Intergeneric Protoplast Fusion Research Articles

Intergeneric protoplast fusion in Saccharomycopsis fibuligera and Saccharomyces cerevisiae

The amylolytic yeast Saccharomycopsis fibuligera K-33 and Saccharomyces cerevisiae were used to breed a hybrid by protoplast fusion. S. fibuligera K-33 produced glucoamylase and α-amylase. The intergeneric protoplast fusion frequency was 1.95 × 10 −4. Fusant 10-1, which enhanced α-amylase production, produced 3.1% (v/v) ethanol in a 10% soluble starch medium at 3 d. The amylase activity of the fusant was 2.5 times higher than that of the parental strain K-33 at 2 d.

Intergeneric protoplast fusion between Acinetobacter sp. A3 and Pseudomonas putida DP99 for enchanced hydrocarbon degradation

Polyethylene glycol 6000 mediated protoplast fusion between an alkane degrader Acinetobacter sp. A3, and a naphthalene degrader, Pseudomonas putida DP99 , resulted in fusants capable of degrading both hydrocarbons and were morphologically similar to Acinetobacter sp. A3. While fusant F4/13 and Pseudomonas putida DP99 degraded over 98% of naphthalene provided by the end of five days, tetradecane degradation by fusant F4/13 was 82% compared to 77% by Acinetobacter sp. A3 in the same time period. Also, while from naphthalene +tetradecane mixture, fusant F4/13 could degrade 99% and 53% of naphthalene and tetradecane respectively, both the parent strains together could degrade over 99% naphthalene but only about 16% tetradecane.

Intergeneric protoplast fusion between Brassica carinata and Camelina sativa

Camelina sativa is a wild crucifer that is reported to be resistant to Alternaria blight. Polyethylene glycol mediated fusion was attempted between protoplasts from etiolated hypocotyls of Brassica carinata and mesophyll protoplasts of Camelina sativa. The mean frequency of heterokaryons was 6.8%. Three hybrid shoots were regenerated, each from a single fusionderived callus. These shoots failed to produce roots capable of withstanding transplantation. Confirmation of hybridity was obtained from the morphology of in vitro produced leaves, somatic chromosome number in leaf tips, and restriction fragment length polymorphism for a nuclear rDNA probe. Analysis for organelle constitution using RFLPs indicated that the hybrid contained chrloroplasts derived from the wild species and mitochondria from the cultivated Brassica species.

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.

Intergeneric protoplast fusion between Gluconobacter oxydans and Corynebacterium species

Intergeneric protoplast fusion between 2,5-diketo-gluconic acid producing Gluconobacter oxydans (ATCC 9937) and a mutant strain of Corynebacterium species (ATCC 31090), capable of reducing 2,5-diketo-gluconic acid to 2-keto- l-gulonic acid, a penultimate step in vitamin C production) resulted in viable recombinants. Some of the fusion products exhibited the capacity to convert d-glucose to 2-keto- l-gulonic acid, but the conversion rate is low.

Hybrid Cell Lines Produced by Protoplast Fusion between Luffa cylindrica Roem. and Gynostemma pentaphyllum Makino.

Hybrid cell lines were produced by the intergeneric protoplast fusion between Luffa cylindrica ROEM. (L. cylindrica) and Gynostemma pentaphyllum MAKlNO (G, pentaphyllum). Protoplasts derived from the cotyledons of L. cylindrica and protoplasts derived from the cultured cells of G. pentaphyllum were fused according to the method of MENCZEL and WOLFE. Four fusion products were confirmed as hybrid cells based on isozyme analysis

Secretion of thermostable β-glucosidase by an intergeneric bacterial hybrid betweencellulomonas and bacillus subtilis

The intergeneric protoplast fusion hybrid (Bs/C 005) betweenCellulomonas sp. andBacillus subtilisproduced extracellular aryl β-glucosidase that is otherwise intracellular in parentalCellulomonassp. This extracellular aryl β-glucosidase was active at relatively higher temperature (60°C) and lower pH (pH 5.0) conditions than that ofCellulomonas enzyme. It also exhibited increased thermostability and stability over wide range of pH. Cellobiase activity, distinctly different from aryl β-glucosidase detected in bothCellulomonassp. Bs/C 005, was only intracellular. Cellobiase from Bs/C 005, however, was more thermostable than that ofCellulomonassp.

