Protoplast Fusion Technique Research Articles

Assessment of antioxidant, antidiabetic, and antimicrobial properties of hybrid mushroom strains derived through intergeneric hybridization between Pleurotus sajor-caju and Calocybe indica

Our present study aims to evaluate the biological activities of the six hybrid mushrooms (APS) obtained through the protoplast fusion technique between two mushrooms Calocybe indica and Pleurotus sajor-caju, in our previous study. Antioxidant activity of hybridized mushroom samples was determined by the free radical scavenging method, using DPPH radical, Superoxide radical, and Hydroxyl radical. Additionally, all the mushroom extract has shown a good amount of total phenolic content, and total flavonoid content and also has potent Ferric reducing antioxidant power. The Total phenolic content, Total flavonoid content, and Ferric reducing antioxidant power were estimated. Six constituents: gallic acid, catechin, protocatechuic acid, p-hydroxybenzoic acid, p-coumaric acid, and cinnamic acid were identified by UHPLC. The MIC values, zone of inhibition, growth kinetic, and scanning electron microscopy (SEM) were performed to establish the antimicrobial efficacy. The tested samples exhibited antimicrobial activity against selected microorganisms Staphylococcus aureus, Klebsiella pneumoniae, Bacillus subtilis, Pseudomonas aeruginosa. A clear morphological change was observed under SEM when Bacillus subtilis and Pseudomonas aeruginosa were treated with APS-4 and APS-3 mushroom extracts. The ability to inhibit carbohydrate hydrolyzing enzymes such as α-amylase and α-galactosidase has also been studied to explore antidiabetic properties. The inhibitory activity of α-glucosidase was APS-4 reflected a very prominent result with the lowest IC50 value i.e.,118.058 ± 1.515, APS-2 and APS-6 showed almost similar IC50 values. α – galactosidase inhibitory activity of APS-4 and APS-6 exhibit better results than other strains.

Genetic improvement of some microorganisms to increase the effect of bio-control on the potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae)

BackgroundDuring the last five decades, chemical synthetic pesticides have been used extensively to control pests and protect crops. Use of synthetic pesticide has caused some unfortunate consequences like environmental pollution, pest resistance and toxicity to other non-target organisms. Due to the hazardous effects of their chemical residues to human and animal health, several studies have been carried out to determine effective alternative control methods. One of methods is the usage of entomopathogens such as bacteria, virus and fungi. Entomopathogenic bacteria have unique insecticidal properties mainly due to the production of larvicidal proteins that accumulate as parasporal crystalline inclusions within the cell. The bio-insecticidal bacterium Bacillus thuringiensis (Bt) has been widely used in agriculture for the control of pest insects which attack crops.ResultsThirteen genetically stable fusants strains were obtained as a result of protoplast fusion technique between a local Bacillus thuringiensis (Bt) and each of B. subtilis subsp. subtilis strain (Bs1), Bacillus licheniformis strain (Bl) or B. subtilis subsp. spizizeniie (Bs2). Thirteen fusants were obtained, including three fusants (group B) from Bt::Bs1 fusion, six fusants (group C) from Bt::Bl fusion and four fusants (group D) from Bt::Bs2 fusion. All fusants were chosen for bioassay treatments against the potato tuber moth (PTM) Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) which recorded a high mortality percentage of PTM ranged from 75 to 80% in F1 and F2 of first attempt B (Bt::Bs1). The accumulative larval mortality was notation the highest percentage reached to 90% in case of treatment by fusants F4 followed by F7, F8 and F9 which gave 75, 70 and 80% larval mortality, respectively, for second attempt C (Bt::Bl). The third attempt D (Bt::Bs2) fusants F10, F11, F13 achieved the highest mortality percentage up to 60, 60 and 70%, respectively. Expression of apoptosis-related encoding genes in PTM was determined in three fusants B (Bt::Bs1), C (Bt::Bl) and D (Bt::Bs2) and compared with A (Bt) and Control. The results showed a high expression of gene apoptosis to fusants D (Bt::Bs2) to Caspase-16 gene, Dronc and Dredd genes in tissues of the (PTM) treated with different biological pesticides.ConclusionsThe study used protoplast fusion technique between a local Bacillus thuringiensis (Bt) and B. subtilis subsp. subtilis strain (Bs1), B. licheniformis strain (Bl) and B. subtilis subsp. spizizeniie (Bs2). Thirteen fusants were chosen for bioassay treatments against PTM. Bacterial fusant, F1, F2, F4, F7, F9 and F13, achieved the highest mortality rates against PTM ranged 75–90% under laboratory conditions. The highest expression of gene apoptosis to fusants D (Bt::Bs2) to Caspase-16 gene, Dronc and Dredd genes was recorded in insect tissues treated with different bio-insecticides. As a result of the effected on the genes responsible for expression the vital processes (genes of apoptosis) in the insect as a result of treatment with bacteria, this led to deformities and death of PTM.

