PROTOPLAST ELECTRO-FUSION TECHNOLOGY AS A TOOL FOR SOMATIC HYBRIDISATION BETWEEN STRAWBERRIES AND RASPBERRIES

Abstract

Inter-generic breeding between Fragaria × ananassa and Rubus in order to develop new Phytophthora resistance into Fragaria has been investigated. The study describes the use of the electro-fusion technique allowing to obtain Fragaria × ananassa var. 'Elsanta' (+) Rubus heterocaryons and microcalli. Protoplasts isolation was carried out following the Durieu and Ochatt (2000) procedure. Protoplasts of each species were identified using fluorochromes. Fluorescein diacetate (FDA) and rhodamine B isothiocyanate (RBi) were respectively used for Rubus and Fragaria protoplasts. Under UV light, protoplasts with FDA staining gave a yellow-green fluorescence allowing evaluation of density and viability evaluation while those with RBi gave a red fluorescence. Density and viability were determined for each species. Electro-fusion was achieved using 2 ml cuvettes of an Electro cell Manipulator ECM ® 630 (BTX, California) with electrodes 1 mm apart. Three pulses at 250, 500, 750 or 1000 V·cm -1 were delivered at 10 s intervals (capacitor of 75 μF and variable resistance of 201, 281 or 1540 Ω). The efficiency of protoplast fusion was evaluated under UV light, as the fluorochromes are linked to different parental protoplasts, whereby heterokaryons can be observed and counted through their double fluorescence, green and red. Protoplasts were cultured at 105 cm -3 on a KM medium (Kao and Michayluk, 1975). After one week a dilution was performed with the same medium and, as soon as the majority of cells had regenerated their wall, weekly dilutions (adding weekly 1 ml media per ml of initial protoplast culture) were carried out. Large numbers of heterokaryons have been produced using different Rubus genotypes and different electrical parameters for fusion. Both divisions of heterokaryons and the formation of heterokaryon-derived microcalli were observed.