Low Co-Cultivation Temperature at 20°C 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

Guiying Su, Sunjung Park, Seokhyun Lee, Norimoto Murai

Department of Plant Pathology and Crop Physiology, Louisiana State University and LSU AgCenter, Baton Rouge, USA.

DOI: 10.4236/ajps.2012.34064                

Abstract

The importance of controlled temperature during the four-days co-cultivation period was evaluated under the most physiologically relevant conditions for Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cv. Xanthi (nn, Smith)) leaf disks. We compared the effect of temperatures ranging from 15°C, 18°C, 20°C, 22°C to 25°C on the stable expression of β-glucuronidase (GUS) activity of 14 days old hygromycin-selected leaf disks, and on the increase in the fresh weight yield of 28 days old kanamycin-selected calli. The highest average of GUS activity was obtained at 20°C among the five temperatures tested although the difference between the 18°C and 20°C treatment was not statistically significant. The GUS activity at 15°C was statistically lower than those at 18°C and 20°C. The GUS activity in 22°C treatment was an intermediate between the highest (18/20°C) and second highest averages (15°C), and was not statistically significantly different. The lowest average of GUS activity was observed at 25°C. The highest increase in the plate average of fresh weight yield was obtained at 20°C among the five temperature tested. The 20°C treatment was statistically significantly better than the 15°C and 18°C treatments. The 20°C co-cultivation treatment resulted in the higher FW yield than 22°C and 25°C even though the differences were not statistically significant. In conclusion, low co-cultivation temperature at 20°C resulted in the reproducible maximum increase in both the fresh weight yield and stable expression of GUS activity after transformation of tobacco leaf disks.

Keywords

Agrobacterium tumefaciens; Co-Cultivation Temperature; Fresh Weight Yield; Stable GUS Gene Expression; Tobacco Leaf Disks; Transformation