Greener approach to the comprehensive utilization of algal biomass and oil using novel Clostridial fusants and bio-based solvents

A greener method has been tested to utilize algal biomass as a feedstock to produce bio-oil in addition to acetone, butanol, and ethanol (ABE) products. Various hydrolysis treatments were used prior to fermentation including combination of thermal, chemical, and enzymatic, which resulted in maximum sugar release of 27.78 g/L. Bio-based terpenes was used instead of common toxic chemicals together with Clostridial fustants to produce bio-alcoholic fuels. Protoplast fusion technique were used to produce the novel Clostridia fusants (C. beijernickii + C. thermocellum and C. acetobutylicum + C. thermocellocum). Fused strains were then subjected to UV radiation for strain enhancement. Final fusansts showed clear improvement in thermal stability and resistance to biobutanol toxicity. Fermentation experiments showed maximum biobutanol final production of 7.98 g/L using CbCt versus 7.39 g/L using CaCt. Oil extraction from virgin algae was tested using a green, bio-based approach using terpenes with ultrasonication and green Bligh and Dyer method, separately. In preliminary study on algal biomass, the combinations of ultrasonication followed by the green Bligh and Dyer have resulted in oil yield of 46.27% (d-limonene) and 39.85% (p-cymene). Oil extraction from an algae sample following fermentation using the combined extraction method resulted in significantly higher oil yield of 65.04%.

New Saccharomyces cerevisiae-Kluyveromyces marxianus fusant shows enhanced alcoholic fermentation performance.

Kluyveromyces marxianus has a promising enological potential because of strong extracellular pectinase activity and the copious production of specific higher alcohols and esters. However, K. marxianus is unable to complete alcoholic fermentation on its own. For this reason it is only used in co- or sequential fermentation in conjunction with Saccharomyces spp. Here we applied the protoplast fusion technique to two commercial Saccharomyces cerevisiae strains (Lalvin EC1118® and Lalvin QA23®) and K. marxianus isolate IWBT Y885, to obtain fusants that possess pre-selected, enologically relevant features of K. marxianus, but with improved fermentation performance. After an extensive selection process, performed using non-auxotrophic markers, we identified one fusant, BF2020, that shows enhanced fermentation performance compared to the parental strain K. marxianus Y885, but that still exhibits strong pectinase activity and may be suitable for industrial production. The genome of the selected hybrid was further investigated and characterized by RAPD-PCR analysis and whole genome sequencing.

Protoplast fusion between Blakeslea trispora 14,271 (+) and 14,272 (-) enhanced the yield of lycopene and β-carotene.

Blakeslea trispora, a heterothallic Zygomycota with two mating types (termed "plus" and "minus"), is an ideal source of lycopene and β-carotene. The lycopene and β-carotene yields when the two type strains are used for fermentation separately are lower than those when they are joint together. To enhance the yield of lycopene and β-carotene in B. trispora, protoplast fusion technology was carried out between ATCC 14,271 (+) and ATCC 14,272 (-). After protoplast preparation, protoplast fusion, fusion sorting, fusion regeneration, and high-throughput screening, two fusions (Fu-1and Fu-2) with high lycopene and β-carotene yields were obtained. The lycopene yields of Fu-1 and Fu-2 were increased to 0.60mg/gDW and 0.90mg/gDW, which were respectively 3.62- and 5.44-fold those of 14,271 and 1.76- and 2.64-fold those of 14,272. The β-carotene yields of Fu-1 and Fu-2 were increased to 22.07mg/gDW and 36.93mg/gDW, which were respectively 1.72- and 2.89-fold those of 14,271 and 1.23- and 2.06-fold those of 14,272. In this study, the protoplast fusion technique was successfully used in Blakeslea trispora, providing new ideas for improving lycopene and β-carotene production.

