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.     

Fertility and chromosome stability in Brassica napus resynthesised by protoplast fusion

Summary Fertile somatic hybrids between Brassica campestris and B. oleracea have been produced by protoplast fusion. Fusion products were identified by their intermediate protoplast morphology. Heterokaryons were isolated either with micropipettes using a micromanipulator or by flow sorting. About 2% of the obtained calli differentiated to shoots. Of the shoots obtained from manually selected heterokaryons, 100% were true hybrids as confirmed by isozyme analysis while 87% of the flow sorted ones showed a hybrid pattern. Ploidy level of the hybrid plants was determined by chromosome counting and relative DNA-content analysis. The sum of the chromosome number (38) from the two fusion partners were found in 30% of the hybrids; 9% had fewer and 61% had more chromosomes. Pollen viability and seed set varied with ploidy level. Compared to natural B. napus, a pollen viability of 52%–93% and a fertility of 1%–40% was found for the somatic hybrids with normal chromosome number. Restriction enzyme analysis of chloroplast-DNA showed that either B. campestris or B. oleracea chloroplasts were present in the somatic hybrid plants. Of 11 hybrid plants 5 had the campestris and 6 had the oleracea type (11 ratio).     

Somatic hybrids between Brassica juncea (L). Czern. and Diplotaxis harra (Forsk.) Boiss and the generation of backcross progenies

An attempt to transfer genes from droughttolerant Diplotaxis harra, a wild relative of Brassica species, to an elite oil-yielding cultivar, B-85, of mustard (Brassica juncea) was made through protoplast fusion, as the two plant systems are sexually incompatible. By following the standard protocol for PEG-mediated protoplast fusion followed by high pH, high Ca⁺⁺, DMSO treatment and appropriate cell-culture technique, 16 presumptive somatic hybrid plants could be regenerated. Chromosomal analysis of four such somatic hybrids revealed that three of them were asymmetric. Analysis of morphological characters, meiotic chromosomes, and esterase isoenzyme pattern revealed that all the somatic hybrids were different from each other. Furthermore four chromosomes of each genome could undergo homoeologous pairing at meiosis indicating the possibilities for genetic recombination and chromosomal rearrangements. Irregular distribution of chromosomes at anaphase-II at meiosis has been a consistent feature of these plants. Eventually, pollen of all the somatic hybrids showed complete infertility preventing the recovery of any selfed seed. Nevertheless, ovule fertility of one somatic hybrid was not totally impaired as it had set some seeds upon backcrossing with the B. juncea parent. The esterase isoenzyme banding pattern of 24 individual progeny plants of this backcross provided evidence for their recombinant nature. It was thus confirmed that a transfer of genetic traits from Diplotaxis harra to B. juncea had indeed taken place. Furthermore, it was conceptualised that a transfer of alien genes through the protoplast-fusion technique is primarily possible in situations where meiotic pairing of the chromosomes of the two participating genomes generates recombinant gametocytes which can pass through subsequent filial generations.     

Somatic hybrids between Brassica oleracea and B. campestris: selection by the use of iodoacetamide inactivation and regeneration ability

Summary An efficient procedure for obtaining somatic hybrids between B. oleracea and B. campestris has been developed. Hypocotyl protoplasts of B. oleracea were fused with mesophyll protoplasts from three different varieties of B. campestris by the polyethylene glycoldimethylsulfoxide method. The selection of somatic hybrids utilized the inactivation of B. oleracea protoplasts by iodoacetamide (IOA) and the low regeneration ability of B. campestris. The efficiency of recovery of somatic hybrids depended upon the IOA concentration, and when 15 mM IOA was used, 90% of the regenerated plants were found to be hybrid. The somatic hybrids were examined for i) leaf morphology, ii) leucine aminopeptidase (LAP) isozyme and iii) chromosome number. All the hybrids had intermediate leaf morphology and possessed LAP isozymes of both parental species. The chromosome analysis revealed a considerable variation in chromosome number of somatic hybrids, showing the occurrence of multiple fusion and chromosome loss during the culture. Some of the hybrids flowered and set seeds.     

