African Journal of
Agricultural Research

  • Abbreviation: Afr. J. Agric. Res.
  • Language: English
  • ISSN: 1991-637X
  • DOI: 10.5897/AJAR
  • Start Year: 2006
  • Published Articles: 6730

Full Length Research Paper

Optimisation of protoplast isolation protocols using in vitro leaves of Dendrobium crumenatum (pigeon orchid)

Chong Siang Tee1* Pui Sinn Lee1, Anna Ling Pick Kiong1 and Maziah Mahmood2
1Department of Biological Science, Faculty of Science, Universiti Tunku Abdul Rahman, Malaysia. 2Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
Email: [email protected]

  •  Accepted: 08 September 2010
  •  Published: 04 October 2010


Protoplast isolation from in vitro leaves of Dendrobium crumenatum was carried out. Factors affecting protoplast isolation, sorbitol concentration, enzyme combinations and concentration and incubation time for isolation were studied. Results obtained showed that 0.5 M sorbitol was the most effective concentration to isolate the protoplast (25.62×104 protoplasts/g FW). High yield of protoplasts, 20.40×104 protoplasts/ g FW, could also be obtained by using combination of cellulase and pectinase instead of using the enzyme individually. In addition, efficacy of protoplast isolation was improved by 20.12×104 protoplasts/g FW, when 2% (w/v) cellulase and pectinase were used; while incubation for 4 h during the isolation process recorded highest protoplast yield, 28.66×104protoplasts/gFW. Based on the results obtained, we suggest that protoplasts isolation of D. crumenatum from in vitro leaves could be achieved using 0.5 M sorbitol with combination of 2% (w/v) cellulase and pectinase for both enzymes and 4 h incubation time.


Key words: Dendrobium crumenatum, cellulase, pectinase, protoplast, sorbitol.