Plant Tissue Culture- A New Tool for Vegetable Improvement (Indian scenario): A Review

In vitro culture of plant cells/tissues is now routine using a range of explant types from many of the important vegetable and fruit crops. Successful technologies include isolation, culture of tissues, cells, protoplasts, organs, embryos, ovules, anthers and microspores and regeneration from them of complete plantlets. The development of plant tissue culture technology represents one of the most exciting advances in plant sciences. For example, the prospect of being able to introduce, develop, produce, transfer and conserve the existing gene pool of plant sciences by using tissue culture methods opens up new opportunities for researches and entrepreneurs. The term plant tissue culture should denote in vitro cultivation of plant cells or tissues in an unorganized mass, i.e., callus culture. Plant tissue culture techniques, in combination with recombinant DNA technology, are the essential requirements for the development of transgenic plants. However, culture techniques like anther/pollen/ovule culture, meristem culture can themselves be utilized for crop improvement or may serve as an aid to conventional breeding. In recent, isolated microspore culture has developed as a breeding tool and an experimental system for various genetic manipulations. The inherent potentiality of a plant cell to give rise to a whole plant, a capacity which is often retained even after a cell has undergone final differentiation in the plant body, is described as ‘cellular totipotency’. On the other hand, production of virus-free plants via meristem culture can reduce losses caused by phyto-pathogens. Embryo culture has many potential uses ranging from overcoming seed dormancy to facilitation of inter-specific hybridization. Protoplast fusion technique can be used for the transfer of cytoplasmic male sterility from one species to another in a short period of time. In cabbage, male sterile cybrids are being utilized by seed companies to produce hybrid seeds on commercial scale and at competitive rates. Plant tissue culture and cell culture are providing useful methods for germplasm storage either by low temperature storage of organized tissue, or cryopreservation of cell or embryo culture.

Construction of engineering bacteria degrading residual polyacrylamide in coal slime water by protoplast fusion technique

Polyacrylamide (PAM) is widely used as flocculant in coal preparation. A protoplast fusant with enhanced degradation ability of PAM was obtained by intergeneric protoplast fusion between Rhodopseudomonas spheroids and Bacillus subtilis. The protoplast preparation was optimised and inactivation of both parents was performed in different ways. The degradation abilities of domesticated R. spheroides, B. subtilis and mixed bacteria (v/v, 1.00) were compared with protoplast fusant. The results showed that the optimal dosage for R. spheroides protoplast preparation was determined to be 20.00% sucrose, 1.00 mg/mL lysozyme, 0.20% EDTA and 60.00 min. Under such conditions, the protoplast formation rate and the protoplast regeneration rate were 63.70% and 8.90%, respectively. The optimal dosage for B. subtilis protoplast preparation was determined to be 20.00% sucrose, 1.50 mg/mL lysozyme and 60.00 min. Under such conditions, the protoplast formation rate and the protoplast regeneration rate were 58.50% and 9.45%, respectively. The protoplast inactivation of R. spheroides and B. subtilis were optimally conducted by ultraviolet radiation (70.00 s) and by heat at 55.00°C (50.00 min), respectively. The degradation ability of protoplast fusant was obviously improved. Furthermore, using microscopic observation, it was found that both parents form protoplasts by the way of apical release.