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 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     

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.     

The rapid evolution of self-fertility in Spartina hybrids (Spartina alterniflora × foliosa) invading San Francisco Bay, CA

Plant hybridization can lead to the evolution of invasiveness. We wished to determine whether hybrids between the largely self-sterile Atlantic Spartina alterniflora and California native S. foliosa had evolved self-fertility during their ca 30 year existence in San Francisco Bay, CA. In pollination experiments we found that neither of the parental species was self-fertile, nor were early generation hybrids. A large fraction of later generation hybrids were profusely self-fertile. Inbreeding depression was high in the parental species and early generation hybrids, but was much reduced in later generation hybrids—some even showed outbreeding depression. We found that populations of later generation hybrids and their seedling progeny were almost two-fold more homozygous than early generation hybrids, consistent with the evidence of increased selfing shown by our parentage analyses based upon 17 microsatellite markers. We posit that evolved self-fertility has contributed substantially to the rapid spread of hybrid Spartina in San Francisco Bay.     

The use of genetic markers selected in vitro for the isolation and genetic verification of intraspecific somatic hybrids of Nicotiana tabacum L.

Summary A scheme employing genetic markers obtained by in vitro selection was developed for the stringent isolation of hybrid somatic cells of Nicotiana tabacum. Mesophyll protoplasts that carried two dominant alleles of nuclear genes conferring resistance to the herbicide picloram (pmR1) and the ability to utilize glycerol as the sole source of carbon (Gut) were fused with suspension-culture protoplasts that were marked with the dominant nuclear allele (HuR9) conferring resistance to hydroxyurea. Putative somatic hybrid cell lines were identified by selecting for the Gut and HuR9 markers, followed by an assay for the unselected marker PmR1. Plants regenerated from six of these cell lines were proved to be true somatic hybrids by demonstrating the segregation of each of the three parental markers in the progeny of crosses of those plants with normal seed-derived plants.     

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.     

Rare symmetric and asymmetric Nicotiana tabacum (+) N. megalosiphon somatic hybrids recovered by selection for nuclear-encoded resistance genes and in the absence of genome inactivation

Following protoplast fusion between Nicotiana tabacum (dhfr) and N. megalosiphon (nptII) somatic hybrids were selected on the basis of dual resistance to kanamycin and methotrexate. Despite strong selection for parental nuclear-encoded resistances, only nine N. tabacum (+) N. megalosiphon somatic hybrids were obtained. A preferential loss of the parental N. tabacum nuclear and organelle genome was apparent in some plants in spite of the lack of genomic inactivation by the irradiation or chemical treatment of the parental protoplasts. Only six of the nine hybrids recovered possessed both parental profiles of nuclear RFLPs and isoenzymes. The remaining three hybrids were highly asymmetric with two being identical to N. megalosiphon except for minor morphological differences and rearranged or recombined mitochondrial DNAs (mtDNA), while the other one was distinguishable only by the presence of a rearranged or recombined mtDNA, and was therefore possibly a cybrid. Overall, eight somatic hybrids possessed rearranged or recombined mtDNAs and chloroplast inheritance was non-random since eight possessed N. megalosiphon-type chloroplasts and only one had N. tabacum chloroplasts. In contrast, using the same selection approach, numerous morphologically similar symmetric somatic hybrids with nuclear RFLPs and isozymes of both the parental species were recovered from control fusions between N. tabacum and the more closely related N. sylvestris. In spite of the low frequency of recovery of symmetric N. tabacum (+) N. megalosiphon hybrids in this study, one of these hybrids displayed a significant degree of self-fertility allowing for back-crosses to transfer N. megalosiphon disease-resistance traits to N. tabacum. Plant Research Centre Contribution No. 1579     

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.     

