasts. Stylar incompatibilities were observed when the E+BT somatic hybrids were used as pollen donors in crosses with S. tuberosum cultivars. Reciprocal crosses did not show this incompatibility. The progeny were vigorous and had improved tuber traits when compared to the maternal E+BT parent. RFLP analyses of three sexual progeny lines confirmed the presence of all 12 S. etuberosum chromosomes. In two of these lines, RFLPs that marked each of the 24 chromosome arms of S. etuberosum were present. However, RFLP markers specific for regions on chromosomes 2, 7, and 11 were missing from the third clone. Because other markers for these chromosomes were present in the progeny line, these results indicated the likelihood of pairing and recombination between S. etuberosum and S. tuberosum chromosomes.     

Genetic mapping from field tests of qualitative and quantitative resistance to Phytophthora infestans in a population derived from Solanum tuberosum and Solanum berthaultii

Under controlled field conditions, a Solanum backcross population segregated for resistance to Phytophthora infestans. The population (`BCT') had been derived previously by crossing the Solanum tuberosum dihaploid USW2230 × Solanum berthaultii PI473331 to obtain the hybrid M200-30, and then backcrossing the hybrid to the S. tuberosum dihaploid HH1-9. Resistance was assessed from analyses of epidemics in small plots of each individual genotype, and data were recorded as area under the disease progress curve (AUDPC). The parents of the original cross (USW2230 and a selection from PI473331) were not included in the test, but the hybrid was incompatible and HH1-9 was compatible with the tester strain of P. infestans (US-8 lineage). Somewhat more than half of the progeny also were incompatible with the tester strain, indicating the presence of an R gene. This gene segregated from the S. berthaultii parent and mapped 4.8 cm from the RFLP marker TG63 on chromosome 10. We deduce that the R gene is not R-1, R-2, R-3, R-6, or R-7 and is probably not R-4, R-5, or R-10. Among the remaining, compatible progeny, there was a wide range of quantitative resistance. All were more resistant than the susceptible cultivar Superior, and most individuals were much more resistant than the moderately resistant cultivar Kennebec. AUDPC values among the sub-population of compatible genotypes ranged from about 400 to 1500 units the first year and from 400 to 1760 units the second year. At least five quantitative trait loci (QTLs) were detected in this sub-population in both 1997 and 1998, including one detected through segregation of alleles from both the hybrid parent and the recurrent S. tuberosum parent. A model of main and epistatic effects explained 56% and 66% of the variation observed for quantitative resistance to late blight in 1997 and 1998, respectively. Several of the QTLs for late blight resistance were located in regions of the genome to which QTLs for late maturity have previously been mapped.     

Somatic hybrid plants between the forage legumes Medicago sativa L. and Medicago arborea L.

Interspecific somatic hybrid plants were obtained by symmetrical electrofusion of mesophyll protoplasts of Medicago sativa with callus protoplasts of Medicago arborea. Somatic hybrid calli were picked manually from semi-solid culture medium after they were identified by their dual color in fluorescent light. Twelve putative hybrid calli were selected and one of them regenerated plants. The morphogenesis of the somatic hybrid calli was induced by the synthetic growth regulator 1,2 benzisoxazole-3-acetic acid. Somatic hybrid plants showed intensive genome rearrangements, as evidenced by isozyme and RFLP analysis. The morphology of somatic hybrid plants was in general intermediate between the parents. The production of hybrids by protoplast fusion between sexually incompatible Medicago species is related to the in vitro respon siveness of the parental protoplasts. The possibility of using somatic hybrid plants in alfalfa breeding is discussed.     

黑果枸杞原生质体培养及植株再生

该研究以黑果枸杞(Lycium ruthenicum)无菌苗为材料,建立了愈伤组织来源的原生质体再生体系,采用ISSR和FCM技术对再生植株进行了遗传稳定性分析。结果表明：(1)黑果枸杞叶片愈伤组织是产生原生质体的最好材料,在含0.5 mg·mL^-1甘露醇的酶液中,继代1次的叶片愈伤组织中原生质体产量为7.77×10⁶个·g^-1,活力为92%。(2)改良MS培养基 固体液体双层培养(MS₂ 固液双层)是培养原生质体的最好方式,培养10 d的原生质体分裂频率为45.9%,培养20 d的细胞团形成频率为22.9%。(3)在1.5 mg·mL^-1 6 BA+0.1 mg·mL^-1 IBA+MS培养基中,叶片愈伤组织产生的原生质体可分化获得再生植株。(4)ISSR分析显示,再生植株的平均遗传相似系数为0.88;FCM显示再生植株为二倍体,与亲本植株一致。该研究结果为进一步研究枸杞体细胞杂交技术转移野生植物抗逆遗传性状提供科学依据,为枸杞优良品种的选育奠定了基础。     

Allotriploid somatic hybrids of diploid tomato (Lycopersicon esculentum Mill.) and monoploid potato (Solanum tuberosum L.)

