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

Abstract

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.

Keywords: CBF/DREB1 genes; PVS3; cryopreservation; duckweed; the operating efficiency of photosystem II; vitrification.

Figure 1

Combined pulsed illumination regime used for duckweed regrowth after cryopreservation. PPFD—photosynthetic photon flux density.

Figure 2

Spin-drying of duckweed fronds using a perforated aluminum insert: (A) collecting duckweed fronds from culture using perforated foil insert; (B) transferring the insert with the duckweeds into the test tube; (C) spin-drying by centrifugation; (D) transferring the insert with spin-dried duckweed from centrifugal tube into a cryo-tube filled with 1 mL of PVS3. White arrow indicates a cotton wool at the bottom of the centrifugal test tube.

Figure 3

Effect of pre-culture medium on regrowth (calculated according Section 3.7.2) of different duckweed accessions at day 21 after rewarming. Error bars indicate standard deviations (n = 3).

Figure 4

Regrowth of 21 accessions from 4 Lemnaceae genera (21 days after rewarming) obtained after applying the newly developed protocol for duckweed cryopreservation. Blue line indicates average regrowth of all tested duckweed accessions. Error bars indicate standard deviations (n = 3).

Figure 5

Dynamics of minimal chlorophyll fluorescence F₀ (A,B) and operating efficiency of photosystem II Φ_PSII (C,D) of the rewarmed duckweed fronds depending on pre-culture (A,C) and duckweed accessions (B,D). For (A,C), data presented as average values for four accessions of Le. gibba (7742, 7796, 7922, 9602). Error bars indicate standard deviations (n = 3).

Figure 6

Relative mRNA abundance of duckweed CBF1 and CBF2 genes (A) after different pre-culture treatment 5% glycerol and/or 0.4 M sucrose (see Figure 3 for corresponding regrowth data); (B) during preparation of duckweed to cryopreservation, including PVS3 treatment. The start points were used as controls. The relative mRNA abundance was normalized against the mRNA abundance of histone H3 (LgH3). Error bars indicate standard deviations (n = 5).