A study on the isolation of protoplasts from mesophyll cells of dendrobium queen pink. | [Pakistan Journal of Botany • 2016]

Author(s):

1. Ramsha Aqeel: Department of Biotechnology, University of Karachi, Karachi, Pakistan; Biotechnology Wing, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, Pakistan; International Center for Chemical and Biological Sciences ICCBS, University of Karachi, Pakistan

2. Marium Zehra: Department of Biotechnology, University of Karachi, Karachi, Pakistan; Biotechnology Wing, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, Pakistan; International Center for Chemical and Biological Sciences ICCBS, University of Karachi, Pakistan

3. Syeda Kahkashan Kazmi: Biotechnology Wing, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, Pakistan

4. Saifullah Khan: Biotechnology Wing, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, Pakistan; International Center for Chemical and Biological Sciences ICCBS, University of Karachi, Pakistan; Department of Agriculture and Agribusiness Management, University of Karachi, Karachi, Pakistan

5. Hammad Afzal Kayani: Biotechnology Wing, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, Pakistan; International Center for Chemical and Biological Sciences ICCBS, University of Karachi, Pakistan;Shaheed Zulfiqar Ali Bhutto Institute of Science and Technology, Pakistan

6. Ameer Ahmed Mirbahar: Biotechnology Wing, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, Pakistan; International Center for Chemical and Biological Sciences ICCBS, University of Karachi, Pakistan

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.

Page(s): 693-697

DOI: DOI not available

Published: Journal: Pakistan Journal of Botany, Volume: 48, Issue: 2, Year: 2016

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