Cryopreservation of in vitro-cultured Axillary Shoot Tips of Japanese Bead Tree (Melia azedarach) using Vitrification Technique

Cryopreservation of in vitro-cultured Axillary Shoot Tips of Japanese Bead Tree (Melia azedarach) using Vitrification Technique

In vitro-grown axillary buds of Melia aredarach were successfully cryopreserved by vitrification. On the MS medium supplemented with BA 1 mg/L, multiple shoots were developed within 4∼5 weeks. Plantlets of Melia azedarach were cold-hardened at 10∘C for a 16-hr photo-period for 6 weeks. Excised axillary shoot-tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at 25∘C. Axillary shoot-tip meristems wert dehydrated using a highly concentrated vitrification solution (PVS2) for 60 min at 0∘C prior to a direct plunge into liquid nitrogen (LN). The PVS2 vitrification solution consisted of 30% glycerol (w/v), 15% ethylene glycol (w/v), 15% DMSO (w/v) in MS medium containing 0.4M sucrose. After short-term warming in a water bath at 40∘C, the meristems were transferred into 2 ml of MS medium containing 1.2M sucrose for 15 min and then planted on solidified MS culture medium. Successfully vitrified and warmed meristems resumed growth within 2 weeks and directly developed shoots without intermediary callus formation. The survival rate of cold-hardened plantlets for 3 and 4 weeks was 90%. We did not find any difference in PCR-band patterns between control and cryopreserved plants. This method appears to be a promising technique for cryopreserving axillary shoot-tips from in vitro-grown plantlets of Medicinal plants.