In this study, an anther culture system was developed for two Citrus varieties known for their genetic value: blood orange (Moro) and mandarin (Lee). Anthers were inoculated on N6 solid medium containing thidiazuron (TDZ, 0.44 mg/L), 6-benzylaminopurine (0.8 mg/L), zeatin (0.43 mg/L), kinetin (0.44 mg/L), 1-naphthaleneacetic acid (0.2 mg/L), 2,4-Dichlorophenoxyacetic acid (0.2 mg/L), and malt extract (500 mg/L). The inoculated anthers were treated with N6 liquid medium containing spermidine (200 mu M) and gibberellic acid (GA3, 1 mg/L), and cultured for 6 weeks. Thereafter, the swollen anthers were transferred to a Murashige and Skoog (MS) basal medium enriched with malt extract (500 mg/L), sucrose (50 g/L), TDZ (0.5 mg/L), GA3 (1 mg/L), and gelrite (0.2%), which induced callus and somatic embryos. These somatic embryos, from both varieties, were then transferred to a germination medium (MS basal medium containing sorbitol [0.05 M], galactose [0.05 M], malt extract [500 mg/L], GA3 [0.5 mg/L], and gelrite [2 g/L]) to develop into normal plants. However, Lee exhibited significantly slower shoot and root growth compared to Moro. Genetic analysis using barley microsatellite-derived cleaved amplified polymorphic sequence markers indicated that Lee likely originated from haploid plants, whereas Moro retained heterozygosity similar to the parent. Ploidy analysis confirmed Lee as a diploid, identical to the control. Internal transcribed spacer region analysis confirmed that Lee was an anther-cultured haploid-derived plant, estimated to be a homozygous diploid carrying recessive genes. These findings highlight potential applications in marker development for haploid-derived plants focused on recessive trait-associated phenotypes and genotypes. The doubled haploid mandarin 'Lee' exhibits dwarfism, as evidenced by poor elongation of new shoots, and can serve as a model to study dwarfism mechanisms in citrus species