Differential Expression of Xylanases and Endoglucanases in the Hybrid Derived from Intergeneric Protoplast Fusion between a Cellulomonas sp. and Bacillus subtilis

A stable hybrid obtained by protoplast fusion between a Cellulomonas sp. and Bacillus subtilis exhibits an altered pattern of enzyme induction with different cellulosic substrates. Unlike in the Cellulomonas sp., xylanase was induced in the hybrid organism specifically by xylan, and endoglucanase was induced by carboxymethyl cellulose. The amount and specific activity of xylanase produced by the hybrid were more than those produced by the Cellulomonas sp. beta-Glucosidase which is cell bound or intracellular in the Cellulomonas sp. was secreted by the hybrid organism, and relative amounts of extracellular beta-glucosidase were high. Furthermore, this extracellular beta-glucosidase activity was dependent on the nature of the cellulosic substrate. Endoglucanases synthesized in the hybrid differed in their electrophoretic mobilities as compared with the parental enzymes.

Intergeneric protoplast fusion between agrobacterium tumefaciens and Bacillus thuringiensis subsp. kurstaki

Intergeneric protoplast fusion betweenA.tumefaciens andB.thuringiensis was performed. The fusants exhibited some properties of both the parental strains. One of the Gram positive fusants with most of theBacillus properties showed tumor inducing capacity in pigeonpea (Cajanascajan).

Escherichia coli Spheroplast-Mediated Transfer of pBR322 Carrying the Cloned Ruminococcus albus Cellulase Gene into Anaerobic Mutant Strain FEM29 by Protoplast Fusion

Intergeneric protoplast fusion between Escherichia coli HB101 with pBR322 carrying the cloned o-(carboxymethyl)cellulase (CMCase) gene of Ruminococcus albus (Pro Leu Ap Km) and an anaerobic mutant strain, FEM29 (Trp His Ap Km), with dehydrodivanillin-degrading activity was performed in the presence of 40% polyvinyl alcohol 300 under aerobic and anaerobic conditions to transfer the cloned cellulase gene into the mutant. The mutant FEM29 had a unique property. When it was incubated in liquid medium with 1% glucose and sucrose, protoplasts could be produced autogenously and regenerated on the agar slant. E. coli spheroplasts formed from a plasmid-amplified overnight culture after 10 min of treatment with lysozyme (20 mug/ml) in a hypertonic solution (0.01 M Tris hydrochloride [pH 7.5], 0.4 M mannitol). Protoplast regeneration rates of FEM29 and HB101 were 30 and 83%, respectively, on the agar-yeast extract medium. Ap Km fusants were obtained at high frequency: 1.7 x 10 anaerobically and 8.2 x 10 aerobically. These fusants showed 23 to 57% of CMCase and dehydrodivanillin-degrading activities, respectively, as compared with parental strains. All the fusants isolated were gram-negative rods with main phenotypes such as urease and catalase activities as in HB101 and esterase and chymotrypsin activities as in FEM29. Southern hybridization experiments suggested that pBR322 with the cloned CMCase gene existed autonomously in the fusant cells. This is the first report describing transfer of pBR322 with a cloned cellulase gene into an anaerobic mutant by polyvinyl alcohol-mediated fusion with an E. coli spheroplast.

Intergeneric Protoplast Fusion between Ruminococcus albus and an Anaerobic Recombinant, FE7

Intergeneric protoplast fusion between Ruminococcus albus, a cellulolytic, gram-positive, anaerobic bacterium (Pc Sm Km), and an anaerobic recombinant, FE7 (Pc Sm Km), having lignin-related compound-degrading activities, was performed under strictly anaerobic conditions to introduce cellulase genes into strain FE7. The fusion frequency varied with different selected markers from 3.0 x 10 to 3.3 x 10. Two fusants, obtained from a synthetic medium with selective pressures of penicillin and streptomycin and with cellooli-gomer as the sole carbon source, were gram-negative rods. One of them, named FE7R2, showed 45 to 47% of the beta-glucosidase and cellobiosidase activities of its parent R. albus and still maintained a level of degradation activity against dehydrodivanillin, a lignin-related compound, of up to 87% of that of the parent strain FE7. To verify that the cellulolytic activities expressed in the fusant FE7R2 originated from R. albus cellulase genes, the beta-glucosidase gene of R. albus was cloned into Escherichia coli HB101 with plasmid pBR322. Cells bearing a recombinant plasmid, pRAII, produced high enzyme activities against both p-nitrophenyl-beta-d-glucoside and p-nitrophenyl-beta-d-cellobioside and could degrade cellobiose to glucose. Southern blot results showed that the cloned DNA fragment could hybridize with chromosomal DNAs of both R. albus and FE7R2, but did not with the chromosomal DNA of FE7, indicating that the beta-glucosidase gene fragment was introduced into the chromosome of FE7R2 from R. albus via the protoplast fusion. The fusant FE7R2 could utilize simultaneously both cellobiose and dehydrodivanillin. These results gave evidence that the fusion product FE7R2 is a recombinant strain between its parents R. albus and FE7. This recombinant has stably kept the above properties for about 2 years.

