Induction and genetic verification of homologous double haploid plants obtained through the anther culture of Citrus cultivars

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 µM) 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