Production of mandarin + pummelo somatic hybrid citrus rootstocks with potential for improved tolerance/resistance to sting nematode

Sting nematode (Belonolaimus longicaudatus Rau) has become a primary factor limiting citrus production in localized regions of the central Florida sandridge citrus production area, making the development of resistant rootstocks a new breeding objective. In efforts to develop a replacement rootstock for the widely adapted sour orange, our focus has been on somatic hybridization of selected mandarin + pummelo combinations [Grosser, J.W., Gmitter, Jr., F.G., 1990. Protoplast fusion and citrus improvement. Plant Breed. Rev. 8, 339-374; Ananthakrishnan, G., Calovic, M., Serrano, P., Grosser, J.W., 2006. Production of additional allotetraploid somatic hybrids combining mandarins and sweet oranges with pre-selected pummelos as potential candidates to replace sour orange rootstock. In Vitro Cell. Dev.: Plant 42, 367-371], since sour orange is probably an introgression hybrid of mandarin and pummelo as suggested by molecular marker analyses [Nicolosi, E., Deng, Z.N., Gentile, A., La Malfa, S., Tribulato, E., 2000. Citrus phylogeny and genetic origin of important species as investigated by molecular markers. Theor. Appl. Genet. 100, 1155-1166; Gulsen, O., Roose, M.L., 2001. Lemons: diversity and relationships with selected Citrus genotypes as measured with nuclear genome markers. J. Am. Soc. Hort. Sci. 126, 309-317]. Somatic hybrid plants were produced from four new mandarin (C. reticulata Blanco) + pummelo (C. grandis L. Osbeck) parental combinations by fusing embryogenic suspension culture-derived protoplasts isolated from selected mandarins with leaf protoplasts of pummelo seedlings previously selected for tolerance/resistance to the sting nematode (B. longicaudatus Rau) as follows: Amblycarpa mandarin + 'Liang Ping Yau' (seedling) pummelo seedling SN7; Amblycarpa mandarin + 'Hirado Buntan Pink' (HBP) pummelo seedling SN3; Murcott tangor + pummelo seedling SN3; and Shekwasha mandarin + pummelo seedling SN3. Somatic hybridization was verified by ploidy analysis (via flow cytometry) and RAPD analyses. Mandarin parents were selected for wide soil-adaptation and ability to produce friable embryogenic callus lines. Pummelo seedlings used as leaf parents were identified from a previous screen of large seed populations (200 each) from four pummelos for resistance to sting nematode as follows: 'Hirado Buntan Pink'; 'Red Shaddock'; 'Large Pink Pummelo' and a seedling pummelo of 'Liang Ping Yau'. Ten resistant/tolerant pummelo seedlings were selected from the 800 pummelo seeds planted in the screen for further study. The four new somatic hybrids have been propagated to evaluate their horticultural performance and resistance to the sting nematode. These potential somatic hybrid rootstocks should also have potential to control tree size due to polyploidy.