Cryopreservation of viroid-infected chrysanthemum shoot tips

Chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd) are the two viroids most frequently infecting chrysanthemum. This study attempted to cryopreserve in vitro shoot tips of Chrysanthemum morifolium 'Piato' infected with high or low titers of CSVd, and co-infected with CSVd and CChMVd. By optimizing several key factors including the time and light regimes during cold-hardening of stock shoots, and the size of shoot tips, an encapsulation-vitrification procedure was established for cryopreservation of viroid-infected shoot tips. Viroid-infected stock shoots were cold-hardened in vitro at 4 °C in a 16-h photoperiod for 6 weeks. Shoot tips (1.5 mm in size) containing 3-4 LPs were excised from the cold-hardened stock shoots and subjected to encapsulation-vitrification cryopreservation. With this protocol, about 65%, 45% and 42% of shoot regrowth levels were obtained in cryopreserved shoot tips derived from in vitro stock shoots infected with low or high titers of CSVd, and co-infected with CSVd and CChMVd, respectively. All regenerants from cryopreservation maintained their viroid-infected status, identical to those of their in vitro stock shoots. Histological observations and in situ hybridization elucidated why cryopreservation could maintain viroids in cryopreserved shoot tips. Cryopreservation of viroid-infected plant materials has potential applications to all types of viroid-related basic and applied studies.