Breeding by protoplast fusion for glucoamylase production

Summary Protoplast fusion was carried out between two strains ofAspergillus niger 8-2, a fast growing culture and poor producer of glucoamylase enzyme andA.niger 8-7, a slow growing culture and good producer of the enzyme. The nonconidiating fused mass in presence of benomyl, produced fast growing segregants showing various combinations of the two parental gene markers. Some of the segregants produced up to 68% more glucoamylase than the better yielding parent 8-7.     

Amino acid accumulation by an analogue sensitive mutant ofCorynebacterium glutamicum

Summary A fluoroacetate/fluoropyruvate-sensitive mutant was derived from the parent strainCorynebacterium glutamicum ATCC 21513. Accumulation of various amino acids in the fermentation broth using the two strains was compared. The FA/FP-sensitive mutant accumulated about 26.5 g/L L-lysine and 2.2 g/L aspartic acid which was about 3-fold and 10-fold respectively, more than the amount produced by the parent strain.     

Use of the method of protoplast fusion in the selection of a nisin producer

Experimental data on selection of Streptococcus lactis producing the polypeptide antibiotic nisin with the method of protoplast fusing, one of the modern methods of cell engineering are presented. Four strains of Streptococcus lactis differing in their nisin-producing levels and difficult for protoplasting were used in the study. It was shown possible to transfer them to the protoplast form when respective conditions for their preliminary cultivation and regeneration are provided. Distinctive features of these strains with respect to the antibiotic resistance, sugar fermentation and growth component requirements were revealed. The protoplast fusing yielded hybrids differing from the parent strains by a number of phenotypical features and nisin-synthesizing activity.     

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     

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.     

Selective production of phenylalanine from phenylpyruvate using growing cells ofCorynebacterium glutamicum

Summary Phenylalanine was produced from phenylpyruvate by growing cells of a mutant strain ofCorynebacteriumglutamicum over-producing valine. A defined medium was used to minimize the accumulation of valine. The maximum phenylalanine concentration achieved was 44.5 mM (7.5 g/l) with a phenylpyruvate molar conversion of 75%.     

Strain improvement ofArthrobacter simplex by protoplast fusion

Anl-tryptophan auxotroph and milky mutants were derived from an inducible cholesterol oxidase-producing bacterium,Arthrobacter simplex USA18, via UV-mutagenesis. Protoplasts of these mutants and a constitutive cholesterol oxidase producer, strain US3011, were prepared by growing cells in the presence of ampicillin (20g ml^–1) followed by digestion with lysozyme. Protoplast fusion between tested strains with complementary characteristics was achieved in the presence of 20–40% polyethylene glycol 6000. The fusion frequency was about 1.5–1.7×10^–3. The cholesterol oxidase activity of four fusants in a cholesterol-containing medium was 20–60% higher than that of parental strains. This study demonstrated that protoplast fusion is applicable to strain improvement ofArthrobacter strains for enzyme production.     

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.     

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.     

Breeding of starch-utilizing and itaconic-acid-producing koji molds by interspecific protoplast fusion between Aspergillus terreus and Aspergillus usamii

Interspecific protoplast fusion between␣Aspergillus terreus, an itaconic acid producer, and A.␣usamii, a glucoamylase producer, was done to breed new koji molds producing itaconic acid from starch. Protoplast fusion between auxotrophic mutant strains by poly(ethylene glycol) treatment produced prototrophic fusants with a fusion frequency of 10⁻⁵−10⁻⁴. The stabilities of some fusants obtained were confirmed by successive subcultures. Conidial analyses of DNA contents and the number of nuclei indicated that the fusants obtained were haploids like the parental strains. One of the stable fusants, F-112, morphologically resembled A. terreus, and produced maximally 35.9 mg/ml itaconic acid from soluble starch (120 mg/ml) at day 6 of cultivation. This productivity from soluble starch was five times as high as that of A. terreus and 70 % of that of A. terreus from glucose (120 mg/ml). Received: 28 June 1996 / Received revision: 3 September 1996 / Accepted: 29 September 1996     

