Tetracycline-Based Binary Ti Vectors pLSU with Efficient Cloning by the Gateway Technology for Agrobacterium tumefaciens-Mediated Transformation of Higher Plants


We constructed small high-yielding binary Ti vectors with a bacterial tetracycline resistance gene to facilitate efficient cloning afforded by the Gateway Technology (Invitrogen) for Agrobacterium tumefaciens-mediated transformation of higher plants. The Gateway Technology vectors are kanamycin-based, thus tetracycline-based destination and expression vectors are easily selected for the antibiotic resistance in the Escherichia coli media. We reduced the size of the tetracycline resistance gene TetC from pBR322 to 1468 bp containing 1191 bp of the coding region, 93 bp of 5’-upstream, and 184 bp 3’-downstream region. The final size of binary Ti vector skeleton pLSU11 is 5034 bp. pLSU12 and 13 have the kanamycin resistance NPTII gene as a plant-selectable marker. pLSU13 and 15 contain the hygromycin resistance HPH gene as a selection marker. pLSU13 and 15 also have the β-glucuronidase (GUS) reporter gene in addition to the plant selection marker. We also constructed a mobilizable version of tetracycline-based binary Ti vector pLSU16 in which the mob function of ColE1 replicon was maintained for mobilization of the binary vector from E. coli to A. tumefaciens by tri-parental mating. The final size of binary Ti vector skeleton pLSU16 is 5580 bp. New tetracycline- based binary Ti vectors pLSU12 were found as effective as kanamycin-based vector pLSU2 in promoting a 10-fold increase in fresh weight yield of kanamycin-resistant calli after A. tumefaciens-mediated transformation of tobacco leaf discs. Using the Gateway Technology we introduced the plant-expressible GUSgene to the T-DNA of binary Ti vector pLSU12. Expression of the β-glucuronidase enzyme activity was demonstrated by histochemical staining of the GUS activity in transformed tobacco leaf discs.

Share and Cite:

