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Stable efficient genetic transformation of Lemna aequinoctialis. (a) Frond sterile culture; (b) Callus induction; (c) Agrobacterium tumefaciens infection; (d) Callus screening; (e) GUS staining of callus; (f) Callus differentiation; (g) Frond regeneration; (h) GUS staining of transgenic frond after 70 days cultivation; and (i) Regeneration frond liquid cultivation.
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The fast growth, ease of metabolic labeling and potential for feedstock and biofuels production make duckweeds not only an attractive model system for understanding plant biology, but also a potential future crop. However, current duckweed research is constrained by the lack of efficient genetic manipulation tools. Here, we report a case study on g...
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Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) technologies are revolutionizing biological and biomedical research. These affordable and easily programmable molecular modalities enable virtually unlimited genetic manipulations in most organisms and can cure human genetic disorders. CRISPR–Ca...
Citations
... These genetic transformations, utilizing precise techniques such as Agrobacterium-mediated transformation, rapid gene insertion methods, and targeted gene overexpression, not only improve the productivity and sustainability of duckweed but also expand its applications in biotechnology, environmental management, and medical therapeutics. Furthermore, advances in genome editing technologies, such as CRISPR-Cas9, have enabled precise and targeted modifications of duckweed genomes, accelerating the development of transgenic lines with desired traits [68]. Through these transgenic development studies, duckweed can emerge as a promising platform for sustainable biotechnological applications, including bioenergy production, wastewater treatment, and environmental remediation. ...
Duckweed (Lemnaceae) rises as a crucial model system due to its unique characteristics and wide-ranging utility. The significance of physiological research and phytoremediation highlights the intricate potential of duckweed in the current era of plant biology. Special attention to duckweed has been brought due to its distinctive features of nutrient uptake, ion transport dynamics, detoxification, intricate signaling, and stress tolerance. In addition, duckweed can alleviate environmental pollutants and enhance sustainability by participating in bioremediation processes and wastewater treatment. Furthermore, insights into the genomic complexity of Lemnaceae species and the flourishing field of transgenic development highlight the opportunities for genetic manipulation and biotechnological innovations. Novel methods for the germplasm conservation of duckweed can be adopted to preserve genetic diversity for future research endeavors and breeding programs. This review centers around prospects in duckweed research promoting interdisciplinary collaborations and technological advancements to drive its full potential as a model organism.
... Further studies could involve the use of different configurations (e.g., a low-concentration "pool" for duckweed growth before transplantation to a high-concentration "pool" for rapid accumulation before harvesting) or genetic modification of duckweed for heavy metal tolerance (with genetic modification already of interest due to duckweed's relative biological simplicity). 101 The metal contents of the pulp, lignin, and liquor were then determined following pretreatment, allowing the partitioning behavior to be determined. Results are displayed in Figure 8. Large differences were observed in the partitioning behavior of both metals, as well as moderate differences between the performance of both ILs. ...
This study establishes a foundation for the ionic liquid (IL) pretreatment of duckweed biomass. An optimized IL-based process was designed to exploit the unique properties of duckweed including efficient metal removal, potential starch accumulation, and protein accumulation. Two ILs, namely, dimethylethanolammonium formate ([DMEtA][HCOO]) and N,N-dimethylbutylammonium hydrogen sulfate ([DMBA][HSO4]), were investigated for the pretreatment of two duckweed species (Spirodela polyrhiza and Lemna minor). The evaluation focused on starch recovery, sugar release, protein recovery, and metal extraction capabilities. [DMEtA][HCOO] demonstrated near-quantitative starch recoveries at 120 °C, while [DMBA][HSO4] showed similar performance at 90 °C within a reaction time of 2 h. Saccharification yields for most pulps exceeded 90% after 8 h of hydrolysis, outperforming “traditional” lignocellulosic biomasses such as miscanthus or sugarcane bagasse. Approximately 50 and 80 wt % of the protein were solubilized in [DMEtA][HCOO] and [DMBA][HSO4], respectively, while the remaining protein distributed between the pulp and lignin. However, the solubilized protein in the IL could not be recovered due to its low molecular weight. Regarding metal extraction, [DMEtA][HCOO] demonstrated higher efficiency, achieving 81% removal of Ni from Lemna minor’s pulps, whereas [DMBA][HSO4] extracted only 28% of Ni with slightly higher pulp concentrations. These findings indicate the need for further optimization in concurrent metal extraction using ILs.
