Recent biotechnological tools for citrus improvement include somatic hybridization by protoplast fusion. The optimization of protoplast isolation, platting and culture, essential for hybrid regeneration, was evaluated in 11 citrus varieties. The enzymatic solutions tested were: 1. cellulase Onozuka RS, 1%; macerozyme R-10, 1%, pectoliase Y-23, 0.2%; 2. celullase Onozuka R-10, 0.2 %; macerozyme R-10, 0.3%; driselase, 0.1%; 3. celulase Onozuka R-10, 1%; macerase R-10, 0.2%; driselase, 0.1%. Protoplasts were cultured in EME 0.7 M at densities of 2 x 10(4); 5 x 10(4); 10(5); 2 x 10(5) e 3 x 10(5) protoplasts.mL-1, in darkness, at 25 ± 1°C. The enzymatic solution 1 resulted in better protoplast isolation for most of the varieties studied, except for Rangpur lime, which presented higher isolation efficiency on enzymatic solution 3, and for 'Valencia' and 'Succari' sweet oranges, with better results were obtained on enzymatic solution 3. Final platting efficiency, evaluated 90 days after culture, was higher at the densities of 10(5) e 2 x 10(5) protoplasts.mL-1, for all varieties. Somatic embryogenesis was observed for all varieties, except for 'Murcott' tangor.
citrus; protoplast; somatic hybridization; variety improvement
