Interspecific Hybridization Between Trichoderma harzianum and Trichoderma viride by Protoplast Fusion for Improving
Biocontrol Activity
E. Sagadevan, K. Theninian and N. Mathivanan*
Biocontrol and Microbial Metabolites Lab
Centre for Advanced Studies in Botany
University of Madras, Guindy Campus, Chennai – 600 025.

296 different strains of Trichoderma were isolated from different soil samples collected from various states of India, among them 30 strains of T. viride and 32 strains of T. harzianum isolates were selected this study based on their antagonistic activity against R. solani. Among the Trichoderma isolates, T. harzianum MML3001 and T. viride MML3116 were chosen as parent strains based on chitinase production and carbendazim tolerance, respectively. Protoplasts were isolated from 16 h old cultures of T. harzianum MML3001 and T. viride MML3116 using Lysing enzymes at 8 mg ml -1 concentration with 0.6 M KCl as osmotic stabilizer. The viability of isolated protoplasts was estimated as 81% and 78%, respectively for T. harzianum and T. viride. Protoplast fusion between T. harzianum and T. viride was carried out using 80% Polyethylene glycol (MW 3500) prepared in STC buffer (0.6 M sorbitol; 10 mM Tris-HCL; 10 mM CaCl2, pH 6.5). The fusion protoplasts were regenerated on regeneration (PDA amended with osmotic stabilizer) medium. Among the 20 fast growing fusants, six were selected based on their enhanced growth on selective media and also on the integrated morphological features of both the parents. Among six fusants four (Th+Tv1, Th+Tv2, Th+Tv3 and Th+Tv5) exhibited fast growth than the parents. All the six selected fusants exhibited morphological variation such as mycelial growth, pigmentation, sporulation and spore colouration among themselves but they also possess common morphological characteristics of the parent strains.