Tissue culture protocol for the new Citrus hybrid cultivar “Minihyang”

Abstract Background Citrus hybrid “Minihyang” is a novel citrus cultivar created through the hybridization of Citrus kinokuni ex Tanaka “Kishu mikan” and Citrus reticulate “Ootaponkan.” The fruits of the domestic cultivar are small and seedless, with a high sugar content. Therefore, Citrus “Minihyang” has a niche market for ornamental and commercial purposes with a high value. Additionally, it is a valuable genetic resource for superior variety development because of its unique characteristics. However, a tissue culture system for “Minihyang” is not available, thereby constraining the utilization of bioengineering techniques for its cultivation. Therefore, in this study, we aimed to develop a regeneration system for “Minihyang” using cultured cells of unfertilized and undeveloped seeds from mature fruits. Results We used an induction-modified Murashige and Tucker medium (70 g·L − 1 lactose, 500 mg·L − 1 malt extract, and 16 g·L − 1 agar added to the basic Murashige and Tucker medium) to generate somatic embryogenic cells under semi-dark conditions at 25 ± 2°C and 4-week intervals. Propagated cells were obtained through subculture at 4-week intervals in a modified Murashige and Skoog (EMS) medium containing 50 g·L − 1 sucrose, 500 mg·L − 1 malt extract, and 8 g·L − 1 agar. Rather than normal cotyledon-stage embryos, abnormal globular embryos were observed after 4 weeks of culture in EMS medium containing 70 g·L − 1 lactose and 16 g·L − 1 agar. By regenerating plants from these abnormal globular embryos, we obtained somatic embryos with developing shoots and roots within 3 weeks of culture in EMS medium containing 0.05 M sorbitol and 0.05 M galactose as carbohydrate sources, 0.5 mg·L − 1 gibberellic acid, and 2 g·L − 1 gelrite. These embryos were transferred to and cultured in EMS medium containing 50 g·L − 1 sucrose, 500 mg·L − 1 malt extract, and 8 g·L − 1 agar to produce normal plants. Furthermore, phylogenetic analysis confirmed that “Minihyang” was closely related to “Kishu mikan.” Conclusions We successfully developed a tissue culture system for “Minihyang,” enabling its cultivation and utilization for bioengineering most citrus tissues. Thus, “Minihyang” can serve as a genetic resource for developing bioactive cultivars using biotechnological techniques and be used to breed cultivars that can be entirely consumed.