Low rate of protoplast regeneration of Pleurotus spp. is one of the limiting factor for strain improvement through protoplast technology. A method for overcoming this barrier and isolation of higher frequency of regenerating protoplasts both from mycelium and sporophore capable of producing fruit bodies is reported in this communication.
Pleurotus sajorcuju, a tropical edible mushroom, is popular for its tastes and easy cultivation. However, development of allergic reaction due to inhalation of spores of Pleurotus among some cultivators is one of the major limitations of its cultivation (MITTAR er al. 1993). Interspecific crosses have, therefore, been attempted for the development of low spore yielding fusants in Pleurotus (Yo0 et al. 1984). The major problem in this area is the low regeneration percentage of protoplasts from Pleurotus species which varies from 0.01 % to 2.02% (Yo0 et al. 1993). In this communication we describe a detailed process for the development of efficiently regenerating protoplasts both from mycelium and sporophore capable of producing fruit bodies.
Methods
Strain and culture media: A mycelial culture of F! sajorcaju provided by Govt of West Bengal, India was maintained on potatodextrose medium (pH 7.0) containing (in 1 1): Dextrose 20 g, potato extract 200 g, agar 20 g at 30 "C. The vegetative culture does not produce any spores even on prolonged growth on artificial medium. For protoplasting the strain was grown in liquid malt-yeast extract-glucose (MYG) medium (pH 7.0) containing (in I 1): malt extract 10 g, yeast extract 4 g, glucose 10 g in a rotary shaker (200 cycleslmin) at 30Β°C. Mycelia that had grown for 4 d were used for protoplast formation. Protoplast preparation: For protoplast liberation mycelia were washed three times with distilled water and then twice with osmotic stabilizer solution. Lytic enzyme solution was prepared in osmotic stabilizer and filter sterilized. A 0.5 g (wet-weight) of mycelia was incubated at 30 "C for 3 h with 0.5 ml of lytic enzyme solution. For protoplast preparation from fruit body freshly harvested sporophores were used. Gill tissues were removed by dissection and 1 g (wet-weight) of tissue was washed by sterilized distilled water and osmotic stabilizer respectively. 1 ml of filter sterilized lytic enzyme was then added to the tissue and incubated for 4 h. The protoplasts liberated were purified following the method described earlier (MUKHERJEE and SENGUFTA 1986). Regeneration: Media used for regeneration of protoplasts either contained (in 11): malt extract, 10 g; yeast extract, 4 g and glucose 10 g (MYG) or (NH4I2HPO4, 1.5 g ; KHzP04, 0.5 g; KzHP04, 1 .O g, MgSO, ' 7 H,O, 0.05 g; glucose, 20 g and thiamine was added at the concentration of 12 pg per 100 ml (MM). Media were prepared in osmotic stabilizer with or without 2% (wt./vol) agar. For regeneration in solid media 0.1 ml of protoplast suspension containing 30 to 35 protoplasts was spread out on regeneration medium. Regeneration of mycelial colonies was observed after 7 days of incubation at 30 "C. Regeneration frequency were taken as the ratio of the number of colonies developed to the number of protoplasts added per plate as determined by haemocytometer count of diluted protoplasts