Improved Method for Cryopreservation of Embryogenic Callus of Fraxinus mandshurica Pupr. by Vitrification

Abstract

In order to simplify the experimental procedure and treatment procedure, we preserved the embryonic callus (EC) of Fraxinus mandshurica more efficiently. In this paper, we established a method for cryopreservation of EC of F. mandshurica by vitrification. EC was subcultured for 7–10 days (d). Vigorous EC with good growth conditions were selected, and cryopreservation was performed by vitrification. The best pre-culture method was to pre-culture EC on 0.5 mol·L−1 sucrose medium for 3 d, load and culture in the liquid woody plant medium (WPM) supplemented with 2 mol·L−1 glycerol and 0.4 mol·L−1 sucrose for 60 min, then dehydrate in 2 mL of plant vitrification solution 2 (PVS2) (30% glycerol + 15% dimethyl sulfoxide (DMSO) + 15% ethylene glycol + 0.4 mol·L−1 sucrose + liquid WPM). EC was rewarmed in a 40 °C water bath for 2 min after cooling in liquid nitrogen. The procedure for cryopreservation of F. mandshurica EC by the vitrification method established in this experiment is relatively reliable. The results from the present study provide a technical reference for improving the cryopreservation of F. mandshurica EC.