Intraspecific Protoplast Fusion Research Articles

Characterization and magnification of hybrid strain production by intraspecific protoplast fusion of Monascus ruber and Pleurotus ostreatus

Characterization and magnification of hybrid strain production by intraspecific protoplast fusion of Monascus ruber and Pleurotus ostreatus

New Hybrid Strains via Intraspecific Protoplast Fusion of the Entomopathogenic Fungi Lecanicillium spp.

The entomopathogenic fungal genus Lecanicillium Gams and Zare (formerly classified as the species Verticillium lecanii) includes species that are highly pathogenic to many insect genera. In this study, we identified six Lecanicilliumspp. isolated strains (designated as V1-V6) belonging to L. lecanii (V1, V3 and V5) and L. attenuatum (V2, V4 and V6). In addition, these strains were used to obtain new strains via protoplast fusion, and nit mutants were used for protoplast selection. Genetic recombination of the hybrid strains was determined using the random amplified polymorphic DNA(RAPD) technique. We obtained nine stable fusant strains from 176 new hybrid strains, which were termed V12-10, V14-3, V16-4, V23-6, V25-8, V34-14, V36-5, V45-16 and V56-7. Morphological characteristics varied between the hybrid and parental strains. Genomic DNA analysis of the fusants also showed genetic recombination. The median lethal concentration (LC50) for the fusants were lower than that for parental strains, and the median survival time (LT50) for the fusants were reduced compared with that for parental strains. Thus, these results showed that we produced new, more virulent hybrid Lecanicillium spp. strainsas biological control agents via intraspecific protoplast fusion.

Enhanced production of fructosyltransferase in Aspergillus oryzae by genome shuffling.

To breed Aspergillus oryzae strains with high fructosyltransferase (FTase) activity using intraspecific protoplast fusion via genome-shuffling. A candidate library was developed using UV/LiCl of the conidia of A. oryzae SBB201. By screening for enzyme activity and cell biomass, two mutants (UV-11 and UV-76) were chosen for protoplast fusion and subsequent genome shuffling. After three rounds of genome recombination, a fusion mutant RIII-7 was obtained. Its FTase activity was 180Ug-1, approximately double that of the original strain, and RIII-7 was genetically stable. In fermentation culture, FTase activity of the genome-shuffled strain reached a maximum of 353 Ug-1 using substrate-feeding method, and this value was approximately 3.4-times higher than that of the original strain A. oryzae SBB201. Intraspecific protoplast fusion of A. oryzae significantly enhanced FTase activity and generated a potentially useful strain for industrial production.

Enhancement of yellow pigment production by intraspecific protoplast fusion of Monascus spp. yellow mutant (ade−) and white mutant (prototroph)

To breed industrially useful strains of a slow-growing, yellow pigment producing strain of Monascus sp., protoplasts of Monascus purpureus yellow mutant (ade−) and rapid-growing M. purpureus white mutant (prototroph) were fused and fusants were selected on minimal medium (MM). Preliminary conventional protoplast fusion of the two strains was performed and the result showed that only white colonies were detected on MM. It was not able to differentiate the fusants from the white parental prototroph. To solve this problem, the white parental prototroph was thus pretreated with 20mM iodoacetamide (IOA) for cytoplasm inactivation and subsequently taken into protoplast fusion with slow-growing Monascus yellow mutant. Under this development technique, only the fusants, with viable cytoplasm from Monascus yellow mutant (ade−), could thus grow on MM, whereas neither IOA pretreated white parental prototroph nor yellow auxotroph (ade−) could survive. Fifty-three fusants isolated from yellow colonies obtained through this developed technique were subsequently inoculated on complete medium (MY agar). Fifteen distinguished yellow colonies from their parental yellow mutant were then selected for biochemical, morphological and fermentative properties in cassava starch and soybean flour (SS) broth. Finally, three most stable fusants (F7, F10 and F43) were then selected and compared in rice solid culture. Enhancement of yellow pigment production over the parental yellow auxotroph was found in F7 and F10, while enhanced glucoamylase activity was found in F43. The formation of fusants was further confirmed by monacolin K content, which was intermediate between the two parents (monacolin K-producing yellow auxotroph and non-monacolin K producing white prototroph).

