Interspecific hybridization between Penicillium chrysogenum and Penicillium cyaneo-fulvum following protoplast fusion

Summary Slow-growing interspecific heterokaryons were isolated on minimal medium following the induced fusion of protoplasts from auxotrophic mutants of Penicillium chrysogenum and Penicillium cyaneo-fulvum. After 5–7 days cultivation the heterokaryons produced vigorously growing sectors which on transfer gave genetically stable colonies. Cultivation of these colonies on a complete medium supplemented with p-fluorophenylalanine or benomyl broke down this stability and several different prototrophic and auxotrophic colony types were isolated. Many of these behaved as diploids or aneuploids showing sectoring either spontaneously, or in the presence of an haploidizing agent. Some of the latter isolates were recombinants for parental spore colour and auxotrophic markers.     

Recombination between chloroplast genomes of Trachystoma ballii and Brassica juncea following protoplast fusion

We document here the presence of a recombinant plastome in a cytoplasmic male sterile (CMS) line of Brassica juncea developed from the somatic hybrid Trachystoma ballii + B. juncea. Restriction endonuclease digestion of the chloroplast (cp) DNA has revealed that the recombinant plastome gives rise to novel fragments in addition to the parent-specific fragments. Analysis of the 16S rRNA region by Southern hybridization shows no variation between B. juncea, T. ballii and the CMS line. The rbcL gene region of the recombinant plastome is identical to that in T. ballii. Analysis with probes for psbA and psbD using single and double DNA digests indicates that the hybridization patterns of the recombinant plastome are identical to those of the parents in digests obtained with some restriction enzymes, while novel bands hybridize to probes in other digests. In the psbA region, a B. juncea-specific PstI site and a T. ballii-specific EcoRI site are found in the recombinant plastome. The psbD region of the recombinant plastome contains a B. juncea-specific HindIII site and T. ballii-specific BamHI and HpaII sites. These results indicate the occurrence of intergenomic recombination between the chloroplasts of T. ballii and B. juncea in the somatic hybrid from which the CMS line was developed. The recombined plastome appears to be a mosaic of fragments specific to both parents and the recombination event has occurred in the single-copy regions. These recombinational events have not caused any imbalance in the recombinant plastome in terms of chloroplast-related functions, which have remained stable over generations. Received: 3 March 1998 / Accepted: 5 August 1998     

Organelle segregation following plant protoplast fusion

Organelle segregation and genome recombination can lead to the production of novel nuclear—cytoplasmic genome combinations following plant protoplast fusion. These unique hybrids are potentially useful in crop improvement.     

Zygote formation and recombination between like mating types in the yeast Saccharomycopsis lipolytica by protoplast fusion

Summary Protoplasts from auxotrophic strains of the alkane yeast, Saccharomycopsis (Candida) lipolytica, will hybridize despite identity in mating type. Fusion products following regeneration and selection form stable prototrophic diploids, and recombinant progeny can be obtained either through the parasexual or the sexual cycle. These results confirm that mating type alleles of this yeast control only the initial steps in the mating sequence, cell recognition and agglutination, but not karyogamy and meiosis.     

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.     

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     

Transmission and recombination of mitochondrial genes in Saccharomyces cerevisiae after protoplast fusion

Summary Protoplasts of auxotrophic strains of Saccharomyces cerevisiae of opposite and identical mating types carrying different mitochondrial drug-resistance markers, with both homosexual and heterosexual mitochondrial backgrounds, were induced to fuse by polyethylene glycol. After selective regeneration of prototrophic fusion products, the transmission and recombination frequencies of mitochondrial genes in populations of cells were determined and compared with those obtained in mating processes. The frequencies obtained in the fusion experiments proved very similar to those found in the zygote clones. The behavior of mitochondrial genes was apparently affected neither by nuclear mating type background nor by the method of transfer of mitochondrial genomes (i.e., protoplast fusion or mating), making possible mitochondrial genetic studies by protoplast fusion irrespective of the mating type barrier of yeast strains.     

Diploid formation of Candida tropicalis via protoplast fusion.

Haploid auxotrophic mutants were produced from Candida tropicalis, and protoplast usion was induced by polyethylene glycol. The resulting nutritional complementation was due to heterokaryon formation and, at a much lower frequenty, to spontaneous diploidization. During cultivation, heterokaryotic clones regularly gave rise to heterozygous diploids from which, in turn, haploids could be isolated. The technique of protoplast fusion gives an opportunity for genetic analysis of this and similarly asexual fungal species.     

