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.     

Interspecific hybrid plant formation by electrofusion in Nicotiana

Summary We obtained complete hybrid plants by electrofusion of mesophyll protoplasts from Nicotiana glauca and N. langsdorffii. Electrofocusing analysis of Fraction I proteins isolated from the leaves of these plants confirmed their hybridity. Cytological analysis indicated that the chromosome number (2n) of these plants is between 60 to 66, suggesting that they are the products of triple fusion. All plants were fertile and set viable seeds after self pollination. As we did not use an AC field for electrofusion, the present results indicate that an AC field is not essential for obtaining hybrid plants with electrofusion.     

Production of interspecific somatic hybrid plants in the genus Medicago through protoplast fusion

Summary Symmetric somatic hybrid plants have been produced by electrofusion of leaf protoplasts of Medicago sativa and callus protoplasts of Medicago coerulea. The selection of hybrid individuals has been performed at the cellular level by recording the positions of single heterocaryons immobilized in a semisolid culture medium. The hybrid nature of the heterokaryons was assessed in fluorescent light on the basis of their color. Hybrid minicalli were picked up manually and grown first on propagating, and then on regenerating, media. Six putative hybrid calli were selected and two of them regenerated several plants. The hybrid nature of the regenerants was confirmed by cytological and isozyme analysis. Among the several morphological traits taken into account for the characterization of somatic hybrid plants, some were intermediate, some lower, and some higher, with respect to the parents. The somatic hybrid plants were fertile and set seed. The production of somatic hybrid plants in the genus Medicago is discussed in relation to the regenerating capability of parental protoplasts.This research was supported by the National Research Council of Italy, Special Project RAISA, Subproject N. 2 paper N. 347     

Morphological and molecular characterization of fertile tetraploid somatic hybrids produced by protoplast electrofusion and PEG-induced fusion between Lycopersicon esculentum Mill. and Lycopersicon peruvianum Mill.

Summary Mesophyl protoplasts of two genotypes of cultivated tomato (Lycopersicon esculentum Mill.) and one of its wild relative species (Lycopersicon peruvianum Mill.) were fused by using electrofusion and polyethyleneglycol-induced fusion. Forty-three fertile tetraploid somatic hybrid plants, each deriving from separate calli, were recovered from both fusion procedures. Electrofusion appeared more efficient than chemical fusion for the production of somatic hybrids. These plants appeared morphologically similar, whatever the fusion procedure and tomato genotype. They had intermediate leaf, inflorescence, and flower morphology. After self-pollination, the hybrids set fruit of intermediate size and color. The hybrid nature of these plants was confirmed by isoelectric focusing of the Rubisco small subunits used as nuclear markers. L. esculentum and L. peruvianum were distinguished by means of two chloroplast markers: CF1-ATPase subunit as analyzed by isoelectro-focusing and ct DNA restriction patterns. All hybrids displayed both ct markers of only one parent with no biased transmission. Mitochondrial (mt) DNAs were prepared from flower buds by using miniaturized CsCl gradients. Preliminary analysis indicated that mt genomes from the hybrids all differed from those of both parents. mt DNA Sall restriction enzyme analysis revealed that all but two hybrids contained one novel fragment of 13.5 kb. Gene mapping experiments showed that the mt apocytochrome b and ATPase subunit 9 homologies in the somatic hybrid mt DNA resembled L. esculentum and L. peruvianum, respectively; the mt nad5 probe distinguished at least four distinct patterns in the hybrids. These results indicated that mt DNA rearrangements involving intergenomic recombinations occurred through protoplast fusion. A greater mt DNA polymorphism was induced with chemical fusion than with electrofusion.     