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	E. F. Smith and C. O. Towsend, “A Plant Tumor of Bacterial Origin,” Science, Vol. 25, No. 643, 1907, pp. 671-673. doi:10.1126/science.25.643.671
[2]	I. Zaenen, N. Van Larebeke, H. Teuchy, M. Van Montagu and J. Schell, “Supercoiled Circular DNA in Crown-Gall Inducing Agrobacterium Strains,” Journal Molecular Biology, Vol. 86, No. 1, 1974, pp. 109-127. doi:10.1016/S0022-2836(74)80011-2
[3]	M. D. Chilton, M. H. Drummond, D. J. Merlo, D. Sciaky, A. L. Montoya, M. P. Gordon and E. W. Nester, “Stable Incorporation of Plasmid DNA into Higher Plant Cells: The Molecular Basis of Crown Gall Tumorigenesis,” Cell, Vol. 11, No. 2, 1977, pp. 263-271. doi:10.1016/0092-8674(77)90043-5
[4]	J. R. Zupan and P. Zambryski, “Transfer of T-DNA from Agrobacterium to the Plant Cell,” Plant Physiology, Vol. 107, No. 4, 1995, pp. 1041-1047. doi:10.1104/pp.107.4.1041
[5]	J. Sheng and V. Citovsky, “Agrobacterium-Plant Cell DNA Transport: Have Virulence Proteins, Will Travel,” Plant Cell, Vol. 8, No. 10, 1996, pp. 1699-1710. doi:10.1105/tpc.8.10.1699
[6]	S. B. Gelvin, “Agrobacterium-Mediated Plant Transformation: The Biology behind the ‘Gene-Jockeying’ Tool,” Microbiology Molecular Biology Reviews, Vol. 67, No. 10, 2003, pp. 16-37. doi:10.1128/MMBR.67.1.16-37.2003
[7]	P. Zambryski, H. Joos, C. Genetello, J. Leemans, M. Van Montagu and J. Schell, “Ti Plasmid Vector for the Introduction of DNA into Plant Cells without Alteration of Their Normal Regeneration Capacity,” EMBO Journal, Vol. 2, No. 12, 1983, pp. 2143-2150.
[8]	N. Murai, D. W. Sutton, M. G. Murray, J. L. Slightom, D. J. Merlo, N. A. Reichert, C. Sengupta-Gopalan, C. A. Stock, R. F. Baker, J. D. Kemp and T. C. Hall, “Phaseolin Gene from Bean Is Expressed after Transfer to Sunflower via Tumor-Inducing Plasmid Vectors,” Science, Vol. 222, No. 4623, 1983, pp. 476-482. doi:10.1126/science.222.4623.476
[9]	A. Hoekema, P. R. Hirsch, P. J. J. Hooykas 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
[10]	R. Hellens, P. Mullineaux and H. Klee, “A Guide to Agrobacterium Binary Ti Vectors,” Trends in Plant Science, Vol. 5, No. 10, 2000, pp. 446-448. doi:10.1016/S1360-1385(00)01740-4
[11]	T. Komori, T. Imayama, N. Kato, Y. Ishida, Y. Ueki and T. Komari, “Current Status of Binary Vectors and Superbinary Vectors,” Plant Physiology, Vol. 145, No. 4, 2007, pp. 1155-1160. doi:10.1104/pp.107.105734
[12]	S. J. Clough and A. F. Bent, “Floral Dip: A Simplified Method for Agrobacterium-Mediated Transformation of Arabidopsis thaliana,” Plant Journal, Vol. 16, No. 6, 1998, pp. 735-743. doi:10.1046/j.1365-313x.1998.00343.x
[13]	M. Burow, C. A. Chlan, P. Sen, A. Lisca and N. Murai, “High Frequency Generation of Transgenic Tobacco Plants after Modified Leaf Disk Cocultivation with Agrobacterium tumefaciens,” Plant Molecular Biology Reporter, Vol. 8, No. 2, 1990, pp. 153-168. doi:10.1007/BF02669766
[14]	M. Burow, P. Sen, C. A. Chlan and N. Murai, “Developmental Control of the β-Phaseolin Gene Requires Positive, Negative, Temporal Seed-Specific Transcription Regulatory Elements and a Negative Element for Stem and Root Expression,” Plant Journal, Vol. 2, No. 4, 1992, pp. 534-548. doi:10.1111/j.1365-313X.1992.00537.x
[15]	W. Dillen, J. De Clercq, J. Kapila, M. Zambre, M. Van Montagu and G. Angenon, “The Effect of Temperature on Agrobacterium tumefaciens-Mediated Gene Transfer to Plants,” Plant Journal, Vol. 12, No. 6, 1997, pp. 1459-1463. doi:10.1046/j.1365-313x.1997.12061459.x
[16]	R. Chakrabarty, N. Viswakarma, S. R. Bhat, P. B. Kirti, B. D. Singh and V. L. Chopra, “Agrobacterium-Mediated Transformation of Cauliflower: Optimization of Protocol and Development of Bt-Transgenic Cauliflower,” Journal Bioscience, Vol. 27, No. 5, 2002, pp. 495-502. doi:10.1007/BF02705046
[17]	G. Sunilkumar and K. Rathore, “Transgenic Cotton: Factors Influencing Agrobacterium-Mediated Transformation and Regeneration,” Molecular Breeding, Vol. 8, No. 1, 2001, pp. 37-52. doi:10.1023/A:1011906701925
[18]	S. Jin, X. Zhang, S. Liang, Y. Nie, X. Guo and C. Huang, “Factors Affecting Transformation Efficiency of Embryogenic Callus of Upland Cotton (Gossypium hirsutum) with Agrobacterium tumefaciens,” Plant Cell, Tissue & Organ Culture, Vol. 81, No. 2, 2005, pp. 229-237. doi:10.1007/s11240-004-5209-9
