Direct RT-PCR method for detecting two chrysanthemum viroids using minimal amounts of plant tissue

A direct reverse transcription-polymerase chain reaction (RT-PCR) method for detecting the chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd) to screen for a viroid-free chrysanthemum plant at a small plant size was established and named microtissue direct RT-PCR. A razor or syringe needle was used for RNA template preparations. Under a stereoscopic microscope, a razor or syringe needle was used to pierce, a tissue sample to a depth of 0.1-0.2mm, and the sample was directly transferred to the RT mixtures. Methods using razors or needles for the preparation of templates could detect CSVd and CChMVd with a high sensitivity. The most sensitive method used a razor or syringe needle to acquire template from the shoot tips. Using the microtissue direct RT-PCR method, both viroids could be detected from the high- and low-viroid-concentration plants. The microtissue direct RT-PCR method was more sensitive than a conventional template preparation method. Using the microtissue direct RT-PCR method established in this study, the laborious subculture step could be omitted because detecting viroids and screening for viroid-free plants even at a small plant size before the subculture could be possible. In addition, the microtissue direct RT-PCR method could also be a powerful tool for clarifing the viroid distribution among microtissues, such as shoot apical meristems.