Factors promoting sustained divisions of mesophyll protoplasts isolated from dihaploid clones of potato (Solanum tuberosum L.) and a cytological analysis of regenerated plants

A culture protocol has been developed for mesophyll protoplasts isolated from various dihaploid clones of potato. A special effort was made to promote the growth of initially dividing cells to form cell colonies and calli. An increase in plating efficiency in 3 different dihaploid clones and one doubled dihaploid clone was obtained after serial dilution of cultures with a suitable amount and type of medium at different stages of cell colony development. Plating on a refined semi-solid medium after 14 days of culture further improved both the yield and the quality of calli obtained. The refined plating medium also enhanced shoot regeneration ability from 67 to 90% in one of the dihaploid clones (67:9). The refined culture protocol could also be used without causing a decrease in plating efficiency at a low population density adjusted after 3 days of culture. The ploidy level of plants regenerated from dihaploid protoplasts were determined by chromosome counting and DNA analysis by flow cytometry. Most of the plants were aneuploid or tetraploid although, some dihaploid plants were obtained after protoplast culture of 2 dihaploid clones derived from the same cultivar (cv. Stina).Abbreviations BA benzyladenine - 2,4-D dichlorophenoxyacetic acid - GA₃ gibberellic acid - NAA naphthaleneacetic acid     

Colorado potato beetle resistant somatic hybrid potato plants produced via protoplast electrofusion

Leptines are natural glycoalkaloids found only in certain selections of the wild potato speciesSolanum chacoense. These rare glycoalkaloids have been identified to be phytochemical defensive agents against insect herbivores such as the Colorado potato beetle (CPB). In an attempt to introduce this CPB resistance into the cultivated potatoS. tuberosum, interspecific somatic hybrid plants were developed between a dihaploid ofS. tuberosum and a high leptine-producing germplasm selection ofS. chacoense. The somatic hybrid was fused using protoplast electrofusion and regeneration techniques. Selection of interspecies fusion cell lines was based on hybrid vigor in protoplast-callus (p-callus) growth, on shoot regeneration from p-calli, and on characteristic appearance of anthocyanin pigment. This selection system was highly efficient and 12 of 13 fully regenerated plants were identified as somatic interspecies hybrids, as determined by the analyses of morphologic biochemical, and isozyme markers. In vitro insect bioassays demonstrated that the hybrids averaged a threefold reduction in leaf consumption by the CPB when compared to cultivated potatoes.     

Microspore embryogenesis and fertile plantlet regeneration in a salt susceptible × salt tolerant rice hybrid

Shed microspore embryogenesis and fertile plantlet regeneration were observed in a salt susceptible × salt tolerant indica rice F₁ hybrid involving IR 24 and CRM 30. The in vitro culture response and regeneration of green plantlets in the hybrid were superior to those of the parents. Direct embryogenesis and plantlet regeneration with multiple tillers were observed in shed microspore embryos. In intact anther culture, plantlet development from microspore involved a callus phase. The number of multiple tillers developed through secondary embryogenesis was almost equal in both the cases. However, the results indicate that regeneration of green plantlets was higher in case of shed microspore culture in liquid medium containing the synthetic polymer Ficoll 400 than from intact anthers cultured on a semi-solid system. Shed microspore culture produced a number of double haploids, which may result in far reaching consequences in genetic improvement of rice. This revised version was published online in June 2006 with corrections to the Cover Date.     

A simple method for the isolation of plant protoplasts

A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO₄), and a protectant (CaCl₂ 2H₂O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.     

Cytogenetics of protoplast cultures of Brachycome dichromosomatica and Crepis capillaris and regeneration of plants

Summary Callus derived protoplasts of Brachycome dichromosomatica (2n=2x=4) and Crepis capillaris (2n=2x=6) have been regenerated into karyologically normal plants, i.e. plants without visible alterations of the diploid chromosome set. However, metaphase analysis of protoplast cultures derived from both callus as well as mesophyll cells showed karyological changes in the overwhelming majority of cells in both species leading to multinucleated, polyploid and aneuploid cells. Furthermore, callus derived protoplasts sometimes exhibited changes at the chromosome level as indicated by translocations. The vast majority of aberrant karyotypes arose from failures during mitosis and cytokinesis, pointing to inadequate microtubules as a possible underlying cause. Karyological events of the kind described herein greatly affect the plating efficiency of isolated protoplasts and the viability of protoplast derived calli. Plant regeneration, although demonstrated in this study for the first time in both species, seems to be limited to rarely occurring, protoplast-derived colonies with a relatively stable genome. Our experiments, performed with chromosomal model species, emphasize the need for controlled, non-mutagenic culture conditions.     

