Pak. J. Bot., 48(2): 693-697, 2016.

A STUDY ON THE ISOLATION OF PROTOPLASTS FROM MESOPHYLL CELLS OF DENDROBIUM QUEEN PINK

RAMSHA AQEEL1,2, MARIUM ZEHRA1,2, SYEDA KAHKASHAN KAZMI2, SAIFULLAH KHAN2,3*, HAMMAD AFZAL KAYANI2,4 AND AMEER AHMED MIRBAHAR2

Abstract: Protoplasts were successfully isolated from one month old In vitro grown plantlets of Dendrobium cultivar Queen pink. The enzyme solution used was composed of 1% Cellulase Onozuka R-10, 0.5% Macerozyme R-10, 0.1% Pectinase, 0.3 M mannitol, 10 mM CaCl2.2H2O and 10 mM 2 (N-morpholino)-ethanesulfonic acid (MES) at pH 5.8. Protoplast highest yield with 15.7x104 protoplasts per 1.5 gm freshly chopped leaves were obtained when digested in enzyme solution for 4 hrs on a rotary shaker with an agitation speed of 45 rpm in dark conditions. Protoplasts were filtered with 45μm nylon sieve and washed with 0.3 M mannitol solution supplemented with 10 mM CaCl2.2H2O and 10 mM MES, and purified with 0.3 M sucrose solution gradient. Purification of protoplasts on a sucrose mannitol gradient yielded clean protoplasts that were free from debris.

Key words: Protoplast isolation protocol, Dendrobium Queen Pink, Protoplast yield, Protoplast viability.

1Department of Biotechnology, University of Karachi, Karachi, Pakistan

2Biotechnology Wing, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, Pakistan

International Center for Chemical and Biological Sciences (ICCBS), University of Karachi-75270, Pakistan

3Department of Agriculture and Agribusiness Management, University of Karachi, Karachi, Pakistan

4Shaheed Zulfiqar Ali Bhutto Institute of Science and Technology, Pakistan

*Corresponding author’s e-mail: drsaif65@gmail.com