Citrus cybrids: production by donor-recipient protoplast-fusion and verification by mitochondrial-DNA restriction profiles

Abstract

Embryogenic nucellar-callus from three Citrus types was used for protoplast isolation. The protoplasts were fused by the ‘donor-recipient’ procedure by which the nuclear-division of the donor-protoplasts was arrested by gamma-irradiation and the metabolism of unfused recipient-protoplasts was transiently inhibited by iodoacetate. The following fusion combinations were performed: (1) Poorman x Poncirus trifoliata with Villafranca lemon; (2) Poorman x P. trifoliata with Sour orange; (3) Sour orange with Villafranca. Combinations 1, 2 and 3 resulted in 120 (125), 70 (5) and 19 (89) calli (regenerated plants), respectively. The mitochondrial-DNA (mtDNA) restriction profiles of Poorman x P. trifoliata obtained by fragmentation with Bcl I, Bam H1 or Sal I differed from the respective profiles of Villafranca and Sour orange but no differences in mtDNA restriction profiles were detected between Villafranca and Sour orange. When Southern blots of Villafranca and Sour orange mtDNAs were hybridized with radiolabelled heterologous mtDNA probes, the mtDNAs of these two Citrus types could be differentiated. The fusion-derived plants from all three donor-recipient combinations had the recipients morphological (nuclear-coded) features. MtDNA restriction profiles, with and without hybridization to heterologous probes, indicated that the analysed plants were cybrids.