Development of a highly efficient system for assessing recombinant gene expression in plant cell suspensions via <i>Agrobacterium tumefaciens</i> transformation

Fuentes, Alejandro; Ramos, Pedro; Ayra, Camilo; Rodríguez, M.; Ramírez, Nadia; Pujol, Merardo

doi:10.1042/ba20030192

Cited by 12 publications

References 37 publications

(42 reference statements)

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A transformation procedure for recalcitrant tomato by addressing transgenic plant‐recovery limiting factors

Fuentes

Ramos

Sánchez

et al. 2008

Biotechnology Journal

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Agrobacterium tumefaciens technology is the battle horse for tomato genetic transformation. However, tomato varieties with low regeneration capacity are very difficult to transform. In the past, tomato transformation through Agrobacterium infection was focused on varieties capable of high regeneration yield, while successful transformation of low regenerable cultivars has not been reported. The genotype response to tissue culture conditions is believed to drive the frequency of regeneration of transgenic plant, whereas the capacity for cell proliferation could determine the transformation efficiency through this technology. The Campbell-28 cultivar is an example of constraints arising from a high morphogenetic potential with low conversion compared to normal plants. In the present work the roles that contribute to improved transgenic plant recovery from this recalcitrant variety were explored for factors like Agrobacterium concentration and antibiotics for bacterial removal and transformant selection. Analysis of the efficiency from independent transformation experiments revealed a more than twofold increase of transformant regeneration after selection on ammonium glufosinate compared to kanamycin selection, showing a transformation efficiency of 21.5%.

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A transformation procedure for recalcitrant tomato by addressing transgenic plant‐recovery limiting factors

Fuentes

Ramos

Sánchez

et al. 2008

Biotechnology Journal

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Predictive models for the accumulation of a fluorescent marker protein in tobacco leaves according to the promoter/5′UTR combination

Buyel

Kaever

Buyel

et al. 2012

Biotech & Bioengineering

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The promoter and 5'-untranslated region (5'UTR) play a key role in determining the efficiency of recombinant protein expression in plants. Comparative experiments are used to identify suitable elements but these are usually tested in transgenic plants or in transformed protoplasts/suspension cells, so their relevance in whole-plant transient expression systems is unclear given the greater heterogeneity in expression levels among different leaves. Furthermore, little is known about the impact of promoter/5'UTR interactions on protein accumulation. We therefore established a predictive model using a design of experiments (DoE) approach to compare the strong double-enhanced Cauliflower mosaic virus 35S promoter (CaMV 35SS) and the weaker Agrobacterium tumefaciens Ti-plasmid nos promoter in whole tobacco plants transiently expressing the fluorescent marker protein DsRed. The promoters were combined with one of three 5'UTRs (one of which was tested with and without an additional protein targeting motif) and the accumulation of DsRed was measured following different post-agroinfiltration incubation periods in all leaves and at different leaf positions. The model predictions were quantitative, allowing the rapid identification of promoter/5'UTR combinations stimulating the highest and quickest accumulation of the marker protein in all leaves. The model also suggested that increasing the incubation time from 5 to 8 days would reduce batch-to-batch variability in protein yields. We used the model to identify promoter/5'UTR pairs that resulted in the least spatiotemporal variation in expression levels. These ideal pairs are suitable for the simultaneous, balanced production of several proteins in whole plants by transient expression.

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Transient expression of antibodies in suspension plant cell suspension cultures is enhanced when co‐transformed with the tomato bushy stunt virus p19 viral suppressor of gene silencing

Boivin

Lepage

Matton

et al. 2010

Biotechnology Progress

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Two distinct transient expression approaches were compared with assess the impact of the viral suppressor p19 on a recombinant protein production performed in Nicotiana benthamiana suspension culture. A parental N. benthamiana cell line was transiently transformed with either an Agrobacterium containing a gene construct for a murine IgG1 (R514) or concurrently with two Agrobacteria containing R514 or p19. In addition, a stably transformed N. benthamiana cell line that constitutively expresses p19 was transformed with R514-containing Agrobacterium. The parental N. benthamiana cell line that had been co-cultivated with both p19 and R514 achieved the highest yield of IgG1 (1.06 mg IgG1/kg FW; 0.024% TSP) compared with that obtained without p19 (0.61 mg IgG1/kg FW; 0.014% TSP). The N. benthamiana cell line that had been stably transformed with p19 only reached 0.25 mg IgG1/kg FW (0.009% TSP) when co-cultured with R514-containing Agrobacterium. Dual agroinfiltration of N. benthamiana leaves with p19 and R514 was also performed to assess for Agrobacteria efficiencies and 147.7 mg IgG1/kg FW were obtained. Therefore, our results demonstrate that transient co-transformation of plant cell suspension culture with two transformation vectors is feasible and that the use of the viral suppressor of silencing p19 significantly raises the production of the protein of interest.

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Development of a highly efficient system for assessing recombinant gene expression in plant cell suspensions via Agrobacterium tumefaciens transformation

Cited by 12 publications

References 37 publications

A transformation procedure for recalcitrant tomato by addressing transgenic plant‐recovery limiting factors

A transformation procedure for recalcitrant tomato by addressing transgenic plant‐recovery limiting factors

Predictive models for the accumulation of a fluorescent marker protein in tobacco leaves according to the promoter/5′UTR combination

Transient expression of antibodies in suspension plant cell suspension cultures is enhanced when co‐transformed with the tomato bushy stunt virus p19 viral suppressor of gene silencing

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