ScienceAsia 27 (2001) : 029-034 |doi: 10.2306/scienceasia1513-1874.2001.27.029

Isolation and Fusion of Protoplasts from Mesophyll Cells of Dendrobium Pompadour

Kamnoon Kanchanapoom^a,*, Saowarath Jantaro^b and Duangkae Rakchad^b

ABSTRACT: Protoplasts were isolated from Dendrobium Pompadour leaves grown in vitro. Yields of 3x10⁵- 2.5x10⁶ mesophyll protoplasts per gram fresh weight were obtained depending on factors such as digesting enzyme, osmoticum concentration, and leaf size. Small leaves (2.5-3 cm in length and 0.3 g in weight) were digested with 5 ml of enzyme solution containing 1% Cellulase Onozuka R-10, 1% Macerozyme R- 10, 0.5% Driselase, and 0.4 M mannitol. The mixture of leaves and enzyme solution was incubated at 30 C for 3 h on a gyr otary shaker with an agitation speed of 80 rpm. Purification of protoplasts on a sucrose gradient yielded very clean protoplasts that are free of raphide crystals. The fusion of protoplasts was carried out using 40% polyethylene glycol. Video microscopic studies revealed that fusion occurred when osmotic gradient was lowered.

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a Department of Biology, Faculty of Science, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand.
b Department of Biotechnology, Faculty of Science, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand.

* Corresponding author, E-mail: kkamnoon@ ratree.psu.ac.th

Received 16 May 2000, Accepted 30 Nov 2000