Isolasi Protoplas Tanaman Kacang Panjang secara Enzimatis - Imron Riyadi (Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian, 2006)
The technique, kind and concentration of enzyme that were appro-priate and optimum affected the isolation process and rendement result of plant protoplasts. A research was conducted to enhance the protoplast rendements of long bean (Vigna sinensis, L.) that was isolated by enzyme Cellulase RS and Macerozyme R-10 as single and combination in a solution. Concentrations of enzyme were used as much as 2.0-3.0% w/v for Cellulase RS and 0.4-0.6% w/v for Macerozyme R-10. Those solutions contain mannitol 25 mM as osmotycum. Isolation process was done on shaker with 50 rpm (rotation per minute) speed in dark room for 3 hours. Results show that C3 treatment (concentration of Cellulase RS enzyme as much as 3.0% w/v) yielded protoplasts density 17.40 x 105 protoplasts/ g fresh weight of mesophyl and M2 treatment (concentration of Macerozyme R-10 enzyme as much as 0.5% w/v) resulted 17.46 x 105 protoplasts/g. As a whole, the best treat-ment was achieved by C2M2 (combination between Cellulase RS as much as 2.5% and Macerozyme R-10 enzyme as much as 0.5% w/v) which resulted protoplasts density 32.67 x 105 protoplasts/g fresh weight of mesophyl AbstrakTeknik, jenis, dan konsentrasi enzim yang tepat dan optimum berpengaruh dalam proses isolasi dan hasil rendemen protoplas tanaman. Penelitian ini bertujuan untuk meningkatkan rendemen protoplas kacang panjang (Vigna sinensis L.) yang diisolasi dengan enzim Cellulase RS dan Macerozyme R-10 secara individu dan penggabungan dua enzim dalam satu larutan. Konsentrasi enzim yang digunakan adalah 2,0-3,0% b/v untuk Cellulase RS dan 0,4-0,6% b/v untuk Macerozyme R-10. Zat osmotikum yang digunakan adalah mannitol 25 mM. Proses isolasi dilakukan di atas gyotoric shaker dengan kecepatan 50 ppm (putaran per menit) dalam kondisi gelap selama 3 jam. Hasil penelitian menunjukkan bahwa perlakuan C3 (konsentrasi enzim Cellulase RS 3,0% b/v) menghasilkan densitas 17,40 x 105 protoplas/g dan perlakuan M2 (konsentrasi enzim Macerozyme R-10 0,5% b/v) menghasilkan densitas 17,46 x 105 protoplas/g berat segar mesofil daun. Secara keseluruhan, perlakuan terbaik dicapai oleh C2M2 (konsentrasi enzim Cellulase RS 2,5% dan enzim Macerozyme R-10 0,5% b/v) yang menghasilkan densitas 32,67 x 105 protoplas/g berat segar mesofil daun.