Cryopreservation of vanilla (Vanilla planifolia) root-tips: a new alternative for in vitro long-term storage of its germplasm

Abstract

Vanilla planifolia (Orchidaceae) is the natural source of vanillin, which is the most widely appreciated flavour compound in the world. Although vanilla is cultivated throughout the tropics, the species' areas of natural distribution are being severely reduced due to anthropogenic effects. As a result, ex situ preservation actions are necessary to safeguard the threatened diversity of this species. In that sense, cryopreservation of vanilla shoot-tips has been previously reported using the droplet-vitrification technique. However, survival and plant regeneration following this protocol were low and not very reproducible. In this study, we evaluated a new alternative for the cryopreservation of vanilla germplasm by using root-tips as explants and following the vitrification and droplet-vitrification protocols. Survival (~60%) and further regeneration (~40%) were obtained using the droplet-vitrification technique by preconditioning root-tips from in vitro propagated plants on semi-solid MS with 0.3 M sucrose (7 days), exposing to loading solution consisting of 0.4 M sucrose plus 2 M glycerol (20 min) followed by glycerol-sucrose plant vitrification solution PVS3 (45 min on ice), and direct plunging into liquid nitrogen in droplets of PVS3 deposited on aluminium foils strips. Tissues were rewarmed by plunging the aluminium foils directly in liquid MS enriched with 1.2 M sucrose (15 min) at room temperature. Growth recovery and bud induction were efficiently achieved by culturing cryostored root-tips on MS added with 1 mg L-1 KIN or BAP. Plant regeneration was reached by transferring the induced buds to MS media. This protocol has great potential for long-term conservation of V. planifolia germplasm and of other vanilla relatives.