Cryopreservation of Ashe magnolia shoot-tips by droplet-vitrification

The Magnoliaceae is one of the oldest families of the angiosperms, and although they are mainly known for their ornamental and horticultural uses. They also have medicinal properties. Half of the Magnolia species are considered to be endangered on a global scale, two-thirds of data deficient species are included. Therefore, an efficient cryopreservation method for the specimens of interest that are maintained in living collections or collected in the wild would help to ensure their long-term conservation. A study was performed comparing three different pre-culture conditions before cryopreservation. In vitro shoots from Ashe magnolia (Magnolia macrophylla var. ashei (Weath.) D.L. Johnson), were cultivated onto a basal medium (CTRL) or sucrose supplemented medium (SUC) at 25 degrees C, and onto the basal medium at 13 degrees C during one week (CLD). Shoot-tips from in vitro donor plants were dissected and cryopreserved using the droplet-vitrification technique. Survival, shoot-tip regrowth, callus formation, oxidation and hyperhydric shoot-tips were recorded four weeks after rewarming from liquid nitrogen. Shoot recovery was recorded twelve weeks after the cryopreservation. Survival and shoot-tip regrowth after cryopreservation were improved by treating the donor plants with sucrose and cold (survival: CTRL 60%, SUC 93%, CLD 100%; shoot-tip regrowth: CTRL 30%, SUC 50%, and CLD 93%). Normal shoot recovery was only obtained when shoot-tips were dissected from donor plants that were cold treated (30%). These results showed that cold acclimation was essential for the plant regeneration from shoot-tips of Ashe magnolia that were submitted to droplet-vitrification. Further studies are ongoing to gain more insight into the regeneration process and improve the recovery rates.