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International Journal of Current Microbiology and Applied Sciences (IJCMAS)
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National Academy of Agricultural Sciences (NAAS)
NAAS Score: *5.38 (2019)
[Effective from January 1, 2019]
For more details click here

ICV 2018: 95.39
Index Copernicus ICI Journals Master List 2017 - IJCMAS--ICV 2018: 95.39
For more details click here

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PRINT ISSN : 2319-7692
Online ISSN : 2319-7706
Issues : 12 per year
Publisher : Excellent Publishers
Email : editorijcmas@gmail.com / submit@ijcmas.com
Editor-in-chief: Dr.M.Prakash
Index Copernicus ICV 2018: 95.39
NAAS RATING 2020: 5.38

Int.J.Curr.Microbiol.App.Sci.2016.5(11): 891-897
DOI: http://dx.doi.org/10.20546/ijcmas.2016.511.102


Fast Isolation and Regeneration Method for Protoplast Production in Trichoderma harzianum
Manika Sharma1*, Pratibha Sharma2, Ranbir Singh1, M. Raja2 and Pankaj Sharma3

1Division of Plant Pathology, FOA, main campus Chatha of SKUAST-Jammu, India

 2Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, India

3Division of Biotechnology, Jammu University, Jammu, India
*Corresponding author
Abstract:

Trichoderma is one of the most important filamentous fungi used as a biocontrol agent. Because of the absence of sexual reproduction in this fungus, other methods of genetic improvement have been developed such as protoplast fusion to enhance its biocontrol potential. In order to develop an unique effective strain as a single source of vital enzyme, it was intended to integrate two biocontrol species for high chitinase production by fusing their protoplasts. Protoplasts were isolated from Trichoderma harzianum fungus using Sigma Lyticase with 0.6 M KClas osmotic stabilizer. Intra-strain T. harzianum protoplast fusion has been carried out using polyethylene glycol with STC (sorbitol, Tris – HCl, CaCl2) buffer. The maximum  number  of  protoplasts (2.0x103/ml) was obtained from 16 h culture at pH 6,28°C for 3h. Five self-fusant strains (Th3fu1, Th3fu2, Th3fu3, Th3fu4, Th3fu5) were selected. Most of the fusants exhibited fast and vigorous mycelial growth on 2 % colloidal chitin agar. The fused protoplasts were regenerated on chitin agar selective medium. In dual culture biocontrol experiments, five selected fusants showed growth inhibition against three pathogens namely; Sclerotinia sclerotiorum, Fusarium oxysporum and Altenaria brassicicola. However, Fus 5 indicated the best ability of inhibition the growth of the three pathogens. Molecular characterization of five fusants using RAPD profile indicated the presence of novel fragments which may be due to recombination events .Results  indicated a fast method to generate protoplast fusants  to be used  to enhance biocontrol potential  intraspecifically as well as interspecifically.


Keywords: Trichoderma harzianum, Protoplast fusion, Fusants
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How to cite this article:

Manika Sharma, Pratibha Sharma, Ranbir Singh, M. Raja and Pankaj Sharma. 2016. Fast Isolation and Regeneration Method for Protoplast Production in Trichoderma harzianum.Int.J.Curr.Microbiol.App.Sci. 5(11): 891-897. doi: http://dx.doi.org/10.20546/ijcmas.2016.511.102