ELECTROFUSION OF PROTOPLASTS IN SELECTED VEGETABLES OF BRASSICA, CUCUMIS AND SOLANUM GENERA

Abstract

The aim of this study is using protoplast electrofusion for somatic hybridization, especially for increasing of genetic variability in selected important vegetable species of Brassica, Cucumis and Solanum genus and possibility of including this method into breeding approaches to obtain new breeding materials. Mesophyll protoplasts were isolated from in vitro cultured plants of Brassica olearacea (B. oleracea var. capitata, B. oleracea var. botrytis), Cucumis spp. (C. metuliferus, C. melo) and Solanum tuberosum dihaploid plants and wild Solanum species (S. pinnatisectum, S. bulbocastanum). Hypocotyl protoplasts were isolated from etiolated seedlings of Brassica oleracea (cabbage, cauliflower) and callus protoplasts of Cucumis melo. Enzymatic mixture of 1% or 2% Cellulase Onozuka R-10 and 0.25% or 1% Macerozyme R-10 in rinsing solution Pgly/W5 (2,1) was used for isolation of mesophyll and hypocotyl protoplasts or callus protoplasts. Leaves, hypocotyls and/or calli were segmented and put into a thermostat at 25°C for 16-18 hours. Protoplasts were isolated using a conventional method (filtration, centrifugation and purification with 20% sucrose gradient). Purified protoplasts were rinsed with pre-fusion solution (0.4 M manitol and 1 mM CaCl 2 ) and fusion solution (0.4 M manitol), after centrifugation their required density was obtained by dilution with fusion solution. Viability of isolated protoplasts used for electrofusions was more than 80% (determined using FDA). Fusing partner components were mixed in the ratio of 1:1. There were fused protoplasts of Brassica genus: mesophyll and hypocotyl, Cucumis genus: mesophyll and callus, Solanum genus: mesophyll and mesophyll. Protoplast fusion was performed using apparatus ECM 2001 and in electroporation chamber for 20-30 μl with the 0.5 mm electrode distance. Following parameters were used fbr fusions: for protoplast alignment - 5V AC and 1 MHz frequency with individual duration of action, for fusion - 1 pulse 10V DC of length 80 μs (3). Following electrofusions protoplasts were viable and undamaged. Homofusion and heterofusion products and also unfused protoplasts could be observed. Subsequent culturing was done in medium LCM1 (2) or SW11 (1) in a thermostat at 25°C for 2 weeks in the dark. Regeneration of cell walls was recorded and first division was found in 6-8 days of culture, in Solanum genus microcolonies were found in 15 days. The experiments have not been finished yet.