Production Of Somatic Hybrids Research Articles

Efficient Production of Somatic Hybrids between Raphanus sativus and Brassica oleracea.

Efficient Production of Somatic Hybrids between Raphanus sativus and Brassica oleracea.

In situ hybridization with species-specific DNA probes gives evidence for asymmetric nature of Brassica hybrids obtained by X-ray fusion

We have previously reported production of somatic hybrids between B. oleracea and B. campestris by fusion of B. oleracea protoplasts with X-irradiated B. campestris protoplasts, in order to transfer a part of the B. campestris genome into B. Oleracea. Our previous analysis of morphology, chromosome number, and isozyme patterns of the hybrids suggested that they are asymmetric in nature. To obtain further evidence for the asymmetric nature of the hybrids, we isolated B. campestris-specific repetitive sequences and used them for in situ hybridization of the chromosomes of the hybrids. The repetitive DNA probes could specifically identify 8 out of 20 chromosomes of the B. campestris genome, and analysis of the hybrids indicates that 1-3 chromosomes of B. campestris are lacking in all five hybrids examined, giving clear evidence for the asymmetric nature of the hybrids. Furthermore, in situ hybridization revealed that some of the abnormal chromosomes observed in the hybrids are generated by rearrangements of B. Campestris chromosomes caused by X-irradiation. Altogether, our study indicates that in situ hybridization using species-specific repetitive sequences is a useful tool to analyze chromosomal compositions of various types of hybrids obtained by cell fusion or conventional methods.

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.

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 β 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.

Production of somatic hybrids between Solanum melongena L. and S. integrifolium Poir.

In order to transfer resistance to Pseudomonas solanacearum from Solanum integrtfolium Poir. (Hiranasu) to S. melongena L. (cv. Shironasu), somatic cell fusion was carried out. The protoplasts derived from cultured cells of S. melongena were treated with 1.0 mM iodoacetamide solution for 15 min. at 5°C, and fused with mesophyll protoplasts of S. integrifolium by dextran method. The protoplasts, which were cultured in MS medium (MURASHIGE and SKOOG, 1962) containing 6-benzylaminopurine (1 mg/l), naphthaleneacetic acid (1 mg/l) and glucose (50 g/l) developed to 4 cell colonies after about 8 weeks. Each colony regenerated plants 2 to 3 weeks later. The characteristics of the regenerated plants were confirmed by morphological studies and isoelectric focusing of acid phosphatase. Resistance to P. solanacearum was tested by culturing offsprings from self-pollinated fertile plants (RW-4) in the solution added P. solanacearum. Some of the offsprings from RW-4 were more resistant than parents. From the present results, it was demonstrated that the selection of somatic hybrids by utilizing the inactivation of S. melongena protoplasts with iodoacetamide and the low cell division ability of S. integrifolium was effective and resistance to P. solanacearum could be transfcrred to S. melongena through protoplast fusion.

Variation in Net Photosynthesis, Rubisco Activity and Chloroplast Ultrastructure among Somatic Hybrids ofSolanum tuberosumandS. brevidens

In the production of somatic hybrids by chloroplast fusion, both nucleus and cytoplasm are combined. Moreover, the occurrence of somaclonal variation during culture and assortment of the cytoplasmic organelles can result in a wide range of hybrid types regarding their nuclear and cytoplasmic constitutions. Somatic hybrids therefore provide unique opportunities for investigating the effect of parental genome dosage, ploidy and nuclear and cytoplasmic interaction on physiological traits.

Intergeneric somatic hybrid production through protoplast fusion between Brassica juncea and Diplotaxis muralis

The need to transfer genetic traits from Diplotaxis muralis (2n=42) to Brassica juncea (2n=36), a major oil seed crop of the tropical world, was realised. Since the two plant types are sexually incompatible, attempts were made to evolve parasexual hybrids as the result of protoplast fusion. Protoplasts of hypocotyl-derived calli of two cultivars of B. juncea were fused with normal and γ-irradiated mesophyll protoplasts of Diplotaxis muralis. Regeneration of 110 plants from the fused products was successfully achieved. Upon analysis of some of them, we realised that true somatic hybrids and partial somatic hybrids had been generated. Thus the primary goal of evolving intergeneric hybridisation products between these two plant types was fulfilled.

Production of somatic hybrids by electrofusion in Solanum

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.

Improved isolation of dihaploidsolanum tubersoum protoplasts and the production of somatic hybrids between dihaploidS. tuberosum andS. brevidens

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.

Production of somatic hybrid plants between cabbage and chinese cabbage through protoplast fusion.

Protoplasts from leaf mesophyll cells of cabbage (Brassica oleracea L.) and chinese cabbage (B. catnpestris L.) were fused by dextran. Calli exhibiting vigorous growth were selected from the dextran treated protoplasts and plants were regenerated from them after about 3 months of culture. The data of plant and leaf morphology, chromosome number and acid phosphatase isozyme patterns indicate that these plants are interspecific hybrids. This is the first example of production of somatic hybrids between heading type vegetables.

A method for production of interspecific hybrids within Brassiceae via somatic hybridization, using resynthesis of Brassica napus as a model

A general method for the production of somatic hybrids within Brassiceae has been developed using mesophyll protoplasts from one parent and hypocotyl protoplasts from the other. Fusion products were easily identified by their intermediate phenotype between the parental protoplasts. They could be isolated 24 h after fusion with micropipettes using a micromanipulator. At that time their frequency was about 15%. They were cultured in small volumes, 10 μl, and a plating efficiency of 14% was obtained. Hybrid calli were obtained from the fusion products, which was confirmed by isozyme analysis. Ploidy level of one hybrid shoot was determined by flow cytometric DNA analysis.

