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

Abstract

-In vitro-grown axillary buds ofMelia azedarachwere successfully cryopreserved by vitrification. On the MS mediumsuplemented with BA1 mg/L, multiple shots were developed within 4 5 weks. Plantlets of～ Melia azedarachwere cold-hard-℃ -idified Murashige & Skog agar medium (MS) suplemented with 0.7 M sucrose for 1 day at 25 . Axillary shot-tip meristems℃weredehydratedusingahighlyconcentratedvitrificationsolution(PVS2)for60minat0 priortoadirectplungeintoliquidnitro℃ -gen (LN). The PVS2 vitrification solution consisted of 30% glycerol (w/v), 15% ethylene glycol (w/v), 15% DMSO (w/v) in MSmediumcontaining0.4Msucrose. Aftershort-termwarminginawaterbathat40 , themeristemsweretransferedinto2mlofMS℃meristems resumed growth within 2 weeks and directly developed shoots without intermediary callus formation. The survival rateof cold-hardened plantlets for 3 and 4 weeks was 90%. We did not find any diference in PCR-band patterns between control andcryopreserved plants. This method apears to be a promising technique for cryopreserving axillary shot-tips fromin vitro-grownplantlets of Medicinal plants.Key words -Cryopreservation, Masspropagation,Melia azedarach, Vitrification technique