Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

Genetic properties of abortive products resulting from the protoplast fusion in yeasts

Summary Protoplast fusion was carried out by using a routine technique with various auxotrophic strains of Saccharomyces diastaticus and Schizosaccharomyces pombe, and abortive fusion products were observed as small colonies which appeared more frequently than large prototrophic colonies. Sixty abortive fusion products retained one or more auxotrophic characters derived from S. diastaticus, one of the strains used in the protoplast fusion. Several hybrids were obtained between the abortive products and S. cerevisiae, and the segregants of these hybrids showed many aberrant tetrads with regard to some genetic markers. These segregation patterns would be likely to result if the segregating characters were in the trisomic condition +/+/-. The results indicate that (1) the abortive fusion products are an alien monosome additional (AMA) haploid containing the genome of S. diastaticus and only one chromosome of S. pombe. (2) The additional chromosome of S. pombe, which is integrated with the genome of Saccharomyces, can be stably transmitted to the progeny.     

Somatic hybrids produced by protoplast fusion between S. tuberosum and S. brevidens: phenotypic variation under field conditions

Summary Phenotypic and flowering characteristics of hybrid plants generated by protoplast fusion between a tetraploid S. tuberosum line and diploid S. brevidens were assessed under field conditions. Hybrids were compared to both clonal parental material and protoplast-derived plants of each parent. Almost all of the hybrids were hexaploid. A wide range of variation in morphological characters was observed for hybrids and protoclones. Flowering was markedly reduced in protoclones. The majority of hybrids flowered, had viable pollen and set tubers. Tuber and pollen characteristics of hybrids produced from individual fusion calli also varied. The potential usefulness of fusion hybrids in potato improvement is discussed.     

Plant regeneration from protoplasts of Solanum species with potential agricultural value (S. hjertingii,S. polyadenium,S. capsicibaccatum)

Protoplasts have been isolated from three tuber-bearing Solanum species, S. hjertingii, S. polyadenium and S. capsicibaccatum, that are sexually incompatible with S. tuberosum, but possess potentially useful characters. For isolating protoplasts from leaves of in vitro shoot cultures of S. hjertingii and S. capsicibaccatum growth was improved by including silver thiosulfate in the medium. However, for S. polyadenium, leaves of pot-grown plants were the best source for protoplasts. Following protoplast division and culture, plants were regenerated from protoplasts of each of the species. The pattern of chromosome variation in regenerants was similar to that observed for other diploid and tetraploid Solanum species. The results indicate that it should be possible to introduce the potentially useful germplasm from these wild species into somatic hybrids with S. tuberosum by protoplast fusion.Abbreviations STS silver thiosulfate - BAP benzylaminopurine - GA₃ gibberellic acid - NAA naphthalene acetic acid - IAA indole-3-acetic acid     

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.     

Metabolic complementation for a single gene function associated with partial and total loss of donor DNA in interspecific somatic hybrids

Summary We report here on the obtainment of interspecific somatic, asymmetric, and highly asymmetric nuclear hybrids via protoplast fusion. Asymmetric nuclear hybrids were obtained after fusion of mesophyll protoplasts from a nitrate reductase-deficient cofactor mutant of N. plumbaginifolia with irradiated (100 krad) kanamycin resistant leaf protoplasts of a haploid N. tabacum. Selection for nitrate reductase (NR) and/or kanamycin (Km) resistance resulted in the production of three groups of plants (NR⁺, NR⁺, Km^R, and NR^-Km^R). Cytological analysis of some hybrid regenerants showed the presence of numerous tobacco chromosomes and chromosome fragments, besides a polyploid N. plumbaginifolia genome (tetra or hexaploid). All the regenerants tested were male sterile but some of them could be backcrossed to the recipient partner. In a second experiment, somatic and highly asymmetric nuclear hybrids were obtained after fusion of mesophyll protoplasts from the universal hybridizer of N. plumbaginifolia with suspension protoplasts of a tumor line of N. tabacum. Selection resulted in two types of colonies: nonregenerating hybrid calli turned out to be true somatic hybrids, while cytological analysis of regenerants obtained on morphogenic calli did not show any presence of donor-specific chromosomes. Forty percent of the hybrid regenerants were completely fertile, while the others could only be backcrossed to the recipient N. plumbaginifolia. Since the gene we selected for is not yet cloned, we were not able to demonstrate the transfer of genetic material at the molecular level. However, since no reversion frequency for the nitrate reductase mutant is known, and due to a detailed cytological knowledge of both fusion partners, we feel confident in speculating that intergenomic recombination between N. plumbaginifolia and N. tabacum has occurred.     