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     

Gynogenesis in the vine cacti <Emphasis Type="Italic">Hylocereus</Emphasis> and <Emphasis Type="Italic">Selenicereus</Emphasis> (Cactaceae)

Gynogenesis was investigated on the allotetraploid Selenicereus megalanthus and the diploid Hylocereus polyrhizus and Hylocereus undatus vine cactus species. Unpollinated ovules from developing flower buds containing microspores at middle uninucleate developmental stage were cultured on MS basal medium containing 2,4-D/TDZ with different sucrose concentrations. Ovule size increased under dark culture conditions in all the three species and the level of response was species and sucrose concentration dependent. The best responses were achieved in the two S. megalanthus accessions, E-123 and J-80, at 0.18 and 0.26 M sucrose. Only ovule enlargement was obtained in H. undatus and both ovule enlargement and callus were obtained in H. polyrhizus. Development in both species ceased and embryoids were not formed. Plant regeneration was directly and indirectly obtained in both S. megalanthus accessions. Ploidy level was determined for a total of 29 S. megalanthus gynogenic plants using flow cytometry: 15 were found to be dihaploid (plants with the gametophytic chromosome number) and the other 14 were found to have higher ploidy levels. This is the first report of successful gynogenesis in Cactaceae. The dihaploids of S. megalanthus successfully produced by ovule culture techniques opens new perspectives in vine cacti breeding.     

Inheritance of competencies for leaf disc regeneration,anther culture,and protoplast culture in Solanum phureja and corrleations among them

Competence for leaf disc regeneration, anther culture, and protoplast culture was examined in the parental, F₁, and F₂ generations of a population of the diploid, cultivated, primitive potato, S. phureja (2n=2x=24). The parental pair consisted of AM3-8, an anther culture derived homozygous diploid, and NBP2, a heterozygous, field selected line. AM3-8 produced embryos in anther culture, and shoots on cultured leaf discs, but its cells did not divide after protoplast isolation. Cells of NBP2 divided to form calli and shoots in protoplast culture, but the clone did not respond to anther culture or leaf disc regeneration. All the individual plants in the F₁ generation were responsive to both anther and protoplast culture; however, there was segregation for the ability to regenerate shoots from leaf discs. The F₂ population, the result of a sib-cross, segregated for all three tissue culture competencies. Segregation data fit a one gene model for anther culture competence with the homozygous dominant genotype expressing the highest response, the heterozygote resulting in a marginal response, and the homozygous recessive resulting in no response. A two-gene model applied to the protoplast culture data, with a dominant allele at both loci required for division to occur after protoplast isolation. Leaf disc regeneration data could only be explained by a two gene model with recessive alleles at each locus required for the highest response, a dominant allele at either of the loci resulting in a marginal response, and dominant alleles at both loci resulting in no response. No significant correlation was found among these traits, implying three separate genetic mechanisms which segregate independently.Abbreviations BA N⁶-benzyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

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.     

Colchicine-induced microspore embryogenesis in coffee

A protocol for the induction of androgenesis and plant regeneration from C. arabica cv. Caturra isolated microspores in vitro using colchicine pretreatment has been developed. Microspores were mechanically isolated and then carefully purified. Before colchicine pretreatment, microspores were cultured in a semi-solid medium for further develop and regeneration. Different times of colchicine exposure as well as different concentrations were tested. The best androgenic response was found when microspores were precultured in 100 mg l^–1 colchicine for 48 h. The microspore developmental stages responsive to colchicine were late-uninucleated and early binucleated pollen. Flow cytometry and morphological analyses revealed that 95% of regenerated plants were dihaploids (2n=2x=22). However, some doubled dihaploid plants (2n=4x=44) were also obtained, suggesting that not only androgenic induction but also chromosome duplication could be expected as result of colchicine exposure of coffee microspores. This report represents a new approach in the coffee pollen culture, as well as a major step forward to the utilization of haploid technology in coffee breeding.     

Plant regeneration from protoplasts isolated from seedling cotyledons of Stylosanthes guianensis,S. macrocephala and S. scabra

Plant regeneration from protoplasts is a prerequisite to the production of modified plants using somatic hybridization and transformation. Whole plant regeneration was achieved from protoplasts isolated from seedling cotyledons of Stylosanthes guianensis, S. macrocephala and S. scabra, three economically important species of this tropical forage legume genus. The effects of both protoplast density and protoplast culture method on cell division and plating efficiency are presented.Abbreviations BAP 6-benzylaminopurine - MES 2-(N-Morpholino) ethanesulfonic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthalenacetic acid On leave from: Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Brasil     

Plant cell and biotechnology studies in <Emphasis Type="Italic">Linum usitatissimum</Emphasis> – a review

