Generation of transgenic cell suspension cultures of the model legume Medicago truncatula: a rapid method for Agrobacterium mediated gene transfer

Santos, Rita B.; Pires, Ana Sofia; Hoorn, Renier A. L. van der; Schiermeyer, Andreas; Abranches, Rita

doi:10.1007/s11240-018-1525-3

Cited by 7 publications

(11 citation statements)

References 19 publications

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“…Bright Yellow 2 (BY-2) and Medicago truncatula cv. Jemalong A17 were maintained as described in previous reports from our group (Santos et al, 2019;Rebelo et al, 2020, for Medicago and BY-2, respectively).…”

Section: Plant Materials and Plasmid Constructsmentioning

confidence: 99%

“…Four-day-old tobacco BY-2 cultures were transformed by co-cultivation with recombinant A. tumefaciens as described by An (1985), with slight modifications as described previously (Folgado and Abranches, 2021). Medicago cultures were transformed following a protocol previously established by our group (Santos et al, 2019). Two days after co-culture, tobacco cells were transferred to MS (Duchefa) solidified medium (Rebelo et al, 2020) and Medicago cells were transferred to CIM solidified medium (Santos et al, 2019).…”

Section: Stable Transformation Of By-2 and Medicago Cell Culturesmentioning

confidence: 99%

“…Medicago cultures were transformed following a protocol previously established by our group (Santos et al, 2019). Two days after co-culture, tobacco cells were transferred to MS (Duchefa) solidified medium (Rebelo et al, 2020) and Medicago cells were transferred to CIM solidified medium (Santos et al, 2019). Both media contained 0.4% (w/v) of gelrite (Duchefa) and were supplemented with 500 mg/L ticarcillin disodium/clavulanate potassium (Timentin, Duchefa) to eliminate Agrobacterium and 100 mg/L kanamycin to select positive transformants.…”

Section: Stable Transformation Of By-2 and Medicago Cell Culturesmentioning

confidence: 99%

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Production of the SARS-CoV-2 Spike protein and its Receptor Binding Domain in plant cell suspension cultures

Rebelo¹,

Folgado²,

Ferreira³

et al. 2022

Front. Plant Sci.

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The COVID-19 pandemic, caused by the worldwide spread of SARS-CoV-2, has prompted the scientific community to rapidly develop efficient and specific diagnostics and therapeutics. A number of avenues have been explored, including the manufacture of COVID-related proteins to be used as reagents for diagnostics or treatment. The production of RBD and Spike proteins was previously achieved in eukaryotic cells, mainly mammalian cell cultures, while the production in microbial systems has been unsuccessful until now. Here we report the effective production of SARS-CoV-2 proteins in two plant model systems. We established transgenic tobacco BY-2 and Medicago truncatula A17 cell suspension cultures stably producing the full-length Spike and RBD recombinant proteins. For both proteins, various glycoforms were obtained, with higher yields in Medicago cultures than BY-2. This work highlights that RBD and Spike can be secreted into the culture medium, which will impact subsequent purification and downstream processing costs. Analysis of the culture media indicated the presence of the high molecular weight Spike protein of SARS-CoV-2. Although the production yields still need improvement to compete with mammalian systems, this is the first report showing that plant cell suspension cultures are able to produce the high molecular weight Spike protein. This finding strengthens the potential of plant cell cultures as production platforms for large complex proteins.

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Section: Plant Materials and Plasmid Constructsmentioning

confidence: 99%

Section: Stable Transformation Of By-2 and Medicago Cell Culturesmentioning

confidence: 99%

Section: Stable Transformation Of By-2 and Medicago Cell Culturesmentioning

confidence: 99%

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Production of the SARS-CoV-2 Spike protein and its Receptor Binding Domain in plant cell suspension cultures

Rebelo¹,

Folgado²,

Ferreira³

et al. 2022

Front. Plant Sci.

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“…Plant cells suspension cultures are the valuable and renewable resource of biological material that can be used for several applications, including production of potential secondary metabolites (Yue et al 2016;Salehi et al 2018;Santos et al 2019). The plant cells suspension cultures are gaining popularity as a host system for the production of recombinant proteins (Tekoah et al 2015;Yue et al 2016) and have several advantages such as post-translational modi cations, a slight risk of viral contamination, low cost of plant culture media, and cost-e ciency of bacterial expression systems (Santos et al 2016;Zagorskaya and Deineko 2017;Permyakova et al 2019).…”

Section: Introductionmentioning

confidence: 99%

“…The suspension cells can be developed from any explant type; however, most preferably, an embryogenic callus is used. Several successful cell suspension cultures were reported from various plants such as Artemisia annua (Salehi et al 2019), Catharanthus (Saiman et al 2018), Taxus (Wilson et al 2018), Capsicum (Brito-Sanchez et al 2019, Medicago (Santos et al 2019), Arabidopsis (Permyakova et al 2019), Gentiana (Rybczynski andWojcik 2019), andPanicum (Ondzighi-Assoume et al 2019). Agrobacterium-mediated genetic transformation depends on factors like types of explants, bacterial density, co-cultivation duration, the temperature of co-cultivation, concentration of acetosyringone, etc.…”

Section: Introductionmentioning

confidence: 99%

Agrobacterium-Mediated Genetic Transformation and Cloning of Reference Genes in Suspension Cells of Artemisia Pallens

Jogam

Sandhya

Alok

et al. 2021

Preprint

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A reliable and stable Agrobacterium-mediated genetic transformation system has been developed using cell suspension cultures derived from Artemisia pallens cotyledon explants. Cotyledon, attached cotyledon, and compound leaf were found to be suitable for the induction of callus among five different types of explants tested. Yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2.4-dichlorophenoxyacetic acid (2,4-D) and in combination with different concentrations of Zeatin (ZEA). Among the two different shock treatments, cold shock (at 4 oC) for 20 minutes and heat shock (at 45oC) treatment for 5 minutes, heat shock treatment increased the transformation efficiency. Supplementation of chemical additives such as Silwet L-77 (0.05%) and Pluronic F-68 (0.05%) significantly enhanced suspension cultures' transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity was measured as 879.4±113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant callus derived from micro-calli showed intense blue colour in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The cloned and mRNA expression levels of three candidate reference genes ADP-ribosylation factor (Arf), β-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) along with transgene (uidA) were evaluated in transgenic callus lines. The present Agrobacterium-mediated genetic transformation protocol could help in better understand the metabolic pathways of this medicinally important plant and its genetic improvement.

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Rapeseed Transformation with aroA Bacterial Gene Containing P101S Mutation Confers Glyphosate Resistance

2021

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Generation of transgenic cell suspension cultures of the model legume Medicago truncatula: a rapid method for Agrobacterium mediated gene transfer

Cited by 7 publications

References 19 publications

Production of the SARS-CoV-2 Spike protein and its Receptor Binding Domain in plant cell suspension cultures

Production of the SARS-CoV-2 Spike protein and its Receptor Binding Domain in plant cell suspension cultures

Agrobacterium-Mediated Genetic Transformation and Cloning of Reference Genes in Suspension Cells of Artemisia Pallens

Rapeseed Transformation with aroA Bacterial Gene Containing P101S Mutation Confers Glyphosate Resistance

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