Multiple transformation of Saccharomyces cerevisiae by protoplast fusion

A technique for the multiple transformation of yeast by protoplast fusion is described. This involved the PEG-induced fusion of protoplasts from cells which had been treated with chromosome-fragmenting agents (in this case cupferron and hydroxylamine) with protoplasts of triply auxotrophic cells. The recovery of transformants was increased significantly if one of the amino acid requirements of the recipient strain was included in the selection medium. Transformants isolated on supplemented media remained auxotrophic for that requirement. Prototrophic, uninucleate transformants had a DNA content and cellular volume similar to that of the parental strains. Possible mechanisms of gene transfer are discussed. This technique offers the possibility of transferring desirable characteristics from one yeast strain to another without altering the ploidy level of the recipient strain.     

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.     

Interspecific T-DNA transfer through plant protoplast fusion

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

Genetic 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     

Hybrids obtained by protoplast fusion with a salt-tolerant yeast

Summary An industrial strain ofSaccharomyces cerevisiae was fused with an osmotolerant yeast,Debaryomyces hansenii, to obtain hybrids having increased tolerance to elevated salt concentrations. The hybrids were intermediate to parent species in production of ethanol and polyols.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Analysis of hybrids of Candida albicans formed by protoplast fusion

Abstract A number of hybrids obtained by fusion of protoplasts of complementary auxotrophic strains of Candida albicans were analysed for growth rates, cell volume, DNA content, stability and adherence to human buccal epithelial cells. SDS-PAGE analysis of the cell wall proteins indicated that the hybrid cell walls contained many more of the proteins associated with one parent than the other. The adherence values of the hybrids were closest to the parent with which they shared most cell wall proteins and it is suggested that the hybrids contain the genome of this parent along with one or more of the chromosomes of the other.     

Effect of Bacillus subtilis BsuM restriction-modification on plasmid transfer by polyethylene glycol-induced protoplast fusion

Polyethylene glycol (PEG)-induced cell fusion is a promising method to transfer larger DNA from one cell to another than conventional genetic DNA transfer systems. The laboratory strain Bacillus subtilis 168 contains a restriction (R) and modification (M) system, BsuM, which recognizes the sequence 5'-CTCGAG-3'. To study whether the BsuM system affects DNA transfer by the PEG-induced cell fusion between R(+)M(+) and R(-)M(-) strains, we examined transfer of plasmids pHV33 and pLS32neo carrying no and eight BsuM sites, respectively. It was shown that although the transfer of pLS32neo but not pHV33 from the R(-)M(-) to R(+)M(+) cells was severely restricted, significant levels of transfer of both plasmids from the R(+)M(+) to R(-)M(-) cells were observed. The latter result shows that the chromosomal DNA in the R(-)M(-) cell used as the recipient partially survived restriction from the donor R(+)M(+) cell, indicating that the BsuM R(-)M(-) strain is useful as a host for accepting DNA from cells carrying a restriction system(s). Two such examples were manifested for plasmid transfer from Bacillus circulans and Bacillus stearothermophilus strains to a BsuM-deficient mutant, B. subtilis RM125.     

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.     

Intertrubal chloroplast transfer by protoplast fusion between Nicotiana tabacum and Salpiglossis sinuata

Summary Chloroplast tranfer was achieved by protoplast fusion between Nicotiana tobacum (Cestreae, Cestroideae) and Salpiglossis sinuata (Salpiglossideae, Cestroideae) in the family Solanaceae. Isolation of cybrid clones was facilitated by irradiation of the cytoplasm donor protoplasts, and the use of appropriate plastid mutants, streptomycin-resistant as donor, or light-sensitive as recipient. Cybrid colonies were selected by their green colour against the background of bleached (light-sensitive or streptomycin-sensitive) colonies. In the Nicotiana (Salpiglossis) cybrid plants possessing normal tobacco morphology and chromsome number, the presence of Salpiglossis, plastids was verified by restriction analysis of the chloroplast DNA. A similar analysis of the mitochondrial DNA of these lines revealed unique, recombinant patterns in the case of both fertile and sterile plants. Progeny showed no appearance of chlorophyll-deficiency in F₁ and an additional back-cross generation. Attempts at transfer of entire chloroplasts between Nicotiana tabacum and Solanum nigrum (Solaneae, Solanoideae) did not result in any cybrid cell lines in a medium suitable for green colony formation of both species. These results suggest that fusion-mediated chloroplast transfer can surmount a considerable taxonomical distance, but might be hampered by a plastome-genome incompatibility in more remote combinations.     

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.     

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     

Intraspecific hybridisation of Trichoderma pseudokoningii by anastomosis and by protoplast fusion

Double auxotrophic and morphological mutants of Trichoderma pseudokoningii Rifai were fused by anastomosis and by protoplast fusion. The recovery of recombinants from heterokaryons on different selective media and from heterokaryotic colonies indicated the occurrence of parasexual events. Prototrophic colonies growing on minimal medium produced binucleate spores, green in colour, revealing a non-autonomous system for conidial pigmentation. Recombinants were obtained from these dikaryotic colonies suggesting the occurrence of a highly unstable diploid phase.     

