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Abstract
Duckweeds (family Lemnaceae), the smallest and fastest-growing flowering plants, are gaining traction as model systems in plant biotechnology due to their simplified morphology, rapid vegetative propagation, and compact genomes. However, their potential remains underutilized in the context of synthetic biology. This project has three complementary aims. The first aim is to develop a novel nanomaterial-based gene delivery approach—termed the “Duckweed Dip”—in which Spirodela polyrhiza passively uptakes plasmid-wrapped carbon nanotubes (DNA-CNTs) directly from the growth medium. This facile, Agrobacterium-free method enables high-efficiency transgene expression without physical infiltration or high nanomaterial concentrations, offering a streamlined, scalable platform for genetic transformation. In our second aim, to complement the non-invasive strategy, we established a robust tissue culture and transformation pipeline for S. polyrhiza. Among the six tested media, callus induction was most efficient (92%) using 0.1 mg/L 2,4-D and 0.1 mg/L TDZ. Regeneration was achieved on media containing either TDZ or zeatin, depending on the callus origin. Transient and stable transformations were successfully conducted using Agrobacterium tumefaciens strains GV3101 (35S::GUS) and LBA4404 Thy⁻ (ZmUbi::GFP), with transient expression efficiencies up to 95%, and stable transformation resulting in an average of 95 transgenic calli per 100 calli. Transgene expression was confirmed via GFP fluorescence and PCR. As a proof of concept for genome editing, successful gRNA cloning and transformation were demonstrated using a CRISPR/Cas9 vector. Our third aim is to extend these methodologies, we developed a high resolution and high throughput phenotyping platform for Wolffia australiana, the fastest growing and rootless duckweed species. Callus induction using 0.1 mg/L 2,4-D and 0.1 mg/L TDZ achieved a 95% success rate. Stable transformation using Agrobacterium tumefaciens strain LBA4404 Thy⁻ carrying the ZmUbi::GFP construct resulted in approximately 20% transformation efficiency. Out of 100 treated calli, around 20 developed antibiotic resistance and exhibited stable GFP expressions, indicating successful integration of the transgene. For downstream propagation and phenotyping, we fabricated custom PDMS devices containing 28 parallel microchannels (8 mm × 0.5 mm) using high-resolution 3D-printed molds. Floated on hydroponic media, these devices supported robust clonal propagation, with each mother plant generating 2–4 daughter plants per week. Collectively, these integrated platforms facilitate scalable genetic transformation, regeneration, and high-throughput phenotyping in duckweeds unlocking new possibilities for functional genomics, synthetic biology, and studies of plant microenvironment interactions. Keywords: Duckweed, Lemnaceae, Spirodela polyrhiza, Wollfia australiana, Carbon Nanotubes, Transient Expression, Tissue Culture, Stable Transformation, Transient Transformation, Gene editing, CRISPR/Cas9, Morphogenic Regulators, 3D printing, Phenotyping