Robust Agrobacterium-Mediated Transient Expression in Two Duckweed Species (Lemnaceae) Directed by Non-replicating, Replicating, and Cell-to-Cell Spreading Vectors

Abstract

Plant-based transient expression systems have recognized potential for use as rapid and cost-effective alternatives to expression systems based on bacteria, yeast, insect, or mammalian cells. The free-floating aquatic plants of the Lemnaceae family (duckweed) have compact architecture and can be vegetatively propagated on low-cost nutrient solutions in aseptic conditions. These features provide an economically feasible opportunity for duckweed-based production of high-value products via transient expression of recombinant products in fully contained, controlled, aseptic and bio-safe conditions in accordance with the requirements for pharmaceutical manufacturing and environmental biosafety. Here, we demonstrated Agrobacterium-mediated high-yield transient expression of a reporter green fluorescent protein using deconstructed vectors based on potato virus X and sweet potato leaf curl virus, as well as conventional binary vectors, in two representatives of the Lemnaceae (Spirodela polyrhiza and Landoltia punctata). Aseptically cultivated duckweed populations yielded reporter protein accumulation of >1 mg/g fresh biomass, when the protein was expressed from a deconstructed potato virus X-based vector, which is capable of replication and cell-to-cell movement of the replicons in duckweed. The expression efficiency demonstrated here places duckweed among the most efficient host organisms for plant-based transient expression systems, with the additional benefits of easy scale-up and full containment.

The “Duckweed Dip”: Aquatic Spirodela polyrhiza Plants Can Efficiently Uptake Dissolved, DNA-Wrapped Carbon Nanotubes from Their Environment for Transient Gene Expression

“…It is notable that S. polyrhiza can not only uptake plasmid-wrapped CNTs but also then express genes encoded on that DNA without requiring the usual direct delivery of large doses of Agrobacterium tumefaciens or DNA-loaded nanomaterials directly into plant tissue. While there had been previous reports of transient expression using agrobacterium-mediated approaches in S. polyrhiza , the phenomenon reported here appears to be a significantly more efficient method for transient gene expression. The presence of the introns in the GUS gene strongly suggests that the plasmid DNA is being transcribed, the resulting RNA is processed and spliced properly, and transgenes are expressed within the S. polyrhiza fronds.…”

“…This can be performed in plants using transient expression systems via agrobacterium-mediated infiltration of whole plants, including for S. polyrhiza . However, agrobacterium-mediated approaches tend to have low success rates (in S. polyrhiza ), can exhibit plant toxicity, and require delicate handling of the duckweeds, including removal of the fragile plantlets from water onto solid media and back again. , In terrestrial plants, an alternative strategy for gene delivery that uses DNA-wrapped carbon nanotubes (CNTs) can also be an effective method to deliver plasmid DNA for transient expression. , In terrestrial plants, these methods typically require manual infiltration of the nanotubes into plant tissue by wounding and injection with a blunt-tipped syringe, which would be difficult in the case of duckweed given its small size and fragility. We hypothesized that we could develop a system whereby simply incubating duckweeds with the DNA-wrapped single-wall CNTs (DNA-CNTs) would result in successful gene delivery, because they are so good at extracting material from their aquatic environments.…”

Duckweeds for Phytoremediation of Polluted Water

“…Simultaneously, rapid advances in genome sequencing have revealed genes and metabolites responding to a growing number of specific pollutants and have provided valuable new information regarding the biochemical pathways underlying the uptake and assimilation of major pollutants by duckweed that are discussed in this review. This information, together with the optimized protocols for the genetic transformation of many duckweed species [ 165 , 166 , 167 , 168 ], should complement traditional selection efforts aimed at developing duckweed into an even more effective tool for protecting our environment and making our water as clean.…”

Biodiversity of Duckweed (Lemnaceae) in Water Reservoirs of Ukraine and China Assessed by Chloroplast DNA Barcoding

“…Prior to genotyping, most specimens from China were sterilized and kept under aseptic conditions on agar medium according to previously described methods [ 43 ]. These specimens were kept at the duckweed in vitro collection recently organized at Huaiyin Normal University, Hui’an, China [ 47 ], barcoded and used in our previous studies [ 11 , 18 , 52 , 53 ]. The Ukrainian duckweed specimens were collected from water reservoirs, sorted according to their morphological characteristics, and directly subjected to DNA extraction for further chloroplast DNA barcoding.…”

“…Duckweeds have also been studied for their use in wastewater treatment [ 10 , 11 ], biosensing [ 12 , 13 ], and phytoremediation of water reservoirs contaminated with various toxic chemicals [ 14 , 15 ]. Several duckweed species have been genetically engineered with the eventual aim of producing pharmaceutical proteins such as antigens, peptide hormones, and antibodies [ 16 , 17 , 18 ].…”