Cleaning cassava genotypes infected with cassava frogskin disease via in vitro shoot tip culture

This study aimed to develop a methodology for eliminating cassava frogskin disease (CFSD) from in vitro shoot tip culture by associating thermotherapy and tetracycline. Cuttings from different accessions (BGM0232, BGM0315, BGM0464, BGM584, BGM0841, and BGM1342), infected with CFSD according to visual inspection of the disease symptoms, were used for cleaning. To verify the absence of other diseases, the plants were indexed for Cassava common mosaic virus - CsCMV (by ELISA) and Cassava vein mosaic virus - CsVMV (by polymerase chain reaction, PCR), proving that the accessions were free of these viruses, except for BGM0315 and BGM0464, which were infected with CsVMV. Subsequently, the cuttings were submitted to different tetracycline concentrations for 3 min, and then subjected to thermotherapy under different temperatures (35°, 38°, 40°, 45°, and 55°C). Shoots of 2 cm were harvested, and their surfaces were sterilized in a laminar flow chamber. Subsequently, the shoot tips of different sizes were removed (0.2, 0.4, 0.5, and 1.0 mm) for inoculation in a culture medium with tetracycline at the same concentrations in which the cuttings were dipped. After 60 days of cultivation, the explants were transferred to a multiplication medium without antibiotics. Thirty days after the transfer, the viability of the regenerated plants was evaluated, which were then acclimatized for 70 days in a greenhouse and transferred to the field. After 7 months, a visual analysis of the symptomatic roots and a PCR analysis were held to prove the elimination of CFSD and CsVMV from the accessions infected with these viruses (BGM0315 and BGM0464), respectively. Most of the treatments resulted in 100% cleaning of CFSD-infected plants. From accessions that were also infected with CsVMV, only 2% of the plants remained infected, also demonstrating the cleaning efficiency of this protocol for this disease.