... Studies on transgenic development in Lemnaceae have showcased its potential as a versatile platform for genetic engineering and biotechnological applications. The transgenic Duckweed lines with enhanced traits for biomass production, nutrient uptake, stress tolerance, and biofuel production have been developed through different transformation approaches [43][44][45]. Genetic engineering approaches have been employed to modulate key metabolic pathways, regulatory genes, and signaling pathways in Duckweed, aiming to improve its productivity and sustainability. For instance, transgenic Duckweed lines with increased expression of genes involved in starch biosynthesis have been developed to enhance starch accumulation for bioethanol production [46]. ...
Duckweed (Lemnaceae) rises as a crucial model system due to its unique characteristics and wide-ranging utility. The significance in physiological research and phytoremediation highlights the intricate potential of Duckweed in the current era of plant biology. Special attention to Duckweed has been brought due to its distinctive features of nutrient uptake, ion transport dynamics, detoxification, intricate signaling, and stress tolerance. In addition, Duckweed can alleviate environmental pollutants and enhance sustainability by participating in bioremediation processes and wastewater treatment. Furthermore, insights into the genomic complexity of Lemnaceae species and the flourishing field of transgenic development highlight the opportunities for genetic manipulation and biotechnological innovations. The novel methods for germplasm conservation of Duckweed can be adopted to preserve genetic diversity for future research endeavors and breeding programs. This review centers on prospects in Duckweed research promoting interdisciplinary collaborations and technological advancements to drive its full potential as a model organism.
... Several technical advancements have improved our understanding of the molecular mechanisms of duckweed HM tolerance. Efficient genetic transformation systems have been developed for several duckweed species, including S. polyrhiza, L. aequinoctialis, L. minor, L. gibba, W. globosa, and Wolffia arrhiza (Yang et al., 2018b(Yang et al., , 2018cLiu et al., 2019). Genomic information is also becoming increasingly abundant for duckweeds, with data released for S. polyrhiza, Spirodela intermedia, L. minor, L. gibba, and Wolffia australiana Michael et al., 2020;Park et al., 2021b). ...
... As mentioned above, the availability of abundant genomic data and efficient genetic transformation methods for duckweeds makes the genetic optimization of duckweeds for phytoremediation a realistic goal. In particular, clustered regularly interspaced short palindromic repeats/ CRISPR-associated nuclease 9 (Cas9)-mediated gene editing now makes it possible to precisely modify the duckweed genome, as demonstrated in L. aequinoctialis (Liu et al., 2019). The modification of key regulatory elements, overexpression or knockouts of genes related to HM uptake, intracellular accumulation, and volatilization in duckweeds can increase their adaptive, accumulation, and tolerance abilities toward HMs. ...
... The increasing availability of genetic resources should greatly benefit such efforts. In recent years, high quality genomes have been published for the duckweed Spirodela polyrhiza [8][9][10][11][12] and other Lemnaceae species (Lemna gibba, Lemna minor, Wolffia australiana) are currently in draft form [13,14]. Furthermore, improved and highly efficient methods for stable genetic transformation and CRISPR/ Cas9-mediated genome editing in duckweed species have been reported recently [15][16][17]. ...
Background
Duckweeds are small, rapidly growing aquatic flowering plants. Due to their ability for biomass production at high rates they represent promising candidates for biofuel feedstocks. Duckweeds are also excellent model organisms because they can be maintained in well-defined liquid media, usually reproduce asexually, and because genomic resources are becoming increasingly available. To demonstrate the utility of duckweed for integrated metabolic studies, we examined the metabolic adaptation of growing Lemna gibba cultures to different nutritional conditions.
