Citrus plants are important fruit tree species; however, the breeding of high-quality varieties of citrus species is a time-consuming process. Using haploid-derived plants from anther culture may reduce the time required for obtaining purebred lines. This study aimed to genetically verify whether anther culture-derived sour orange (Citrus aurantium L.) plants developed from somatic embryos or haploid tissues. Sour orange anthers were cultured in N6 and MS media to induce calli and somatic embryos. N6 liquid medium supplemented with 1 mg·L⁻¹ gibberellic acid and 200 µM spermidine resulted in a 10% increase in callus and embryo induction rates. Regenerated plants were validated using simple sequence repeat markers. Out of the 109 regenerated plants, ploidy analysis identified 99 diploids, two haploids, and eight putative aneuploids; out of the 99 diploid plants, 33 were haploid-derived homozygous diploids. The chromosomal analysis confirmed most plants as diploids, whereas some were identified as aneuploids (19–21 chromosomes). Furthermore, phylogenetic analysis confirmed that the resultant homozygous or heterozygous plants were haploid-derived. This is the first report of haploid-derived homozygous diploid and aneuploid sour orange plants obtained through anther culture. Moreover, the anther cultivation technique described herein can be applied to other citrus varieties.

This study discusses the challenge of distinguishing between two high-quality mandarin cultivars, ‘Asumi’ and ‘Asuki’, which have been introduced and cultivated in Korea after being developed through crossbreeding in Japan. Owing to genetic similarities resulting from crossbreeding between the same parent cultivars, it is challenging to differentiate them morphologically at the seedling stage. This difficulty poses challenges for cultivation and harvesting on farms. To address this issue, we developed a method using sequence characteristic amplification region (SCAR) markers for rapid and accurate differentiation between the two cultivars. We selected specific primer sets from random amplified polymorphic DNA–SCAR combinations and sequence-related amplified polymorphism contrast markers. The multiplex PCR system using these molecular markers was able to identify 16 mandarin cultivars, including ‘Asumi’ and ‘Asuki’, among 30 cultivars. The use of these SCAR markers is expected to enhance citrus cultivation by accurately identifying mixed cultivars and facilitating proper harvest timing for citrus distribution. Additionally, the markers can help identify the genetic traits of hybrid varieties at the seedling stage.

Min Ju Kim, Mi Sun Kim, Kihye Shin, Sukman Park, Cheolwoo Choi, Su Hyun Yun, and Seong Beom Jin. Korean J. Breed. Sci. 2021;53:16-31. https://doi.org/10.9787/KJBS.2021.53.1.16

Background Citrus ‘Minihyang’ is a novel citrus cultivar created through the hybridization of Citrus kinokuni and Citrus poonensis. This domestic cultivar has a small fruit size and high sugar content, making it highly valuable in the niche market both for ornamental and commercial purposes. Additionally, as a genetic resource, ‘Minihyang’ is highly valuable. However, the lack of a tissue culture system for ‘Minihyang’ could potentially constrain 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 in mature fruits.Results Somatic embryogenic cells were obtained on somatic embryogenesis induction modified Murashige and Tucker (SIME) medium, and the propagated cells were obtained through subculture at 4-week intervals in a modified Murashige and Skoog (EMS) medium. Rather than normal cotyledon-stage embryos, abnormal globular embryos were observed after 4 weeks of culture in an EMS medium containing 70 g·L^− 1 lactose and 16 g·L^− 1 agar. Through the regeneration of plants from these abnormal globular embryos, somatic embryos with developing shoots and roots were obtained within 3 weeks of culture in an EMS medium containing 0.05 M sorbitol and 0.05 M galactose as carbohydrate sources, 0.5 mg·L^− 1 GA₃, and 2 g·L^− 1 gelrite. These embryos were transferred to and cultured in a basic MS medium enriched with 500 mg·L^− 1 malt extract, 50 g·L^− 1 sucrose, and 8 g·L^− 1 agar to produce normal plants.Conclusions ‘Minihyang’ can serve as a promising genetic resource for developing bioactive cultivars using biotechnological techniques and can be used to breed cultivars that can be consumed whole.

Late-maturing citrus cultivars widely cultivated in Korea, such as ’Shiranuhi’, ’Kanpei’, ’Beni Madonna’, and ’Setoka’, are typically grown as grafted trees on trifoliate orange (Poncirus trifoliata) rootstock. Scion-derived root formation at the graft union of P. trifoliata rootstocks results in reduced fruit set and the deterioration of fruit quality. However, in commercial orchards, accurately distinguishing between rootstock- and scion-derived roots solely by morphological characteristics is a considerable challenge. Here, we aimed to accurately differentiate between scion-derived and normal rootstock roots. Accordingly, primers were designed within the promoter region of the LEAFY gene of Poncirus trifoliata clone 1; we subsequently selected two trifoliate orange-specific markers that were exclusively amplified in trifoliate orange. Further, five previously developed sequence-characterized amplified region markers for the identification of scion-derived roots, along with the two newly selected trifoliate orange-specific markers, were applied either individually or in multiplex polymerase chain reaction assays, enabling reliable discrimination between rootstock and scion-derived roots. The results indicated that growers of late-maturing citrus cultivars can accurately identify and selectively remove only scion-derived roots while maintaining normal trifoliate orange roots, thereby ensuring normal fruit set and minimizing economic losses. Additionally, the developed markers successfully distinguished roots in cultivars grafted onto ’Flying Dragon’ rootstock, as well as trifoliate orange. Therefore, the molecular markers established in this study can be effectively applied in citrus orchards to precisely identify and remove damage caused by scion rooting, thereby reducing fruit-set decline and facilitating efficient recovery and management at the farm level.