Enhancement of nodulation by some arid climate strains of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> using protoplast fusion

Fourteen randomly clover indigenous nodulated Rhizobium strains were isolated from different locations in Saudi Arabia. They were identified as different strains of the genus Rhizobium leguminosarum biovar trifolii and characterized for their intrinsic antibiotic resistance against a range of antibiotics, nodulation capability and plasmid profiles. Results revealed the presence of high molecular weight plasmids (megaplasmids) in all the selected strains. Based on the ability for nodulation production, two weak strains (RtI1 and RtI2) and one efficient strain (RtA1) were selected for protoplast fusion and the numbers of nodules produced by the intra-specific protoplast fusion strains were investigated. Results clearly confirmed the effective role of the protoplast fusion in enhancing both nodulation production capacity of Rhizobium species and their range of antibiotic resistance. Protoplast fusion of the local Rhizobium species resulted in 1.93- to 5.67-fold increase in nodulation number compared to their parental strains, which was considered an excellent result concerning agricultural practices, especially the formation of nitrogen-fixing root nodules on legume crop plants. Protoplast fusion also produced fusants with a wide range of antibiotic resistance, another advantage added to the new strains against environmental stresses. In conclusion, protoplast fusion proved its efficiency as a tool for constructing a second generation of Rhizobia with much better characteristics for efficient applications in arid land.     

Selection methods in bacilli for recombinants and transformants of intra- and interspecific fused protoplasts

The intraspecific fusion frequencies obtained with the direct selection method on a semi-synthetic regeneration medium between strains of B. subtilis and B. licheniformis were distribution from 9.9×10^-2 to 4.5×10^-3, which was one or two orders higher than those of interspecific recombinations between B. subtilis and B. licheniformis.The regeneration media were also useful for selecting interspecific transformants from plasmid carrier to non-carrier. This selection could be used as a primary selection method for inter-and intraspecific recombinants obtained by protoplast fusion.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - Cm chloramphenicol - SMM 0.5 M sucrose-0.02 M MgCl₂-0.02 M maleate buffer, pH 6.5     

Genome composition of asymmetric hybrids in relation to the phylogenetic distance between the parents. Nucleus-chloroplast interaction

Summary A series of fusion experiments were performed between protoplasts of a cytoplasmic albino mutant of tomato, Lycopersicon esculentum (ALRC), and gamma-irradiated protoplasts of L. hirsutum and the Solanum species S. commersonii, S. etuberosum and S. nigrum. These species were chosen for their different phylogenetic relationships to tomato. In all fusion combinations except from those between ALRC and S. nigrum, green calli were selected as putative fusion products and shoots regenerated from them. They were subsequently analyzed for their morphology, nuclear DNA composition and chloroplast DNA origin. The hybrids obtained between ALRC and L. hirsutum contained the chloroplasts of L. hirsutum and had the flower and leaf morphology of L. esculentum. After Southern blot analysis, using 13 restriction fragment length polymorphisms (RFLPs) randomly distributed over all chromosomes, all hybrids showed L. esculentum hybridization patterns. No chromosomes of L. hirsutum were found. These results indicate that these hybrids were true cybrids.The putative asymmetric hybrids, obtained with S. commersonii and S. etuberosum, showed phenotypic traits of both parents. After hybridization with species-specific repetitive nuclear DNA probes it was found that nuclear material of both parents was present in all plants. In the case of S. nigrum, which combination has the greatest phylogenetic distance between the fusion parents, no hybrid plants could be obtained. The chloroplast DNA of all hybrid plants was of the donor type suggesting that chloroplast transfer by asymmetric protoplast fusion can overcome problems associated with large phylogenetic distances between parental plants.     

Further studies on recombination in diploid clones from Bacillus subtilis protoplast fusion

Summary Diploid prototrophs were obtained from protoplast fusion of Bacillus subtilis strains. They are unstable but upon further cultivation they stabilize retaining diploidy but are genetically inactive. It has been suggested that recombination between the parental chomosomes is involved in the production of stable prototrophs and recombinants. In this work the occurrence of this recombination was searched for by determining genetic linkages in transformation experiments. In prototrophs two alleles: hisH2 and trpE8 carried originally on each parental chromosome, were shown to be 48% co-transformable in a stable clone whereas they were only cotransformed in 10% of the unstable colonies. For Trp^- recombinants (the most frequent type of a Leu^- Met^- Thr^- x Ade^- Ura^- Trp^- fusion pair) lysed protoplasts were used as donor DNA for the transformations. High values of co-transfer for Ura⁺ Met⁺ were obtained. These results confirm the occurrence of recombination in stable diploid clones, prototrophs or recombinants.     

