Abstract

A standard method for isolation, fusion and regeneration of protoplasts fromTrichoderma harzianum and Trichoderma viride was developed. The protoplasts fromT. harzianum and T. viride were isolated using Novozym 234 as lytic enzyme and potassium chloride as osmotic stabilizer. The maximum number of protoplasts (9.37 × 10⁵/ml) was obtained from 16 h old mycelium of T. harzianum and 11.1 × 10⁵/ml for T. viride from 14 h old mycelium at pH 5.5, 28ºC for 3 h. The interspecific and intraspecific fusion frequency was determined using 40% polyethylene glycol (PEG) as fusogen.  The intrafusants were selected based on their growth, sporulation, pigmentation on chitin and cellulose amended media, where as the interfusants were selected on fungicide resistance as a marker. The protoplast fusion frequency was found to be 1.92% for interspecific fusion. In the case of intraspecific fusion it was about 6.2 and 7.2%, respectively, for T. harzianum and T. viride. The protoplast regeneration frequency of intrafusant was 17% for T. harzianum on chitin medium and 19.2% for T. viride on cellulose medium after two days. The regeneration frequency of 11% for interfusants on fungicide amended medium was observed after three days. The regenerated fusants morphology, growth, sporulation and pigmentation were compared with parental strains.

Keywords: Fusion, isolation, regeneration, Trichoderma harzianum, Trichoderm viride.