Freeze-preservation of pea meristems in liquid nitrogen and subsequent plant regeneration

Abstract

A procedure has been developed for the freeze-preservation of pea ( Pisum sativum L.) meristems and subsequent plant regeneration. The meristems were precultured in the presence of 5% DMSO for 48 h followed by freezing at cooling velocities varying from 0.5 to 1.0°C/min up to a terminal freezing temperature of −40°C and subsequently stored in liquid nitrogen (−196°C). Maximum survival as determined by the regrowth of meristems into whole plants was obtained at a cooling rate of 0.6°C/min after 1 h storage in liquid nitrogen (73%). The meristems were in storage in liquid nitrogen for up to 26 weeks and upon reculture more than 60% of the meristems differentiated into whole plants. Cold conditioning of meristems at 4°C prior to freezing without preculture in the presence of DMSO resulted in lower survival rates. Glycerol and ethylene glycol were ineffective cryoprotectants. Ultra-rapid freezing of meristems by direct immersion in liquid nitrogen was also lethal.