Abstract
Experiments were conducted to investigate the factors influencing mesophyll protoplast isolation in `Fuji' apple. Half an hour pretreatment in 0.6M mannitol gave the highest protoplast yield.The enzyme solution containing 2% Cellulase Onozuka R-10 and 0.5% macrozyme R-10 with CPW 0.6M mannitol at pH 5.5 was most effective for protoplast isolation from leaf. Effective incubation time for the enzyme treatment was found to be 15-20 hrs at 25°C in the dark. Use of 1.0-2.0% PVP and 0.5mM MES was essential for higher yield and viability of protoplast. Supplementation of BA and IBA to the shoot culture media gave the higher yield of viable protoplast. From these protoplast, new cell walls were regenerated and 4 cell structures developed from one protoplast by cell division in K8P medium supplemented with 3A and NAA. Planting density higher than 10 protoplasts/ml was required for cell division from protoplast in liquid or 0.5% agarose culture.
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