Somchai Chuan-udom and Luddawan Moosikapala

pp. 701 - 706

Abstract

Friable callus induced from young leaves of Ma-phut on Murashige and Skoog (MS) medium containing 3% sucrose, 1 mg/l 2,4 dichlorophenoxyacetic acid (2,4-D), 0.5 mg/l benzyladenine (BA) and 500 mg/l polyvinylpyrrolidone (PVP), was cultured in liquid medium with the same components. Various ages of cell suspension at weekly intervals were then incubated in various kinds and concentrations of cell wall digestion enzymes combined with 1% macerozyme R-10 on a rotary shaker at 100 rpm under 1500 lux illumination at 26±4°C. Purified protoplasts were cultured at various densities in MS medium (adjusted osmoticum to 0.4 M by mannitol) supplemented with 3% sucrose and two types of auxin, 2,4-D and NAA at four concentrations (1, 2, 3 and 4 mg/l) together with 1 mg/l BA. The results revealed that a four-day old cell suspension culture incubated in 2% cellulase Onozuka R-10 (CR10) in combination with 1% macerozyme R-10 gave an
optimum result in both yield and viability of protoplasts at 5.7x10⁶/1 ml PCV and 80%, respectively. Embedding protoplasts at a density of 2.5x10⁵
/ml in 0.2% phytagel containing MS medium supplemented with 3 mg/l NAA and 1 mg/l BA promoted the most effective division of the protoplasts (20%). The first division of the protoplasts was obtained after 2 days of culture
and further divisions to form micro- and macro-colonies could be observed after 7-10 days of culture. However, callus formation and plantlet regeneration was not obtained.