ScienceAsia 20 (1994): 063-072 |doi: 10.2306/scienceasia1513-1874.1994.20.063

CONDITIONS FOR ISOLATION AND CULTURE OF ARANDA CHARK KUAN MESOPHYLL PROTOPLASTS

KAMNOON KANCHANAPOOM AND PATWADEE TONGSEEDAM

ABSTRACT: Aranda Chark Kuan buds were cultured on Vacin and Went (1949) agar medium for 1 month. After removing the leaves and the apex from the bulging bud and transferring it to a liquid medium, protocorm-likebodies (plb) were obtained. When these plb were transferred onto the same agar medium, complete plantlus developed. Protoplasts were isolated from young leaves (1-3 cm) of these plantlets. One gram fresh weight of the leaves was digested with 10mlofenzyme solution containing 2% (w/v) Cellulase Onozuka R-10,O.7 Msorbitol,10mM CaCI₂.2H₂0, 10mM MES. The mixture of leaves and enzyme solution was incubated at 30^oC for 7 hours on a gyrotary shaker with an agitation speed of 40 rpm. The average yield of isolated protoplasts was 3.6x10⁵ per gram fresh weight. The protoplast culture was investigated according to different types of media and culture methods. The best result found was 1x10⁵ initial protoplasts/ml cultured in Nagata and Takebe (1970) liquid medium supplemented with 1 mg/l 2,4-D (2,4-dichlorophenoxy acetic acid) and 1 mg/l BA (N⁶-benzyladenine) under dark condition. After 3 days in culture, wall formation was clearly visible; however, cell division was not evident.

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Department of Biology, Faculty of Science, Prince ofSongkla University, Haad-Yai, Songkla 90112, Thailand.

Received July 5, 1994