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.

Cyprinid herpesvirus-3 (CyHV-3) is highly contagious and lethal to cyprinid fish, necessitating development of sustainable vaccination strategies. Our study explores duckweed as platform for vaccine production and as vehicle for oral administration which is considered most suitable for mass vaccination of fish. Using a deconstructed potato virus X-based transient expression system, serologically recognizable recombinant CyHV-3 antigens (ORF25, ORF81, ORF136, ORF72) and fusion proteins (ORF136::T2A::ORF72, ORF25::T2A::ORF81) were accumulated in the duckweed, Landoltia punctata. The specific immune response of fish on immunization by the freeze-dried antigen-expressing duckweed biomass was proved by the serum neutralization test. Uptake of green fluorescent protein fused to the adjuvant cholera non-toxic subunit B (CTB::GFP) from duckweed biomass by fish intestinal cells was demonstrated after intubation of fish with CTB::GFP-expressing biomass. Therefore, transient expression of recombinant proteins in duckweed is a promising tool for antigen production and can facilitate the development of oral vaccines for veterinary application.