Sexual reproduction of cordgrass hybrids (Spartina foliosa x alterniflora) invading tidal marshes in San Francisco Bay

We genetically analysed cordgrass plants and seedlings throughout the San Francisco, California, USA, estuary and found that hybrids between exotic Spartina alterniflora and native Spartina foliosa are the principal cordgrass invaders and colonizers. We hypothesized that this was due to higher seed set and siring ability by hybrids relative to the native species; too few alien parents remained in San Francisco Bay for our comparative studies. Hybrid seed comprised 91% to 98% of that set in the marsh study plants over the 2 years of the study. Total viable pollen production by hybrid plants was 400 times that of the native plants. Seed and pollen production were highly skewed towards a few hybrid genotypes. In addition to seed produced by hybrid plants, hybrid seed was produced by S. foliosa due to hybrid backcrossing. While the greatest advantage for hybrids was in pollen and seed production, hybrid seeds germinated, and seedlings survived and grew as well or better than the native species. As native S. foliosa becomes increasingly rare, hybrid seed floating on the tides will predominate, overwhelming recruitment sites and resulting in further colonization by hybrids. In an evolutionary context, hybrids with exceptional pollen and seed production will be initially favoured by natural selection, leading to the evolution of even more fertile hybrid genotypes.     

Cytogenetic characterization and fae1 gene variation in progenies from asymmetric somatic hybrids between Brassica napus and Crambe abyssinica.

Sexual progenies of asymmetric somatic hybrids between Brassica napus and Crambe abyssinica were analyzed with respect to chromosomal behavior, fae1 gene introgression, fertility, and fatty-acid composition of the seed. Among 24 progeny plants investigated, 11 plants had 38 chromosomes and were characterized by the occurrence of normal meiosis with 19 bivalents. The other 13 plants had more than 38 chromosomes, constituting a complete chromosomal set from B. napus plus different numbers of additional chromosomes from C. abyssinica. The chromosomes of B. napus and C. abyssinica origin could be clearly discriminated by genomic in situ hybridization (GISH) in mitotic and meiotic cells. Furthermore, meiotic GISH enabled identification of intergenomic chromatin bridges and of asynchrony between the B. napus and C. abyssinca meiotic cycles. Lagging, bridging and late disjunction of univalents derived from C. abyssinica were observed. Analysis of cleaved amplified polymorphic sequence (CAPS) markers derived from the fae1 gene showed novel patterns different from the B. napus recipient in some hybrid offspring. Most of the progeny plants had a high pollen fertility and seed set, and some contained significantly greater amounts of seed erucic acid than the B. napus parent. This study demonstrates that a part of the C. abyssinica genome can be transferred into B. napus via asymmetric hybridization and maintained in sexual progenies of the hybrids. Furthermore, it confirms that UV irradiation improves the fertility of the hybrid and of its sexual progeny via chromosomal elimination and facilitates the introgression of exotic genetic material into crop species.     

Molecular,cytological and morpho-agronomical characterization of hexaploid somatic hybrids in Medicago

Somatic hybrid plants produced by protoplast fusion between tetraploid Medicago sativa (2n= 4x=32) and the diploid species Medicago coerulea (2n= 2x=16) have been RFLP fingerprinted to establish their nuclear composition. Although all of the chromosomes were present, molecular analysis revealed an incomplete incorporation of the alleles of the diploid parent in the fusion products. In the polycross progeny the alleles of both parents segregated in a Mendelian mode. Cytological observations indicated that in the somatic hybrid population minor abnormalities are present; these are restricted mainly to the formation of univalents and lagging chromosomes. Meiosis appeared to be more stable than has been previously reported in the hexaploids of alfalfa. The somatic hybrids grown in the field had a rather vigorous aspect, particularly with respect to the vegetative organs. Forage yield was comparable to that of thmore productive parent. The results are discussed with a view to utilizing the somatic hybrids as starting material for breeding alfalfa at the hexaploid level.This paper was supported by the National Research Council of Italy, Special Project RAISA, Sub-project No.2 paper No. 1911     

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

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

Transfer of resistance to the beet cyst nematode (Heterodera Schachtii Schm.) from Sinapis alba L. (white mustard) to the Brassica napus L. gene pool by means of sexual and somatic hybridization

