Elimination of chrysanthemum stunt viroid and chrysanthemum chlorotic mottle viroid from infected chrysanthemum by cryopreservation

Chrysanthemum morifolium ‘Borami’ and ‘Secret Pink’ showing symptoms of stunt disease caused by chrysanthemum stunt viroid (CSVd) and ‘Yellow Cap’ showing chlorotic mottle disease caused by chrysanthemum chlorotic mottle viroid (CChMVd) were confirmed to be infected by the respective viroids by using reverse transcription polymerase chain reaction (RT-PCR). Real-time PCR results showed that the viroid concentrations in the infected cultivars varied between the different regions of origin (Chilgok, Gumi, and Gyeongsan). We applied a cryopreservation protocol for elimination of CSVd from naturally infected ‘Borami’ collected from Gumi, showing the lowest concentration of CSVd, by varying several factors such as plant vitrification solutions (PVS2 and PVS3), duration of exposure to liquid nitrogen, shoot-tip size, and low-temperature treatment. The solution (PVS2) and low-temperature treatment were found to be critical factors determining the efficacy of viroid elimination. We optimized the protocol by combining of all resulted optimal factors and tested the applicability of the protocol in ‘Borami’ collected from Chilgok and Gyeongsan and in ‘Secret Pink’ from Chilgok, Gumi, and Gyeongsan, which displayed different viroid concentrations. We found that the elimination rates varied depending on the cultivar and region of origin. Similar results were observed when the protocol was applied to eliminate CChMVd from the ‘Yellow Cap’ collected from the same regions. Finally, we found that nested PCR is more reliable for viroid detection than RT-PCR. Overall, cryopreservation can be used to eliminate viroids from infected chrysanthemums; however, the efficacy depends on genotype and initial viroid concentration.