The optimization mesophyll protoplast isolation for Phalaenopsis amboinensis

Indonesia has 25 species of month orchids and 10 of them are endemic to Indonesia. Plant Conservation Center (LIPI) conserved 15 species of orchids which means that it has been conserving as much as 23.44% of the orchids in the world (Rahayu, 2015). Orchid conservation efforts in Phalaenopsis amboinensis, both ex vitro and in vitro continue to be made in order to increase biodiversity. One of them is made by protoplast fusion approach that begins with the isolation of protoplast Phalaenopsis amboinensis. This study aimed to produce viable protoplast of Phalaenopsis amboinensis by developing procedure for mesophyll protoplast isolation. Protoplast is isolated from young orchid leaf. This experiment was conducted on six combinations of enzymes (Cellulase concentrations of 1%, 1.5%, 2% and macerozyme 1%, 1.5%) and two incubation in the dark (4 h and 6 h), so the total combinations become 12. The method of analysis used a completely randomized design. The viable protoplast result was evaluated. The result showed that the best achieved enzyme combination was Cellulase 2.0% + Macerozyme 1.0% and incubation time of 6 h; compared to other treatments of forming spherical protoplasts and chloroplasts it tends to clump together and solid. The number of protoplasts 1.1 × 10⁶ and the percentage of viable protoplasts 83.88%.