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Aim: To develop new Trichoderma strains, capable of removing toxic heavy metal ions from polluted environments, via protoplast fusion.Methodology: Trichoderma parental strains (T. viride and T. koningii) along with their ten fusants (Tk+Tv 1, Tk+Tv 2, Tk+Tv 3, Tk+Tv 4, Tk+Tv 5, Tk+Tv 6, Tk+Tv 7, Tk+Tv 8, Tk+Tv 9 and Tk+Tv 10) were obtained from the Department of Plant Pathology, Junagadh Agricultural University, Junagadh. The strains obtained by protoplast fusion were examined for their ability to remove toxic heavy metal ions, especially zinc ion. Fourier-transform infrared spectroscopy (FTIR) was conducted to detect the zinc uptake mechanism of Trichoderma parental and their fusant strains. Results: FTIR results demonstrated the Zn ion uptake capacity of fusant strains was found to be higher than that of the parental strains (12.8 to 10.7 mg g-1 on a dry weight basis at 1300 ppm). The highest Zn ion mobility observed was 62.1 mg. kg-1 and the highest Zn ion mobility observed per strain was 12.4% in Tk+Tv 3, followed by 11.86 % in Tk + Tv 7, 11.84% in Tk + Tv 9 and 11.28% in Tk + Tv 10. Parental and fusant strains Tk + Tv 3, Tk + Tv 8 and Tk + Tv 10 confirmed the involvement of different functional groups for different concentrations of zinc during adsorption by the fungus. Interpretation: FTIR results identified greater metal removal capacity in the fusant strains, particularly for soil Zn ion. Zinc tolerance was higher in the fusant strains than in the parental strains. Thus, protoplast fusion is an effective and feasible method for constructing new strains that can be used for bioremediation of contaminated environments.
ABSTRACT
ABSTRACT Protoplasts are microbial or vegetable cells lacking a cell wall. These can be obtained from microalgae by an enzymatic hydrolysis process in the presence of an osmotic stabilizer. In general, protoplasts are experimentally useful in physiological, genetic and biochemical studies, so their acquisition and fusion will continue to be an active research area in modern biotechnology. The fusion of protoplasts in microalgae constitutes a tool for strain improvement because it allows both intra and interspecific genetic recombination, resulting in organisms with new or improved characteristics of industrial interest. In this review we briefly describe the methodology for obtaining protoplasts, as well as fusion methods and the main applications of microalgal platforms.
RESUMEN Los protoplastos son células microbianas o vegetales que carecen de pared celular. Estos pueden obtenerse a partir de microalgas por un proceso de hidrólisis enzimática en presencia de un estabilizador osmótico. En general, los protoplastos son experimentalmente útiles en estudios fisiológicos, genéticos y bioquímicos, por lo que su obtención y fusión continuarán siendo un área de investigación activa en la biotecnología moderna. La fusión de protoplastos en microalgas constituye una herramienta para el mejoramiento de cepas pues permite la recombinación genética intra e interespecífica, logrando así organismos con nuevas características de interés industrial. En esta revisión, describimos brevemente la metodología para obtener protoplastos, métodos de fusión y las principales aplicaciones de las plataformas basadas en microalgas.
ABSTRACT
La fusión de protoplastos ha facilitado la obtención de nuevas cepas de levaduras con propiedades biotecnológicas muy interesantes. El principal objetivo de esta investigación fue obtener una levadura híbrida intergénica con potencialidades enológicas características de dos géneros diferentes. Para ello se fusionaron protoplastos de Saccharomyces cerevisiae autóctona de la región zuliana con Hanseniaspora guillermondii CECT 11102 de origen comercial. Saccharomyces es una levadura que produce altas concentraciones de etanol pero el perfil aromático es sencillo y común. Hanseniaspora no resiste las concentraciones de etanol, pero puede generar aromas agradables e intensos. La identificación de las levaduras antes y después de la fusión de protoplastos se realizó con la técnica PCR-RFLP del gen 5.8S rADN y las regiones intergénicas adyacentes ITS1 e ITS2 del ADN extraído, sometiendo los productos amplificados a un análisis de restricción con las enzimas HinfI, HaeIII, CfoI y DdeI. El polietilenglicol fue usado para inducir la fusión de protoplastos. La cepa híbrida presentó características de ambas levaduras parentales debido a que resistió altas concentraciones de etanol como S. cerevisiae y fue capaz de metabolizar el salicín como H. guillermondii. El análisis molecular PCR-RFLP de la levadura híbrida mostró un patrón de bandas diferente al de las levaduras parentales.