Characterization of the multiple resistance traits of somatic hybrids between Solanum cardiophyllum Lindl. and two commercial potato cultivars

Interspecific somatic hybrids between commercial cultivars of potato Solanum tuberosum L. Agave and Delikat and the wild diploid species Solanum cardiophyllum Lindl. (cph) were produced by protoplast electrofusion. The hybrid nature of the regenerated plants was confirmed by flow cytometry, simple sequence repeat (SSR), amplified fragment length polymorphism (AFLP), microsatellite-anchored fragment length polymorphism (MFLP) markers and morphological analysis. Somatic hybrids were assessed for their resistance to Colorado potato beetle (CPB) using a laboratory bioassay, to Potato virus Y (PVY) by mechanical inoculation and field trials, and foliage blight in a greenhouse and by field trials. Twenty-four and 26 somatic hybrids of cph + cv. Agave or cph + cv. Delikat, respectively, showed no symptoms of infection with PVY, of which 3 and 12, respectively, were also resistant to foliage blight. One hybrid of cph + Agave performed best in CPB and PVY resistance tests. Of the somatic hybrids that were evaluated for their morphology and tuber yield in the field for 3 years, four did not differ significantly in tuber yield from the parental and standard cultivars. Progeny of hybrids was obtained by pollinating them with pollen from a cultivar, selfing or cross-pollination. The results confirm that protoplast electrofusion can be used to transfer the CPB, PVY and late blight resistance of cph into somatic hybrids. These resistant somatic hybrids can be used in pre-breeding studies, molecular characterization and for increasing the genetic diversity available for potato breeding by marker-assisted combinatorial introgression into the potato gene pool.     

Production and characterization of somatic hybrids between the Japanese radish and cauliflower

Summary Somatic hybrids between the Japanese radish and cauliflower (Brassica oleracea) were produced by protoplast electrofusion in order to introduce clubroot disease resistance in the Japanese radish (Raphanus sativus) into Brassica crops. After electrofusion of iodoacetamide-treated cauliflower protoplasts with untreated radish ones, culture was performed under conditions, that allowed only cauliflower protoplasts to regenerate. Out of 40 regenerated plants, 37 were morphologically of a hybrid type and 3 of a cauliflower type. On the basis of isozyme and RFLP analysis, all of the hybrid-type plants tested proved to be true hybrids. Of the 10 true hybrids tested, 9 were found to contain chloroplasts similar to those found in the Japanese radish, while only 1 contained those of the cauliflower. Using two mitochondrial genes as probes, we were able to show that 3 hybrids contained mitochondria of the Japanese radish, with some modification, while 7 hybrids had either parental or new patterns. All of the hybrid-type plants showed resistance to clubroot disease as high as that found in the Japanese radish. Some hybrids were self-fertile. All of the self-fertile hybrids were found to contain 36 chromosomes, indicating that they were amphidiploids. In addition, a few seeds were obtained from a backcross of the self-fertile hybrids to both parents.     

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.     

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.     

Regeneration of intergeneric somatic hybrid plants between Lycopersicon esculentum and Solanum muricatum

Summary Mesophyll protoplasts of tomato (Lycopersicon esculentum) and pepino (Solanum muricatum) were fused by using an electrofusion method and cultured in modified MS medium supplemented with naphthaleneacetic acid and kinetin, in which only pepino and somatic hybrid protoplasts could divide. Somatic hybrid plants showing intermediate characteristics in morphology were regenerated from the calli exhibiting vigorous growth in contrast with those of pepino. The hybrid nature of these plants was confirmed by cytological observation and biochemical analyses of phosphoglucomutase isozymes and the fraction-1-protein. The regenerated somatic hybrids grew to flowering stage and set fruits.     

Somatic hybrid plants produced by electrofusion between Solanum melongena L. and Solanum torvum Sw

Summary Somatic hybrid plants between eggplant (Solanum melongena) and Solanum torvum have been produced by the electrofusion of mesophyll protoplasts in a movable multi-electrode fusion chamber. Using hair structure as a selection criteria, we identified a total of 19 somatic hybrids, which represented an overall average of 15.3% of the 124 regenerated plants obtained in the two fusion experiments. Several morphological traits were intermediate to those of the parents, including trichome density and structure, height, leaf form and inflorescence. Cytological analyses revealed that the chromosome numbers of the somatic hybrids approximated the expected tetraploid level (2n=4x=48). Fifteen hybrid plants were homogeneous and had relatively stable chromosome numbers (46–48), while four other hybrids had variable chromosome numbers (35–48) and exhibited greater morphological variation. The hybridity of these 19 somatic hybrid plants was confirmed by analyses of phosphoglucomutase (Pgm) and esterase zymograms.     

