Protoplast isolation and culture are important factors for adequate in vitro culture of this type of explant to further genetic manipulations. The composition of the enzymatic solution, protoplast platting density, and plant genotype are important variables in these steps. Therefore, this work aimed to evaluate the isolation efficiency of protoplasts related to three enzymatic solutions and platting efficiency of protoplasts based on five cell densities in different culture media compositions of sweet oranges cultivars. The enzymatic solutions tested were: 1. celulase Onozuka RS 1%, macerase R-10 1% and pectoliase 0,2%; 2. celulase Onozuka RS 1%, macerase R-10 1% ; 3. celulase Onozuka R-10 4% , macerase R-10 1%. Protoplasts were cultured at densities of 2 x 10(4); 5 x 10(4); 10(5); 2x 10(5) e 3 x 10(5) protoplasts.mL-1 in EME 0,7M, BH3 0,7M e BH3 + EME 0,7M, in darkness, at 25 ± 1 ºC. The enzymatic solution 2 provided higher yield for 'Hamlin', 'Natal' and 'Pêra' sweet orange cultivars, and enzymatic solution 1 resulted in better protoplast isolation for 'Westin' sweet orange. For 'Lima Verde' sweet orange, enzymatic solution 3 was the most efficient. Final platting efficiency, evaluated 90 days after culture, was higher at the densities of 3 x 10(5) e 2 x 10(5) protoplasts.mL-1 for 'Hamlin', 'Natal', and 'Lima Verde' sweet orange cultivars, and at the density of 2 x 10(5) e 10(5) protoplasts. mL-1 for 'Westin' sweet orange.
carbohydrate; Citrus sinensis; enzyme; plant tissue culture
