Establishment of a Regeneration System for ‘Minihyang’, a Domestic Citrus Variety With High Utility as a Genetic Resource

ABSTRACT ‘Minihyang’, a novel citrus cultivar, was developed through the hybridization of Citrus kinokuni ex Tanaka ‘Kishu mikan’ and Citrus reticulata ‘Ootaponkan’. This study aimed to establish a tissue culture system for ‘Minihyang’ using cells from unfertilized and undeveloped seeds of mature fruits. Somatic embryogenic cells were induced on somatic embryogenesis induction modified Murashige and Tucker (SIME) medium and propagated through subculture on modified Murashige and Skoog (EMS) medium containing sucrose, malt extract and agar. Abnormal globular embryos were observed after 4 weeks in the EMS medium with lactose and agar. Normal plants were regenerated from somatic embryos by culturing in the EMS medium supplemented with sorbitol, galactose, gibberellic acid and gelrite. Genetic and phylogenetic analyses confirmed the hybrid origin of ‘Minihyang’, demonstrating closer genetic relatedness to its seed parent ‘Ootaponkan’ than to its pollen parent. Additionally, regenerated plantlets were identified as nucellar tissue derivatives, genetically identical to ‘Minihyang’. This tissue culture system facilitates the use of ‘Minihyang’ as a genetic resource for breeding bioactive cultivars.