Systematic optimization enables high-efficiency stable and transient transformation of Spirodela polyrhiza (Greater Duckweed)

The in vitro transformation of plants, or the delivery of foreign genetic material that is incorporated into their genomes, represents a powerful tool both for elucidating genotype-phenotype relationships and for generating plant cultivars which have desirable traits. However, outside of a few model species, the processes involved in transformation are often inefficient, taking months to perform for many plant species, with several bottlenecks at the different stages of calli induction, genetic transfection, and plant regeneration. While duckweeds – aquatic monocots that are the smallest and fastest-growing flowering plants – have distinguished themselves with several emerging biotechnological applications, they too are the subject of conflicting reports regarding their transformation potential. Here, we synthesized and optimized the protocols for in vitro transformation of duckweed Spirodela polyrhiza (Greater Duckweed) from start-to-finish: achieving > 90% − 100% efficiencies for each of calli induction; transient and stable genetic transformation; visual marker-free selection of transformants; and regeneration of genetically modified plants with stable transgene expression for over 100 generations – and which in S. polyrhiza can be achieved over the course of weeks instead of months. These approaches overcome many bottlenecks and help to pave the way for functional genomics studies and synthetic biology applications in this biotechnologically important species. An optimized transformation protocol achieves nearly 100% efficiency in Spirodelapolyrhiza, establishing duckweed as a robust, reliable platform for genetic studies andadvancing plant biotechnology applications.