Intraspecific protoplast fusion of Brettanomyces anomalus for improved production of an extracellular β-glucosidase

Improvement of production of an extracellular β-glucosidase with high activity by Brettanomyces anomalus PSY-001 was performed by using recursive protoplast fusion in a genome-shuffling format. The initial population was generated by ultraviolet irradiation, ultrasonic mutagenesis and, then, subjected to recursive protoplast fusion. Mutant strains exhibiting significantly higher β-glucosidase activities in liquid media were isolated. The best mutant strain showed increased cell growth in a flask culture, as well as increased β-glucosidase production. A recombinant strain, F3-25, was obtained after three rounds of genome shuffling and its production of β-glucosidase activity reached 4790 U L−1, which was a nearly eightfold increase compared to the original strain B. anomalus PSY-001. The subculture experiments indicated that F3-25 was genetically stable.

English

  In an attempt to construct superior Trichoderma harzianum isolates for improving β-glucosidase productivity, protoplast fusion technique was applied. After application of different mutagenic treatments, twenty mutants were chosen to be tested for their resistance or sensitivity against four antifungal agents. Out of them, four isolates were selected on the basis of their response to antifungal agents and their productivities of carboxymethylcellulase (CMCase) and β-glucosidase to be introduced into intraspecific protoplast fusion experiments using two different methods (PEG and electrofusion). Three crosses were carried out among the selected four isolates. Results showed that, the number of fusants obtained after electrofusion were more than those obtained after polyethylene glycol (PEG) method. In addition, high productivities of CMCase and β- glucosidase were obtained after electrofusion in the three crosses. The applied protoplast electrofusion method proved to be a good and effective method for obtaining T.  harzianum fusants with higher productivity of β- glucosidase enzyme.   Key words: Trichoderma, protoplast fusion, electroporation, β-glucosidase.

Chitinolytic enzyme production and genetic improvement of a new isolate belonging to Streptomyces anulatus

Thirty bacterial isolates were obtained from different sources and sites at Jeddah, Saudi Arabia, on chitin agar medium; 9 of the 30 isolates were cultured in liquid medium containing chitin as sole carbon and nitrogen sources. Isolate SM21, which was isolated from shrimp shells, showed the best growth and chitinase production in liquid medium. According to its morphological, physiological and biochemical characteristics, SM21 belongs to the genus Streptomyces and was identified as Streptomyces anulatus SM21. Identification was confirmed using 16S rDNA analysis. The chitinase enzyme was precipitated with 80% NH4SO4 and purified using DEAE-cellulose ion exchange chromatography followed by Sephadex G-100 gel filtration. The molecular weight determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis was 28 kDa. Genetic improvement using the protoplast fusion technique was carried out between the identified Streptomyces isolate and Streptomyces coelicolor SM1. These two species, which have different resistance profiles to streptomycin and tetracycline (400 μg/ml and 10 μg/ml, respectively), were used in an intraspecific protoplast fusion using PEG 6000. The percentage of real protoplasts that could regenerate successfully was 71% for S. coelicolor SM1 and 80% for S. anulatus SM21. Out of three recombinant fusants obtained, one (named Fu3) showed higher chitinase production compared to both parents (5 fold increase).

Enhancement of nodulation by some arid climate strains of Rhizobium leguminosarum biovar trifolii using protoplast fusion

Fourteen randomly clover indigenous nodulated Rhizobium strains were isolated from different locations in Saudi Arabia. They were identified as different strains of the genus Rhizobium leguminosarum biovar trifolii and characterized for their intrinsic antibiotic resistance against a range of antibiotics, nodulation capability and plasmid profiles. Results revealed the presence of high molecular weight plasmids (megaplasmids) in all the selected strains. Based on the ability for nodulation production, two weak strains (RtI1 and RtI2) and one efficient strain (RtA1) were selected for protoplast fusion and the numbers of nodules produced by the intra-specific protoplast fusion strains were investigated. Results clearly confirmed the effective role of the protoplast fusion in enhancing both nodulation production capacity of Rhizobium species and their range of antibiotic resistance. Protoplast fusion of the local Rhizobium species resulted in 1.93- to 5.67-fold increase in nodulation number compared to their parental strains, which was considered an excellent result concerning agricultural practices, especially the formation of nitrogen-fixing root nodules on legume crop plants. Protoplast fusion also produced fusants with a wide range of antibiotic resistance, another advantage added to the new strains against environmental stresses. In conclusion, protoplast fusion proved its efficiency as a tool for constructing a second generation of Rhizobia with much better characteristics for efficient applications in arid land.

