Induction and Genetic Verification of Haploid-Derived Plants Using Anther culture of Citrus Aurantium L. (Sour Orange)

Abstract This study aimed to genetically verify whether Citrus aurantium L. (sour orange) plants grown using anther culture were obtained from somatic embryos or haploids. The ploidy of the individuals was assessed using ploidy measuring instruments and simple sequence repeat (SSR) molecular markers. The anthers of sour oranges were cultured onto a medium supplemented with 0.44 mg/L thidiazuron (TDZ), 0.8 mg/L 6-benzylaminopurine (BA), 0.43 mg/L Zeatin, 0.44 mg/L Kinetin, 0.2 mg/L a-naphthalene acetic acid (NAA), 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 500 mg/L malt extract. Somatic embryos and callus were obtained using dentin in N6 medium supplemented with malt extract (500 mg/L) and N6 liquid medium supplemented with spermidine (200 µM), and gibberellic acid (GA3, 1 mg/L) was added to the anther surface. Regenerated plants were obtained from Murashige and Skoog medium supplemented with 0.5 mg/L TDZ, 1.0 mg/L GA3 (or single use 0.5 mg/L) and 500 mg/L malt extract. For the genetic verification of the obtained plants, haploid-derived plants were investigated by polymerase chain reaction using two SSR markers. In addition, verification with a ploidy meter confirmed that approximately 80–90% of the regenerated plants were triploids, and two plants among the investigated plants were haploids. This study was the first to report these results among haploid and triploid studies obtained from an anther culture of sour oranges. The study findings suggested that the anther culture technique can produce seedless triploid cultivars of domestic citrus varieties.