Allotriploid somatic hybrids were obtained from fusions between protoplasts of diploid tomato and monohaploid potato. The selection of fusion products was carried out in two different ways: (1) The fusion of nitrate reductase-deficient tomato with potato gave rise only to hybrid calli if selection was performed on media lacking ammonium. Parental microcalli were rarely obtained and did not regenerate. (2) The fusion of cytoplasmic albino tomato with potato gave rise to albino and green hybrid calli and plants. Allotriploids were identified from the two somatic hybrid populations by counting chloroplast numbers in leaf guard cells and by flow cytometry of leaf tissue. Although some pollen fertility of allotriploids and pollen-tube growth of tomato, potato andLycopersicon pennellii into the allotriploid style were observed, no progeny could be obtained. The relevance of allotriploid somatic hybrids in facilitating limited gene transfer from potato to tomato is discussed.     

Studies of the gene expression in a somatic hybrid of dihaploid Solanum tuberosum and diploid S. brevidens using two-dimensional protein electrophoresis

Two-dimensional protein electrophoretic patterns of leaf, stem and microtuber were compared between a somatic hybrid (Solanum tuberosum + S. brevidens) and parental plants. Polypeptide spots observed in leaf of the somatic hybrid (BT-1) were similar to those of S. brevidens. In the stem of BT-1, the spots characteristic for each parental plants were also observed. Three specific spots (W, X, Y) found in BT-1 were identical to those of S. tuberosum, however their appearance in S. brevidens depended on the culture conditions (observed at 16h daylength regime, but not in the dark with high sucrose concentration). Potato tuber storage protein patatin was observed in small amounts in the microtubers of BT-1. The data indicated that gene expression unique to each parental plants also existed in the somatic hybrid.Abbreviations BAP 6-benzylaminopurine - 2,4-D (2,4-dichlorophenoxy) acetic acid - IAA indole-3-acetic acid - MES 2-(N-morpholino) ethanesulfonic acid - NAA 1-naphthaleneacetic acid - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

Asymmetric fusion between wild and cultivated species of potato (Solanum spp.) –detection of asymmetric hybrids and genome elimination

 The objective of this study was to evaluate the suitability of different techniques for a simple and rapid identification of asymmetric hybrids, without the use of selection markers and independent of the fusion partners used. Additionally, the degree of donor DNA elimination was determined. Among 473 viable plants obtained from asymmetric fusion experiments between three di-haploid breeding lines of potato (Solanum tuberosum) and diploid wild species (S. bulbocastanum, S. circaeifolium; X-ray treatment of the wild species) the most promising ones were investigated with three different methods: flow cytometry, RFLP analysis with an oligonucleotide probe (GATA)₄, and with single-copy probes. Flow cytometry, which combines a high screening capacity with detailed information about the DNA content and allows a distinction between asymmetric hybrids and chimeras, detected 31 hypo-tetraploid and 42 hypo-hexaploid regenerates among 224 plants. With the oligonucleotide probe (GATA)₄ only a few asymmetric hybrids were detected among all regenerates. More than 50% of these asymmetric regenerates were chimeras. Concerning the degree of DNA elimination, the results obtained by RFLP analysis with 17 single-copy probes were correlated with the results obtained by flow cytometry. The maximum DNA elimination of the donor genome was 52%. As a trend, an irradiation dosage of 210 Gy caused a higher DNA elimination in the wild species than a dosage of 70 Gy. No calli were obtained after irradiation of the wild species with 420 Gy. Received : 6 December 1996/Accepted: 24 January 1997     

The use of cross prediction methods in a practical potato breeding programme

Summary Most previous studies on cross prediction methods have examined relatively few crosses, particularly in relation to the numbers involved in most breeding programmes. In this paper the feasibility of using cross prediction methods was examined in a practical potato (Solanum tuberosum) breeding scheme by the analyses of progeny from 52 crosses. The variate considered was breeder's preference, a visual assessment made of the harvested tubers to estimate their commercial potential. The results showed that it was possible to identify the superior crosses. Cross prediction based simply on the mean preference scores, averaged over scorers and clones within progenies, estimated on seedlings or first clonal year plants, provided the best estimate of a progeny's performance in the third clonal generation. Predictions based on the expected proportion of clones that would transgress a given target value also provided a good indication of a progeny's potential. The poorest prediction was obtained by using the observed frequency of desirable clones in a progeny sample. The implications for potato breeding are discussed.