New somatic hybrid mandarin tetraploids generated by optimized protoplast fusion and confirmed by molecular marker analysis and flow cytometry

Authors: CALOVIC, MILICA - University Of Florida; Chen, Chunxian; YU, OIBIN - University Of Florida; ORBOVIC, VALDIMIR - University Of Florida; GMITTER JR, FRED - University Of Florida; GROSSER, JUDE - University Of Florida

Submitted to: Journal of the American Society for Horticultural Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/20/2019
Publication Date: 5/1/2019
Citation: Calovic, M., Chen, C., Yu, O., Orbovic, V., Gmitter Jr, F.G., Grosser, J.W. 2019. New somatic hybrid mandarin tetraploids generated by optimized protoplast fusion and confirmed by molecular marker analysis and flow cytometry. Journal of the American Society for Horticultural Science. 144(3):151-163. https://doi.org/10.21273/JASHS04563-18.
DOI: https://doi.org/10.21273/JASHS04563-18

Interpretive Summary: Seedlessness and easy-peeling are highly desired traits for mandarin cultivars primarily for fresh citrus fruit consumption. Somatic tetraploid (2n = 4x) mandarin hybrids can be used in crosses with diploid (2n = 2x) mandarin parents to produce triploids (2n = 3x) hybrids that usually are seedless and likely easy to peel. Sixty-five somatic mandarin hybrids, presumably tetraploids are generated from heterokaryon fusion using six known diploid mandarin cultivars. Their ploidy level and parental sources are determined by flow cytometry and nuclear DNA markers, respectively. These mandarin-derived tetraploids are valuable as potential breeding parents for interploid crosses to generate seedless and easy-peeling triploid hybrids.

Technical Abstract: Six mandarin cultivars, Ponkan (Citrus reticulata), Willowleaf (Citrus deliciosa), Kinnow (Citrus nobilis × C. deliciosa), Murcott (purported C. reticulata × Citrus sinensis), W. Murcott [purported (C. reticulata × C. sinensis) × C. reticulata)], and Snack (purported C. reticulata hybrid), were used in protoplast fusion with different parental combinations to generate somatic hybrids. Sixty-five somatic regenerants were obtained using optimized formulation of enzymes and molecular weight of polyethylene glycol for improved protoplast yield and heterokaryon fusion rate, respectively. Flow cytometry was used to determine the ploidy level of somatic regenerants, and nuclear expressed sequence tag–simple sequence repeat (EST-SSR) markers to determine their parental source. Of the 65 somatic regenerants, 46 were identified as autotetraploids, 18 allotetraploids, and one undefined. The EST-SSR markers also revealed that some ‘W. Murcott’ embryogenic callus lines that were presumed to be of nucellar origin were actually derived unexpectedly from individual ovules of zygotic origin. These mandarin-derived tetraploids are valuable as potential breeding parents for interploid crosses with an aim at seedlessness and easy-peeling traits.