Cryopreservation by Vitrification for the Aquatic Plant Family Lemnaceae

Cryopreservation provides the conditions necessary for the long-term storage of plant germplasm, ensuring its viability and genetic stability over potentially limitless time periods. To enable regrowth of the propagated vegetatively plant germplasm, the plant material must undergo cryoprotection treatment before cryo-cooling and rewarming, followed by washing and revitalization procedures. Dehydration of plant tissue during cryoprotection often results in a substantial decrease or complete inhibition of regrowth after cryopreservation. Here, we describe a vitrification-based cryopreservation protocol for the genera Spirodela, Landoltia, Lemna, and Wolffia, which are mostly vegetatively propagating flowering plants belonging to the Lemnaceae family (duckweeds), and serve as a model for aquatic plants. Our protocol involves cryo-cooling whole duckweed plants and overcomes their inherent high desiccation sensitivity and exceptional buoyancy, which are characteristics of their aquatic lifestyle. Additionally, we describe the introduction and maintenance of duckweed in an aseptic in vitro culture. Overall, this protocol has been successfully applied to over 25 duckweed accessions from four genera.