The Elimination of Viroids through In Vitro Thermotherapy and a Meristem Tip Culture from a New Limonime Hybrid (Citrus x limon var. limon (L.) Burm. f. x Citrus latifolia var. latifolia)

Viruses and viroids pose a significant challenge in citriculture, and their control is crucial for plant health. This study evaluated the effectiveness of in vitro thermotherapy combined with a meristem tip culture for eliminating citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd) from a new limonime hybrid (<i>Citrus</i> x <i>limon</i> var. <i>limon</i> x <i>Citrus latifolia</i> var. <i>latifolia</i>). The elimination success was confirmed by RT-PCR assays. The in vitro elimination rate for CEVd during the shoot proliferation stage (43%) was higher than for HSVd (21%). Accordingly, in the subsequent rooting stage, the in vitro elimination rate for CEVd (50%) was higher than for HSVd (33%). Successful CEVd and HSVd eradication at a 100% rate was confirmed in the ex vitro acclimatized plants in the greenhouse. The study also established an efficient micropropagation protocol. The optimal treatment for in vitro shoot induction was 0.5-2 mg L^-1 benzyladenine (BA) + 0.5 mg L^-1 gibberellic acid (GA₃) + 0.25 mg L^-1 naphthalene acetic acid (NAA), while for shoot elongation, it was 0.5 mg L^-1 BA + 0.5 mg L^-1 kinetin (KIN) + 0.5 mg L^-1 GA₃ + 0.25 mg L^-1 NAA. Rooting was best promoted by 1 mg L^-1 NAA. This study provides valuable insights for the mass production of viroid-free propagation material in this new lemon x lime hybrid, contributing to the conservation of genetic resources in citrus breeding programs through the combined application of in vitro thermotherapy and an in vitro meristem tip culture, a novel and highlighted achievement reported for the first time in this study.