FACTORS INFLUENCING PROTOPLAST ISOLATION FROM COFFEA-ARABICA CELLS

Cultured plant cells such as Coffea arabica L. cells, accumulate low c oncentration of secondary metabolites. One way to obtain high-producin g plant cell cultures is to prepare single cell clones by using protop last systems. Identification of limiting factors should facilitate the development of an isolation procedure that can generate adequate yiel ds of intact and viable protoplasts Coffea arabica L. suspension cells . The most suitable conditions for protoplasting were as follows: 6 g of fresh tissue were plasmolysed in 100 ml of K3 salts (Nagy & Maliga 1976) containing 0.5 M sucrose for 1 h at 24-degrees-C. Then, 1 g of p replasmolysed cells were incubated in 10 ml of cellulase R10 (1%), mac erozyme R10 (0.8%) and driselase (0.5%) in preplasmolysis medium. The protoplasts were collected and purified after 15 h of lytic reaction i n the dark, at 28-degrees-C. More than 75% and 95% of the cells were c onverted into protoplasts when 5 and 8 day-old suspensions respectivel y were used for the release step. A1 number of viable protoplasts rang ing from 3.5 x 10(6) to 4.6 x 10(6) p g-1 fresh weight was obtained co rresponding to an increase by a1 factor 10 to 15 of the protoplast yie ld obtained by Acuna & De Pena (1991).