Factors affecting healthy protoplast isolation from mesophyll tissue of 60 to 90-day old Nicotiana sanderae seedling was identified, and optimal conditions for high plating efficiency was also tested. As a result, it could be possible to be used as basic informations for obtaining much more plantlets which were originated from protoplast-derived calli.
When leaves which were grown in green house were enzymatically digested in 0.1% macerozyme R-10, 2.0% cellulase Onozuka R-10, 0.01M MES buffer, 0.2% BSA and 0.7M mannitol containing enzyme mixture with CPW inorganic salts which was adjusted pH to 5.8, more healthy protoplasts were obtained. On the other hand, protaplast yield of in vitro cultured mesophyll tissue was more increased in enzyme mixture containing 0.1% macerozyme R-10, 1.5% cellulase Onozuka R-10, 0.01M MES buffer, 0.2% BSA, 0.3M mannitol and CPW inorganic salts. Crude protoplasts were purified on 0.6M or 0.3M, mannitol-sucrose-sucrose solution, once every 2-2-1 hour´s time interval by centrifugation.
After 7days when protoplasts from in vitro cultured mesophyll were cultured in 8p-KM medium containing 0.1M glucose and 0.2M mannitol in a 5 × 10⁴/㎖ cell density at 25℃ and dim light, freshly prepared 8p-KM medium with 0.1M glucose and mannitol were supplemented to protoplast cultures, and subsequently, micro callus formation was observed.
After another 7days, Murashige-Skoog´s medium containing 2.0㎎/ℓ NAA, 0.5㎎/ℓ BA and 30g/ℓ sucrose substituted for the former medium.
When the calli which was formed in the latter medium were cultured in Murashige-Skoog´s medium with 2.0㎎/ℓ IAA, 1.0∼2.0㎎/ℓ BA, 30g/ℓ sucrose and 8g/ℓ agar, shooting frequency was highly increased.
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