Protoplast Isolation and Callus Formation in Oroxylum Indicum (L.) Vent

A simple protoplast isolation protocol from leaf of a medicinally important endangered tree Oroxylum indicum (L.) Vent. was designed to recover totipotent plant protoplasts. The key elements of the protocol are, use of protoplast releasing enzymes (cellulase, pectinase and hemicellulose) alone and in combination in different concentrations for different incubation period of time. Study revealed that protoplast yield was obtained maximum (6x105) in the 13S CPW provided with two combinations of enzymes (hem1+ cel 0.1+pec 0.1 and hem1+ cel 0.5 + pec 0.5).Whereas maximum protoplast viability (70.51%) obtained on hem1+ cel 0.1+pec 0.1 (13S CPW). The optimum time of incubation was observed 4 hrs (25 ± 1°C temp). The isolated protoplast grown gradually when the suspension of protoplast was inoculated on MS media containing (BAP 1 mgL-1and AgNO3 2 mgL-1). The protocol has ensured reproducibility, higher yields and is gentle, as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plantlets were regenerated form the callus developed from isolated protoplast.