Hybrid Calli Research Articles

An Intergeneric Hybrid Cell Line of Duboisia hopwoodii and Nicotiana tabacum by Protoplast Fusion

Summary Fusion of protoplasts from Nicotiana tabacum mesophyll cells and protoplasts from Duboisia hopwoodii suspension culures was induced by polyethylene glycol treatment. Heterokaryons were isolated wih a micropipette and were cloned individually in small nylon mesh chambers, around which mesophyll protoplasts of tobacco were culured as nurse cells. One putative intergeneric hybrid cell line was obtained, and the hybrid nature of this line was confirmed by isozyme analyses, analysis of fluorescent compounds, and cytological investigations. The chromosome numbers in the hybrid varied from ca. 70 to ca. 240. Shoots with heterogeneous morphology were induced from the hybrid callus.

Electrofusion of biochemically well characterized nitrate reductase deficient Nicotiana plumbagnifolia mutants. Studies of optimization and complementation

Electrofusion was carried out using biochemically well characterized NR − Nicotiana plumbaginifolia mutants ( Cnx 20, Nia 26, NA 36). In analytical experiments, optimal conditions for mesophyll-mesophyll and callus-callus protoplast fusions were assessed. Subsequently, in large scale experiments NR + somatic hybrids were obtained after mesophyll protoplast fusions between Cnx 20 + Nia 26 as well as after callus protoplast fusions between Cnx 20 + Nia 26 and between Cnx 20 + NA 36. In addition, complementation of the nia mutants Nia 26 and NA 36, each characterized by a distinct biochemical phenotype, was studied using electrofusion. In these experiments no completing NR + somatic hybrid callus was obtained. As fusion conditions were optimal and fusion products were observed to be formed it was concluded that the nia mutations, although leading to distinct biochemical phenotypes, are allelic. We also studied complementation in short term experiments. NR activity in vivo was assayed 3–4 weeks after fusion. Plants could be regenerated from the majority of the NR + somatic hybrid calli, resulting from the fusions between Cnx 20 + Nia 26 and Cnx 20 + NA 36. Chromosome numbers of shoot tip cells of glass house grown plants varied between 32–58, the majority having the normal tetraploid number (2 n = 40). Most of the plants appeared to be sterile.

An Improved Culture Method of Mechanically Isolated Heterokaryons of Potato

AbstractFused protoplasts between different dihaploid genotype of potato were cultivated in small droplets of liquid medium. It was observed that divisions and sustained development of these protoplasts could only be achieved it they had contact via gas phase to other living cells. The factor being responsible for this effect could not be identified. Nevertheless it was possible under such a culture condition to regenerate somatic hybrid calli from heterokaryons of various fusion combinations of potato.

Somatic hybridization in the Gramineae: Saccharum officinarum L. (sugarcane) and Pennisetum americanum (L.) K. Schum. (pearl millet)

Somatic hybrid cell lines with embryogenic capacity were obtained by fusion of protoplasts isolated from an embryogenic cell line of sugarcane with inactivated protoplasts of an S-(2-aminoethyl)-L-cysteine (Aet-Cys)-resistant cell line of pearl millet. Initial selection for putative hybrids was performed by plating protoplast-derived microcolonies onto medium containing 300 mug of Aet-Cys per ml. Calli growing in the presence of Aet-Cys were screened with respect to their electrophoretic pattern of phosphogluconate dehydrogenase (6-phospho-D-gluconate; EC 1.1.1.44), a dimeric enzyme. The somatic hybrid calli showed both parental homodimer bands plus an intermediate heterodimer band. Somatic hybrid lines were characterized further by electrophoretic patterns of shikimate dehydrogenase (shikimate; EC 1.1.1.25) and arylesterase (aryl-ester hydrolase; EC 3.1.1.2). All cell lines expressed the nuclear genomes of both parents. Use of a maize ribosomal DNA probe allowed the identification of diagnostic ribosomal DNA fragments in Xba I digests of total DNA in both parents and the somatic hybrids. The somatic hybrid lines carried both sugarcane and pearl millet ribosomal DNA fragments. The somatic hybrid calli formed somatic embryos. The banding pattern of phosphogluconate dehydrogenase from the somatic embryos was similar to that of the somatic hybrid calli.