Summary Sexual and somatic hybrid plants have been produced between Sinapis alba L. (white mustard) and Brassica napus L. (oil-seed rape), with the aim to transfer resistance to the beet cyst nematode Heterodera schachtii Schm. (BCN) from white mustard into the oil-seed rape gene pool. Only crosses between diploid accessions of S. alba (2n = 24, S_a1S_a1) as the pistillate parent and several B. napus accessions (2n = 38, AACC) yielded hybrid plants with 31 chromosomes. Crosses between tetraploid accessions of S. alba (2n = 48, S_a1S_a1S_a1S_a1) and B. napus were unsuccessful. Somatic hybrid plants were also obtained between a diploid accession of S. alba and B. napus. These hybrids were mitotically unstable, the number of chromosomes ranging from 56 to more than 90. Analysis of total DNA using a pea rDNA probe confirmed the hybrid nature of the sexual hybrids, whereas for the somatic hybrids a pattern identical to that of B. napus was obtained. Using chloroplast (cp) and mitochondrial (mt) DNA sequences, we found that all of the sexual F₁ hybrids and somatic hybrids contained cpDNA and mtDNA of the S. alba parent. No recombinant mtDNA or cpDNA pattern was observed. Three BC₁ plants were obtained when sexual hybrids were back-crossed with B. napus. Backcrossing of somatic hybrids with B. napus was not successful. Three sexual hybrids and one BC₁ plant, the latter obtained from a cross between a sexual hybrid and B. napus, were found to show a high level of BCN resistance. The level of BCN resistance of the somatic hybrids was in general high, but varied between cuttings from the same plant. Results from cytological studies of chromosome association at meiotic metaphase I in the sexual hybrids suggest partial homology between chromosomes of the AC and S_a1 genomes and thus their potential for gene exchange.     

Somatic hybrids between unilateral cross-incompatible Petunia species

Summary Somatic hybrid plants regenerated following the fusion of leaf mesophyll protoplasts of Petunia parodii with those isolated from a cell suspension of albino P. inflata. These two species exhibit a unilateral cross-incompatability with a pre-zygotic mode of reproductive isolation preventing hybridizations with P. inflata as the maternal parent. Selection of somatic hybrids relied on the fact that unfused or homokaryon protoplasts of P. parodii did not develop beyond the cell colony stage while those of the putative somatic hybrids and albino P. inflata parent produced callus. Green somatic hybrid calluses were readily identified against the white background of P. inflata following complementation to chlorophyll synthesis proficiency and continued growth in hybrid cells. Shoots, and ultimately flowering plants, were identified as somatic hybrids based on their floral morphology and colour, chromosome number and the fact that they segregated for parental characters. The frequency of somatic hybrid production was comparable to that previously established for two sexually compatible Petunia species.     

T-DNA-tagged chromosome 12 in donor Lycopersicon esculentum × L. pennellii is retained in asymmetric somatic hybrids with recipient Solanum lycopersicoides

Summary Asymmetric somatic hybrid plants were recovered after fusing irradiated mesophyll protoplasts of donor Lycopersicon esculentum × L. pennellii (EP) interspecific hybrid with callus-derived protoplasts of recipient Solanum lycopersicoides. EP plant A54 had been previously transformed by an agrobacterium vector, and the T-DNA insert mapped to the L. esculentum chromosome 12. The T-DNA insert conferred kanamycin resistance to EP that was subsequently used to select cell fusion products and recover asymmetric hybrid plants that retained tagged chromosome 12. Doses of 50- and 100-Gy irradiation promoted the elimination of only a few donor chromosomes. At 200 Gy, the regenerated plants had ploidy levels higher than tetraploid. However, the T-DNA tagged chromosome 12 was always retained in the asymmetric hybrid plants tested. Likewise, all plants from the 100-Gy series, with the exception of number 160, were mixoploid in the root-tip cells. Such mixoploid asymmetric somatic hybrids could be stabilized by inducing adventitious shoots on leaf strips cultured on shoot regeneration medium containing kanamycin. The asymmetric hybrid plants did not produce viable seed when self-pollinated or backcrossed to tomato or S. lycopersicoides. Present address: Department of Biology, University College of London, Gower Street, London, UK     

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.