Factors affecting the protoplast isolation and culture of aquatic plant Anubias nana Engler, 1899
2006
Kanchanaree Pongchawee(Department of Fisheries, Bangkok (Thailand). Aquatic Plant and Ornamental Fish Research Institute) | Uthairat Na-Nakorn(Kasetsart University. Bangkhen Campus, Bangkok (Thailand). Faculty of Fisheries. Dept. of Aquaculture) | Siranut Lamseejan(Gamma Irradiation Service and Nuclear Technology Research Center, Bangkok (Thailand)) | Supawadee Poompuang(Kasetsart University. Bangkhen Campus, Bangkok (Thailand). Faculty of Fisheries. Dept. of Aquaculture) | Salak Phansiri(Kasetsart University, Bangkok (Thailand). Kasetsart University Research and Development Institute. Scientific Equipment Center)
Effects of various factors on the isolation and culture of Anubias nana Engler protoplast were investigated. Several factors, such as kind and concentration of enzymes, mannitol concentration in digestion solution, incubation time, plant material age and sucrose concentration in purifying solution were extremely important to obtain highest number and viability of protoplasts. The highest yield (4.79+-0.48*10**(6) per gram fresh weight) and viability (82.90+-4.31 percent) was obtained when protoplasts were digested from in vitro six-week-old leaves with 2 percent Cellulase Onozuka RS, 0.2 percent Pectolyase Y-23, 0.6 M mannitol, 2.5 mM CaCl2.2H2O and 5 mM morpholinoethane sulfonic acid (MES), pH 5.6 for 4 hours in the dark and purified with 18 percent sucrose gradient centrifugation. Purified protoplasts were cultured on KM8P (Kao and Michayluk, 1975) medium supplemented with 0.2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg/l alpha-naphthalene acetic acid (NAA), 0.5 mg/l zeatin by agarose-bead with thin layer liquid culture. The protoplasts regenerated cell walls within 24 hours. First cell division was observed after culturing for 7-9 days, and micro-colonies were formed within 30 days. The culture medium, culture method and plant growth regulator were significant factors influencing cell division and survival of protoplast.
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