Results
To establish a framework for quantitative metabolic research in duckweeds we derived a central carbon metabolism network model of Lemna gibba based on its draft genome. Lemna gibba fronds were grown with nitrate or glutamine as nitrogen source. The two conditions were compared by quantification of growth kinetics, metabolite levels, transcript abundance, as well as by ¹³C-metabolic flux analysis. While growing with glutamine, the fronds grew 1.4 times faster and accumulated more protein and less cell wall components compared to plants grown on nitrate. Characterization of photomixotrophic growth by ¹³C-metabolic flux analysis showed that, under both metabolic growth conditions, the Calvin-Benson-Bassham cycle and the oxidative pentose-phosphate pathway are highly active, creating a futile cycle with net ATP consumption. Depending on the nitrogen source, substantial reorganization of fluxes around the tricarboxylic acid cycle took place, leading to differential formation of the biosynthetic precursors of the Asp and Gln families of proteinogenic amino acids. Despite the substantial reorganization of fluxes around the tricarboxylic acid cycle, flux changes could largely not be associated with changes in transcripts.
Conclusions
Through integrated analysis of growth rate, biomass composition, metabolite levels, and metabolic flux, we show that Lemna gibba is an excellent system for quantitative metabolic studies in plants. Our study showed that Lemna gibba adjusts to different nitrogen sources by reorganizing central metabolism. The observed disconnect between gene expression regulation and metabolism underscores the importance of metabolic flux analysis as a tool in such studies.
... These range from phytoremediation of wastewater [4][5][6], to use for human nutrition and animal feeding [7][8][9][10], to the production of starch for bioalcohol conversion [11,12] and to biogas production [12]. Moreover, as a result of whole genome sequencing of increasing numbers of duckweed species and clones, e.g., in Spirodela polyrhiza [13,14], and the feasibility of application of other molecular methods such as genetic transformation using CRISPR/Cas9 [15], members of the Lemnaceae became "a model plant system in the genomics and postgenomics era" [16]. ...
A spontaneous mutant of the duckweed Lemna gibba clone no. 7796 (known as strain G3, WT) was discovered. In this mutant clone, L. gibba clone no. 9602 (mt), the morphological parameters (frond length, frond width, root length, root diameter) indicated an enlarged size. A change in the frond shape was indicated by the decreased frond length/width ratio, which could have taxonomic consequences. Several different cell types in both the frond and the root were also enlarged. Flow cytometric measurements disclosed the genome size of the WT as 557 Mbp/1C and that of the mt strain as 1153 Mbp/1C. This represents the results of polyploidisation of a diploid clone to a tetraploid one. The mutant clone flowered under the influence of long day-treatment in half-strength Hutner’s medium in striking contrast to the diploid WT. Low concentration of salicylic acid (<1 µM) induced flowering in the tetraploid mutant but not in the diploid plants. The transcript levels of nuclear-encoded genes of the photosynthetic apparatus (CAB, RBCS) showed higher abundance in light and less dramatic decline in darkness in the mt than in WT, while this was not the case with plastid-encoded genes (RBCL, PSAA, PSBA, PSBC).
... This ability will enable rapid molecular studies for genes of interest that have been defined from a rapidly growing database of transcriptomics data being generated by our community. A reliable and facile method that can overcome the often-observed strain and species dependence of transformation protocols [35] to investigate gene functions and pathways will truly open the door to the fascinating world of duckweeds in the coming decade. ...
The 6th International Conference on Duckweed Research and Applications (6th ICDRA) was organized at the Institute of Plant Genetics and Crop Plant Research (IPK) located in Gatersleben, Germany, from 29 May to 1 June 2022. The growing community of duckweed research and application specialists was noted with participants from 21 different countries including an increased share of newly integrated young researchers. The four-day conference focused on diverse aspects of basic and applied research together with practical applications of these tiny aquatic plants that could have an enormous potential for biomass production.
... CRISPR/Cas9-mediated targeted mutagenesis has been successfully optimized in the duckweed Lemna aequinoctialis via EHA105 Agrobacterium transformation with a rice ubiquitin promoter within the vector . Liu et al. (2019) discussed that the increased transformation efficiency in Lemna aequinoctialis over Lemna minor was due to an optimized sonication and vacuum filtration protocol. This method has also been successful in cowpea (Vigna unguiculata), chickpea (Cicer arientinum), and banana (Musa cv. ...
