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.     

Enrichment for Nicotiana heterokaryons after protoplast fusion and subsequent growth in agarose microdrops

Protoplasts isolated from Nicotiana tabacum L. leaves and Nicotiana suaveolens Lehm. cell suspensions have been fused with polyethylene glycol (PEG). Enrichment for heterokaryons was based on a Percoll flotation protocol which allowed a preparation with 50% heterokaryons to be obtained. The heterokaryons developed into calli whose hybrid nature was shown by polyacrylamide gel electrophoresis of esterase isoenzymes. Sensitivity of the mesophyll protoplasts to PEG and different buoyant densities of the heterokaryon and cell-suspension protoplasts contribute to the enrichment. The 50%-fusion figure following purification is an improvement on standard PEG procedures.Heterokaryons obtained were embedded in 20l drops of agarose and placed in a liquid nurse culture that allows optimum growth of the heterokaryons and maintains a physical boundary between the heterokaryons and the nurse culture. Once colonies develop, the agarose microdrop is removed from the nurse culture and placed on shoot-induction medium. Agarose microdrops containing the heterokaryons can be readily removed at any stage and processed for electron microscopy to follow the early stages of colony development.The procedures we have utilised provide a robust physical selection method that allows the total variation from a heterokaryon population to be expressed.Abbreviations BAP N⁶-benzylaminopurine - BM basal medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthalene acetic acid - PEG polyethylene glycol - PKM modified Kao (1977) medium for protoplast culture     

Fate of introduced genetic markers in transformed root clones and regenerated plants of monohaploid and diploid potato genotypes

Summary Agrobacterium transformation of stem internodes of four monohaploid (839-79, 849-7, 851-23, 855-1) and two diploid (M9 and HH260) potato genotypes using hairy root-inducing single (LBA 1020, LBA 9365, LBA 9402) and binary (LBA 1060KG) vectors is reported. Various media and successive culture steps were tested for plant regeneration from different transformed root clones. The fate of introduced genetic markers in root clones and regenerated plants (hairy root phenotype, hormone autotrophy, opine production, kanamycin resistance, -glucuronidase activity), the ploidy stability and protoplast yield were analysed. The transformation efficiency of stem internodes (hairy root production) and the regeneration capacity of the transformed root clones greatly differed within and between the various potato genotypes. The regenerated plants obtained after transformation with both types of vectors often showed the absence of one or more genetic markers. However, transformation with the binary Agrobacterium vector generally resulted in the stable presence of the opines in all transformed root clones and most regenerated plants. In HH260, transformation efficiency, plant regeneration of transformed root clones, protoplast yield and ploidy stability were the highest as compared to the other genotypes. The application of these transformed plants as marker lines in gene mapping and gene expression studies is indicated.     

Production of potato monohaploids (2n=x=12) through prickle pollination

Summary Data are presented on the potential of gynogenesis for the production of monohaploids and on factors affecting their frequency and relative vigour. Diploid Solanum tuberosum L. and S. tuberosumxS. phureja Juz et Buk hybrids were used as maternal parents and selected S. phureja clones as prickle pollinators with embryo-spot and nodal band as dominant seed and plant marker. About 2 million seeds were screened for absence of embryo-spot. After raising plants from phenotypically spotless seeds, further screening for absence of nodal bands and for ploidy level was carried out. Finally more than 500 monohaploid plants from three genetically different groups of maternal parents were obtained. Frequency and vigour of the monohaploids were clearly dependent on their maternal genotypes. The data also indicated an effect of the pollinator genotype, the physiological stage of the maternal plant and the environment on monohaploid frequency. On the basis of these results the possibility of breeding for a higher monohaploid production rate and for more stable and vigorous monohaploids is discussed. Furthermore, gynogenesis and androgenesis are compared. It is suggested that both should be used in order to obtain monohaploids from sufficiently various diploid breeding material.     

Transformation of plant mitochondria with mitochondrial DNA plasmids via protoplast fusion

Summary Brassica napus cybrid plants which contain novel nucleus-mitochondria-chloroplast combinations have been constructed, via protoplast fusion. Such fusions resulted in mitochondrial DNA plasmids being lost (at a frequency of 12.5%) or, more surprisingly, being transferred from mitochondria of one protoplast population to mitochondria of the other population (at a frequency of 6.1%). Mitochondria containing their new DNA complement became the dominant organelle population in regenerated plants and were faithfully maternally inherited through successive sexnal generations. No concomitant alterations in mitochondrial chromosome organization or nuclear chromosome number occurred. Protoplast fusion can, therefore, cure plant mitochondria of extrachromosomal DNA and, more importantly, be used to transform plant mitochondria with naturally occurring mitochondrial plasmids. The potential for mitochondrial transformation with recombinant vectors is discussed.     

