Plant regeneration of meristematic callus of white clover (Trifolium repens L.) cooled to −196°C by vitrification

Abstract

A callus line of white clover capable of forming numerous meristemoids (meristematic cell masses) has been selected and subcultured on agar B5 medium containing 0.5 mg/l 2,4-D and 0.5 mg/l kinetin for three years. The meristematic callus was successfully cryopreserved by vitrification and subsequently regenerated plants. Preculturing callus in liquid B5 medium containing 0.6 M sorbitol at 25°C for 16 hr was essential to the process. Precultured samples (50 mg) were transferred to a 1.8 ml plastic cryotube and then 1 ml of a highly concentrated cryoprotective solution (designated PVS2) was added and mixed. After treatment with PVS2 at 25°C for 7 min or 0°C for 20 min, the sample was directly plunged into LN. After rapid warming, PVS2 was drained from the cryotubes and replaced twice with liquid B5 medium containing 1.2 M sucrose. Samples were transferred onto filter disc over agar B5 medium. Some surviving cells in the cryopreserved meristematic callus proliferated and produced new meristemoids. After 30 days the meristematic callus was transferred onto hormone-free MS agar medium. The meristemoids developed directly into shoots and spontaneously formed roots. Plant regeneration efficiency expressed as a percent of control amounted to about 90%. This vitrification method appears promising as a routine method for cryopreserving meristematic callus of white clover.