Cryopreservation of Shoot-Tips of Grevillea scapigera (Proteaceae): a Rare and Endangered Plant From Western Australia

Abstract

Cryopreservation of leaf, petiole, stem pieces and shoot-tips was investigated as a means for long-term maintenance of germplasm of the rare and endangered species, Grevillea scapigera A.S. George. Crypreservation was only achieved using shoot-tips or axillary buds and a slow-cooling regime with the aid of an improvised freezing device. The highest survival of thawed explants (20%) was obtained with 4-week-old in vitro shoot-tips cultured for 48 h in a prefreezing liquid culture medium supplemented with 5% dimethylsulfoxide. The pretreated shoot-tips were frozen in a liquid medium containing 10% dimethylsulfoxide cooled at a rate of 0.5°C/min to -40°C and held at this temperature for 30 min before being plunged into liquid nitrogen. Shoot-tips that survived the freeze-thaw cycle produced callus followed by shoot production 22 weeks after thawing. All shoots regenerated from thawed tissues and transferred to soil appeared phenotypically identical to untreated control shoots and plants. Rapid methods for assessing post-thaw tissue viability and explant recovery using triphenyltetrazolium chloride were tried but these methods were inadequate for determining the capacity of thawed tissues to recover from freeze damage.