Genetic recombination by protoplast fusion inNocardia asteroides

Summary Fusion and regeneration of protoplasts ofNocardia asteroides strains ATCC 3318, IMRU W3599 and HIK B971 have been used to study genetic recombination in this species. Protoplasts were produced by treatment with lysozyme, following incubation with glycine. Mutants of ATCC 3318 were grown in peptone yeast extract medium at 32°C prior to protoplast production to maximize protoplast frequency, whereas mutants of IMRU W3599 and HIK B971 were grown in trypticase-soy broth. Glycine concentrations favoring protoplast formation varied from 1.5% to 5% depending on strain. For all strains, protoplast formation was complete 1 h after addition of 5 mg/ml lysozyme. Protoplasts were fused by addition of 50% polyethylene glycol-1000. In general, 25% of the protoplasts could be regenerated. The incidence of recombinant recovery was increased up to 750-fold. The distribution of recombinant phenotypes in matings was similar for protoplast fusion and conventional crosses.     

Isolation and characterization of potato diploid clones generating a high frequency of monohaploid or homozygous diploid androgenetic plants

Summary Cultured microspores of diploid potato clones lead with high frequency to diploid regenerants. In this paper we report on the genetic variability for in-vitro monohaploid production from anthers of diploid plants. Three diploid genotypes have been isolated which combine the capacity to regenerate monohaploid plants with outstanding embryoid production. A trait of the anther-donor clones associated with the generation of monohaploid plants is the low production of 2n pollen grains. When present in anthers of diploid genotypes, diploid unreduced microspores are, in fact, derived mainly from a first division restitution mechanism leading to high heterozygosity of the derived embryoids, a state which apparently supports superior growth in-vitro. Also, reduced microspores have been found capable of generating diploid regenerants and the adoption of the RFLP technique allowed the isolation of such diploid plants, which can be considered to be pure lines. Donor clones with a low capacity to generate monohaploids are, as expected, poor producers of homozygous diploid plants. The selection of an anther donor producing a sufficient number of monohaploid or homozygous diploid regenerants fulfills the requirements of the first part of the analytical breeding scheme, i.e., the production of homozygous diploid clones.     

Brassica carinata resynthesized by protoplast fusion

Brassica carinata (bbcc) was resynthesized by protoplast fusion betweenB. nigra (bb) andB. oleracea (cc). In two fusion experiments 64 hybrid plants were obtained and identified to be true hybrids by isoenzyme analysis, nuclear DNA content, chromosome number, and intermediate morphology. Of these plants 56% were normal amphidiploids with 2n=34 chromosomes and a DNA content equivalent to that of naturalB. carinata. The remaining plants were polyploid, morphologically abnormal, and infertile. The majority of the hybrids contained both chloroplasts and mitochondria fromB. nigra, but some plants combined chloroplast and mitochondria from the different progenitors. Hybrids with a DNA content equivalent to that ofB. carinata had a wide range of male fertility (4–98%), but consistently low female fertility. Only a few selfed seed were produced, but these germinated and grew into vigorous plants.Salaries and research support provided by State and Federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Journal Article No. 296-92     

Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana

The electrical fusion of protoplasts has been studied in order to maximize the formation of heterokaryons for culture. Heterokaryons of Nicotiana tabacum L. mesophyll protoplasts and N. plumbaginifolia Viviani supension-cell protoplasts were identified in fixed and stained as well as living material; a quantitative fusion index was thereby developed. With this index the efficiencies of various electric fields and fusion-chamber designs have been determined. Optimal fusion was obtained with an alternating-current (AC) field of 150 V/cm and direct-current (DC) square-wave pulses of 1000 V/cm. A new, simple-to-use, largescale fusion chamber is described in which batches of up to 5·10⁵ protoplasts (0.5 ml of cells at 10⁶/ml) can be fused in 5–7 min with efficiencies approaching 40%. Half of the fusion products are heterokaryons, thus fusion is random. Of the fusion products, 60% are bi- or trinucleate. Using fusion procedures similar to those described here Bates and C. Hasenkampf (1985, Theor. Appl. Genet., in press) have recovered viable somatic hybrids which have been regenerated.Abbreviations AC alternating current - DC direct current - PEG polyethylene glycol     

Plant regeneration from protoplast fusion inPassiflora spp.