Protoplast fusion has facilitated the development of new yeast strains with very interesting biotechnological properties. The main objective of this research was to obtain hybrid yeast with potentialities of two different genera, that could be used in the wine manufacture. Saccharomyces cerevisiae protoplasts from the Zulian region were fused with Hanseniaspora guillermondii CECT 11102 of commercial origin. Saccharomyces is a yeast that produces high levels of ethanol but the aromatic profile is simple and common. Hanseniaspora does not withstand ethanol concentrations, but it can generate pleasant and intense aromas. Identification of yeast before and after the fusion of protoplasts was performed using the PCR-RFLP technique of the 5.8S rDNA gene and the adjacent ITS1 and ITS2 intergenic regions of the extracted DNA, subjecting the amplified products to a restriction with the enzymes HinfI, HaeIII, CfoI and DdeI. Polyethylene glycol was used to induce fusion of protoplasts. The hybrid strain showed characteristics of both parental yeasts because it resisted high concentrations of ethanol as S. cerevisiae and was able to metabolize the salicin as H. guillermondii. PCR-RFLP molecular analysis of hybrid yeast showed a different band pattern than those pertaining to parental yeasts.
ABSTRACT
Protease hyper producing recombinant strains were produced by intergeneric protoplast fusion of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae. β-glucuronidase and KCl were used as lysing enzyme and osmotic stabilizer. Along with inter-generic fusion, intra-strain and inter-strain fusions were also carried out using polyethylene glycol (PEG) as fusogen. When the fused protoplasts were regenerated on Czapekdox agar medium, they exhibited fast mycelial growth and abundant sporulation when compared to non-fusants. Pr1 and Pr2 specific activities were found to be increased by two- fold in recombinant strains than the nonfusants and the parental strains.
ABSTRACT
Yeast strains with improved ethanol yield are important for efficient bioconversion of lignocellulosic biomass for fuel ethanol.Candida shehatae CICC1766 was adapted to 4%(v/v)ethanol,and then subjected to UV mutagenesis.One respiration deficient mutant Rd-5 with improved xylose fermentation capability was selected.Protoplasts of Rd-5 were inactivated by UV treatment,followed by the PEG-mediated protoplast fusion with a Saccharomyces cerevisiae strain with good ethanol-fermenting capability.The xylose fermenting capability of the fusants was investigated,and the fusant F6 demonstrated good ethanol fermentation performance,producing 18.75g/L ethanol from 50g/L xylose with an ethanol yield of 0.375 or 73.4% of its theoretical value of 0.511.Comparing with its parent Candida shehatae strain,the ethanol yield of F6 was increased by 28%.
ABSTRACT
Protoplast fusion is a useful technique for establishing fungal hybrids to overcome the natural barriers. The ultrastructure of protoplast and its fusion process were observed using a scanning electron microscopy(SEM) and a transmission electron microscopy(TEM). The protoplasts were variable in size from 0.5~15microm in diameter, and the mean diameter was about 3~5microm. It was impossible to discriminate protoplasts of Lentinula edodes from protoplasts of Coriolus versicolor by size and surface structure. Big aggregates of the dehydrated protoplasts were observed, after polyethylene glycol 4000 treatment. Nucleus, mitochondria, lipid granules and various vesicles having granules were scattered in the cytoplasm. The vesicles were heterogeneous in size and vary from one protoplast to another. The fused membrane layer of the two protoplasts was observed. Time protoplast membrane contact and reorganization of membrane components were essential condition for protoplast fusion. Transmission electron micrograph showed fused protoplasts and flattening of the cells in the area of the membrane contact. We hope that our electron microscopic observations provide some insights into the understanding of the fusion process of protoplast in fungi.
Subject(s)
Cytoplasm , Fungi , Hope , Lentinula , Membranes , Mitochondria , Polyethylene Glycols , Protoplasts , Shiitake MushroomsABSTRACT
Penconazole-resistant and cabendazim-resistant mutants of Venturiu inaequalis were developed by chemical (MNNG) mutagenesis. Protoplasts of these mutants were isolated and fused using polyethylene glycol as the fusogen. Fusants were classified into parental, non-parental and recombinant types. The recombinants were resistant to penconazole and carbendazim. The double resistant strains were stable and exhibited pathogenicity on fungicide-sprayed and unsprayed apple twigs.
Subject(s)
Humans , Mutagenesis , Parents , Polyethylene Glycols , Protoplasts , VirulenceABSTRACT
Object To explore the protoplast fusion of Dendrobium candidum Wall ex. Lindl. and Gynostemma pentaphyllum. (Thunb) Makino. Methods The two kinds of protoplasts were fused by PEG method, and then cultured in the modified liquid medium containing 2 mg/L BA and 1 mg/L NAA. Results High yield, viability, and pure mesophyll protoplasts were isolated from D. candidum and G. pentaphyllum. Conclusion The first cell division occurres within three days after fusion. And some of the cells are divided three times.