"Minihyang" presents considerable value as a genetic resource for developing superior citrus varieties because the fruit is small and seedless, with a high sugar content. However, given that “Minihyang” is the first reported Citrus hybrid variety, a tissue culture system had not been established. Consequently, its use in cultivar breeding using biotechnology is limited. Therefore, in this study, we have developed a tissue culture protocol applicable to most citrus fruits, including “Minihyang,” the first domestic cultivar in South Korea, possessing substantial breeding resource utilization potential. Phylogenetic analysis using the ITS regions confirmed that “Minihyang” plants used in this study were genetically related to “Kishu mikan,” used as the seed parent, not to “Ootaponkan,” used as the pollen parent. This corroborates that the “Minihyang” cultivar used in this study was a Citrus hybrid selected through crossbreeding of “Kishu mikan” and “Ootaponkan.”
In general, the induction of somatic embryogenic callus cells for citrus fruits involves the culture of ovules from immature fruits [16] or unfertilized and undeveloped seeds from mature fruits [13]. We employed the method described by Jin et al. [13] to ensure the induction and propagation of somatic embryogenic callus cells, i.e., culture of unfertilized and undeveloped seeds of “Minihyang.” Previous studies have demonstrated that somatic embryogenesis is mainly promoted by stress-inducing factors, which are promoted by the effects of osmotic stress and uridine diphosphate glucose–glucose synthesis inhibition on plant growth [18–20], which include carbohydrate type and concentration and agar concentration [13, 16, 21, 22]. Thus, to select optimal somatic embryogenic cells from propagated cells of “Minihyang,” cells isolated from the 40% Percoll layer [13] were cultured for 4 weeks in the basic MT medium containing 500 mg·L− 1 malt extract, 70 g·L− 1 lactose, and 14 g·L− 1 agar, resulting in green globular embryos. However, these globular embryos failed to develop into normal plants unless the medium contained plant growth hormones and diverse carbohydrate sources owing to an abnormality that prevents cotyledon formation, similar to what occurs in abnormal globular embryos of the satsuma mandarin variety C. unshiu “Miyagawa wase” reported in previous studies [13, 16]. Although some somatic embryos showed shoot formation, that number was exceedingly low, constituting a mere 0.5% of the total embryo population, and the time required for shoot formation was long.
According to previous studies [13, 16], somatic embryos of some citrus cultivars can be induced using a culture medium containing 0.1 M sorbitol and 0.1 M galactose as carbohydrate sources, 500 mg·L− 1 malt extract, 1.0 mg·L− 1 GA3 as the plant growth hormone, and 2 g·L− 1 gelrite. After the obtained somatic embryos were transferred to EMS medium containing 500 mg·L− 1 malt extract, 50 g·L− 1 sucrose, and 8 g·L− 1 agar without added hormones and cultured for 10 weeks, seedling was achieved at > 60% [16]. In contrast, somatic embryos from the satsuma mandarin variety developed into normal plants after the generation of abnormal plants in medium with GA3, followed by 4 weeks of culture in medium without GA3 [16]. Herein, somatic embryos of “Minihyang” and sweet blood orange “Moro” were cultured in MS medium containing 0.05 M sorbitol, 0.05 M galactose, 500 mg·L− 1 malt extract, 0.5 mg·L− 1 GA3, and 2 g·L− 1 gelrite. Regarding “Minihyang,” this led to the formation of shoots or roots from the abnormal globular embryos or the co-occurrence of shoots and roots after 3 weeks. However, using increased concentrations of the plant growth hormone and carbohydrate sources, similar to those used by Jin et al. [16], resulted in chlorophyll pigment loss, leading to a browning phenomenon after 4 weeks of culture. Thus, the optimal conditions for the regeneration of plants using somatic embryos of “Minihyang” were 0.05 M sorbitol and 0.05 M galactose as carbohydrate sources and 0.5 mg·L− 1 GA3 as the plant growth hormone. “Moro” plants (which were used as the control group) obtained through anther culture also exhibited optimal regeneration under the same culture conditions as those for “Minihyang.” The results of this study are consistent with those of Jin et al. [16].
Notably, approximately 9 months were required for plant regeneration from cultured cells of unfertilized and undeveloped seeds of “Minihyang,” similar to the time required for the regeneration of plants from cultured cells of ovules in the study by Jin et al. [16]. Therefore, our tissue culture system established for “Minihyang” is expected to enable the acquisition of plant materials to develop outstanding citrus cultivars using bioengineering techniques.