Summary Protoplasts were isolated from leaf explants ofPassiflora edulis var.flavicarpa (the yellow passion fruit) and from cell suspensions of fivePassiflora species. Chemical fusion was performed using polyethylene glycol and the microcolonies obtained were transferred to growth medium to produce calli. Electrophoresis of soluble proteins and analysis of isoenzymes from calli produced from the fusion experiments were performed to select somatic hybrids. Specific polypeptide bands allowed the identification of somatic hybrids betweenP. edulis var.flavicarpa (+)P. alata, P. edulis var.flavicarpa (+)P. amethystina, P. edulis var.flavicarpa (+)P. cincinnata, P. edulis var.flavicarpa (+)P. giberti andP. edulis var.flavicarpa (+)P. coccinea. An average of 3 to 5% hybrid calli were obtained. With the exception of theP. edulis var.flavicarpa (+)P. coccinea, whole plants were recovered from all hybrids. These somatic hybrids showed 4n=36 chromosomes, which represents a further evidence of their hybridity.     

The first and second backcross progeny of the intergeneric fusion hybrids of potato and tomato after crossing with potato

Somatic fusion hybrids between the diploid potato and tomato were backcrossed to several genotypes of potato. Two ploidy levels of fusion hybrids, 4x and 6x, were used as female parents in backcrosses with five clones of 4x-potato. An estimate of the berry set and seed set in immature berries harvested 14–21 days after pollination indicated that crosses between certain combinations of 6x-fusion hybrids and male parents were more successful than others. The culture of over 4000 young seeds from berries harvested 2–2.5 weeks after pollination gave rise to a single seedling, 93.6701, from the cross between the 6x-fusion hybrid C 31-17-1 and the 4x-potato AM 66.42. This seedling was found to possess a pentaploid chromosome number, which was expected of a 6x × 4x cross. Isozyme analysis and DNA hybridisation studies confirmed that the seedling 93.6701 was indeed a backcross (BC₁) progeny. Morphologically, this BC₁ plant resembled potato with respect to plant habit, leaf shape, stolons and tuber characteristics, while some of the characters, such as floral morphology and the fragrance of the crushed leaves (typical of tomato), were intermediate. It was male sterile but could be successfully hybridized with 4x-potato through in vitro culture of yound seeds; thus, BC₂ plants were obtained. The possibilities of backcrossing and the potential use of BC₁ and BC₂ plants in genetics and breeding are discussed.     

Genetic mapping by means of protoplast fusion in Bacillus subtilis

Summary A new mapping method involving protoplast fusion in Bacillus subtilis is described. Protoplasts from an isogenic standard marker strain containing purA and from a strain containing both purB and the marker, x, to be mapped were fused with polyethylene glycol, and purA ⁺ purB ⁺ fusants were selected. After isolation of single colonies and determination of unselected markers, marker x was mapped between two standard markers. This method was fully applicable to PBS1-resistant strains (e.g., lyt strains). The results obtained by protoplast fusion, conventional transformation and/or lysed protoplast transformation indicated that a lyt strain, Ni15, contained two new autolysin-minus mutations (lyt-151 and lyt-152). The properties of lyt-15 are also discussed.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - SMM 0.5 M sucrose, 0.02 M MgCl₂, 0.02 M maleate buffer, pH 6.5     

Male-sterile chicory cybrids obtained by intergeneric protoplast fusion

Male-sterile chicory plants were obtained by fusion of chicory mesophyll protoplasts and hypocotyl protoplasts derived from male-sterile sunflower plants. The protoplasts of both species were fused by the PEG method and the products were selected manually and cultivated at very low density in a liquid medium. Three to twenty percent of the heterokaryocytes divided and evolved into microcalli, then into calli where budding could be induced. The mitochondrial genome of ten male-sterile or totally sterile plants was studied. Restriction endonuclease profiles of mitochondrial DNA and molecular hybridization with specific genes of the mitochondrial genome used as probes indicated that mitochondrial DNA rearrangement had occurred between sunflower and chicory and the intensity of the rearrangements correlated with the degree of sterility of the different plants.     

Characterization of chromosome instability in interspecific somatic hybrids obtained by X-ray fusion between potato (Solanum tuberosum L.) and S. brevidens Phil

Summary Asymmetric somatic hybrids between Solanum tuberosum L. and S. brevidens Phil. have been obtained via the fusion of protoplasts from potato leaves and from cell suspension culture of S. brevidens. The wild Solanum species served as donor after irradiation of its protoplasts with a lethal X-ray dose (200 Gy). Selection of the putative hybrids was based on the kanamycin-resistance marker gene previously introduced into the genome of Solanum brevidens by Agrobacterium-mediated gene transfer. Thirteen out of the 45 selected clones exhibited reduced morphogenic potential. The morphological abnormalities of the regenerated plantlets were gradually eliminated during the extended in vitro culture period. Cytological investigations revealed that the number of chromosomes in the cultured S. brevidens cells used as protoplast source ranged between 28–40 instead of the basic 2n=24 value. There was a high degree of aneuploidy in all of the investigated hybrid clones, and at least 12 extra chromosomes were observed in addition to the potato chromosomes (2n=48). Interand intraclonal variation and segregation during vegetative propagation indicated the genetic instability of the hybrids, which can be ascribed to the pre-existing and X-ray irradiation-induced chromosomal abnormalities in the donor S. brevidens cells. The detection of centromeric chromosome fragments and long, poly-constrictional chromosomes in cytological preparations as well as non-parental bands in Southern hybridizations with restriction fragment length polymorphism (RFLP) markers revealed extensive chromosome rearrangements in most of the regenerated clones. On the basis of the limited number of RFLP probes used, preferential loss of S. brevidens specific markers with a non-random elimination pattern could be detected in hybrid regenerants.     

