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

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.