Protocol for model-guided alternating high-temperature thermotherapy to eliminate major viruses from diverse sugarcane cultivars.

Vegetatively propagated sugarcane can accumulate multiple viruses that compromise planting-material health. Here, we present a model-guided alternating high-temperature thermotherapy (AHTT) protocol to eliminate major viruses from diverse sugarcane cultivars. We describe preparation and disinfection of single-bud axillary-bud cuttings, planting, thermal preconditioning, and a 12-day AHTT regimen. We then detail post-thermotherapy recovery, health scoring, leaf sampling, total-RNA extraction, control-validated one-step RT-PCR, result interpretation, and the detection of additional sugarcane viruses. For complete details on the use and execution of this protocol, please refer to Huang et al.1.