Articles
CRYOPRESERVATION OF DENDRANTHEMA MORIFOLIUM ‘JAPANESE RED’ SHOOT TIPS BY DROPLET-VITRIFICATION
Article number
1039_24
Pages
187 – 192
Language
English
Abstract
Chrysanthemum is one of the most popular flower crops worldwide.
Development and availability of cryopreservation technique is necessary for long-term conservation of genetic resources.
In the present study, we describe a droplet-vitrification procedure for cryopreservation of Dendranthema morifolium Japanese Red. In vitro stock shoots were maintained on Murashige and Skoog (1962) medium supplemented with 30 g/L sucrose and 7 g/L agar (pH=5.8) and kept at 24±2°C under a 16-h photoperiod with a light intensity of 50 µmol m-2 s-1 provided by fluorescent tubes.
Shoot tips (1-2 mm in size with 3-4 leaf primordia) were excised from 4-week-old in vitro shoots and precultured on solidified MS medium containing 0.5 M sucrose for one day.
Precultured shoot tips were loaded with a loading solution made of MS containing 2 M glycerol and 0.4 M sucrose for 20 min at room temperature, followed by dehydration with PVS2 for 30 min at 0°C. Each shoot tip was transferred onto a 2.5 µl PVS2 on aluminum foils (2 × 0.8 cm), prior to a direct immersion in liquid nitrogen (LN). After immersed in LN for a few minutes, the foils with shoot tips were transferred into 2 ml cryotubes for cryostorage.
Frozen shoot tips were thawed by incubation in a solution composed of MS containing 1.2 M sucrose for 20 min at room temperature, and then post-cultured on MS medium containing 0.05 mg/L GA3 for shoot recovery.
With this procedure, 100% of shoot tips survived and 67% of them regenerated shoots following cryopreservation.
The droplet-vitrification protocol developed in the present study is being tested for its potential applications to cryopreservation of other four genotypes and cryotherapy for virus eradication of chrysanthemum.
Development and availability of cryopreservation technique is necessary for long-term conservation of genetic resources.
In the present study, we describe a droplet-vitrification procedure for cryopreservation of Dendranthema morifolium Japanese Red. In vitro stock shoots were maintained on Murashige and Skoog (1962) medium supplemented with 30 g/L sucrose and 7 g/L agar (pH=5.8) and kept at 24±2°C under a 16-h photoperiod with a light intensity of 50 µmol m-2 s-1 provided by fluorescent tubes.
Shoot tips (1-2 mm in size with 3-4 leaf primordia) were excised from 4-week-old in vitro shoots and precultured on solidified MS medium containing 0.5 M sucrose for one day.
Precultured shoot tips were loaded with a loading solution made of MS containing 2 M glycerol and 0.4 M sucrose for 20 min at room temperature, followed by dehydration with PVS2 for 30 min at 0°C. Each shoot tip was transferred onto a 2.5 µl PVS2 on aluminum foils (2 × 0.8 cm), prior to a direct immersion in liquid nitrogen (LN). After immersed in LN for a few minutes, the foils with shoot tips were transferred into 2 ml cryotubes for cryostorage.
Frozen shoot tips were thawed by incubation in a solution composed of MS containing 1.2 M sucrose for 20 min at room temperature, and then post-cultured on MS medium containing 0.05 mg/L GA3 for shoot recovery.
With this procedure, 100% of shoot tips survived and 67% of them regenerated shoots following cryopreservation.
The droplet-vitrification protocol developed in the present study is being tested for its potential applications to cryopreservation of other four genotypes and cryotherapy for virus eradication of chrysanthemum.
Authors
Ren-Rui Wang, Xiao-Xia Gao, Long Chen, Qiao-Chun Wang
Keywords
Dendranthema morifolium, cryopreservation, droplet-vitrification, shoot tips
Online Articles (39)