Construction of engineering bacteria degrading residual polyacrylamide in coal slime water by protoplast fusion technique

Construction of engineering bacteria degrading residual polyacrylamide in coal slime water by protoplast fusion technique

Novel fusants of two and three clostridia for enhanced green production of biobutanol

The objective of the present study is to improve biobutanol production by utilizing renewable resources of agriculture residues (i.e. wheat straws) in simultaneous saccharification and fermentation (SSF). Three strains of anaerobic gram-positive clostridia (Clostridium beijerinckii, Clostridium thermocellum, and Thermoanaerobacterium saccharolyticum) were fused through a protoplast fusion technique. Results show that protoplast fusion of thermophilic and mesophilic clostridia led to improved thermostability in a fermentation medium at 45 °C. This represents the optimum temperature for enzymatic hydrolysis. Results also show that the fused strain produced essential hydrolysis enzymes, which eliminated the need to add any enzymes during the hydrolysis step. Furthermore, results in the present study demonstrate that the fused culture of bacteria was able to tolerate the elevated concentration of acetone, butanol, and ethanol during production, which resulted in higher biobutanol production of 13.8 g/L. This study includes a comparison with the co-culture as a benchmark to account for the effects of protoplast fusion.

Molecular and biological characterization of a novel botybirnavirus identified from a Pakistani isolate of Alternaria alternata

Mycoviruses ubiquitously infect a wide range of fungal hosts in the world. The current study reports a novel double stranded RNA (dsRNA) virus, termed Alternaria alternata botybirnavirus 1 (AaBbV1), infecting a Pakistani strain, 4a, of a phytopathogenic ascomycetous fungus Alternaria alternata. A combined approach of next generation and conventional terminal end sequencing of the viral genome revealed that the virus is a distinct member of the genus Botybirnavirus. This virus comprised of two segments (dsRNA1 and dsRNA2) of sizes 6127 bp and 5860 bp respectively. The dsRNA1-encoded protein carrying the RNA-dependent RNA polymerase domain showed 61% identity to the counterpart of Botrytis porri botybirnavirus 1 and lower levels of amino acid similarity with those of other putative botybirnaviruses and the fungal dsRNA viruses such as members of the families Totiviridae, Chrysoviridae and Megabirnaviridae. The dsRNA2-encoded protein showed resemblance with corresponding proteins of botybirnaviruses. Electron microscopy showed AaBbV1 to form spherical particles of 40 nm in diameter. Biochemical analyses showed that two structural proteins encoded by dsRNA1 and dsRNA2 underwent processing to some extent during particle purification, resulting in the appearance of multiple smaller products. Phylogenetic analyses of structural proteins suggested that their coding region might have been duplicated once and maintained without recombination. Protoplast fusion technique allowed for the introduction of AaBbV1 into a virus free Japanese strain of A. alternata and demonstrated its symptomless infection by the virus. Interesting similarities and dissimilarities between AaBbV1 and other previously reported botybirnaviruses are also discussed.

Isolation, identification, and improving nematotoxicity of rhizobacterial strains against Meloidogyne incognita

Background and objectives Root-knot nematodes, Meloidogyne spp., have been reported to cause severe losses to field, horticultural, and medicinal plants throughout the world. Chemical nematicides are routinely applied for protection of the aforementioned crops. Residues of these toxic nematicides are carried to the final consumed products, which can be quite hazardous to human health on consumption. Biocontrol by using antagonistic organisms to inhibit the pathogens and reduce diseases is an ecofriendly management tool. This study was planned to isolate, identify, and improve or increase the activity of two isolated rhizobacterial strains known to have nematicidal properties − based on preliminary studies − using protoplast fusion technique and assessing the nematicidal potential of the fusants against root-knot nematode, Meloidogyne incognita J2. Materials and methods Certain rhizobacterial colonies isolated from the rhizosphere soil of cucumber and banana plants were identified by 16S rDNA to Pseudomonas aeruginosa (accession number LC187271) and Bacillus licheniformis (accession number LC187270). They were manipulated using protoplast fusion technique to improve their nematicidal potentials. The fused protoplasts generated and 10 fusant isolates were assessed against M. incognita J2. Results and conclusion All the tested fusants exhibited increase in their nematicidal activity than their parents against root-knot nematode, M. incognita J2, under laboratory conditions. The percentages of nematode mortality after 72 h of exposure were 85 and 86% by B. licheniformis and P. aeruginosa suspensions, respectively, whereas the percentages of mortality caused by fusants ranged between 91 and 99% as compared with control. The obtained results indicated that protoplast fusion technique is a successful tool to enhance the lethal effect of the isolated rhizobacteria strains against root-knot nematode. The genetically engineered bacteria can play a dual role under field conditions as a biocontrol agent against nematode and subsequently improve plant productivity.

