Cryopreservation of Lilium species and cultivars

Cryopreservation is the method of choice for long-term preservation of valuable germplasm of plants for which a tissue culture method exists. It can be cheaper than field or tissue culture preservation and the risk of loss of specimen by diseases or climate conditions is almost non-existent. We developed a cryopreservation protocol for lily shoot meristems using a vitrification method. It consisted of: i. a preculture period of cold hardening and acclimation for ca 5 days of the meristems; on 0.3 M sucrose at 5 degreesC, ii. a loading step of 20 min with 2 M glycerol with 0.4 M sucrose, iii. a cryoprotectant treatment of 80 min with PVS 2 solution, and iv. fast freezing by plunging. For regrowth fast thawing is necessary at 37 degreesC followed by an unloading/washing step for 20 min in 1.2 M sucrose, or, alternatively, by direct thawing in the unloading solution. Further recovery and regrowth was on normal lily medium in the dark. This method was successfully applied to a number of cultivars and lily species with survival rates from 60 to 90 %. We made a calculation model which showed that cryopreservation is cheaper than tissue culture preservation if more than ca 90 admissions are involved. With a slightly modified protocol we were able to cryopreserve root tips with survival rates of 90 % or more for all tested cultivars. This root tip cryopreservation method could have high potential, because of the easiness to obtain the starting material. The same method was successfully applied to meristems of some hyacinth cultivars, and calli of lily and iris.