Improved technique for isolation and culture of protoplasts from young leaves of mangosteen (Garcinia mangostana L.)

Abstract

Improved technique for isolation of protoplasts from young leaves of mangosteen was developed using dark treatment and varying ages of in vitro-grown leaves. In this experiment different kinds and concentrations of cellulase Onozuka R-10, macerozyme R-10 and pectolyase Y-23 were used. One gram fresh weight of leaf tissue was incubated in a 10 ml of enzyme solution and placed on a gyratory shaker at 40-50 rpm under darkness for 12 hours. Yield and viability of protoplasts were compared among those treatments, then the density was adjusted and cultured in MS medium supplemented with different kinds and concentrations of growth regulators. The results showed that 8 week-old leaves (after adding liquid culture medium) gave released protoplasts at 1.9 × 105/gram fresh weight (g fr wt.) This result was obtained when 2% cellulase Onozuka R-10, 1% macerozyme R-10 and 0.1% pectolyase Y-23 were used. Viability of the protoplasts was 77.63%. Pretreatment the leaves in the dark for 24 hours before being subjected to protoplast isolation resulted in the greatest release of protoplasts at 1 × 106/g fr wt. Viability of the protoplasts was also the highest (91.35%). The protoplasts at density of 5 × 105/ml could promote cell division at 3.41% in a thin layer of liquid MS with 0.5 mg/l BA and 0.5 mg/l TDZ.

Keywords

• improved technique
• isolation protoplasts
• mangosteen