Comparative Analysis of Genetic Diversity Among Bacillus Thuringiensis and Bacillus Sphaericus and Their Fusants Using Molecular Markers

Ten genetically stable fusants strains were obtained as a result of protoplast fusion technique between Bacillus sphaericus (Bs) and Bacillus thuringiensis (Bt), six of them produced from, Bs GHAI Bt 66 Ia experiment (fusants no. 1, 2, 16, 26, 31 and 34) and four fusants produced from Bs GHAI Bt I977experiment (fusants no.1, 2, 17 and 25). Two molecular marker systems, four ISSR primers (Inter-Simple Sequence Repeat) and seven SCoT primers (Start Codon Targeted Polymorphism) were employed for detection of genetic variation between the parents and their fusants. Four ISSR primers and seven SCoT detected 55and 97 amplicons, among which 92.696% and 81.948% bands were polymorphic respectively. The genetic similarity values among the species were calculated based on ISSR and SCoT profiles which ranged from 0.50 to 0.83 and 0.59 to 0.90 respectively. Based on the marker analysis, the three parents and the ten fusants were clustered into three major groups for ISSR primer and SCoT primers. Cluster analysis were performed to construct a dendrogram of the Bacillus Strains and their fusants and also reveals high genetic variation among the parents and their fusants. It could be concluded that each type of the two molecular marker approaches of DNA analysis could identify the different between the parents, and all of their fusants under investigation have probably originated from closely related ancestors and possess high degree of genetic similarity.

Construction of novel cold-tolerant strains of Volvariella volvacea through protoplast fusion between Volvariella volvacea and Pleurotus eryngii

Volvariella volvacea, an edible mushroom that grows at high temperature and humidity, has attracted growing attention because of its high nutrient value, short cropping duration, unique aroma and texture. However, it is difficult to store fresh because it is highly perishable in low-temperature. To expend its cultivation region and season, this study aims to improve the low-temperature tolerance ability and the biological efficiency of this macrofungus. For this purpose, interfamily hybridization between V. volvacea and Pleurotus eryngii was studied through PEG-mediated protoplast fusion technique. Two low-temperature resistant strains, namely, VP1 and VP2 were successfully obtained through protoplast fusion with biological characteristics screening and molecular determination. The hybrid strain VP1 showed a higher biological efficiency (31.53%), compared with the 9.38% in the parental strain. Fruiting bodies of both VP1 and VP2 had a longer storage life than that of control at 16 °C. The size and amplified fragments length polymorphism (AFLP) pattern of fusants were similar, but differed from the parental V138 species, which implied that fusants are novel strains. This research has achieved interfamily protoplast fusion, which provides an effective way and the theoretical basis for the distant hybridization breeding of edible fungi.

Improvement of Bioconversion of Vitamin D3 into Calcitriol by Actinomyces hyovaginalis through Protoplast Fusion and Enzyme Immobilization

Protoplast fusion and enzyme immobilization techniques were applied to increase calcitriol production from vitamin D 3 using Actinomyces hyovaginalis , a local isolate recovered from Egyptian soil, that has a potential bioconversion activity of vitamin D 3 into calcitriol. A total of sixteen protoplast hybrids, formed between Actinomyces hyovaginalis isolate and two Bacillus species ( B. thuringiensis and B. weihenstephanensis ) were screened for vitamin D 3 bioconversion activity. Compared to wild type isolate, four hybrids (formed between Actinomyces hyovaginalis isolate and B. thuringiensis ) were found to preserve the bioconversion activity; out of which, three hybrids coded V2B, V3B and V8A exhibited higher calcitriol production. The hybrids coded V2B and V8A produced, per 1 L culture medium, about 0.5 and 0.4 mg calcitriol corresponding to 350% and 280%, respectively, increase compared to the wild type isolate. Among different alginate concentrations applied, immobilization of cell lysate of Actinomyces hyovaginalis isolate using 2% alginate showed 140% increase in calcitriol production from vitamin D 3 compared to the free cell lysate. Activity of the immobilized form was preserved for five repetitive uses over a period of 15 days but with a 50% decline in production occurring at the fifth use.

