Isolation and culture of leaf protoplasts from tunisian grapes

Experimental conditions for leaf protoplast isolation and culture were optimised for in vitro plants deriving from shoot culture of two Tunisian grape varieties, Sakasly and Muscat d’Alexandrie ( Vitis vinifera L.). The best yields were obtained from leaves of 4 to 5 weeks old in vitro plants, digested for 13 hours under 25 rpm agitation with an enzymatic mixture containing 0.25 % cellulase of Aspergillus niger , 0.25 % cellulase of Penicillium funiculosum , 0.5 % cellulysin of Trichoderma viridae , and 0.2 % macerozyme R-10 of Rhizopus sp. More than 50 % of the purified protoplasts had a diameter of 30-40 μm and were rich in chloroplasts. Best aptitude for cell division was found in protoplasts immobilised in sodium alginate layers at a density of 0.5x10 6 cell/ml, cultivated in CPW-13 medium containing 4 mg/l of NOA and 0.88 mg/l of TDZ. The variety Muscat d’Alexandrie gave better yield whereas Sakasly showed better cell division rates. Formation of micro and macrocallus have been obtained, but the oxidation of the medium has to be solved in order to promote plant regeneration.