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 104; 5 x 104; 105; 2 x 105 e 3 x 105 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 105 e 2 x 105 protoplasts.mL-1, for all varieties. Somatic embryogenesis was observed for all varieties, except for 'Murcott' tangor.
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机译：用于柑橘改良的最新生物技术工具包括通过原生质体融合的体细胞杂交。在11个柑桔品种中评估了原生质体分离，培养和培养的优化，这对于杂种再生至关重要。测试的酶溶液为：1.纤维素酶Onozuka RS，1％;核酶R-10，1％，果胶酶Y-23，0.2％; 2. celullase Onozuka R-10，0.2％;核酶R-10，0.3％;干燥酶，0.1％; 3. celozase Onozuka R-10，1％;蛋白酶R-10，0.2％;干燥酶，0.1％。原生质体在EME 0.7 M中以2 x 104的密度培养; 5 x 104; 105; 2 x 105 e 3 x 105原生质体.mL-1，在黑暗中，在25±1°C下。酶溶液1使得大多数研究的品种的原生质体分离效果更好，除了Rangpur石灰对酶溶液3的分离效率更高，“ Valencia”和“ Succari”甜橙的酶分离效果更好。解决方案3.对于所有品种，培养90天后评估的最终接种效率在密度为105 e 2 x 105原生质体.mL-1时更高。除了'Murcott'探戈之外，所有品种均观察到了体细胞胚发生。
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