Tetraploid somatic hybrids of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) obtained from diploid breeding lines

Intraspecific somatic hybrids between 16 different diploid breeding lines of Solanum tuberosum L. were produced by PEG-induced fusion. Manually selected heterokaryons were cultured in a Millicells-CM using a post-fusion protoplast mixture. Plants were regenerated from calli derived from heterokaryons obtained from 10 out of 38 combinations of diploid lines. Of the tested putative somatic hybrids, 14.2% were diploid, 72.8% were tetraploid and 13% pentaploid. The DNA amplification pattern obtained with RAPD or semi-random primers confirmed that 6 fusion combinations were hybrids. In most cases, the morphological traits were intermediate to those of the diploid fusion partners. About 23.0% of the tested somatic hybrids showed variation in their morphology. Of the tested somatic hybrids, 78.0% flowered and 86.0% tuberized. The cytoplasm of 9 diploid lines and 6 somatic hybrid combinations was analysed. Two of the diploid lines had W/S chloroplasts and α or ε mitochondria; the remainder contained T chloroplasts and β mitochondria. All the analysed somatic hybrids carried T chloroplasts and β mitochondria.     

Interspecific T-DNA transfer through plant protoplast fusion

Summary An attempt was made to transfer the T-DNA of Agrobacterium tumefaciens, previously introduced into plant cells, via protoplast fusion from one species into another. For the experiments two cell lines were used: firstly, a Nicotiana paniculata cell line transformed with the Agrobacterium strain B6S3. This cell line exhibits both hormone independent growth and synthesis of octopine as a result of the incorporated T-DNA from Agrobacterium. These two markers are dominant. The second cell line was the nitrate reductase deficient cnx-68 cell line of N. tabacum which contains an intracellular calcium oxalate druse. These two markers are recessive. Isolated protoplasts of the donor cell line N. paniculata B6S3 were mitotically inactivated by X rays and fused with protoplasts of the cell line cnx-68. Asymmetric somatic hybrids were selected on hormone free agar medium supplemented with 50 mM KClO₃. This compound is toxic for cells possessing nitrate reductase activity. From about 1.1×10⁷ cultivated protoplasts 18 cell lines survived the selection treatment. Of these seven exhibited the two dominant and the two recessive markers, whereas the others showed either only one or none of the recessive or only one of the dominant markers. In dot-blot experiments using species specific DNA clones of the donor and the recipient plant species it was confirmed that besides the T-DNA other nuclear genomic DNA of the donor species had also been transferred in various amounts. The possible consequences of these results for plant breeding programmes are discussed.     

Genetic analysis of the yeastCandida maltosa by means of induced parasexual processes

The usefulness of hybridization by protoplast fusion and mitotic segregation for the genetic analysis of the imperfect fodder yeastCandida maltosa was tested. Mitotically stable fusion hybrids were obtained with frequencies between 10^–6 and 10^–7. Complementation tests were performed by protoplast fusion. Substances that are known to induce frequent mitotic segregation in other yeast species such as benomyl, p-fluorophenylalanine, and acriflavine were ineffective inC. maltosa. UV irradiation induced mitotic segregation in up to 10%. This agent induced mainly mitotic crossing over inC. maltosa. Our data enabled the construction of the linkage group I with the sequenceCEN-ade-26-pro-1.     

Comparison of penicillins produced by inter-species hybrids fromPenicillium chrysogenum

Summary From inter-species heterokaryons produced following induced protoplast fusion betweenPenicillium chrysogenum andP. stoloniferum, P. patulum orP. verrucosum var.cyclopium, hybrid progeny with different stability and morphology could be isolated.Analysis of penicillins excreted from the hybrid progeny showed that antibiotic production was influenced both quantitatively and qualitatively. In most instances, and except forP. chrysogenum+P. stoloniferum hybrids, penicillin yield was adversely affected after inter-species hybridization. Investigations of the different natural penicillins produced, including pentyl-, benzyl- and heptylpenicillin, indicated that the relative amount of pentylpenicillin was considerably decreased in favour of benzyl- and/or heptylpenicillin. In addition, as observed forP. chrysogenum+P. verrucosum var.cyclopium, genes from different species and coding for different biochemical properties could simultaneously be expressed in inter-species hybrids.     

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     

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.     