Further studies on the genetics ofCephalosporium acremonium using protoplast fusion methods

Summary Genetic analysis of protoplast fusants inCephalosporium acremonium can be carried out by two complementary approaches: analysis of stable haploid segregants and analysis of unstable heterozygotes of uncertain ploidy. However, segregation may be distorted by physiological as well as genetic phenomena, i.e., cross-feeding, syntrophic growth, allele viability, clonal effects, or parental genome segregation.     

Somatic hybridization between Brassica juncea and Moricandia arvensis by protoplast fusion

Intergeneric somatic hybrids have been produced between Brassica juncea (2n=36, AABB) cv. RLM-198 and Moricandia arvensis (2n=28, MM) by protoplast fusion. Hypocotyl protoplasts of B. juncea were fused with mesophyll protoplasts of M. arvensis using polyethylene glycol. Fusion frequency, estimated on the basis of differential morphological characterstics of parental protoplasts was about 5%. Of the 156 calli obtained, four calli produced shoots intermediate in morphology between the parents. Hybrid nature of the plants was confirmed using wheat nuclear rDNA probe. Hybridization of total DNA with a mitochondrial DNA probe carrying 5s–18s rRNA genes of maize showed that the mitochondria of the somatic hybrids were derived from the wild species M. arvensis. Meiosis in the only hybrid that produced normal flowers revealed the occurrence of 64 chromosomes, the sum of chromosomes of parental species. Inspite of complete pollen sterility, siliquas were produced in this hybrid by back-crossing with B. juncea. These siliquas on in vitro culture produced 12 seeds.     

Improvement of the cell volume ofCandida blankii through protoplast fusion

Summary The yeastCandida blankii ESP-94, capable of utilizing xylose as substrate, was isolated for the production of single-cell protein (SCP) on bagasse hydrolysates. However, the small cell volume of strain ESP-94 would complicate harvesting of the cells during a continuous fermentation process. Auxotrophic mutants of strain ESP-94 were generated and intraspecific protoplast fusion experiments performed in an attempt to increase the cell volume of strain ESP-94. The fusion products were characterised with respect to cell volume, DNA content and genetic stability. Six genetically stable fusants with bigger cell volumes and higher DNA contents were obtained. One such fusant, fusant F17, had a cell volume 3-times that of strain ESP-94, while exhibiting similar growth rates to strain ESP-94 ond-xylose as carbon source.     

Production of Trichoderma strains with pesticide-polyresistance by mutagenesis and protoplast fusion

The sensitivity of two cold-tolerant Trichoderma strains belonging to the species T. harzianum and T.␣atroviride was determined to a series of pesticides widely used in agriculture. From the 16 pesticides tested, seven fungicides: copper sulfate, carbendazim, mancozeb, tebuconazole, imazalil, captan and thiram inhibited colony growth of the test strains significantly with minimal inhibitory concentrations of 300, 0.4, 50, 100, 100, 100 and 50 g/ml, respectively. Mutants resistant to carbendazim and tebuconazole were produced from both wild type strains by means of UV-mutagenesis. The cross-resistance capabilities and in␣vitro antagonistic properties of the mutants were determined. Carbendazim-resistant mutants showed total cross-resistance to benomyl and thiabendazole at a concentration of 20 g/ml. Intraspecific protoplast fusion was carried out between carbendazim- and tebuconazole-resistant mutants of both parental strains, and putative haploid recombinants with stable resistance to both pesticides were produced in the case of T.␣atroviride. These pesticide-polyresistant progenies are potential candidates for application in an integrated pest management system.This work was presented as an oral lecture in section ‘Agriculture, Soil, Forest Microbiology’ at the BioMicroWorld2005 conference.     

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).     

Transfer of organelles of the alga Chlamydomonas reinhardii into carrot cells by protoplast fusion

A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.Abbreviations PEG polyethylene glycol - TEM transmission electron microscopy - SEM scanning electron microscopy This study was undertaken during sabbatical leave in The Research School of Biological Sciences. Australian National University