Production of fertile somatic hybrids of Gossypium hirsutum?+?G. bickii and G. hirsutum?+?G. stockii via protoplast fusion

Fertile somatic hybrids were obtained via symmetric electrofusion of protoplasts from two combinations of tetraploid cotton (G. hirsutum cv. Coker 201, AD genome) and diploid wild cottons G. bickii (G genome) and G. stockii (E genome), respectively. Observation by morphological, flow cytometric analysis, chromosome counting and RAPD analysis of the tested hybrids of Coker 201 + G. bickii and Coker 201 + G. stockii confirmed the regenerated plants as hybrid status. Cytological investigation of the metaphase root-tip cells revealed there were 78 chromosomes in the hybrids. Flow cytometric analysis showed the tested plants had a relative DNA contents close to the total DNA contents of the two parents. RAPD analysis revealed the hybrids contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the hybrids was intermediate between the two fusion partners. The hybrid plants were successfully transferred to the soil, and they bloomed and set bolls. It is sure that the new hexaploids developed by cell fusion would contribute to cotton breeding through backcrossing with the elite genotypes of G. hirsutum.     

Fertile somatic hybrids between Petunia hybrida and a wild species,Petunia variabilis

Somatic hybrid plants were regenerated following electrofusion between leaf mesophyll protoplasts of P. hybrida (2n = 14) and a wild sexually incompatible species, P. variabilis (2n = 18). The selection of hybrids was based on the hybrid vigour, expressed both in the growth of the callus and at the shoot formation stage, resulting from the combination of parental genomes. Calli exhibiting vigorous growth were selected, and upon transfer to regeneration medium gave rise to shoots. Four regenerated plants from three calli had morphological characteristics intermediate between those of the parents. The hybrid nature of these plants was confirmed by chromosome counts as well as isozyme and DNA analyses. They had amphidiploid chromosome numbers (2n = 32) and were fertile. Following self-pollination and backcrossing with P. variabilis, large numbers of F₂ and BC₁ seedlings were obtained.     

Production and identification of somatic hybrids between Solanum tuberosum and S. papita by using the rolC gene as a morphological selectable marker

A successful hybridization of a diploid clone of Solanum tuberosum with a rolC-transgenic, diploid S. papita clone is reported. By using leaf expiants of this S. papita clone, which after transformation expressed kanamycin resistance, intact protoplasts were obtained, but these protoplasts did not develop to microcalli or regenerate to mature plants. However, protoplasts of the S. tuberosum clone showed a high capacity to regenerate plants from isolated protoplasts. On a medium containing Kanamycin only calli regenerated to plants, which revealed a rolC phenotype (reduced apical dominance with a large number of adventitious shoots and a pale green color of leaves) and later on turned out to be true hybrids. Self fusions of S. papita never developed to microcalli and those of S. tuberosum ceased to develop on the kanamycin-containing medium. Identification of somatic hybrids was done by RFLP and RAPD analysis. In the greenhouse, out of four selected hybrids only FK3.1 was successfully crossed with two standard S. tuberosum varieties (Datura, Desirée). Out of all the seeds germinated, only rolC-negative F¹ seedlings were further characterized. Within the seedling population obvious differences were evident in respect of the S. papita and S. tuberosum characteristics.     

An interspecific somatic hybrid between upland cotton (<Emphasis Type="Italic">G. hirsutum</Emphasis> L. cv. ZDM-3) and wild diploid cotton (<Emphasis Type="Italic">G. klotzschianum</Emphasis> A.)