S. Lee, G. Su, E. Lasserre and N. Murai, "Tetracycline-Based Binary Ti Vectors pLSU with Efficient Cloning by the Gateway Technology for Agrobacterium tumefaciens-Mediated Transformation of Higher Plants," American Journal of Plant Sciences, Vol. 4 No. 7, 2013, pp. 1418-1426. doi: 10.4236/ajps.2013.47173.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] A. Landy, “Dynamic, Structural, and Regulatory Aspects of Site-Specific Recombination,” Annual Review of Biochemistry, Vol. 58, 1989, pp. 913-949. doi:10.1146/annurev.bi.58.070189.004405
[2] J. L. Hartley, G. F. Temple and M. A. Brasch, “DNA Cloning Using in Vitro Site-Specific Recombination,” Genome Research, Vol. 10, 2000, pp. 1788-1795. doi:10.1101/gr.143000
[3] D. L. Cheo, S. A. Titus, D. N. R. Byrd, J. L. Hartley, G. F. Temple and M. A. Brasch, “Concerted Assembly and Cloning of Multiple DNA Segments Using in Vitro Site-Specific Recombination: Functional Analysis of Multi-Segment Expression Clones,” Genome Research, Vol. 14, 2004, pp. 2111-2120. doi:10.1101/gr.2512204
[4] M. Karimi, B. De Meyer and P. Hilson, “Modular Cloning in Plant Cells,” Trends in Plant Science, Vol. 10, 2005, pp. 103-105. doi:10.1016/j.tplants.2005.01.008
[5] M. Karimi, A. Depicke and P. Hilson, “Recombinational Cloning with Plant Gateway Vectors,” Plant Physiology, Vol. 145, No. 4, 2007, pp. 1144-1154. doi:10.1104/pp.107.106989
[6] M. Karimi, D. Inze and A. Depicker, “Gateway Vectors for Agrobacterium-Mediated Plant Transformation,” Trends in Plant Science, Vol. 7, 2002, pp. 193-195. doi:10.1016/S1360-1385(02)02251-3
[7] M. Karimi, A. Bleys, R. Vanderheghen and P. Hilson, “Building Blocks for Plant Gene Assembly,” Plant Physiology, Vol. 145, No. 4, 2007, pp. 1183-1191. doi:10.1104/pp.107.110411
[8] M. D. Curtis andU. Grossniklaus, “A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta,” Plant Physiology, Vol. 133, No. 2, 2003, pp. 462-469. doi:10.1104/pp.103.027979
[9] K. W. Earley, J. R. Haag, O. Pontes, K. Opper, T. Juehne, K. Song and C. S. Pikkard, “Gateway-Compatible Vectors for Plant Functional Genomics and Proteomics,” Plant Journal, Vol. 45, No. 4, 2006, pp. 616-629. doi:10.1111/j.1365-313X.2005.02617.x
[10] L.-Y. Lee and S. B. Gelvin, “T-DNA Binary Vectors and Systems,” Plant Physiology, Vol. 146, No. 2, 2008, pp. 325-332. doi:10.1104/pp.107.113001
[11] A. Hoekema, P. R. Hirsch, P. J. J. Hooykaas and R. A. Schilperoort, “A Binary Plant Vector Strategy Based on Separation of vir- and T-Region of the Agrobacterium tumefaciens Ti-Plasmid,” Nature, Vol. 303, No. 5913, 1983, pp. 179-180. doi:10.1038/303179a0
[12] F. Bolivar, “Construction and Characterization of New Cloning Vehicles III: Derivatives of Plasmid pBR322 Carrying Unique EcoRI Sites for Selection of EcoRI Generated Recombinant DNA Molecules,” Gene, Vol. 4, No. 2, 1979, pp. 121-136. doi:10.1016/0378-1119(78)90025-2
[13] J. G. Sutcliffe, “Complete Nucleotide Sequence of the Escherichia coli Plasmid pBR322,” Cold Spring Harbor Symposium of Quantitative Biology, Vol. 43, 1978, pp. 77-90.
[14] J. G. Sutcliffe, “Nucleotide Sequence of the Ampicillin Resistance Gene of Escherichia coli Plasmid pBR322,” Proceedings of the National Academy of Science of the United States of America, Vol. 75, 1978, pp. 3737-3741.
[15] S. P. Chambers, S. E. Prior, D. A. Barstow and N. P. Minton, “The pMTLnic- Cloning Vectors I. Improved pUC Polylinker Regions to Facilitate the Use of Sonicated DNA for Nucleotide Sequencing,” Gene, Vol. 68, No. 1, 1998, pp. 139-149. doi:10.1016/0378-1119(88)90606-3
[16] C. Yanisch-Perron, J. Vieira and J. Messing, “Improved M13 Phage Cloning Vectors and Host Strains: Nucleotide Sequences of the M13mpl8 and pUC19 Vectors,” Gene, Vol. 33, No. 1, 1985, pp. 103-119. doi:10.1016/0378-1119(85)90120-9
[17] F. Barany, “Single Stranded Hexameric Linkers: A System for In-Phase Insertion Mutagenesis and Protein Engineering,” Gene, Vol. 37, No. 1-3, 1985, pp. 111-123. doi:10.1016/0378-1119(85)90263-X
[18] H. C. Birnboim and J. Doly, “A Rapid Alkaline Extraction Procedure for Screening Recombinant Plasmid DNA,” Nucleic Acids Research, Vol. 7, No. 6, 1979, pp. 1513-1523. doi:10.1093/nar/7.6.1513
[19] J. B. Hansen and R. H. Olsen, “Isolation of Large Bacterial Plasmids and Characterization of the P2 Incompatibility Group Plasmids pMG1 and pMG5,” Journal of Bacteriology, Vol. 135, No. 1, 1978, pp. 227-238.
[20] B. Vogelstein and D. Gillespie, “Preparative and Analytical Purification of DNA from Agarose,” Proceedings of the National Academy of Science of the United States of America, Vol. 76, 1979, pp. 615-619.
[21] J. Sambrook and D. W. Russell, “Molecular Cloning,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001.
[22] S. Lee, “New Binary Ti Vectors with the Co-Directional Replicons for Agrobacterium tumefaciens-Mediated Transformation of Higher Plants,” PhD thesis, Louisiana State University, Baton Rouge, 2010.
[23] R. Hofgen R and L. Willmitzer, “Storage of Competent Cells for Agrobacterium tumefaciens,” Nucleic Acids Research, Vol. 16, No. 20, 1988, p. 9822.
[24] G. Su, S. Park, S. Lee and N. Murai, “Low Co-Cultivation Temperature at 20℃ Resulted in the Reproducible Maximum Increase in Both the Fresh Weight Yield and Stable Expression of GUS Activity after Agrobacterium tumefaciens-Mediated Transformation of Tobacco Leaf Disks,” American Journal of Plant Sciences, Vol. 3, 2012, pp. 537-545. doi:10.4236/ajps.2012.34064
[25] S. Lee, G. Su, E. Lasserre, M. A. Aghazadeh and N. Murai, “Smaller High-Yielding Binary Ti Vectors pLSU with Co-Directional Replicons for Agrobacterum tumefaciens-Mediated Transformation of Higher Plants,” Plant Science, Vol. 187,2012, pp. 49-58.
[26] S. B. Levy, L. M. MCMurry, T. M. Barbosa, V. Burdett, P. Courvalin, W. Hillen, M. C. Roberts, J. I. Rood and D. E. Taylor, “Nomenclature for New Tetracycline Resistance Determinants,” Antimicrobial Agents and Chemotherapy, Vol. 43, No. 6, 1999, pp. 1523-1524.
[27] L. Brand, M. Horler, E. Nuesch, S. Vassalli, P. Barrell, W. Yang, R. A. Jefferson, U. Grossniklaus and M. D. Curtis, “A Versatile and Reliable Two-Component System for Tissue-Specific Gene Induction in Arabidopsis,” Plant Physiology, Vol. 14, No. 4, 2006, pp. 1194-1204. doi:10.1104/pp.106.081299
[28] R. Xu and Q. Q. Li, “Protocol: Streamline Cloning of Genes into Binary Vectors in Agrobacterium via the Gateway TOPO Vector System,” Plant Methods, Vol. 4, 2008, p. 4. doi:10.1186/1746-4811-4-4
[29] A. Himmelbach, U. Zierold, G. Hensel, J. Riechen, D. Douchkov, P. Schweizer and J. Kumiehn, “A Set of Modular Binary Vectors for Transformation of Cereals,” Plant Physiology, Vol. 145, No. , 2007, pp. 1192-1200.

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.