[19]	T. Kondo, H. Hasegawa and M. Suzuki, “Transformation and Regeneration of Garlic (Allium sativum L.) by Agro-bacterium-Mediated Gene Transfer,” Plant Cell Report, Vol. 19, No. 10, 2000, pp. 989-993. doi:10.1007/s002990000222
[20]	W. Dang and Z.-M. Wei, “An Optimized Agrobacterium-Mediated Transformation for Soybean for Expression of Binary Insect Resistance Genes,” Plant Science, Vol. 173, No. 4, 2007, pp. 381-389. doi:10.1016/j.plantsci.2007.06.010
[21]	T. Murashige and F. Skoog, “A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures,” Physiologia Plantarum, Vol. 15, No. 3, 1962, pp. 473-497. doi:10.1111/j.1399-3054.1962.tb08052.x
[22]	S. Lee, “New Binary Ti Vectors with the Co-Directional Replicons for Agrobacterium tumefaciens-Mediated Transformation of Higher Plants,” Ph.D. Dissertation, Louisiana State University, Baton Rouge, 2010.
[23]	S. J. Park, “Agrobacterium tumefaciens-Mediated Transformation of Tobacco (Nicotiana tabacum L.) Leaf Disks: Evaluation of the Co-Cultivation Conditions to Increase-Glucuronidase Gene Activity,” M.S. Thesis, Louisiana State University, Baton Rouge, 2006.
[24]	G. Su, “Low Co-Cultivation Temperature at 20°C Improved Agrobacterum tumefaicens-Mediated Transformation of Tobacco Leaf Disks,” M.S. Thesis, Louisiana State University, Baton Rouge, 2010.
[25]	R. A. Jefferson, “Assaying Chimeric Genes in Plants: The GUS Gene Fusion System,” Plant Molecular Biology Reporter, Vol. 5, No. 4, 1987, pp. 387-405. doi:10.1007/BF02667740
[26]	Z. Zheng, K. Sumi, K. Tanaka and N. Murai, “The Bean Seed Storage Protein β-Phaseolin Is Synthesized, Processed, and Accumulated in the Vacuolar Type-II Protein Bodies of Transgenic Rice Endosperm,” Plant Physiology, Vol. 109, No. 3, 1995, pp. 777-786.
[27]	K. J. Fullner, J. C. Lara and E. W. Nester, “Pilus Assembly by Agrobacterium T-DNA Transfer Genes,” Science, Vol. 273, No. 5278, 1996, pp. 1107-1109. doi:10.1126/science.273.5278.1107
[28]	A. J. Riker, B. Henry and B. M. Duggar, “Growth Substance in Crown Gall as Related to Time after Inoculation, Critical Temperature, and Diffusion,” Journal Agriculture Research, Vol. 63, No. 7, 1941, pp. 395-405.
[29]	J. De Clercq, M. Zambre, M. Van Montagu, W. Dillen and G. Angenon, “An Optimized Agrobacterium-Mediated Transformation Procedure for Phaseolius acutifolius A. Gray,” Plant Cell Report, Vol. 21, No. 4, 2002, pp. 333-340. doi:10.1007/s00299-002-0518-0
[30]	A. J. Riker, “Studies in the Influence of Some Environmental Factors on the Development of Crown Gall,” Journal Agriculture Research, Vol. 32, 1926, pp. 83-89.
[31]	A. C. Braun, “Thermal Studies on the Factors Responsible for Tumor Initiation in Crown Gall,” American Journal of Botany, Vol. 34, No. 4, 1947, pp. 234-240. doi:10.2307/2437424
[32]	J. Tempe, A. Petit, M. Holsters, M. Van Montagu and J. Schell, “Thermosensitive Step Associated with Transfer of the Ti Plasmid during Conjugation: Possible Relation to Transformation in Crown Gall,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 74, No. 7, 1977, pp. 2848-2849. doi:10.1073/pnas.74.7.2848
[33]	K. J. Fullner and N. W. Nester, “Temperature Affects the T-DNA Transfer Machinery of Agrobacterium tumefaciens,” Journal Bacteriology, Vol. 178, No. 6, 1996, pp. 1498-1504.
[34]	C. Baron, N. Domke, M. Beinhofer and S. Hapfelmeier, “Elevated Temperature Differentially Affects Virulence, VirB Protein Accumulation, and T-Pilus Formation in Different Agrobacterium tumefaciens and Agrobacterium vitis Strains,” Journal Bacteriology, Vol. 183, No. 23, 2001, pp. 6852-6861. doi:10.1128/JB.183.23.6852-6861.2001
[35]	V. Chandran, R. Fronzes, S. Duquerroy, N. Cronin, J. Navaza and G. Waksman, “Structure of the Outer Membrane Complex of a Type IV Secretion System,” Nature, Vol. 462, No. 7276, 2009, pp. 1011-1015. doi:10.1038/nature08588
[36]	R. Fronzes, E. Schafer, L. Wang, H. R. Saibil, E. V. Orlova and G. Waksman, “Structure of a Type IV Secretion System Core Complex,” Science, Vol. 323, No. 5911, 2009, pp. 266-268. doi:10.1126/science.1166101
[37]	J. Aguilar, J. Zupa, T. A. Cameron and P. C. Zambryski, “Agrobacterium Type IV Secretion System and Its Substrates form Helical Arrays around the Circumference of Virulence-Induced Cells,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, No. 8, 2010, pp. 3758-3763. doi:10.1073/pnas.0914940107