Selection of a universal hybridizer in Sinapis turgida Del. and regeneration of plantlets from somatic hybrids with Brassica species

A double-mutant cell line, which was unable to grow in a medium with NO ₃ ^- as the sole nitrogen source and was resistant to 5-methyl-tryptophan (5MT), was selected from cell suspensions of Sinapis turgida Del. (Brassicaceae) by culturing the cells in AA medium (Toriyama and Hinata, 1985, Plant Sci. 41, 179–183) supplemented with 50 mM chlorate and 229 M 5MT. Protoplasts of this cell line were fused with mesophyll protoplasts of Brassica oleracea L. with dextran, and six somatic hybrids were selected initially by culture in the NO ₃ ^- medium and then by transfer to the NO ₃ ^- medium supplemented with 229 M 5MT. The somatic hybrids produced embryoids, leaves and plantlets on a regeneration medium. The hybrid characters were confirmed by investigations of acid phosphatase (EC 3.1.3.2) and peroxidase (EC 1.11.1.7) isoenzymes, chromosome number, growth on NO ₃ ^- medium, 5MT resistance, and capacity to regenerate plants. Somatic hybrids between S. turgida Del. and B. nigra (L.) Koch were also obtained using this method. These results indicate that the double-mutant cell line established here will be able to serve as a universal hybridizer to select somatic hybrids after protoplast fusion with any other wild-type partner.Abbreviations B5 medium of Gamborg et al. (1968) - MS medium of Murashage and Skoog (1962) - 5MT 5-thethyltryptophan     

Accessing and exploiting genes of breeding value of distant relatives of crop Brassicas

The Brassicas are an important group of crops in India yielding edible oils and many vegetables. For improving cultivated Brassicas, the wild relatives are of considerable value. The Brassica group of seed oil and vegetables comprises six cultivated species, out of which three are diploids and three are digenomic tetraploids. Brassica juncea is the major seed oil crop in India which can be improved for several traits by incorporating genes from its distant relatives. The early work in India relating to genome manipulation consisted of synthesis of B. juncea by crossing B. campestris with B. nigra, experimental resynthesis of Brassica species and non-homologous pairing and genetic exchange at the interspecific level. The alloploid species B. napus and B. carinata have not been successful in India due to agrometereological limitations. However, synthetic forms of B. napus have been produced which have a desirable maturity period with good yield potential. Also, through non-homologous pairing, pod shatter resistant B. napus has been obtained, B. napus ordinarily suffers from pod shattering. Similarly, synthetic forms of B. carinata have been derived from reciprocal crosses between morphotypes of B. oleracea and B. nigra and also through protoplast fusion of B. nigra with B. oleracea. Molecular analysis has revealed that one of the somatic hybrids had a novel cytoplasmic combination which carried B. nigra mitochondrial and B. oleracea chloroplast genomes. A range of wild and weedy species related to crop Brassicas possess extensive genetic variability. Work for utilizing this variability included hybridization between wild and crop species, analysis of chromosome pairing and induction of alloploidy. Among Brassicas of interest to India, protoplast culture and regeneration has been successful in the case of B. oleracea, B. juncea, B. nigra and B. carinata (cultivated species) and Eruca sativa and Diplotaxis muralis (related wild species). Polyethylene glycol mediated protoplast fusion has been the most commonly used method in India for producing somatic hybrids involving Brassicas. The eight somatic hybrids produced and studied showed that in the majority of cases the fusions led to symmetric hybrids combining the complete genomes of the donor species. For developing suitable male sterile lines, B. juncea, B. campestris and B. napus nuclei have been combined with the cytoplasm of six wild species and stable male steriles have been developed. Protoplast fusion methodology has been used extensively for improving these CMS by manipulating cytoplasmic organelles, including production of new combinations of cp and mt.     

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 hybridization between Lycopersicon esculentum and Lycopersicon pennellii

Summary Selection and screening methods were devised which resulted in the identification of a number of somatic hybrid callus clones following fusion of Lycopersicon esculentum protoplasts and L. pennellii suspension culture protoplasts. Visual selection for callus morphology combined with a high fusion frequency and irradiation of one parental protoplast type (¹³⁷Cs source, 1.5 Krads) resulted in selection of a callus clone population containing a high proportion of somatic hybrids. Analysis of a dimeric isozyme for the presence of a heterodimeric form was found to be satisfactory for distinguishing parental-type calli, somatic hybrid calli, and mixed calli derived from both types of unfused parental cells. No somatic hybrid calli produced shoots, although the sexual hybrid between L. esculentum and L. pennellii regenerated well under the culture conditions employed. This result suggests that the non-regenerable growth habit of the L. pennellii suspension culture was dominant in the somatic hybrid. The culture conditions described here are suitable for obtaining regenerated plants from L. esculentum mesophyll protoplasts. L. esculentum protoplast calli from fusion cultures gave rise to shoots with L. esculentum phenotype at higher frequency than calli from control unfused L. esculentum mesophyll protoplast cultures. The use of probes for species-specific organelle DNA fragments allowed identification of organelle DNA restriction fragments in digests of total DNA from small samples of individual callus clones. The callus clones analyzed either carried predominantly one parental plastid DNA type or mixtures of both types. Use of a mitochondrial DNA (mtDNA) probe which distinguishes two parental mtDNA fragments revealed that the L. pennellii-specific fragment was present in all clones examined, but the L. esculentum fragment was absent or in low proportion.