Production of new CMS Brassica oleracea by transfer of `Anand' cytoplasm from B. rapa through protoplast fusion

New types of cytoplasmic male sterility (CMS) in Brassica oleracea would be useful for F₁ hybrid seed production. The `Anand' cytoplasm derives from the wild species B. tournefortii. Rapid cycling stocks of B. rapa and B. oleracea were used in cybridization experiments as donor and recipient of `Anand' (=`tour') CMS, respectively. Prior to fusion with PEG, donor protoplasts were inactivated with 30 krad γ-rays and recipient ones with 3 mM iodoacetate, respectively. No calli were obtained from the pre-treated protoplasts. The frequency of shoot regeneration was 21–43% in untreated B. oleracea controls, but only 0–0.5% in `Anand' B. rapa. Putative cybrids were regenerated from about 3% of the calli from fused protoplasts. Regenerated plants were analyzed for nuclear DNA content, plant and flower morphology, pollen production, female fertility, cold tolerance, and organelle composition. Eighty-one percent of the regenerated controls and 63% of fusion-derived plants were diploid. The rest showed DNA contents corresponding to 2x–4x, 4x, or higher ploidy levels, presumably due to somatic doubling in vitro and/or fusions in which the donor nucleus was not completely eliminated. Sixty-four percent of the cybrids had stamens and petals varying in size and shape and were male-sterile, with indehiscent anthers. Their phenotype was otherwise similar to that of B. oleracea. The remaining plants had normal flowers and were male-fertile. Data from crosses with fertile pollinators indicated good female fertility in some of the sterile lines, both after hand and insect pollinations in cages. Mitochondrial (mt) segregation in the cybrids was slightly biased towards `Anand' mitochondria, and the presence of `Anand' mtDNA fragments was strongly associated with male sterility. Evidence of mtDNA rearrangements was obtained in some cybrids. Segregation of chloroplasts was slightly biased towards B. oleracea. The presence of `Anand' chloroplasts with a B. oleracea nucleus did not result in cold temperature chlorosis, as seen in `Ogura' CMS plants. Received: 22 February 1996 / Accepted: 10 May 1996     

Insertion sequence analysis of protoplast fused strains of Lactococcus lactis ssp. cremoris

Abstract The use of insertion sequence probes for the analysis of fusants obtained following protoplast fusion is described. Hybridization of both total and plasmid DNA from parent and fusant strains with probes to IS 904 and IS S1 showed that of the four protoplast fusions examined, three appeared to involve a rearrangement of genetic material while in the fourth the fusant appeared similar to one of the parental strains. This method of analysis provides more information about the changes induced by protoplast fusion than that obtained by monitoring the acquisition or loss of individual characteristics.     

Somatic transfer of cytoplasmic traits in Brassica napus L. by haploid protoplast fusion

Summary Fusions between protoplasts from haploid cytoplasmic atrazine resistant (CATR) and haploid cytoplasmic male sterile (CMS) Brassica napus plants were used to produce a diploid CMS/CATR cybrid. The hybrid nature of the cytoplasm was confirmed by comparing the EcoRI restriction fragment patterns of chloroplast and mitochondrial DNA from the cybrid with the parental patterns. The advantages of using haploid protoplasts for fusion experiments as well as the utilization of the CMS/CATR cybrid for hybrid seed production are discussed.     

Introduction of plasmid pC194 into Bacillus thuringiensis by protoplast transformation and plasmid transfer

The Staphylococcus aureus plasmid pC194 which codes for resistance to chloramphenicol was introduced into six Bacillus thuringiensis strains representing five varieties by protoplast transformation. Six other varieties could not be transformed. pC194 could be identified in transformed strains as autonomous plasmid. The transformed clones contained in addition a new extrachromosomal element of somewhat lower electrophoretic mobility hybridizing with pC194, and pC194 in multimeric forms. pC194 was also transferred from one B. thuringiensis variety to another and from Bacillus thuringiensis to Bacillus subtilis and vice versa by a conjugation-like process, requiring close cell-to-cell contact.Non-standard abbreviations BSA bovine serum albumin - CAT chloramphenicol acetyltransferase - Cm^R chloramphenicol resistant - PAB Penassay broth - SDS sodiumdodecylsulfate - Tc^R tetracycline resistant     

Activation of ergot alkaloid biosynthesis in prototrophic isolates by Claviceps purpurea protoplast fusion.

Intraspecific protoplast fusions were carried out with active ergocornine-ergokryptine and inactive ergocristine Claviceps purpurea strains and vice versa. The isolated prototrophic strains from both types of crosings produced all three alkaloid types, showing that biosynthesis of distinct alkaloid was activated in an inactive partner strain. The prototrophic isolates were stable on minimal medium but they segregated by subculturing on complete medium. In comparison with the original partner strains, differences in morphological and cytological characteristics were also established.     

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.     

A light sensitive recipient for the effective transfer of chloroplast and mitochondrial traits by protoplast fusion in Nicotiana

Summary A light sensitive mutant was used as a recipient in the transfer of chloroplasts from a wildtype donor. Gamma irradiated (lethal dose) mesophyll protoplasts of Nicotiana gossei were fused with mesophyll protoplasts of a N. plumbaginifolia line carrying light sensitive plastids from a N. tabacum mutant. After fusion, colonies containing wild-type plastids from the cytoplasm donor were selected by their green colour. Most of the regenerated plants had N. plumbaginifolia morphology, but were a normal green in colour. The presence of donor-type plastids was confirmed by the restriction pattern of chloroplast DNA in each plant analysed. These cybrids were fully male sterile with an altered flower morphology typical of certain types of alloplasmic male sterility in Nicotiana. The use of the cytoplasmic light sensitive recipient proved to be suitable for effective interspecific transfer of wild-type chloroplasts. The recombinant-type mitochondrial DNA restriction patterns and the male sterility of the cybrids indicated the co-transfer of chloroplast and mitochondrial traits. On leave from: Department of Genetics, Section of Biosciences, Martin Luther University, Domplatz 1, DDR-4020 Halle/ S., German Democratic Republic