Differential Expression Analysis of Cannabis sativa response to thermotherapy of Hops Latent Viroid (HLVd) infection and clearance of the viroid in Tissue Culture Micropropagation

Increased farming and cultivation of Cannabis sativa is rapidly pushing Cannabis (Cannabis sativa L.) towards becoming a commercial agricultural commodity. Large-scale cultivation facilities maintain thousands of clonal varieties of recreational and medicinal cannabis and there is a strong market-driven motivation to maintain a commercial pipeline of clean healthy vigorously growing plants free of pathogens. However, mass production and high-capacity cultivation create an environment that is susceptible to highly transmissible pathogens and infectious entities such as Hop Latent Viroid (HLVd). From nurseries to cultivation facilities, its become increasingly important to maintain a routine testing regimen and ensure cultivation facilities are HLVd-free environments. One method to address the problem of infected plants is to perform thermotherapy on C. sativa explants under tissue culture conditions and isolate clean tissue to multiply productive and healthy mature plants. We carried out a novel thermotherapy method using tissue culture in 5 varieties of type III cannabis that were HLVd positive to document the degree of success of the treatment at the RNA level. We observed that following thermotherapy treatment we were able to decrease the level of HLVd positive tests in select varieties and observed some varieties were highly susceptible and unable to clear the viroid. Plants were tested using a one-step RT-qPCR method, developed and validated, in part, along with this work, and present the results as well as an exploratory transcriptome analysis of an internally developed variety, AnnaLee, which tested negative for HLVd following thermotherapy treatment, and explore possible genes of interest for viroid infection, clearance, and mitigation. Author SummaryRT-qPCR and transcriptome analysis of Hops latent viroid (HLVd) in infected and non-infected Cannabis varieties. A thermotherapy procedure was conducted on HLVd infected Cannabis sativa meristem tissue maintained through tissue culture micropropagation techniques. Total RNA was isolated from the cultured plantlet stocks and evaluated by a real-time reverse transcriptase assay for HLVd. Infection status post thermotherapy was assessed, and viroid-free plants were maintained and subsequently tested. A single thermotherapy-treated cultivar, Anna Lee was selected for transcriptomics, and an analysis of the genes that were differentially regulated in infected and non-infected treated plants is discussed.