The species Linum usitatissimum (flax/linseed) has been the focus of a great deal of both basic and applied research effort in plant cell and biotechnology studies in recent years. In this review we consider applications of the techniques of plant biotechnology in this species under several distinct headings. Plant cell and tissue regeneration strategies and applications are discussed, and the applications of the techniques of somatic embryogenesis, protoplast isolation, culture and fusion and cell suspension cultures in this species are described. A major area of study is the use of anther and microspore culture where clear advantages to breeding programmes could be applied. In addition, embryo and ovary culture studies have resulted in significant findings. The more recent technologies of gene transfer and expression by genetic transformation are reviewed, and a section on strategies for improvements in technological quality is also included. Finally we propose conclusions and future prospects for this ancient, but still highly relevant crop.     

Production of androgenic dihaploid lines of the disomic tetraploid potato species Solanum acaule ssp. acaule

Over 250 dihaploid lines derived from a disomic tetraploid genotype of Solanum acaule ssp. acaule Bitt. (acc. PI 472655) were produced via androgenesis. The anther donor plant had previously shown immunity to bacterial ring rot caused by Clavibacter michiganensis ssp. sepedonicus (Spieck. and Kotth.) Davis et al., and has now been shown to have high embryogenic capacity in anther culture. In total, 370 shoots were regenerated from 4,011 anthers cultured. The ploidy level of the 287 regenerants was determined from greenhouse-grown plants using flow cytometry. Of these plants, 274 (95%) were dihaploids with an average DNA content of 1.68 pg, approximately half that of the tetraploid anther donor (2.95 pg). The remainder of the anther-derived regenerants (5%) were tetraploid, hexaploid or mixoploid. Chromosome counts confirmed the results obtained by flow cytometry. In the greenhouse, none of the 33 dihaploid lines analysed produced berries but showed low (2%) male fertility. This contrasted with five greenhouse-grown tetraploid anther-derived plants which produced berries and seeds. Comparison of the general leaf morphology and floral characteristics of the tetraploid anther donor, S. acaule, and the dihaploids indicated that little variation exists in this species. Received: 28 August 1997 / Revision received: 22 December 1997 / Accepted: 27 July 1998     

A comparison of tissue culture response between related tetraploid and dihaploid S. tuberosum genotypes

The response to tissue culture of a series of related, agronomically useful, dihaploid (2n=2x=24) and tetraploid (2n=4x=48) S. tuberosum genotypes was assessed by regenerating shoots from leaf explants. Dihaploid genotypes that showed superior responses to their tetraploid parents were identified. Large differences in tissue culture response were also found between dihaploid genotypes derived from the same tetraploid parents. These results indicate that it should be possible to select agronomically useful dihaploid genotypes with good tissue culture responses for use in genetic manipulation experiments. Possible factors determining tissue culture response in S. tuberosum are discussed.     

Isolated microspore culture and plant regeneration in rye (Secale cereale L.)

 An isolated microspore culture and green plant regeneration method for rye (Secale cereale L.) was established. Rye isolated microspore androgenesis was genotype-dependent. PG-96M medium supplemented with 6% maltose gave the highest microspore survival rate after 48 h of culture and the highest embryo/callus yield (930 embryos/calli per 100 anthers from cv. Florida 401). Osmotic pressure in the induction medium played an important role. Pretreatment of the anthers with mannitol was beneficial for the microspore culture. Embryos/calli of a relatively younger age and smaller size had a higher regeneration ability, with the best green plant regeneration rate being 6%. Over 150 microspore-derived green plants have been obtained so far. About 90% of the regenerated plants were spontaneous doubled haploids. This is the first report of isolated microspore culture in true rye resulting in androgenic embryogenesis and plant regeneration. Received: 26 April 1999 / Accepted: 23 November 1999     

Factors influencing plant regeneration from protoplasts isolated from long-term cell suspension culture of recalcitrant Indica rice cultivar IR36

Summary Factors influencing successful establishment of embryogenic cell-suspension cultures and plant regeneration from longterm cell suspension-derived protoplasts of the recalcitrant Indica rice cultivar IR36 were studied. The factors included cell and protoplast culture medium, protoplast culture procedure, the source of nurse cells, and the regeneration procedure. Embryogenic cell suspension cultures could only be established from mature seed-derived callus of IR36 in AA-based medium (Müller and Grafe, 1978). Protoplast-derived colonies could be obtained only using the filter-membrane nurse-culture procedure when Lolium multiflorum suspension cells served as nurse, rather than wild rice (Oryza ridleyi) and Japonica rice (Oryza sativa cv. Taipei 309) cells. The utilization of a two-step regeneration procedure led to regeneration of fertile plants from protoplasts isolated from 2-yr-old cell suspensions of IR36, one of the most important but recalcitrant rice cultivars.     

