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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.

New Somatic Hybrid Mandarin Tetraploids Generated by Optimized Protoplast Fusion and Confirmed by Molecular Marker Analysis and Flow Cytometry