A NOVEL PRECULTURE METHOD FOR THE INDUCTION OF DESICCATION TOLERANCE IN GENTIAN AXILLARY BUDS FOR CRYOPRESERVATION

Axillary buds of in vitro-grown gentian (Gentiana scabra Bunge var. bu ergeri Maxim.) were successfully desiccated and cryopreserved using a novel two-step preculture method: preculturing axillary buds excised f rom in vitro-grown gentian plants on Murashige and Skoog (MS) semi-sol id medium containing 0.1 M sucrose for 8-13 days at 25 degrees C under a 16 h-photoperiod (first step) and the subsequent short period incub ations on 0.4 M sucrose (MS agar medium) for 1 day followed by 0.7 M s ucrose (MS agar medium) for 1 day (second step). After preculturing, t he buds were air-dried down to about 10% water content (fresh weight b asis) without encapsulation, and then immersed in liquid nitrogen (LN) . With this cryogenic protocol, we could obtain 78-90% of survival of axillary buds following desiccation and LN exposure. Most of the buds that survived showed normal shoot and root development. The most impor tant part of this protocol was the preculture which circumvents the co nventionally used lengthy cold-hardening treatment. The results show t hat induction of desiccation tolerance by preculturing was specific to a few species of sugars used in the medium and not merely caused by o smotic effects. The efficient desiccation-cryopreservation protocol sh own here using the new preculture method appears promising for routine ly cryopreserving gentian germplasm. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.