Production of somatic hybrids of moss by electrofusion

Mixtures of protoplasts of two auxotrophic mutants of Physcomitrella patens, one requiring thiamine (aneurine), the other p-aminobenzoic acid, have been subjected to electrofusion. The protoplasts were aligned in an alternating electric field (500 KHz, 20 V RMS/cm) and induced to fuse by a brief DC pulse (800 V/cm, time constant lms). After culture, first on complete medium and then on selective medium, hybrid plants were obtained at a frequency of 3%. One hybrid with a morphology typical of polyploidy was also observed.

Cytogenetics of plant cell and tissue cultures and their regenerates

After a short introduction, the cytogenetics of plant cell and tissue cultures and their regenerates will be discussed. In the first section discussion will focus on cytogenetic conditions “in vivo”, i.e., in the original explant: (I) widespread ocurrence of polysomaty as a consequence of endoreduplication; (2) aneusomaty, an important, though rare, cause of chromosome number variation in vivo; (3) occurrence of chromosome structural changes in differentiated tissues, especially in association with aging; (4) mixoploidy and/or gene mutations, either nuclear or organellar, present as mosaics or periclinal chimeras, especially in vegetatively propagated plants. In section two the discussion will follow with nuclear processes at and during callus induction: (1) mitosis induction in diploid (haploid) and endoreduplicated cells and initiation of cell lines with different ploidy levels; (2) chromosome endoreduplication prior to mitosis induction as a mechanism of polyploidization; (3) nuclear fragmentation (amitosis) followed by mitosis, a mechanism responsible for wide chromosome number variation in cultured cells. In the third section the discussion will consider cytogenetic conditions in medium‐ and long‐term culture: (1) stability at the diploid level with emphasis on the genetic makeup of the species; (2) polyploidy, both existing and originated during culture, and its competitive ability in chromosomally heterogeneous cultures; (3) haploidy: origin and fate; (4) aneuploidy: origin, selective advantage of particular karyotypes, etc.; (5) chromosomal and gene” mutations: preexisting vs. induced during culture. In section four the discussion will focus on cell fusion and somatic hybrid cell lines. The two main aspects of protoplast fusion: (1) for gene (cytoplasm) transfer or exchange; and (2) for the production of somatic hybrids by nuclear fusion, will be treated in detail. Section five will consider cytogenetic conditions in regenerated plants. How much of the genetic variation present in in vitro cultures may be incorporated into regenerated plants? In trying to answer this question, the discussion will concern: (I) cell selection during the regeneration process via adventitious shoots or somatic embryos; (2) polyploidy and aneuploidy in regenerates; (3) chromosome number rnosaicism (aneusomaty) a rather frequent occurrence in plants regenerated via adventitious shoots and intrasomatic cell selection; (4) haploidy, diploidy, and polyploidy in pollen‐derived plants; (5) other genetic variation in regenerates; (6) an analysis of the somatic hybrid plants produced so far. Finally, section six will cover ensuring genetic stability: micropropagation. After stressing the genetic continuity of the meristem cell line in higher plants, papers will be discussed which show maintenance of genetic stability in plants developed in vitro from shoot apex cultures, from axillary buds and latent meristems in some plant parts. The review will end with concluding remarks.

Restriction endonuclease analysis of chloroplast DNA in interspecies somatic Hybrids of Petunia

Restriction endonuclease cleavage pattern analysis of chloroplast DNA (cpDNA) of three different interspecific somatic hybrid plants revealed that the cytoplasms of the hybrids contained only cpDNA of P. parodii. The somatic hybrid plants analysed were those between P. parodii (wild type) + P. hybrida (wild type); P. parodii (wild type)+P. inflata (cytoplasmic albino mutant); P. parodii (wild type) + P. parviflora (nuclear albino mutant). The presence of only P. parodii chloroplasts in the somatic hybrid of P. parodii + P. inflata is possibly due to the stringent selection used for somatic hybrid production. However, in the case of the two other somatic hybrids P. parodii + P. hybrida and P. parodii + P. parviflora it was not possible to determine whether the presence of only P. parodii chloroplasts in these somatic hybrid plants was due to the nature of the selection schemes used or simply occurred by chance. The relevance of such somatic hybrid material for the study of genomic-cytoplasmic interaction is discussed, as well as the use of restriction endonuclease fragment patterns for the analysis of taxonomic and evolutionary inter-relationships in the genus Petunia.

Somatic hybrids between unilateral cross-incompatible Petunia species

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.

Selection procedures for the production of inter-species somatic hybrids of Petunia hybrida and Petunia parodii I. Nutrient media and drug sensitivity complementation selection

Three methods of selection are described for the production of somatic hybrids of Petunia hybrida and Petunia parodii. Using cultured leaf protoplasts of the F 1 sexual hybrid, and the two parents, it was possible to develop media in which only protoplasts of the sexual hybrid would grow. Following fusion treatments, protoplasts were plated in such media in order to assess the frequency of somatic hybrid plant production and selection efficiency.

The production of somatic hybrids by protoplast fusion in the moss, Physcomitrella patens

A technique has been developed for the isolation of large numbers of protoplasts from protonemal tissue of Physcomitrella patens, and for their regeneration to give whole plants. Somatic hybrids have been selected following treatment of mixtures of protoplasts from complementary auxotrophic strains with 50 mM CaCl2 at high pH. The hybrids have a morphology different from that of normal haploid strains, but similar to that of aposporously produced diploids. The progeny resulting from selffertilisation of the hybrids show a segregation which is consistent with their being the products of meioses in an autotetraploid.