Isolasi Protoplas secara Enzimatis pada Tanaman Kecipir

Abstract

The kind and concentration of enzyme that appropriate affected isolation process and result of plant protoplast. The research was conducted to enhance the protoplast rendements of winged bean (Psophocarpus tetragonolobus L.) that was isolated by Cellulase RS and Macerozyme R-10 enzyme as single and its combination in a solution. Concentrations of enzyme used were as much as 2.0-3.0% w/v for Cellulase RS and 0.4-0.6% w/v for Macerozyme R-10. The solution containt mannitol 25 mM as an osmotycum. Isolation process was done on shaker with 50 rpm (rotation per minute) speed in dark room for 5 hours. Results showed that C3 treatment (concentration of Cellulase RS enzyme as much as 3.0% w/v) resulted protoplasts density 3.49 x 105 protoplasts/g fresh weight of hypocotyl and M2 treatment (concentration of Macerozyme R- 10 enzyme as much as 0.5% w/v) resulted 3.19 x 105 protoplasts/g. Whereas the best combined enzymes treatment was achieved by C3M2 (combination between Cellulase RS as much as 3.0% and Macerozyme R-10 enzyme as much as 0.5% w/v) which resulted protoplasts density 4.94 x 105 protoplasts/g fresh weight of hypocotyl. The protoplast was intact and viable.