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Protoplast isolation is one of the important steps for protoplast fusion in pineapple breeding program. Condition for protoplast isolation is different in each plant species. The objective of this study was to establish appropriate condition for protoplast isolation from in vitro leaf of Pineapple cv. Plulae. Mannitol concentrations (0.1 – 0.5 M), cellulase concentrations (Cellulase Onuzuga R-10, 1 – 2.5% (w/v)), macerozyme concentrations (Macerozyme Onozuga R-10, 0.5 – 2% (w/v)), digestion time (4 – 6 h) and centrifugation speeds (700 – 1000 rpm) were investigated. The results showed that 0.4 M mannitol, 2% (w/v) cellulase, 1% (w/v) macerozyme, 6 h digestion time and centrifugation speed at 800 rpm were the appropriate condition for protoplast isolation from in vitro leaf of Pineapple cv. Plulae. The protoplast yield was 3.82x106±0.02/ g FW.
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