Protoplast isolation and growth of Citrus rootstock v. Cravo lime and Cleópatra mandarin were carried out Enzyme mixture was used to digest cell suspensions 7-10 times, following criterious purification. Protoplast culture was carried out in doplets of semi-solid (agarose 0.6% p/v) KM8p medium, in the dark, at 28 ± 1°C following density adjustment to 2 x 10(5) ppts/ml. Osmotic pressure reduction was acomplished for each growth every 10 days with a mixture in equal volumes of KM8p and KM8 medium in ratios of 2:1; 1:1; 1:2 e 0:3. Isolation produced 0.7 x 10(6) ppts/ml for Cleópatra mandarin and 0.5 x 10(6) ppts/ml for Cravo lime, with a viability between 86 to 92% and 73 to 80%, respectively. First cell divisions were recorded at the 8th. day of growth, with an inicial platting efficiency of 9% for Cleópatra mandarin and 5% for Cravo lime. Callus development was achived in 50 days upon transfer of microcalus to semi-solid MT medium.
rootstock; citrus; protoplasts