Application of encapsulation-vitrification in combination with air dehydration enhances cryotolerance of Chrysanthemum morifolium shoots tips

• In vitro shoot tips were successfully used in optimizing cryopreservation protocol.
• Factors affecting cryotolerance by encapsulation-vitrification has been optimized.
• A protocol termed encapsulation-vitrification-dehydration (EVD) is also proposed.
• The EVD protocol was better than optimized encapsulation-vitrification protocol.
• The new EVD protocol showed improved recovery with all the cultivars tested.

In this study, we report an efficient encapsulation-vitrification-dehydration protocol for cryopreservation of Chrysanthemum morifolium shoot tips. Various factors influencing cryopreservation such as explant size (1.0–2.0 mm), carbon source (0.0–0.9 M sucrose, glucose, fructose and maltose), duration of pre-culture (0–3 days), duration of treatment with loading solution (0–300 min), duration of treatment with plant vitrification solution (0–120 min with PVS2 or PVS3), and duration of air-dehydration (0–6 h) were investigated. Shoot tips of 1.0 mm size that were encapsulated, and pre-cultured for 1 day in medium enriched with 0.6 M sucrose, treated with loading solution for 180 min, dehydrated with PVS3 for 60 min, and sequentially air dried for 4 h (33.0% water content) in laminar air flow cabinet prior to exposure with liquid nitrogen (LN) for 1 h was found to be the most suitable regime for cryopreservation of Chrysanthemum shoot tips. Under optimal conditions, the highest survival rate of 91.3%, and shoot regrowth rate of 80.3% was obtained using cultivar ‘Borami’. The method developed was successfully adapted to three more cultivars, ‘Secret pink’, ‘Yellow cap’, and ‘White ND’ which showed a survival rate of 60.3–87.3%, and shoot regrowth rate of 55.3–70.0%. To our knowledge this could be the first report in cryopreservation combining the advantage of encapsulation-vitrification and encapsulation-dehydration. We believe that the developed protocol will be a tool to conserve Chrysanthemum germplasm.