Enhancement and selective production of avermectin B by recombinants of Streptomyces avermitilis via intraspecific protoplast fusion

Among eight components of avermectin, B1 fractions have the most effective antiparasitic activities and the lowest level of toxic side-effects and are used widely in veterinary and agricultural fields. Intraspecific protoplast fusion between two strains of Streptomyces avermitilis, one an avermectin high producer (strain 76-05) and the other a genetically engineered strain containing the mutations aveD− and olmA− (strain 73-12) was performed for enhancement and selective production of avermectin B in the absence of oligomycin. Two recombinant strains (F23 and F29) were isolated and characterized with regards to the parental merits. F23 and F29 produced only the four avermectin B components with high yield and produced no oligomycin. The avermectin production of F23 and F29 was about 84.20% and 103.45% of the parental strain 76-05, respectively, and increased about 2.66-fold and 3.50-fold, respectively, compared to that of parental strain 73-12. F23 and F29 were genetically stable prototrophic recombinants and F29 was quite tolerant of fermentation conditions compared to avermectin high producer parental strain 76-05. The ability to produce avermectin B with high yield without the production of other avermectin components and oligomycin will make F23 and F29 useful strains for avermectin production. Strain F29’s tolerance of fermentation conditions will also make it suitable for industrial applications.

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 microg/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 microg/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.

Antagonistic properties of two recombinant strains of Streptomyces melanosporofaciens obtained by intraspecific protoplast fusion.

Intraspecific protoplast fusion was used to produce stable prototrophic recombinants of Streptomyces melanosporofaciens EF-76, a biocontrol agent of plant disease producing geldanamycin. Two recombinant strains (FP-54 and FP-60) that differed with regard to their antagonistic properties against Bacillus cereus ATCC 14579, Streptomyces scabies EF-35 and Phytophthora fragariae var. rubi 390 were characterized. FP-60 lost the ability to inhibit the in vitro growth of these microbial strains while FP-54 exhibited higher antagonistic activities against them. FP-60 was deficient in geldanamycin biosynthesis whereas FP-54 was shown to produce, in addition to geldanamycin, at least two other antimicrobial compounds that were absent in the culture supernatants of strain EF-76. Like the wild-type strain EF-76, strain FP-54 reduced common scab symptoms on potato tuber but no significant difference was observed between the disease index attributed to tubers treated with strain EF-76 or with strain FP-54. Strain FP-60 showed no protective effect against common scab. The disease index of tubers treated with this recombinant was worse than the index associated with potato tubers from control treatments.

Characterization of Candida albicans colony-morphology mutants and their hybrids.

The collection wild-type strain of Candida albicans was used to obtain auxotrophic and colony-morphology mutants by 'nitrosoguanidine' treatment. Intraspecific protoplast fusion induced by Ca(2+)-poly(ethyleneglycol) was carried out in various pairings between the auxotrophic strain producing smooth colonies and containing blastospores and the colony-morphology mutants containing a mixture of blastospores and pseudohyphae or only hyphae. Hybrids exhibiting full or partial complementation were obtained when mutants producing smooth colonies and colony-morphology variants of different origins were fused. The mutation responsible for the colony-morphology character (if various types of colony morphomutants were crossed) proved to be recessive or semidominant. Representative hybrids exhibited elevated DNA contents as measured by flow cytometry. To illustrate various cell types, and especially the intermediate one (never observed in natural isolates), a preparation method was further developed for scanning electron microscopic studies.