Only a handful of model systems for studying programmed cell death (PCD) exist. The model Arabidopsis thaliana has generated a plethora of knowledge, but it is essential to introduce new models to broaden our understanding of the commonalities of PCD. This review focuses on Aponogeton madagascariensis (the lace plant) as a choice model to study PCD in vivo. PCD plays a key role in plant development and defence. Thus, identifying key regulators across plants is a priority in the field. The formation of perforations in lace plant leaves in areas called areoles is a striking example of PCD. Cells undergoing PCD within areoles can be easily identified from a loss of their anthocyanin pigmentation. In contrast, cells adjacent to veins, non-PCD cells, retain anthocyanins, creating a gradient of cell death. The spatiotemporal pattern of perforation formation, a gradient of cell death within areoles, and the availability of axenic cultures provide an excellent in vivo system to study mechanisms of developmental PCD. The priorities to further develop this model involve sequencing the genome, establishing transformation protocols, and identifying anthocyanin species to determine their medicinal properties. We discuss practical methodologies and challenges associated with developing the lace plant as a model to study PCD.
... 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. ...
Tiny aquatic plants from the Lemnaceae family, commonly known as duckweeds, are often regarded as detrimental to the environment because of their ability to quickly populate and cover the surfaces of bodies of water. Due to their rapid vegetative propagation, duckweeds have one of the fastest growth rates among flowering plants and can accumulate large amounts of biomass in relatively short time periods. Due to the high yield of valuable biomass and ease of harvest, duckweeds can be used as feedstock for biofuels, animal feed, and other applications. Thanks to their efficient absorption of nitrogen- and phosphate-containing pollutants, duckweeds play an important role in the restorative ecology of water reservoirs. Moreover, compared to other species, duckweed species and ecotypes demonstrate exceptionally high adaptivity to a variety of environmental factors; indeed, duckweeds remove and convert many contaminants, such as nitrogen, into plant biomass. The global distribution of duckweeds and their tolerance of ammonia, heavy metals, other pollutants, and stresses are the major factors highlighting their potential for use in purifying agricultural, municipal, and some industrial wastewater. In summary, duckweeds are a powerful tool for bioremediation that can reduce anthropogenic pollution in aquatic ecosystems and prevent water eutrophication in a simple, inexpensive ecologically friendly way. Here we review the potential for using duckweeds in phytoremediation of several major water pollutants: mineral nitrogen and phosphorus, various organic chemicals, and heavy metals.
... Effective callus induction of plants is crucial to molecular farming. In duckweed, the choices of genotypes greatly influence transformation efficiency [25,49]. Previously, we found L. minor strain ZH0403 had a high callus induction rate under the T-1 condition in our screening (date not shown), though it took more than 3 months in regeneration. ...
Molecular farming utilizes plants as a platform for producing recombinant biopharmaceuticals. Duckweed, the smallest and fastest growing aquatic plant, is a promising candidate for molecular farming. However, the efficiency of current transformation methods is generally not high in duckweed. Here, we developed a fast and efficient transformation procedure in Lemna minor ZH0403, requiring 7–8 weeks from screening calluses to transgenic plants with a stable transformation efficiency of 88% at the DNA level and 86% at the protein level. We then used this transformation system to produce chicken interleukin-17B (chIL-17B). The plant-produced chIL-17B activated the NF-κB pathway, JAK-STAT pathway, and their downstream cytokines in DF-1 cells. Furthermore, we administrated chIL-17B transgenic duckweed orally as an immunoadjuvant with mucosal vaccine against infectious bronchitis virus (IBV) in chickens. Both IBV-specific antibody titer and the concentration of secretory immunoglobulin A (sIgA) were significantly higher in the group fed with chIL-17B transgenic plant. This indicates that the duckweed-produced chIL-17B enhanced the humoral and mucosal immune responses. Moreover, chickens fed with chIL-17B transgenic plant demonstrated the lowest viral loads in different tissues among all groups. Our work suggests that cytokines are a promising adjuvant for mucosal vaccination through the oral route. Our work also demonstrates the potential of duckweed in molecular farming.