Improvement of Nutrition Production by Protoplast Fusion Techniques in Chlorella vulgaris

Recent decades showing remarkable development of the biotechnology of microalgae.Valuable product for food, nutrition and other applications will extend into broader area. Natural nutrition production from microalgae are not yet competitive with their synthetic levels. Chlorella is widely used as a health food and feed supplement, as well as in the pharmaceutical and cosmetics industries. Protoplast fusion was found to be an efficient method in improving its nutrition production and diversification in Chlorella vulgaris. The research was carried out by application of protoplast fusion on interspecific microalgae of C. vulgaris. The fusant was subjected for analysis of nutrition content by GCMS methods on C. vulgaris powder from 100 L liquid cultivation of fusant. The research result gained fusant in high mass production level. Nutrition analysis of fusants showed 17 amino acid with high concentration glutamic acid (14495.52 ppm) followed by leucine (10856.97 ppm) and Aspartic acid (10378 ppm). Palmitic acid (1.59%) was showed highest concentration in its lipid acid profile. Lipid analysis also showed polyunsaturated fatty acids (PUFA) with concentration 1.0987% and DHA 0.2%. Surprisingly, the fusant also revealed Omega 9 instead of Omega 3 and Omega 6. The research result showed potential acquisition of improvement nutrition by protoplast fusian application on microalgae.

Dendrobium protoplast co-culture promotes phytochemical assemblage in vitro.

The present study is intended to analyze the occurrence of potent, low produce, naturally occurring stilbenes in protoplasts of wild species and hybrids of Dendrobium. The wild species selected for the study was Dendrobium ovatum, endemic to Western Ghats of India. Protoplasts were isolated from leaves and tepal tissues of all the species and were cultured purely to generate homofusants and cross-cultured to raise heterofusants. Phytochemical composition of protoplast culture with atypical and pure microcolonies was performed using mass spectrometry. Enzyme cocktail of 4% pectinase together with 2% cellulase displayed the highest competence for protoplast isolations. Maximum protoplast density of 30.11×104/ml was obtained from D. ovatum leaves in 2h. Subcellular features such as the presence of partially formed cell wall, the position of the nucleus, chloroplast density, colony existence, and integrity of the plasma membrane were analyzed. Among the pure and cross-cultured protoplasts, the number of heterofusants and homofusants formed were enumerated. The spectral feature extraction of the mass spectrometry indicated the presence of five phenolic marker compounds, viz., tristin, confusarin, gigantol, moscatilin, and resveratrol, some of them in pure and others in assorted protoplast cultures raised from Dendrobium leaves and tepals. The study demonstrated that protoplast fusion technique enabled phytochemical assemblage in vitro as stilbenes tend to get restricted either in a tissue or species specific manner. This is the first report showing the presence of resveratrol, moscatilin, tristin, gigantol, and confusarin in wild and hybrid species from cultured Dendrobium protoplasts in vitro.

Enhanced degradation of lignin by the strain fusion of Pseudomonas putida and Gordonia sp

The protoplast fusion technique was applied to construct a more efficient engineering microbial strain to degrade lignin by fusing two strains, Pseudomonas putida and Gordonia sp. At an initial lignin concentration of 900 mg/L, COD, BOD, TOC removal efficiencies increased from 69–76%, 69–72%, and 70–72% by the parent stains to 83%, 83%, and 83% of the fused strain, respectively. IR and HPLC analyses of the treated solution suggested that the fused strains were more capable of breaking the Cα–Cβ bonds of the benzene ring in lignin compared to its parent strains, yielding syringyls as the main product. GC–MS analysis was used to identify the release of three-types of lower molecular intermediates: ring-opening, monomer, and dipolymer products. The phenolic hydroxyl group in lignin was oxidized to carbonyls, followed by further degradation to acids and esters. The carboxyl group on the ether linkage that maintains the macromolecular structure of lignin was oxidized to acyls, which further led to depolymerization and the opening of benzene ring.

