Isolation and growth of citrus rootstock protoplants

Abstract

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.