Somatic hybridization in the gramineae: Pennisetum americanum (L.) K. Schum. (Pearl millet) +Panicum maximum Jacq. (Guinea grass)

Protoplasts from Pennisetum americanum resistant to S-2-amino-ethyl-l-cysteine (AEC) were fused with protoplasts of Panicum maximum utilizing polyethylene glycol-dimethylsulfoxide after inactivation of the Pennisetum protoplasts with 1 mM iodoacetic acid. The iodoacetate treatment prevented division of Pennisetum protoplasts; therefore, only Panicum protoplasts and heterokaryons potentially could give rise to colonies. A second level of selection was imposed by plating 3–4-week-old colonies on AEC medium. Putative somatic hybrid calli were analyzed for alcohol dehydrogenase, 6-phosphogluconate dehydrogenase, aminopeptidase, and shikimate dehydrogenase isozymes. Three somatic hybrid cell lines (lines 2, 3, and 67) were identified which showed two bands of alcohol dehydrogenase activity representing homodimers of P. maximum and P. americanum as well as a novel intermediate band of activity where Panicum-Pennisetum heterodimers would be expected. Aminopeptidase and shikimate dehydrogenase were useful for identifying presumptive hybrid calli but the isozyme patterns were additive-evidence which would not preclude the selection of chimeric callus. A more complex isozyme pattern which varied among the somatic hybrids was observed for 6-phosphogluconate dehydrogenase. In the hybrid calli, the presence of DNA sequences homologous to both P. maximum and P. americanum sequences was confirmed by hybridization of a maize ribosomal DNA probe to XbaI and EcoRI restriction fragments. Growth of hybrid lines on various concentrations of AEC was either similar to the AEC-resistant parent (hybrid line 2) or intermediate between the resistant and sensitive parents (hybrid lines 3, 67).

Regeneration of somatic hybrid plants formed between Lycopersicon esculentum and Solatium rickii

A single somatic hybrid callus clone was identified following the fusion of Lycopersicon esculentum protoplasts and Solanum rickii suspension culture protoplasts. The hybrid nature of the callus and the plants regenerating from it was determined by assaying phosphoglucomutase-2 isozyme expression. The chloroplast genome present in four somatic hybrid plants was characterized by probing digests of total DNA with nick translated L. esculentum chloroplast DNA(cpDNA). All four somatic hybrid plants had inherited S. rickii cpDNA. Two clones of plant mitochondrial DNA (mtDNA), soybean 18S and 5S rDNA and maize cytochrome oxidase subunit II were used to characterize the mtDNA present in total DNA digests of four somatic hybrid plants. In both cases, the somatic hybrid plants had inherited most but not all of the S. rickii specific fragments, but none of the L. esculentum specific fragments.

Studies on the mode of separation of chloroplast genomes in parasexual hybrid calli. III Random separation of two types of chloroplast genomes in a hybrid callus.

In the parasexual hybrid callus between Nicotiana glauca and N. langsdorfii, the two parental types of chloroplasts separate during callus development. The system to investigate the mode of separation involves the hormone independent growth of the hybrid callus between N. glauca and N. langsdorfii as a selective marker for the parasexal hybrid and the isoelectrofocusing patterns of the large subunit (LS) of Fraction I protein as a chloroplast genetic marker. We improved this system by utilizing N. gossei as a chloroplast donor, because the isoelectrofocusing patterns of LS from N. gossei and that from N. langsdorfii can be distinguished clearly. The hormone independency which is suitable for selecting hybrids in the state of small calli was restored by transmitting the nuclear genome of N. glauca into N. gossei by means of sexual hybridization. Using the improved system, the mode of chloroplast separation was investigated in more detail. It was found that 84% of the cells in a two month old parasexual hybrid callus have only one or the other parental type of chloroplasts and 16% of the cells have two types.We calculated the rate of separation using the formulae based on the assumption that the two types of chloroplasts separate at random by cell divisions in the callus. The result obtained by the calculation turned out to be consistent to the experimental data obtained by the analysis of the two month old parasexual hybrid callus, showing that our assumption is reasonable.

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.

Selection and enrichment of plant protoplast heterokaryons of Brassicaceae by flow sorting

The experimental conditions for an efficient and reproducible enrichment of fusion products by flow cytometry, using protoplasts of different Brassica species as hybridization material, have been investigated. The heterokaryons were identified by the endogenous chlorophyll autofluorescencence of mesophyll protoplasts of one parent and the fluorescense of exogenously supplied carboxyfluorescin to the hypocotyl protoplasts of the other parent. By using a low head drive frequency (11 kHz), a large nozzle (110 μm) and a low nozzle pressure (30–35 kPa) good survival of the protoplasts was obtained after sorting. Heterokaryons were sorted using these parameters and on average 80% of the protoplasts were fusion products as judged by microscopy. They were cultured in small volumes, 150 μl, and started to divide after 3–5 days and regenerated calli easily. Isozyme analysis of the calli confirmed that 81% had the pattern typical for a hybrrid. Differentiation into shoots have been obtained from some of the hybrid calli; these shoots were also confirmed to be true hybrids.

