Asian Journal of Microbiology, Biotechnology & Environmental Sciences Paper

Vol 11, Issue 3, 2009; Page No.(597-603 )

PREPARATION, REGENERATION AND ELECTRON MICROSCOPY OF MYCELIAL PROTOPLASTS OF VOLVARIELLA DIPLASIA

NIVEDITA SINGH AND U.S. BAGDE

Abstract

We studied the formation and regeneration of viable protoplasts from the mycelia of Volvariella diplasia and optimized the experimental conditions. Several commercial lytic enzymes have been compared for their ability to liberate protoplasts from the mycelia. The composition of the liquid medium for cultivation, culture aging of the mycelium, the combination of lytic enzymes and osmotic stabilizers used had important effects on the isolation of the protoplasts. A mixture of commercially available cellulase, chitinase, lyticase and lysing enzymes (from Trichoderma harzianum) yielded protoplasts exceeding 4.9 x 107 protoplasts per ml of the reaction mixture within 3 hours. Transmission electron microscopic studies of the protoplasts revealed that these protoplasts were totally devoid of cell wall material.

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