AbstractThe citrus industry is confronted with significant challenges posed by viral diseases. However, commercially favored seedless cultivars cannot be propagated through seeds to generate virus-free plants. Obtaining virus-free plants from seedless cultivars has been an important goal for research and practical purposes. In this study, virus-free plants were successfully regenerated from undeveloped ovules of seedless citrus cultivars through somatic embryogenesis. Following rigorous sterilization, the undeveloped ovules, collected from fruits harvested at 210 DAF (days after flowering), were cultured in darkness on MGS medium (MS medium containing 0.5 g L−1 malt extract, 1 mg L−1 GA3, 40 mg L−1 SAD [adenine hemisulfate]) to induce embryoid formation. The embryoids were then transferred to a 16-h photoperiod to promote bud regeneration. Thereafter, the regenerated buds were grafted onto in vitro rootstocks to recover entire plants. Four viruses, including Citrus exocortis viroid (CEVd), Citrus yellow vein clearing virus (CYVCV), Citrus tristeza virus (CTV), and Citrus tatter leaf virus (CTLV), were confirmed to be successfully eradicated in the regenerated plants. Both whole-genome resequencing and single-gene sequence alignment analyses confirmed that the regenerated plants retained genetic fidelity to the maternal plants. Furthermore, we established that the regeneration protocol from undeveloped ovules is an effective and practical method for virus elimination across diverse citrus varieties. These findings provide an alternative strategy for combating citrus viral diseases and achieving germplasm conservation.
Read full abstract