Electrofusion of protoplasts from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>, <Emphasis Type="Italic">S. bulbocastanum</Emphasis> and <Emphasis Type="Italic">S. pinnatisectum</Emphasis>

This paper discusses a number of experiments performed, involving the fusion by an electric field of mesophyll protoplasts from Solanum tuberosum cv. Bintje, S. tuberosum dihaploid clones 243, 299 and the wild tuberous disease-resistant species S. bulbocastanum and S. pinnatisectum. Three fusion experiments (S. bulbocastanum + S. tuberosum dihaploid 243, S. pinnatisectum + S. tuberosum cv. Bintje and S. pinnatisectum + S. tuberosum dihaploid 299) yielded 542 calli, the 52 ones of which produced shoots. Obtained regenerants were estimated by the flow-cytometry (FC) and RAPD analysis to determine hybrid plants.The utilisation of the FC as a useful method for detecting somatic hybrids is also discussed in this paper. The combination S. bulbocastanum + S. tuberosum dihaploid 243 led to the creation of eight somatic hybrids, the combination S. pinnatisectum + S. tuberosum cv. Bintje yielded four somatic hybrids and the combination S. pinnatisectum + S. tuberosum dihaploid 299 resulted in no hybrid regenerants. Morphology in vitro, growth vigour and production of tuber-like structures were evaluated in hybrid plants. Plants were transferred in vivo for further estimation (acclimatization, habitus evaluation and tuberization ability).     

Tissue culture response in various wild and cultivated Solanum germplasm accessions for exploitation in potato breeding

The response to different in vitro methods for use in potato breeding has been evaluated in 11 genotypes of 5 Solanum species, S. etuberosum, S. lycopersicoides, S. maglia, S. rickii, and S. tuberosum. Callus induction and growth, and shoot regeneration were strongly influenced by the genotype, explant source, and medium utilized. Furthermore, considerable differences among the 11 genotypes were found both in plating efficiency and shoot regeneration from protoplast culture. Some interesting correlations were found between different tissue culture responses, suggesting linkage and/or pleiotropic effect of genes. The potential application to potato breeding of the in vitro techniques analyzed is discussed.Abbreviations BA 6-benzylaminopurine - GA₃ gibberellic acid - NAA naphthaleneacetic acid - MS Murashige & Skoog (1962) - 2,4-d dichlorophenoxyacetic acid     

Genetic selection and liquid medium conditions improve the yield of androgenetic plants from diploid potatoes

Summary Solatium tuberosum L. diploid strains with superior androgenetic capacity have been selected for from androgenetic progenies of unselected diploid material. The paper also demonstrates that the use of a liquid medium for culturing potato anthers, instead of the conventional solid agar plates, improves the yield of androgenetic embryoids. The new method, associated with two successive cycles of selection for superior androgenetic response, allows the induction and regeneration of microspore derived plants on a large scale. The best genotype (clone 21 in this paper) regenerates androgenetic plants with a frequency around 30 per each anther plated. Over 80% of the regenerated plants are diploid. It is suggested that the androgenetic embryoids mainly originate from unreduced microspores by a mechanism which maintains a heterozygous or a partly heterozygous genetic situation.     

Production and characterization of somatic hybrids between <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. and <Emphasis Type="Italic">S. pinnatisectum</Emphasis> Dun.

Interspecific somatic hybrids between the dihaploid Solanum tuberosum and the wild species S. pinnatisectum Dun. were produced via protoplast fusion. Protoplast isolation, electrofusion, culture of post-fusion products and regeneration of calli/shoots were undertaken following optimized protocols. Regenerants were characterized for hybridity, ploidy and resistance to Phytophthora infestans (Mont.) de Bery, causal fungal pathogen of late blight disease. From a total of 126 regenerated macrocalli, 12 somatic hybrids were confirmed by possessing species-specific diagnostic bands of their corresponding parents as revealed by RAPD, SSRs and cytoplasmic-DNA analyses. Tetraploid status of the 12 hybrids was determined using flow cytometry analysis. Intermediate phenotypes for leaf, flower, and tuber characteristics and high male fertility were observed in field-grown hybrid plants. Hybrids were highly resistant to foliage late blight based on field assessment for two seasons. In contrast, moderate level of resistance to foliage blight was observed in hybrids based on the detached leaf assay under laboratory conditions. Overall, somatic hybrids with moderate levels of resistance to foliage blight were identified, and these will be useful for in situ hybridization in potato breeding efforts.