Factors affecting protoplast isolation and cultivation of <em>Cucumis</em> spp.

Abstract

Protoplasts of Cucumis anguria, Cucumis melo (3 accessions), Cucumis metuliferus and Cucumis sativus were isolated from leaves, growing apices, hypocotyls and calluses. Plants were cultured on 2 concentrations of sucrose. The effect of plant culture medium, explant age and explant type on protoplast viability were investigated. The protoplasts were cultured in 3 types of culture medium and at two temperatures. Optimal age range for protoplast isolation was 1-5 weeks depending on explant type and genotype. Viabilities ranging between 50 % and 80 % were obtained from all explants and genotypes. Increased concentration of sucrose had negative impact on viability of protoplasts. The highest level of regeneration achieved was callus, regenerated from leaf protoplasts of C. melo cv. ‘Charentais’ and C. melo ‘MR-1’. The lowest regeneration capability was observed in hypocotyls. Liquid LCM medium (B5 macro and microelements (1 g · l^-1 CaCl₂), B5 vitamins with 1g· l^-1 myo-inositol, 2 mg· l^-1 ascorbic acid, 0.8 mg· l^-1 glycine, 20 mg· l^-1 glutamine, 100 mg · l^-1 casein hydrolysate, 70 g · l^-1 mannitol, 10 g · l^-1 sucrose, 5 g · l^-1 glucose, 585 mg · l^-1 MES, 5.37 μmol· l^-1 NAA, 2.26 μmol· l^-1 2,4-D, 2.22 μmol · l^-1 BA) was optimal for protoplast regeneration. Agarose-solidified medium caused a decrease in the number of cell divisions (used in C. melo ‘PI 124112’). Cultivation at 25ºC resulted in a higher frequency of cell divisions (tested in C. metuliferus).