Development and validation of a novel and robust cell culture system in soybean (Glycine max (L.) Merr.) for promoter screening

Sultana, Mst Shamira; Frazier, Taylor P.; Millwood, Reginald J.; Lenaghan, Scott C.; Stewart, C. Neal

doi:10.1007/s00299-019-02455-5

Cited by 11 publications

(11 citation statements)

References 48 publications

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“…Plant tissues can be analyzed using scanning fluorometry, while single cells (protoplasts) can be analyzed by flow cytometry. 21 Calibration of flow cytometry to units of equivalent standard dye molecules (e.g., molecules of equivalent fluorescein – MEFL) allows reproducible measurements across laboratories. 22 , 23 This supports better reproducibility of results and improved process debugging by new users.…”

Section: Introductionmentioning

confidence: 99%

Building the Plant SynBio Toolbox through Combinatorial Analysis of DNA Regulatory Elements

et al. 2022

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While the installation of complex genetic circuits in microorganisms is relatively routine, the synthetic biology toolbox is severely limited in plants. Of particular concern is the absence of combinatorial analysis of regulatory elements, the long design-build-test cycles associated with transgenic plant analysis, and a lack of naming standardization for cloning parts. Here, we use previously described plant regulatory elements to design, build, and test 91 transgene cassettes for relative expression strength. Constructs were transiently transfected into Nicotiana benthamiana leaves and expression of a fluorescent reporter was measured from plant canopies, leaves, and protoplasts isolated from transfected plants. As anticipated, a dynamic level of expression was achieved from the library, ranging from near undetectable for the weakest cassette to a ∼200-fold increase for the strongest. Analysis of expression levels in plant canopies, individual leaves, and protoplasts were correlated, indicating that any of the methods could be used to evaluate regulatory elements in plants. Through this effort, a well-curated 37-member part library of plant regulatory elements was characterized, providing the necessary data to standardize construct design for precision metabolic engineering in plants.

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Section: Introductionmentioning

confidence: 99%

Building the Plant SynBio Toolbox through Combinatorial Analysis of DNA Regulatory Elements

et al. 2022

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“…Detecting the promoter activity using transient expression system has been well studied in many plant species [ 11 , 14 , 37 , 59 – 61 ]. For A. annua , due to its odd-pinnately compound leaves with deeply indented margins, it is not appropriate for Agrobacterium injection (Fig.…”

Section: Discussionmentioning

confidence: 99%

A high-efficiency Agrobacterium-mediated transient expression system in the leaves of Artemisia annua L.

Chen

Wang

et al. 2021

Plant Methods

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Background The Agrobacterium-mediated transient transformation, which proved effective in diverse plant species, has been widely applied for high-throughput gene function studies due to its simplicity, rapidity, and high efficiency. Despite the efforts have made on Artemisia annua transient expression, achieving high-throughput gene functional characterization basing on a fast and easy-manipulated transient transformation system in A. annua remains challenging. Results The first pair of true leaves of A. annua is an ideal candidate for Agrobacterium injection. EHA105 was the optimal strain that can be used for the development of the transient expression system. The supplementation of Triton X-100 at a concentration of 0.005% greatly improved the transient expression frequency. According to the histochemical β-Glucuronidase (GUS) staining assay, high transient expression level of the reporter gene (GUS) maintained at least a week. Dual-luciferase (Dual-LUC) transient assays showed that the activity of cauliflower mosaic virus 35S (CaMV35S) promoter and its derivates varied between A. annua and tobacco. In A. annua, the CaMV35S promoter had comparable activity with double CaMV35S promoter, while in tobacco, CaMV35S exhibited approximately 50% activity of double CaMV35S promoter. Otherwise, despite the CaMV35S promoter and double CaMV35S promoter from GoldenBraid Kit 2.0 displayed high activity strength in tobacco, they demonstrated a very low activity in transiently expressed A. annua. The activity of UBQ10 promoter and endogenous UBQb promoter was investigated as well. Additionally, using our transient expression system, the transactivation of AaGSW1 and AaORA on AaCYP71AV1 promoter was confirmed. Dual-LUC assays demonstrated that AaHD8 activated the expression of two glandular secreting trichomes-specific lipid transfer protein genes AaLTP1 and AaLTP2, indicating that AaLTP1 and AaLTP2 might serve as downstream components of AaHD8-involved glandular trichome initiation and cuticle formation, as well as artemisinin secretion in A. annua. Conclusions A simple, rapid, good-reproducibility, high-efficiency and low-cost transient transformation system in A. annua was developed. Our method offered a new way for gene functional characterization studies such as gene subcellular localization, promoter activity and transcription activation assays in A. annua, avoiding the aberrant phenotypes resulting from gene expression in a heterologous system.

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“…The main challenge is achieving protoplast regeneration, which has yet to be reported in soybean. Protoplast-based transfection has been mainly conducted to evaluate gene functions (Yi et al, 2010;Faria et al, 2011;Kidokoro et al, 2015;Xiong et al, 2019), screen promoters (Sultana et al, 2019), and validate vectors for GE (Sun et al, 2015;Demorest et al, 2016;Do et al, 2019;Patil et al, 2022). Recently, Wu and Hanzawa (2018) developed a method to isolate protoplasts from leaves of soybean seedlings and established a PEG-mediated transfection method that can achieve high transfection efficiency compared to other transient assays.…”

Section: Protoplast Transfectionmentioning

confidence: 99%

Progress in Soybean Genetic Transformation Over the Last Decade

Guo

Qiu

et al. 2022

Front. Plant Sci.

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Soybean is one of the important food, feed, and biofuel crops in the world. Soybean genome modification by genetic transformation has been carried out for trait improvement for more than 4 decades. However, compared to other major crops such as rice, soybean is still recalcitrant to genetic transformation, and transgenic soybean production has been hampered by limitations such as low transformation efficiency and genotype specificity, and prolonged and tedious protocols. The primary goal in soybean transformation over the last decade is to achieve high efficiency and genotype flexibility. Soybean transformation has been improved by modifying tissue culture conditions such as selection of explant types, adjustment of culture medium components and choice of selection reagents, as well as better understanding the transformation mechanisms of specific approaches such as Agrobacterium infection. Transgenesis-based breeding of soybean varieties with new traits is now possible by development of improved protocols. In this review, we summarize the developments in soybean genetic transformation to date, especially focusing on the progress made using Agrobacterium-mediated methods and biolistic methods over the past decade. We also discuss current challenges and future directions.

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Development and validation of a novel and robust cell culture system in soybean (Glycine max (L.) Merr.) for promoter screening

Cited by 11 publications

References 48 publications

Building the Plant SynBio Toolbox through Combinatorial Analysis of DNA Regulatory Elements

Building the Plant SynBio Toolbox through Combinatorial Analysis of DNA Regulatory Elements

A high-efficiency Agrobacterium-mediated transient expression system in the leaves of Artemisia annua L.

Progress in Soybean Genetic Transformation Over the Last Decade

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