Cryopreservation of Duckweed Genetic Diversity as Model for Long-Term Preservation of Aquatic Flowering Plants

Vegetatively propagating aquatic angiosperms, the Lemnaceae family (duckweeds) represents valuable genetic resources for circular bioeconomics and other sustainable applications. Due to extremely fast growth and laborious cultivation of in vitro collections, duckweeds are an urgent subject for cryopreservation. We developed a robust and fast DMSO-free protocol for duckweed cryopreservation by vitrification. A single-use device was designed for sampling of duckweed fronds from donor culture, further spin-drying, and subsequent transferring to cryo-tubes with plant vitrification solution 3 (PVS3). Following cultivation in darkness and applying elevated temperatures during early regrowth stage, a specific pulsed illumination instead of a diurnal regime enabled successful regrowth after the cryopreservation of 21 accessions of Spirodela, Landoltia, Lemna, and Wolffia genera, including interspecific hybrids, auto- and allopolyploids. Genome size measurements revealed no quantitative genomic changes potentially caused by cryopreservation. The expression of CBF/DREB1 genes, considered as key factors in the development of freezing tolerance, was studied prior to cooling but was not linked with duckweed regrowth after rewarming. Despite preserving chlorophyll fluorescence after rewarming, the rewarmed fronds demonstrated nearly zero photosynthetic activity, which did not recover. The novel protocol provides the basis for future routine application of cryostorage to duckweed germplasm collections, saving labor for in vitro cultivation and maintaining characterized reference and mutant samples.