Application of Protoplast Fusion Technology to Tansy (Tanacetum vulgare L.): Biodiversity as a Source to Enhance Biological Activity of Secondary Compounds

SUMMARY Tansy (Tanacetum vulgare L., Asteraceae) is an under-exploited species adapted to a northern climate. Its flower heads and leaves are characterized by their terpenoids which are moderately bioactive against insects. A related species, pyrethrum (Tanacetum cinerariifolium Vis.), produces highly bioactive terpenoids, pyrethrins, which are used in commercial biopesticides. The constraint of pyrethrum for further utilization is its cold sensitivity. In this study, tissue culture methods for tansy and pyrethrum were first developed to optimize the best conditions for protoplast isolation from donor tissues. Protoplast fusion between tansy and pyrethrum was developed using polyethyleneglycol. In addition, the genetic and chemical biodiversity of tansy was explored to find new germplasm for the further breeding experiments. Up to 1 × 106 protoplasts per 1 g−1 FW of leaves could be obtained from tansy leaves. The production of protoplast-derived calli varied, but the best time for cell division was late winter or early spring. Protoplast-derived calli of tansy resulted in root and spontaneous shoot formation, although a protoplast-to-plant procedure still needs more studies. The hybridity of intraspecific (tansy × tansy) and interspecific (tansy × pyrethrum) protoplast fusion was successfully tested already at the callus stage. The nuclear DNA content was higher for calli derived from interspecific fusion (10.66 and 31.87 pg) than from intraspecific fusion (8.84, 15.96 and 19.59 pg). The nuclear DNA content of both types of fusion calli was more than the parental level in many fusion calli. The increased content of nuclear DNA in the fusion calli suggests that some degree of protoplast hybridization had occurred. The distance matrices calculated from the RAPD-PCR data with complete linkage cluster analysis showed that the calli derived from the intraspecific fusion were closer to tansy than to pyrethrum. In contrast, the calli derived from interspecific fusion were closer to pyrethrum than to tansy. Compounds such as syringaldehyde, coniferyl alcohol and artedouglasia oxide were only identified from protoplast-fusion derived calli, but no compounds common for parental tissues and fusion calli could be detected. Tansy grown in Finland contains a wide range of genetic, chemical and morphological variation. Especially, among the larger number of volatile compounds detected from tansy, the chemotypes accumulating irregular monoterpenes such as artemisia ketone and davadone D, which are biochemically close to pyrethrins, are an interesting source for further biotechnological applications. In conclusion, biodiversity can be a valuable source of novel germplasm for breeding tansies that better accumulate more bioactive compounds.

Amylase hyper-producing haploid recombinant strains of Thermomyces lanuginosus obtained by intraspecific protoplast fusion.

Amylase hyper-producing, catabolite-repression-resistant, recombinant strains were produced by intraspecific protoplast fusion of thermophilic fungus Thermomyces lanuginosus strains, using well-characterized, morphological, and 2-deoxy-D-glucose resistant markers. The fusant heterokaryons exhibited enhanced amylase activities as compared to the amylase hyper-producing parental strain (T2). Diploids derived from heterokaryons segregated to stable haploid recombinant strains. In the haploid strain (Tlh 4q), approximately 5-fold higher specific activities of alpha-amylase and glucoamylase in the culture filtrate were observed as compared to the wild-type strain (W0).

Combination of resistance to potato late blight in foliage and tubers by intraspecific dihaploid protoplast fusion

Five dihaploid Solanum tuberosum genotypes, encompassing different levels of polygenically inherited resistance to potato late blight disease (Phytophthora infestans (Mont.) de Bary) in foliage and tubers, were used in four intraspecific protoplast fusion combinations. Vigorous growing putative hybrid calli were selected four weeks after electrofusion. Intact plants were regenerated from 6% of the selected calli. Verification of hybridity was accomplished by use of RAPD analysis which revealed that 53% of the regenerated plants were true somatic hybrids. The score of true somatic hybrids in the different fusion combinations ranged within 21% – 100%. The hybrid plants were analysed for resistance to foliage blight in a field trial and assessed for tuber blight resistance by use of a laboratory test. Resistance to late blight in foliage and tubers varied between the hybrids. Very high levels of resistance to both foliage and tuber blight were obtained in some hybrids. However, loss of resistance in some hybrids as compared to the parental plants were also observed. Possible reasons for the phenotypic disappearance of resistance to either foliage or tuber blight are suggested.