Interspecific Plant Hybridization by Electrofusion in Nicotiana

Mesophyll protoplasts from Nicotiana glauca and Nicotiana langsdorffii were fused electrically. From a total of 9 × 104 electrically fused protoplasts (4.5 × 104 each), we obtained more than 96 green hybrid colonies that grew vigorously on hormone–free medium after 10 weeks of culture. Electrofocusing analysis of Fraction I proteins isolated from hybrid calluses confirmed the hybridity of the calluses. The hybrid isolation yield was about 0.1 percent. All hybrid colonies regenerated shoots after about 4 months of culture and some formed roots.

Response of Somatic Hybrid Callus between Rice and Soybean to Streptomycin

Response of Somatic Hybrid Callus between Rice and Soybean to Streptomycin

Electrofusion, a simple and reproducible technique in somatic hybridization of Nicotiana plumbaginifolia mutants

Electrofusion of protoplasts from two complementary nitrate reductase deficient mutants of Nicotiana plumbaginifolia has resulted in somatic hybrid lines. Mesophyll protoplasts isolated from the cofactor mutant CNX 20 and fluorescein diacetate stained protoplasts derived from a cell suspension culture of the NA 36 line, being defective in the apoenzyme, were used in the fusion experiments. In total, 594 lines were recovered which could proliferate on a selective medium with nitrate as the sole nitrogen source. This is including 141 putative hybrid lines which were obtained after transfer of 1048 heterokaryons with a micromanipulator one day after electrofusion. The hybrid character of some of the selected lines was confirmed by nitrate reductase activity measurements. Plants were grown from hybrid calli.

In vitro induction of genetic variability in cotton (Gossypium spp.)

Through the in vitro culture of excised embryos and ovules, interspecific hybrids have been obtained from cultivated and wild species of Gossypium. The hybrids matured upon transfer to the field. The anthers, ovules and embryos from both the diploid (2n=26) and tetraploid (2n=52) species underwent proliferation, and this response was genotypic. The diploid species invariably showed profuse callusing in comparison with the tetraploid. The callus showed various chromosome numbers, ranging from haploids to hexaploids, and from high polyploidy to aneuploidy. Hybrid callus culture may augment the genetic variability by providing a means for obtaining genetic exchange in interspecific hybrids. The implications of the in vitro induction of genetic variability for cotton improvement are discussed.

Expression of nuclear-cytoplasmic genomic incompatibility in interspecific Petunia somatic hybrid plants

Somatic hybrid plants were regenerated following calcium-high pH fusion of the unidirectional, sexually incompatible cross of Petunia parodii wild-type leaf mesophyll protoplasts with protoplasts from a cytoplasmic determined chlorophyll-deficient mutant of P. inflata. Genic complementation to chlorophyll synthesis and sustained growth in the selective medium was used to visually identify hybrid calluses. Hybrid calluses were subsequently regenerated to shoots, rooted, and confirmed as somatic hybrids by their intermediate floral and leaf morphology based on comparison to the 2 n = 4 x = 28 sexual counterpart, dominant anthocyanin expression in the corolla, chromosome number, and peroxidase and maleic dehydrogenase isozyme patterns. Certain cytologically stable somatic hybrids displayed aberrant reproductive and floral morphologies including subtle to moderate corolla and leaf pigment variegation, floral dimension changes and reduced pollen viability. In contrast, cytologically unstable somatic hybrids showed various degrees of aneuploidy coupled with corolla splitting, and irregularities in reproductive organs such as double stigmas and styles in addition to reduced pollen viability. Postulated mechanisms to account for these phenotypic changes in stable and unstable somatic hybrids include nuclear-cytoplasmic genomic incompatibility, chromosome loss in a biparental cytoplasm, or a phenomenon similar to hybrid dysgenesis occurring as a result of somatic fusion.

Somatic hybridization between Lycopersicon esculentum and Lycopersicon pennellii

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 ((137)Cs 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.

Callus formation of somatic hybrid of rice and soybean and characteristics of the hybrid callus.

Induction of calli from rice (strain A-58) and soybean (cv. Harosoy) protoplast mixtures treated by polyethyleneglycol (PEG) was successful and hybrid calli were able to be identified and selected by the observation of a blackish purple pigment of fravonoids in the rice calli and a softness in the soybean calli. However, the blackish purple pigment of hybrid calli disappeared rapidly after one or two subcultures. The peroxidase and esterase isozyme band patterns of all hybrid calli after several subcultures were that of soybean callus. From these results, it can be supposed that rice chromosomes are rapidly eliminated, while soybean nuclear genome may be retained in the hybrid calli. During subcultures of the hybrid calli, yellow, green and white calli have been segregated. The soybean calli were usually yellow, but sometimes turned green. The rice calli were a blackish purple which was so unstable that whitish yellow calli were often obtained. When all these calli were transferred onto the medium containing 1-naphthaleneacetic acid (NAA) and kinetin, the whitish yellow calli of rice and the white calli segregated from the hybrids did not usually turn green. Nevertheless the segregated white calli occasionally produced green parts in a mosaic pattern during long term cultures. On the other hand, soybean calli, and yellow and green calli segregated from the hybrids became green or remained green. Analysis of Fraction I protein showed that the segregated green calli have soybean chloroplasts, while the white calli have both of rice and soybean chloroplasts. Therefore, it may be considered that the segregated green calli have mainly soybean nuclei and soybean chloroplasts, while the white calli have soybean nuclei, and rice and soybean chloroplasts in a mosaic pattern.

