Abstract
This study develops protocols for the micropropagation and cryopreservation of Dracocephalum austriacum (Lamiaceae). It is a perennial herbaceous plant that overwinters with ground-level sprouts and is classified as critically endangered in Europe. In vitro cultures were initiated from seeds on growth-regulator-free Murashige & Skoog (MS) medium after nicking the seed coat. Propagation via shoot culture was achieved on ½ MS medium with 1 µM benzyl adenine (BAP). Rooting on various indole-3-butyric acid (IBA)-media was not reliable, but the rooting success was 80% after 10 weeks on medium with 1 µM BAP. Two starting materials underwent cryopreservation: (1) shoot tips from cold-acclimated in vitro plantlets and (2) axillary buds from winter shoots from field plants. For the cryopreservation of in vitro shoots, plant vitrification solution (PVS)3 and incubation over ice yielded the best results (~ 34% regeneration success). However, regeneration using winter acclimated buds were 100, 76 and 30% for collections in December, February and March, respectively, using the same protocol. Moreover, the ploidy levels of cryopreserved plantlets were estimated using flow cytometry. The use of winter-acclimated field material of temperate herbaceous plants or subshrubs has high potential as explant source for cryopreservation and calls for exploring this technique for other species.
Highlights
Austria’s range of landscapes comprises approximately 2950 vascular plant species (Rabitsch and Essl 2008) and is host to a significant portion of threatened European species
A CV% ≤ 3% is desirable, but in plants containing essential oils such as Dracocephalum, values ≤ 5% are acceptable. This is a rare report on a dicotyledonous herbaceous plant being cryopreserved using winter-acclimated buds from field material
The use of naturally cold-acclimated field material proved to be more successful for the cryopreservation of D. austriacum than using shoot tips from cold-acclimated in vitro plants
Summary
Austria’s range of landscapes comprises approximately 2950 vascular plant species (Rabitsch and Essl 2008) and is host to a significant portion of threatened European species. Alongside all European Union (EU) member states, Austria has committed to the Global Strategy for Plant Conservation. (GSPC), a worldwide initiative for halting biodiversity loss by advancing plant conservation. One GSPC target is to have at least 75% of the threatened plant species in ex situ collections (CBD 2019). Ex situ conservation measures include seed storage in seed banks, in vivo living plant collections, e.g. in botanical gardens, as well as in vitro culture and cryopreservation. D. austriacum is recognized as endangered in Europe and listed in Appendix II and IV of the EU’s Fauna-Flora-Habitat Guidelines, which specify a strict protection regime (The Council of the European Communities 1992). Its native range spreads over Central and Eastern Europe, with outposts in the Spanish and French Pyrenees in the west to populations in Ukraine, the
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