Somatic hybrids were produced through protoplast electrofusion between the tetraploid cotton Gossypium hirsutum L. cv. ZDM-3 and the wild diploid cotton G. klotzschianum. Hybrid plants were generated from 3 out of 24 callus lines that were derived from fused protoplasts. Hybrid plants were initially identified as somatic hybrids by ploidy analysis: the plants from the 3 callus lines had chromosome numbers near to sum of the two parents (78 = 52 + 26). The plants from the 3 lines were subsequently confirmed as hybrids by cytological, molecular, histological and morphological analyses. The morphology of hybrids was distinct from that of the parents, with elongated stigmas and malformed anthers lacking microspores and pollen, leading to male sterility. It is expected that the male sterility resulted from the high number of univalent and irregular multivalent chromosome pairings per meiocyte.     

Metabolic robustness in young roots underpins a predictive model of maize hybrid performance in the field

Heterosis has been extensively exploited for yield gain in maize (Zea mays L.). Here we conducted a comparative metabolomics‐based analysis of young roots from in vitro germinating seedlings and from leaves of field‐grown plants in a panel of inbred lines from the Dent and Flint heterotic patterns as well as selected F₁ hybrids. We found that metabolite levels in hybrids were more robust than in inbred lines. Using state‐of‐the‐art modeling techniques, the most robust metabolites from roots and leaves explained up to 37 and 44% of the variance in the biomass from plants grown in two distinct field trials. In addition, a correlation‐based analysis highlighted the trade‐off between defense‐related metabolites and hybrid performance. Therefore, our findings demonstrated the potential of metabolic profiles from young maize roots grown under tightly controlled conditions to predict hybrid performance in multiple field trials, thus bridging the greenhouse–field gap.     

Molecular and morphological characterization of somatic hybrids between <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. and <Emphasis Type="Italic">S. etuberosum</Emphasis> Lindl.

Interspecific potato somatic hybrids between Solanum tuberosum L. (di)haploid C-13 and 1 endosperm balance number non-tuberous wild species S. etuberosum Lindl. were produced by protoplasts electrofusion. The objective was to transfer virus resistance from this wild species into the cultivated potatoes. Post-fusion products were cultured in VKM medium followed by regeneration of calli in MS₁₃ K medium at 20°C under a 16-h photoperiod, and regenerants were multiplied on MS medium. Twenty-one somatic hybrids were confirmed by RAPD, SSR and cytoplasm (chloroplast/mitochondria) type analysis possessing species-specific diagnostic bands of corresponding parents. Tetraploid nature of these somatic hybrids was determined through flow cytometry analysis. Somatic hybrids showed intermediate phenotypes (plant, leaves and floral morphology) to their parents in glass-house grown plants. All the somatic hybrids were male-fertile. ELISA assay of somatic hybrids after artificial inoculation of Potato virus Y (PVY) infection reveals high PVY resistance.     

Characteristics of fertile somatic hybrids of G. hirsutum L. and G. trilobum generated via protoplast fusion

Fertile somatic hybrids between tetraploid upland cotton G. hirsutum L. cv. Coker 312 and wild cotton G. trilobum were generated by symmetric electrofusion. Comparisons of morphology, combined with flow cytometric, RAPD, SRAP and AFLP analyses confirmed the hybrid nature of the regenerated plants. The hybrids differed morphologically from the parent plants. Flow cytometric analysis showed that the hybrids had DNA similar in amount to the total combined DNA content of the two parents, and the use of molecular markers revealed that the hybrids contained genomic fragments from both fusion parents, further indicating the hybrid nature of the regenerated plants. The stability of the morphological features of the hybrids was examined in following generations. The hexaploid fusion plants showed strong photosynthesis and a high expression level of some photosystem-related genes. Our results suggest that novel traits may be incorporated in cotton breeding programs through the production of somatic hybrids and the backcrossing of these plants with elite cultivars.     

An Improved Protocol for Microcallus Production and Whole Plant Regeneration from Recalcitrant Banana Protoplasts (Musa spp.)