Intraspecific hybridization of Trichoderma reesei by protoplast fusion

Protoplasts obtained from mycelia of a single auxotrophic mutant of Trichoderma reesei QM 9414 were fused with those of T. reesei QM 9136 in the presence of 0.5 M glycine-NaOH buffer, pH 7.5, containing 0.05 M CaCl₂ · 2H₂O and 35% polyethylene glycol 4,000. The regeneration frequency of these protoplasts was 8.9–12.0% on a solid culture medium with soft agar overlay. The fused protoplasts successfully formed heterokaryons showing 3.33% of the fusion frequency. A heterozygous diploid was obtained from conidia of the heterokaryon by treatment with 0.1% d-camphor. The diploid showed a 1.9 fold DNA content per conidial nucleus compared to T. reesei QM 9414. The frequency of diploid formation was about 1.9 × 10⁻⁴ per conidium. Cellulase activities, such as filter paper degrading and CM-cellulose and Avicel saccharifying activities, and the xylanase activity of the diploid showed intermediate values between those of T. reesei QM 9414 and T. reesei QM 9136. However, the β-glucosidase, β-1,3-glucanase and chitinase activities of the diploid increased to levels equal to on above those of T. reesei QM 9414 and T. reesei QM 9136. The existence of a parasexual cycle of T. reesei and the possibility of its application to enhanced enzyme productivity were confirmed using the protoplast fusion technique.     

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.     

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.     

Intergeneric gene transfer mediated by plant protoplast fusion

Summary In attempts at somatic transfer of plant genomes of reduced size, X-irradiated leaf protoplasts of parsley (Petroselinum hortense, 2n=22) were fused with cell culture protoplasts of a nuclear albino mutant of carrot (Daucus carota, 2n=18). Introduction of genes from the irradiated parsley nuclei into the carrot genome was shown by the correction of the albino defect and by the appearance of parsley isoenzymes in selected green tissues and plants. The cytological studies provided information on significant deviation from the amphidiploid chromosome number. The high frequency of cells with 2n=19, 2n=38 and regeneration of plants with 2n=19 chromosomes can indicate that the elimination of parsley chromosomes is incomplete. A correlation was found between the lethality of selected tissues and differentiated or undifferentiated stages of the cells.     

Genetic mapping in Streptomyces clavuligerus by protoplast fusion

The development of a protoplast manipulation protocol for the industrially important bacterium Streptomyces clavuligerus, which produces the beta-lactamase inhibitor clavulanic acid, made possible a preliminary genetic mapping study based on protoplast fusion crosses. A preliminary position for 11 markers on the S. clavuligerus genetic map is proposed. Fusion progeny were characterized by random spore analysis because the markers present in the strains were not amenable to the conventional four-on-four selection procedure. Whilst the resulting map is similar to that derived by conjugation for S. clavuligerus and S. coelicolor, further analysis of the markers is required to confirm these observations.     

Use of the protoplast fusion method in the selection of Streptomyces griseus--the producer of the streptothricin antibiotic grisin

An effective method for protoplast fusion in S. griseus producing grisin was developed. The method requires the use of polyethylene glycol with a molecular weight of 1000. It was demonstrated that protoplasts formed most effectively in this organism, when the mycelium of the streptomycete previously treated with ultrasound in the process of its growth was used for the treatment with lysozyme. The efficacy of protoplast regeneration in the strains with the use of the modified hypertonic medium R2MD was 25-75 per cent. The possibility of using the protoplast fusion method for constructing phage resistant strains producing kormogrisin was shown.     

种间双亲灭活原生质体融合选育高产Monacolin K红曲菌

为获得高产MonacolinK的红曲菌菌株,将经农杆菌介导转化获得的携带潮霉素抗性基因且以甘油为原料液态发酵高产MonacolinK的发白红曲菌H2和以大米为原料固态发酵产Monaco-1inK的烟色红曲菌9908作为亲本,对其原生质体分别进行热灭活及紫外灭活,然后对灭活双亲用PEG作融合剂进行原生质体融合。从融合子中选出有潮霉素抗性的突变株,通过发酵与亲本对比,筛选得到一株以大米为原料固态发酵高产MonacolinK的融合株F12．11,其MonacolinK产量达到8．73mg／g;较发白红曲菌H2与烟色红曲菌9908分别提高了100．23％和48．98％;一株以甘油为原料液态发酵高产MonacolinK的融合株F13-2,其MonacolinK的产量达到1752．46mg／L,较发白红曲菌H2与烟色红曲菌9908分别提高了32．98％和1979．33％。     

Genetic recombination in Actinoplanes brasiliensis by protoplast fusion.

Protoplast formation, fusion, and cell regeneration have been achieved with mutant strains of Actinoplanes brasiliensis. Three-, four-, and five-factor crosses have shown genetic recombination among the markers, and a five-factor cross is analyzed and discussed. Possibilities of using protoplast fusion for gene mapping and strain improvement are suggested.     

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.