Protoplast enzymatic isolation from mesophyll of Dioscorea alata cultivare “Bottle Peak”

Aislamiento enzimático de protoplastos a partir de mesófilo de Dioscorea alata cultivar “Pico de Botella”

A plant protoplast cell lacking a cell wall is surrounded by its plasma membrane and is potentially able to regenerate the cell wall, grow and divide. The aim of this study was to evaluate different commercial enzyme concentrations and times of action in the isolation of protoplasts of D. alata cultivar “Bottle Peak”. The protoplasts were isolated from mesophyll of vitroplants combining concentrations of the enzymes Cellulase Onozuka R10® (0,0 to 1,5% w/v) and Macerozyme R10® (0,0 to 0,5% w/v), to action times of 6 to 18h and pH of 5,6, giving approximately 0,015g of leaf protoplast isolation medium yam at 27 ± 2 °C, 40 rpm, followed by washing in isolation medium without enzymes, purification in 24% sucrose, resuspension in protoplast culture medium yam and counting in Neubauer camera. Most protoplasts were obtained with 1% Cellulase and 0,5% macerozyme in 18h of action, with an average of 18,6 x10⁶ protoplasts g^-1 fresh tissue. Regression analysis (R² = 0,84) yielded the data obtained, optimum conditions for the most isolated protoplasts correspond to a combination of 0,96% Cellulase and 0,36% macerozyme to 13,99 h action, with an value estimated of 15,3 x10⁶ protoplasts g^-1 tissue. The enzymatic method to isolate protoplasts yam, recommended assessing their feasibility for cell wall regeneration, growing, dividing and forming microcolonies and microcallos.

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