An efficient droplet-vitrification cryopreservation procedure for high imperatorin-yielding hairy root clones of Urena lobata

The valuable anti-cancer and anti-inflammatory secondary metabolite, imperatorin, has been found in the hairy roots (HRs) of Urena lobata. However, an increasing number of problems related to cryo-injury and cryoprotectant toxicity could potentially reduce the quality of root clones, highlighting the need to develop a reliable technique for long-term preservation. Based on the impact of the successive steps of the initial droplet-vitrification procedure employed for cryopreservation of HRs using the histological evaluation of plasmolysis, various selected factors were independently investigated. The maximum plasmolysis was observed after the preculture with 0.5 M sucrose (46.59 %) and the dehydration treatment (48.32 %). In the improved cryopreservation procedure, when prolonged preculture for 72 h in liquid WPM with 0.3 M sucrose and dehydration in the appropriate vitrification solution, which included 30 % (w/v) glycerol and 50 % (w/v) sucrose, for 10 min at 0 °C, the plasmolysis in the two steps was significantly reduced in comparison with the untreated control. The cryopreserved HRs could increase their regeneration to 93.3 % and regenerated root length to 4.65 cm. Their growth and imperatorin production were almost the same as those of untreated controls after three to five subsequent subcultures. Our results have demonstrated that our new approach, which only focuses on the key factors that significantly increase the plasmolysis, modifies their level to fit with the HR cells of U. lobata. The early application of the plasmolysis evaluation method may immediately screen the impact of factors on individual root cells instead of spending time and cost evaluating the recovery of root tips at each step to develop an efficient cryopreservation procedure.