Translating theory into practice for conservation of germplasm of recalcitrant-seeded species

Abstract

Recalcitrant seeds, which are metabolically-active and desiccation-sensitive, can be stored in the short-term at non-injurious temperatures under conditions precluding dehydration, but associated problems include fungal proliferation and initiation of germination. The only means for long-term conservation of the germplasm of recalcitrant-seeded species is by cryopreservation in liquid nitrogen (LN), which is not possible for intact seeds. The means to achieve this _ along with attendant problems which are gradually being overcome _ constitute the main subject matter of this paper. Hence, excised embryonic axes (or occasionally, embryos) are the ideal explants to be cryopreserved. As a priori requirements, however, a non-injurious decontamination procedure, and a suitable medium for in vitro germination of the axes must be developed. Thereafter, preparative procedures to condition axes for LN immersion must be refined, implemented and their possible impact on axis viability ascertained at each stage. These include: assessing the effects of cryoprotectants, to which axes of some species are intolerant and determining the responses of axes (whether cryoprotected or not) to flash drying, which involves ascertaining the least amount of water to be very rapidly removed to facilitate cooling to cryogenic temperatures without lethal ice formation. Further pivotal parameters to be optimised are the rate of cooling to cryogenic temperatures and the nature of the medium used for re-warming (thawing) and rehydration of explants after cryopreservation. Each step in the cryopreservation protocol, including excision, is accompanied by generation of free radicals/reactive oxygen species (ROS), the effects of which can be lethal _ and particularly because the consequent damage is cumulative. Hence, for successful cryopreservation, it is imperative that means are developed _ as discussed _ to counteract ROS-mediated damage. Keywords: Cathodic water, cryopreservation, desiccation-sensitive seeds, embryonic axes, reactive oxygen species, ROS-mediated damage.