Construction and analysis of an intergeneric fusion from Pigmentiphaga sp. strain AAP-1 and Pseudomonas sp. CTN-4 for degrading acetamiprid and chlorothalonil.

Pseudomonas sp. CTN-4 degrades chlorothalonil (CTN) but not acetamiprid (AAP), and Pigmentiphaga sp. strain AAP-1 degrades AAP but not CTN. A functional strain, AC, was constructed through protoplast fusion of two parental strains (Pseudomonas sp. CTN-4 and Pigmentiphaga sp. strain AAP-1) in order to simultaneously improve the degradation efficiency of AAP and CTN. Fusant-AC with eight transfers on plates containing two antibiotics and CTN was obtained. For the purpose of identifying and confirming the genetic relationship between fusant-AC and its parents, randomly amplified polymorphic DNA (RAPD), scanning electron microscopy (SEM), and 16S ribosomal DNA (rDNA) analysis were performed. In toto, RAPD fingerprint analysis produced 194 clear bands with 9 primers, which not only had bands in common with strains CTN-4 and AAP-1, but also had its own novel fusant-specific bands. The genetic similarity indices between fusant-AC and parental strains CTN-4 and AAP-1 were 0.40 and 0.69, respectively. The result of SEM indicated that the cell morphology of fusant-AC differed from both its parents. The fusant strain AC possesses a strong capability for AAP and CTN degradation. At AAP concentration (50-300mgL(-1)), the degradation was achieved within 5h. At the initial dose of 50 and 100mgL(-1) CTN, the percentages reached 96 and 91% over a 36-h incubation period. The present study indicates that the protoplast-fusion technique may have possible applications in environmental pollution control.

Influence of Protoplast Fusion in Trichoderma Spp. on Controlling Some Soil Borne Diseases

Protoplasts from Trichoderma harzianum NBAII Th 1 and T. viride NBAII Tv 23 were isolated using lysing enzymes. Protoplast fusion of T. harzianum and T. viride was carried out. The fused protoplasts generated and 21 fusant isolates were used to study their antagonistic activity and RAPD-PCR characterization in comparison with their parents. Among the fusant isolates, F7 isolate produced maximum growth inhibition pathogen (one and half-fold increase as compared to the parent strains). All the tested fusants isolates exhibited increased antagonistic activity against Fusarium oxysporum than the parent strains except fusant F21. Specific results for fingerprinting were obtained by the seven primers of RAPD. These markers produced different fragment patterns with varied number of bands and yielded a total of 79 distinct bands. Polymorphic bands came to be 16.5% whereas monomorphic bands came to be 83.5%. Moreover, the OPO-13 primer has showed the highest polymorphism 35.3%. While, the OPA-16 primer has shown the lowest polymorphism 9.0%. The Dendrogram based on RAPD marker results grouped the two parent strains and twenty-one fusants into two different clusters with about 83% genetic similarity. The first cluster contained the two parent strains and twenty fusants, and the second cluster contained the fusant F2. Our results in this study suggested that the protoplast fusion technique is useful for developing the superior hybrid strains and enhance antagonistic activity of Trichoderma spp. against tested pathogenic fungi.

High-Yield Laccase-Producing Strains Constructed by Protoplast Fusion Between Bacterium and Fungus

This study is based on the construction of high-yield laccase-producing fusant achieved by inter-kingdom protoplast fusion between Pleurotus ostreatus and Escherichia coli. The optimized protoplasts formation and regeneration conditions were demonstrated with the presence of 1.5% cellulase +1.0% snailase and 0.6M mannitol at 30°C for 3h. The fusants were screened for different characteristics between two parental strains and further identified by laccase activity, offering one of the genetically stable fusants, Strain F. The fusant F produced the highest yield of laccase, being about 22% higher than that of the parental strain. The results suggest that the protoplast fusion technique can be considered as a promising technique for the control of environmental pollution.