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Combination of ammonium nitrate, cerium chloride and potassium chloride salts improves Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum

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Abstract

The frequency of plant transformation can be improved by addition of various chemical into transformation media. In the past, we showed that exposure of tobacco, wheat and triticale explants to ammonium nitrate, cerium and lantanium chloride and potassium chloride resulted in an increase in the frequency of transformation. Here, we tested whether a combination of increased concentrations of the aforementioned salts yielded a higher transformation frequency. We found that exposure to 61.8 mM ammonium nitrate caused a 5.0-fold increase in transformation frequency, whereas exposure to 1.0 μM cerium chloride or 47.0 mM potassium chloride resulted in 1.2- and 2-fold increases, respectively. Exposure to 61.8 mM ammonium nitrate and 1.0 μM cerium chloride led to a 4.8-fold increase in transformation frequency, whereas exposure to 61.8 mM ammonium nitrate and 47.0 mM potassium chloride let to a 5.2-fold increase. Finally, exposure to 61.8 mM ammonium nitrate, 1.0 μM cerium chloride and 47.0 mM potassium chloride produced a 5.1-fold increase. The analysis of the intactness of T-DNA borders showed that plants exposed to ammonium nitrate and a combination of ammonium nitrate with other salts had the more intact right borders and the less intact left borders. The best results were observed when all three salts (ammonium nitrate, potassium chloride and cerium chloride) were used. Thus, we concluded that the addition of cerium chloride and potassium chloride does not substantially improve the transformation rate beyond the improvement observed upon treatment with 61.8 mM ammonium nitrate, but may slightly improve the intactness of T-DNA borders.

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Acknowledgments

We would like to thank Valentina Titova for proofreading the text. NSERC Strategic and AARI are acknowledged for financial support.

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Authors and Affiliations

  1. Department of Biological Sciences, University of Lethbridge, Lethbridge, Canada

    Priti Maheshwari & Igor Kovalchuk

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  1. Priti Maheshwari
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  2. Igor Kovalchuk
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Correspondence to Igor Kovalchuk.

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11816_2012_243_MOESM1_ESM.ppt

Figure S1. Schematic presentation of pCAMBIA::NLUC plasmid. Genomic DNA was digested with NcoI and probed with the probe specific to luciferase gene (marked as green arrow). Black arrow indicates the minimum size of the fragment on the Southern blot – 4.32 kb (PPT 101 kb)

11816_2012_243_MOESM2_ESM.ppt

Figure S2. Schematic presentation of the PCRs for the analysis of T-DNA intactness. Four primers were used for the analysis of each border, one going outward from T-DNA (LB + and RB +) and three going inward (LB1-, LB2-, LB3- and RB1-, RB2- and RB3-). PCR on intact T-DNA borders would result in 3 amplicons, whereas PCR on truncated T-DNA would result in 2, 1 or even no amplicons at all, depending on the size of truncations. In the case when no amplicons were observed, internal primers, IP + and IP-, were used to confirm T-DNA presence (PPT 126 kb)

11816_2012_243_MOESM3_ESM.ppt

Figure S3. Analysis of the intactness of the left T-DNA border. The intactness of the left border was checked by PCR using one outward and three inward primers. The presence of three fragments indicates the intactness of the left border, whereas the absence of one or more fragments indicates truncations. − negative control – PCR of a wild type sample. + positive control – PCR from the binary vector containing the T-DNA. Each lane represents an individual plant. The numbers indicate the number of fragments present. A. Representative image of the control 1× nitrate group; B. Representative image of the 3× nitrate group; C. Representative image of the 2.5× KCl group;D. Representative image of the CeCl3″ group;E. Representative image of the 3× nitrate + 2.5× KCl + CeCl3 group (PPT 326 kb)

11816_2012_243_MOESM4_ESM.ppt

Figure S4. Analysis of the intactness of the right T-DNA border The intactness of the right border was checked by PCR using one outward and three inward primers. The presence of three fragments indicates the intactness of the right border, whereas the absence of one or more fragments indicates truncations. -negative control – PCR of a wild-type sample. + positive control – PCR from the binary vector containing T-DNA. Each lane represents an individual plant. The numbers indicate the number of fragments present. A. Representative image of the control 1× nitrate group; B. Representative image of the 3× nitrate group; C. Representative image of the 2.5× KCl group; D. Representative image of the CeCl3 group; E. Representative image of the 3× nitrate + 2.5× KCl + CeCl3 group (PPT 332 kb)

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Maheshwari, P., Kovalchuk, I. Combination of ammonium nitrate, cerium chloride and potassium chloride salts improves Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum . Plant Biotechnol Rep 7, 147–154 (2013). https://doi.org/10.1007/s11816-012-0243-2

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