Correlation between the type species of chloroplast DNA and that of the large subunit of fraction I protein in parasexual hybrid calli.

Nicotiana glauca chloroplasts can be distinguished from N. langsdorffii chloroplasts by a difference in size of the Bam HI restriction endonuclease fragment of chloroplast DNA (ctDNA), and by a difference in the isoelectric focusing pattern of the large subunit of Fraction I proteins. Chloroplast DNA and Fraction I protein were isolated from parasexual hybrid calli derived from cell f usions between N. glauca and N. langsdorffii. Results obtained from 16 calli showed that the expression of the N. glauca type large subunit was correlated with presence of N. glauca ctDNA, N. langsdorffii large subunit with N. langsdorffii ctDNA. Whenever two kinds of ctDNA were present in a callus, both types of large subunit were expressed. Although the Bam HI fragment is distant from the gene for the large subunit, no evidence was obtained for recombination between these two ctDNA markers.

Studies on the mode of separation of chloroplast genomes in parasexual hybrid calli. II. Heterogeneous distribution of two kinds of chloroplast genomes in hybrid callus

Parasexual hybrid callus ( Nicotiana glauca + N. langsdorffii) was separated into eight pieces by marking the relative position of each piece in an original callus, and Fraction I protein in each piece was analyzed by isoelectrofocusing. The results showed that the ratios of two kinds of large subunits, which are coded in chloroplast DNA, are completely different in different positions in the callus. This evidence demonstrates that a hybrid callus is a three-dimensional mosaic of cells containing different ratios of two kinds of chloroplasts. The Fraction I protein of five sub-cloned calli grown from single cells isolated from the hybrid callus was analyzed. Two calli had only the N. langsdorffii type of large subunits and one callus had only the N. glauca type. However, two calli contained both types of large subunits, showing that some cells in the original hybrid callus contained chloroplasts from both sources.

Studies on mode of separation of chloroplast genomes in parasexual hybrid calli. I. fraction I protein composition in unseparated hybrid callus

The mode of chloroplast genome separation after cell fusion between Nicotiana glauca and N. langsdorffii protoplasts was studied by microscopic observation and iso-electrofocusing analysis of Fraction I protein obtained from somatic hybrid calli. Chloroplasts from one species of plant were observed to spread all over the fused cell at 48 h after cell fusion indicating that two kinds of chloroplasts were mixed together within the same fused cell. Analysis of Fraction I protein showed that 70% of the calli contained two kinds of chloroplast genomes and all contained two kinds of nuclear genomes. Reciprocal fusions between protoplasts from leaves of one species with protoplasts from suspension cultures of the other species also showed a majority of the calli to contain two kinds of chloroplast genomes. Selection of a small piece of the callus followed by further growth and further subdivisions and growth periods caused separation of one of the kinds of chloroplast genomes within the callus leading to the conclusion that the callus was already a mosaic of cells where elimination of one of the two kinds of chloroplast genomes had occurred in some cells. Thus, the separation of chloroplast genomes previously found in leaf cells of parasexual hybrid plants could have been the result of shoot differentiation having occurred in a very restricted region of a callus where cells had already undergone separation of the chloroplast genomes.

Restoration of the ability to regenerate shoots by somatic hybridization between Petunia hybrida and P. parodii.

Protoplasts from suspension cultured cells of Petunia hybrida. whlch had lost the ability to regenerate shoots were fused with protoplasts from leaf mesophyll cells of P.parodii which could not grow beyond a small colony stage. Fused protoplasts showed a more vigorous growth pattern than the parental protoplasts, and formed colonies which could regenerate shoots. On the contrary, parental colonies could not regenerate shoots. The hybrid nature of regenerated plants was confirmed by the color of their flower buds, their characteristic isozyme patterns and the morphology of the regenerated plants. Also, from the fact that the plants could be regenerated from hybrid cells between two different protoplasts each of which could not regenerate shoots, it was confirmed that they were somatic hybrids. Cytological analysis revealed that suspension cultured cells had 24 chromosomes in contrast with 14 chromosomes of root tip cells of both parents. On the other hand, somatic hybrid calli mostly consisted of cells which had 44 chromosomes. Somatic hybrid plants had distorted leaves and all flower buds deteriolated prematurely. These phenomena were partially attributed to higher chromosome numbers of cells of somatic hybrid plant compared to those of parental plants.