Transfer of Plasmids between Bacillus subtilis and Streptococcus lactis

The shuttle plasmid pGK12, as well as several Staphylococcus aureus plasmids, was introduced into Streptococcus lactis by intergeneric protoplast fusion with Bacillus subtilis. The S. aureus plasmids were stably inherited in S. lactis, and so they may possibly be used as cloning vectors in lactic streptococci.

Liposome-enhanced transformation of streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of streptococcus lactis and bacillus subtilis

Liposome-enhanced transformation of streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of streptococcus lactis and bacillus subtilis

Intergeneric protoplast fusion between Aspergillus niger and Trichoderma viride.

Intergeneric protoplast fusion between Aspergillus niger and Trichoderma viride.

Intergeneric protoplast fusion between Fusobacterium varium and Enterococcus faecium for enhancing dehydrodivanillin degradation.

Intergeneric protoplast fusion between Fusobacterium varium (Pcs Glu+) and Enterococcus faecium (Pcr Glu-) was performed under strictly anaerobic conditions to improve dehydrodivanillin (DDV) degradation. The fusion frequency obtained from the selective medium (Pc+ Glu-) was about 0.9 X 10(-5) to 1.3 X 10(-5). The seven fusants isolated were all gram-negative anaerobes with rod shapes like that of F. varium and with main phenotypical properties of cocci like those of E. faecium such as esculin and starch hydrolysis, milk clotting, and lactate production. Five fusants showed enhanced DDV degradation activities that were 2 to 4 times higher than those of parental strains. Genetic relatedness between a fusant (FE7) and the parents was estimated by DNA-DNA Southern blot hybridization with 32P-labeled chromosomal DNA fragments of F. varium and E. faecium as respective probes. The fusant FE7 presented a very high cross-hybridization with both probes, indicating a high DNA homology between the fusant and both parental strains. Almost all the fusants obtained here have stably kept the properties described above for about 2 years. These results suggest that intergeneric gene transfer takes place through protoplast fusion and that the fusants that were obtained are stable recombinants.

Production of extracellular debranching activity by amylolytic yeasts

The production of extracellular pullulan-degrading enzymes by several amylolytic yeast strains was studied. The highest activity was obtained with species of Endomycopsis, Lipomyces, Filobasidium, Leucosporidium, and Trichosporon, and also with some amylase-hyperproducing mutants. The most active strains are potentially valuable partners for intergeneric protoplast fusion.

Plant regeneration from intergeneric cell hybrids

Protoplasts isolated from cell suspension culture of an albino mutant of Daucus carota and from leaves of Aegopodium podagraria were fused by polyethylene glycol treatment. Plants have been regenerated from green calli developed after this intergeneric protoplast fusion. The degree of hybridization of RNA synthesized in nuclei of selected plants to Ae. podagraria DNA was significantly higher than that obtained using nuclear RNA from the albino mutant. Restoration of normal chloroplast ultrastructure and of synthesis of photosynthetic pigments, as well as expression of Ae. podagraria markers for root development and for root carotenoids, indicated the presence of Ae. podagraria genes in the genome of selected plants, which possessed only D. carota chromosomes. Intergeneric gene transfer by fusion of protoplasts from dividing and mitotically inactive somatic cells is discussed as a possible method in genetic manipulation of higher plants.

Cell Division of Intergeneric Protoplast Fusion Products

Protoplasts from soybean cell suspension cultures were fused with those isolated from leaves of two Pisum sativum varieties, four Nicotiana species and Colchicum autumnale. Fusion products of all combinations underwent cell division. The first division of fusion products occurred on day 2 or 3 of the culture period. Parental soybean protoplasts started to divide on day 1, leaf protoplasts on day 5 or later. The capacity to divide proved the protoplast combinations tested to be compatible when fused.