One important limitation for routine production of somatic hybrids in banana (Musa spp.) is the difficulty in protoplast regeneration. To facilitate protoplast regeneration in banana, the crucial step of microcallus production was optimised for the following parameters: nurse culture medium, duration of microcalli on nurse culture, differing nurse cells, and filter composition. A comparative study between two nurse cell media, Ma₂ and PCM, significantly affected the number of microcalli produced, which was 90 × 10³ per Petri dish on Ma₂ with 0.5 μM zeatin and 9.0 μM 2,4 D, and 30 × 10³ per Petri dish on PCM. Moreover, continuous production of microcalli was achieved on Ma₂ and the frequency of embryogenic cell aggregates was higher among microcalli on Ma₂-medium. However, no cell division was observed in protoplasts cultured on Ma₂ in which nurse cells were maintained for 2 weeks suggesting a requirement of effective presence of nurse cells for cell division of banana protoplasts. Use of a filter in conjugation with nurse cells resulted in greater than 7-fold increase in the number of microcalli. Flow cytometry analysis of 124 protoplast-derived plants showed the presence of hexaploid plants (mother plant is triploid) at the frequency of 4%. Together, these data are indicative of the complex factors involved in the regulation of plant cell division and growth. Each individual aspect must be optimised for efficient protocol development.     

Somatic hybrids between the cultivated potato Solanum tuberosum L. and the 1EBN wild species Solanum pinnatisectum Dun.: morphological and molecular characterization

Interspecific somatic hybrids between the 1EBN-wild species Solanum pinnatisectum (S. pnt) and four different diploid breeding lines of Solanum tuberosum (S. tbr) were produced by electrofusion. S. pnt exhibits resistance to Phytophthora infestans and Erwinia blackleg. Somatic hybrids were identified by RFLP analysis using the oligonucleotide (GATA)₄ as a probe. In three of four combinations all regenerates obtained were somatic hybrids. All 86 somatic hybrids between the breeding line H256/1 and S. pnt were analyzed in detail with respect to morphological and molecular characters; 50% of the somatic hybrids showed normal intermediate leaf morphology. Tubers of somatic hybrid plants grown in the greenhouse as well as in the field were evenly shaped and remarkably similar to those of the S. tbr breeding line. Analysis of relative DNA content by flow cytometry revealed that 75% of the somatic hybrids were tetraploid, some were hypotetraploid and others polyploid or mixoploid. Slotblot and RFLP analyses were carried out using repetitive and some single-copy DNA probes. The genome portion of the S. tbr breeding line was determined by slot-blot analysis using the species-specific repetitive probe pSA287. Obviously, most somatic hybrids contain the complete genomes of both fusion partners. In some of the somatic hybrids, a significantly lower intensity of the S. pnt-specific hybridization signal indicated a certain degree of asymmetry.Dedicated to Prof. Melchers on the occasion of his 90th birthday     

GISH,AFLP and PCR-RFLP analysis of an intergeneric somatic hybrid combining Goutou sour orange and <Emphasis Type="Italic">Poncirus trifoliata</Emphasis>

Intergeneric somatic hybrids combining Goutou sour orange (Citrus aurantium L.) with trifoliate orange [Poncirus trifoliata (L.) Raf] were produced by electrofusion and their genetic inheritance analyzed by amplified fragment length polymorphism (AFLP), genomic in situ hybridization (GISH), and PCR-restriction fragment length polymorphism (PCR-RFLP). Sixteen mini-calluses were obtained after 20 days of culture; they all developed into embryoids on EME500 medium. Following several subcultures on shoot induction medium for a total culture period of 6 months, shoots regenerated. The plants grew vigorously with a well-developed root system and exhibited the trifoliate leaf character of P. trifoliata. Ploidy analysis verified that all of the regenerates were tetraploids (2n=4x=36) as expected. GISH analysis confirmed that 18 chromosomes came from trifoliate orange and the remaining 18 from Goutou sour orange, as with most symmetric somatic hybrid plants; moreover, chromosome translocations were also observed in one plant. AFLP analysis of 16 regenerates and their fusion parents indicated that all of the somatic hybrids except one were genetically uniform. Analysis of the somatic hybrid cytoplasmic genomes with universal primers revealed that their chloroplast DNA (cpDNA) banding patterns were identical to those of the mesophyll parent trifoliate orange, while their mitochondria (mt) genomes were of the callus parent sour orange. The potential of GISH in Citrus somatic hybrid analysis is discussed.The first two authors contributed equally to this paper.