OPTIMIZATION OF ELECTROFUSION PARAMETERS AND INTERSPECIFIC SOMATIC HYBRID REGENERATION IN CITRUS

This study was primarily attempted to optimize the electrofusion parameters using protoplasts isolated from cell suspension cultures of “Page” tangelo (Citrus reticulata Blanco×C.paradisi Macf) and mesophyll protoplasts of rough lemon(Citrus jambhiri Lush) as fusion partners. It was shown that the binuclear heterokaryons frequency reached 15% with the following parameters: alternate current (AC) 125 V/cm, AC time 60 s, direct current (DC) 1250 V/cm, DC pulse width 50 μs, DC pulse interval 0.5 s, No. of DC pulse 3. Considering the fact that different types of protoplasts have different specific weights, higher frequency of the binuclear heterokaryons was obtained by controlling the centrifugation time after fusion. The fusion products regenerated into plantlets after 3 to 4 months of culture. Chromosome counting of the root tips and morphological observation of the regenerants verified that 78% were tetraploids and the rest were diploids with the leaf morphology of mesophyll parent. Peroxidase (POX) isozyme and RAPD analysis indicated that interspecific somatic hybrids were obtained and an autotetraploid plant of mesophyll parent type was also verified.