2014 ASHS Annual Conference

In cybridization, new combinations of nuclear and cytoplasmic genes result in a unique genotype that may bring cellular, physical, physiological and biochemical changes to the plant. This has been demonstrated in cybrids generated from the fusion of citrus protoplasts in two independent experiments. The first experiment was conducted to potentially generate a seedless triploid by fusing diploid protoplasts of embryogenic ‘Dancy’ tangerine (Citrus reticulata Blanco) suspension cultured cells with haploid ‘Ruby Red’ grapefruit (Citrus paradisi Macf.) protoplasts derived from microspores. Only one triploid was recovered, but several diploid plants with typical grapefruit morphology were also regenerated. In the second experiment, protoplasts derived from embryogenic ‘Dancy’ tangerine suspension culture were fused with non-embryogenic protoplasts from ‘Duncan’ grapefruit leaves. The fruits from the resulting trees resembled grapefruit in morphology and fruit type, and maintained good fruit flavor and quality. Furthermore, fruits obtained from trees in the first experiment retained excellent quality throughout the summer, when commercial grapefruit rapidly lose quality. Fruits on these trees remain firm with exceptional sweetness and good flavor into August, with no seed germination. The regenerants obtained in the protoplast fusion experiments were confirmed as cybrids by genetic marker analysis. The nuclear genotype of the test grapefruit was determined at seven nuclear SSR marker loci, with all alleles identical to those of commercial ‘Ruby Red’ grapefruit. The mitochondrial genome in the test grapefruit trees was shown by mitochondrial intron marker analysis to have originated from the embryogenic ‘Dancy’ cultures, whereas the plastid genomes of individual trees originated from either fusion partner. Apparently in the first experiment, haploid protoplast preparations also contained diploid protoplasts, which fused with the ‘Dancy’ suspension culture protoplasts, resulting in cybrid cells following the loss of the ‘Dancy’ nucleus. Apart from the value of altered fruit quality attributes in the market, these plants provide an opportunity to understand the contributions of cytoplasmic organelle genetics to important citrus fruit breeding objectives.