Diploid state of phenotypically recombinant progeny arising after protoplast fusion in Bacillus subtilis

Summary After fusion of Bacillus subtilis protoplasts the phenotypically recombinant clones isolated, whether immediately or as segregants of complementing diploid clones, have in common the following properties. They appear independently of the recN ⁺ gene, most often as the result of apparently non-reciprocal recombination occurring in genetic intervals encompassing the origin and the terminus of replication. First indicated by reciprocal fusion crosses between 105-lysogenic and 105-sensitive strains, the diploidy of the recombinants was confirmed by studying the transforming activities of their DNA. These experiments establish heterozygosity at eight loci scattered on the chromosome map. By revealing the presence of the rpF ⁺ allele in trpF7 recombinants, the results also strongly suggest that stable phenotypic recombinants may arise by genetic inactivation. Two possible genetic structures for these recombinants are discussed, one implying total inactivation of one recombinant chromosome, the other a segmentary inactivation of one unrecombined chromosome. Whatever the structure, genetic stability is not a reliable sign of haploidy in bacterial clones produced after protoplast fusion.     

Carrot protoplast fusion

Conclusion Carrot protoplasts have been used successfully in many protoplast fusion studies. These studies have demonstrated 1) whether or not the expression of certain resistances is dominant or recessive, 2) the development of a “universla hybridzer,” 3) the use of chemicals to inactivate one parent, 50 the complementation of albino mutants, 5) interspecific, intergeneric, and interkingdom fusions, and 6) the dominant expression of plant regeneration ability, and 7) the occurrence of somatic segregation in fusion hybrids.     

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.     

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

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

Electrochemical protoplast fusion in citrus

We report here the development of a novel protoplast fusion method for citrus somatic hybridization. This new procedure, which we have named electrochemical protoplast fusion, is based on chemically induced protoplast aggregation, using a low concentration of polyethylene glycol, and DC pulse-promoted membrane fusion. Based on the results of nucleus and mitochondria molecular analyses, we were successful in using this method to regenerate both symmetric somatic hybrids and cybrids. Various parameters, including pulse intensity, pulse length, and composition of the fusion media, were tested, and the optimum fusion condition selected consisted of two 100-s pulses of 1,500 V cm^–1. Our conclusion is that electrochemical fusion is a reliable and reproducible method that combines the best features of both the chemical and electrical methods, thereby promoting cell division and high embryogenesis rates of the fused cells. It represents a new approach to citrus somatic hybridization. Various interesting features of this new approach are presented and discussed.     

Laser-induced tobacco protoplast fusion

Laser tweezers can manipulate small particles, such as cells and organdies. When coupling them with laser microbeam selective fusion of two tobacco protoplasts containing some chloroplast was achieved. Physical and biological variables that affect laser trapping and laser-induced fusion were also discussed. The results show that the effect of chloroplast content and distribution on the yield of cell fusion is remarkable.     

Laser-induced tobacco protoplast fusion

Laser tweezers can manipulate small particles, such as cells and organelles. When coupling them with laser microbeam selective fusion of two tobacco protoplasts containing some chloroplast was achieved. Physical and biological variables that affect laser trapping and laser-induced fusion were also discussed. The results show that the effect of chloroplast content and distribution on the yield of cell fusion is remarkable.     

Occurence of reiterated DNA sequences in strains of Streptomyces produced by an interspecific protoplast fusion

Summary DNA isolated from a Streptomyces strain produced by interspecific protoplast fusion of Streptomyces jumonjinensis and Streptomyces lipmanii contained a tandemly repeated six kilobase pair sequence which constituted 15%–18% of the total DNA. Examination of other strains produced by similar fusions showed that they also contained reiterated DNA sequences.     

Isolation and characterization of the nucleoid of non-complementing diploids from protoplast fusion in Bacillus subtilis

Summary Nucleoids of non-complementing diploids (Ncd) from protoplast fusion of B. subtilis were isolated. Their purified DNA banded in neutral CsCl gradient as a single unimodal peak of buoyant density 1.711 g/cm³, a value which is similar to that of the DNA purified from the original parental strains, suggesting that methylation of bases is not a significant factor in chromosome inactivation. Nucleoids released from a Ncd clone give two peaks in a sucrose gradient with a characteristic S value for each nucleoid. That is in contrast to nucleoids from the haploid parents whose sedimental patterns show only one peak.Both nucleoid preparations from Ncd strains assayed for transformation activity show the fast sedimenting nucleoid devoid of transformation activity while the slow nucleoid was active in transformation for the alleles carried by the genome which is expressed in vivo. Both nucleoids of the Ncd strains are transcribed in vivo. The RNA associated with the inactive chromosome is synthesized by the RNA polymerase of the active one.This study provides evidence that inactivation of one parental genome in the Ncd strain may be related with the tertiary organization of its DNA.