Protoplast Electro-Fusion Technology as a Tool for Somatic Hybridisation between Strawberries and Raspberries P. Geerts 1 , A. Henneguez 2 , P. Druart 1 and B. Watillon 1 1 CRA-W Biotechnology, Chaussée de Charleroi 234, B 5030 Gembloux, Belgium 2 Haute Ecole Charlemagne, rue de Verlaine 9, B 5030 Gembloux, Belgium Keywords: ‘Elsanta’, ‘Autumn bliss’, Phytophthora, rhodamine, fluorescein diacetate, intergeneric 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. INTRODUCTION Major production constraints of strawberry plantlet multiplication in European nurseries are linked to soil infection by Phytophthora sp. Methyl Bromide is known as the only effective fumigant to combat this major fungus when soil is already contaminated but its utilization will soon be forbidden worldwide. By this time, the only alternative is to use healthy mother plants derived from meristem culture and in vitro multiplication and to produce healthy runners on non-contaminated nursery soils. As many of the soils are already contaminated due to intensive culture and limited soil rotation, another alternative would be to use new cultivars bred for resistance to Phytophthora. Regarding this situation, it was proposed to look for sources of resistance in an enlarged gene pool: the Rosales. Intergeneric crosses within the Rosales are rarely successful. However, Squirrell et al. (2005) succeeded in the regeneration of Rosa (+) Prunus and Rosa (+) Rubus somatic hybrids. Based on this advanced technology we proposed as a main objective to try to introduce existing Phytophthora tolerance within raspberries into strawberries. The first objective was to achieve the fusion between strawberry × raspberry and to observe the behaviour of the fused product into the formation of microcalli. To initiate our research, the protocol described by Durieu and Ochatt (2000) for intergeneric fusion of pea (Pisum sativum L.) and grass pea (Lathyrus sativus L.) protoplasts was adopted while the regeneration process took into consideration the work of Nyman (1992). 495 Proc. VI th Internat. Strawberry Symposium Ed.: J. López-Medina Acta Hort. 842, ISHS 2009