Evaluation of Candida blankii hybrids for biomass production

The cell size of a diploid Candida blankii isolate (UOVS-64.2), a yeast with potential for use in single cell protein production from hemicellulose hydrolysates, was increased by intraspecific protoplast fusion of auxotrophic mutants produced by UV irradiation followed by nystatin enrichment. Protoplast fusion yielded an isolate (UOVS-P2) which had a mean cell volume of approximately three times that of the parental strain. This isolate was not a true tetraploid but rather an aneuploid strain. Its maximum specific growth rate was only 40% that of the parental strain and the cell and protein yields were also appreciably lower. During continuous cultivation in simulated bagasse hemicellulose hydrolysate, isolate UOVS-P2 proved unstable and a spontaneous reduction in ploidy occurred. A new isolate (UOVS-PB2) originating from this culture had a 43% higher mean cell volume and contained approximately 13% more DNA than the parental strain (UOVS-64.2). In simulated hemicellulose hydrolysate the cell and protein yields of isolate UOVS-PB2 were slightly lower than those of isolate UOVS-64.2, although these yields were similar in shake flask cultures.

Improvement of the cell volume ofCandida blankii through protoplast fusion

The yeastCandida blankii ESP-94, capable of utilizing xylose as substrate, was isolated for the production of single-cell protein (SCP) on bagasse hydrolysates. However, the small cell volume of strain ESP-94 would complicate harvesting of the cells during a continuous fermentation process. Auxotrophic mutants of strain ESP-94 were generated and intraspecific protoplast fusion experiments performed in an attempt to increase the cell volume of strain ESP-94. The fusion products were characterised with respect to cell volume, DNA content and genetic stability. Six genetically stable fusants with bigger cell volumes and higher DNA contents were obtained. One such fusant, fusant F17, had a cell volume 3-times that of strain ESP-94, while exhibiting similar growth rates to strain ESP-94 ond-xylose as carbon source.

Enhanced intraspecific protoplast fusion in yeast

Intraspecific hybrid production from the polyethylene glycol induced fusion of yeast protoplasts was greatly increased when calcium propionate was included as the source of the requisite Ca2+. The use of calcium propionate, as opposed to the more commonly employed calcium chloride, resulted in substantially greater yields of hybrids from intraspecific fusions of protoplasts of Saccharomyces cerevisiae and Candida albicans. It is postulated that the ability of calcium propionate to enhance the fusion frequency is due to the anion binding to the etheric oxygen of PEG and potentiating the fusogenicity of the polymer.

Enhanced intraspecific protoplast fusion in yeast

Intraspecific hybrid production from the polyethylene glycol induced fusion of yeast protoplasts was greatly increased when calcium propionate was included as the source of the requisite Ca2+. The use of calcium propionate, as opposed to the more commonly employed calcium chloride, resulted in substantially greater yields of hybrids from intraspecific fusions of protoplasts of Saccharomyces cerevisiae and Candida albicans. It is postulated that the ability of calcium propionate to enhance the fusion frequency is due to the anion binding to the etheric oxygen of PEG and potentiating the fusogenicity of the polymer.

<I>Aspergillus usamii</I> mut. <I>shirousamii</I> RI-B 11のプロトプラスト融合による種内雑種形成

The protoplast fusion technique was used to obtain useful intraspecific hybrids of Aspergillus usamiimut. shirousamii for making spirits for drinking. Protoplasts were obtained from mycelia of this fungus by using Trichoderma lytic enzyme preparations. Intraspecific hybridization between two auxotrophic mutants of this fungus was achieved by protoplast fusion in the presence of 35% polyethylene glycol as a fusogen. A stable diploid strain was obtained from a heterokaryon strain by treating with 0.1% d-camphor. The diploid strain was treated by benomyl as a haploidizing agent, and many segregants were obtained. Prototrophic segregants were selected and these haploids were confirmed by their conidial size and DNA contents. The diploid strain and some haploids showed high stability and numerous sporulation, producing the same levels of amylase and citric acid as those ofthe original strains. From these results, a technique of intraspecific protoplast fusion was considered to be a useful clue for studying the maintenance of characteristics and homostasis stability and the genetics of parasexual recombination in fungi.