E. coli cultures expressing a synthetic sequence of ptz gene (stz) promoted in vitro direct organogenesis in Nicotiana tabacum L.

  • Alberto Camas-Reyes
  • Ricardo Laguna-Ramírez
  • Alba E. Jofre-Garfias
  • Faviola Cardoso-Martínez
  • Ana Lilia Hernández-Orihuela
  • Jorge Molina-Torres
  • Agustino Martínez-AntonioEmail author
Original Article


In vitro plant organogenesis requires plant regulator cytokinins to be exogenously supplied to the culture media. Cytokinins are either obtained from natural sources, from purified commercial plant extracts or by chemical synthesis. Besides plants, several species of plant pathogenic bacteria also naturally produce cytokinins. For example, Pseudomonas syringae pv. savastanoi (P. savastanoi) produces cytokinins by virtue of its isopentenyl transferase (ptz) gene. Therefore, we asked whether cell cultures of an Escherichia coli (E. coli) strain transformed with a synthetic sequence of the ptz gene (stz) may induce a morphogenetic response in vitro. To address this question, the stz gene was inserted into the pColdI™ DNA cold shock expression vector, and this clone was used to genetically transform cells of the E. coli TOP10 strain. The same strain transformed with an empty expression vector was used as the experimental control. Our results showed that cell-free media and methanolic fractions of the cell-free media prepared from the E. coli TOP10 strain overexpressing the stz gene combined with MS basal medium were able to induce in vitro organogenesis in tobacco bioassays. These cell-free media promoted shoot and root formation in tobacco leaf explants. We propose that these types of cell-free extracts could be used not only for in vitro plant propagation, but also for promoting plant rooting.


Synthetic biology Ptz Escherichia coli Cytokinins In vitro organogenesis Tobacco 













4-Hydroxy-3-methyl-2-(E)-butenyl diphosphate


Indole-3-acetic acid


Isopentenyl adenine


Isopentenyl transferase


Isopropyl β-D-1-thiogalactopyranoside


Lysogeny broth


Murashige and Skoog medium


Phenylacetic acid


Plant growth-promoting rhizobacteria


Sodium dodecyl sulfate-polyacrylamide gel electrophoresis


Standard error




Synthesized tZ


Synthetic IPT gene


Ultra performance liquid chromatography



We would like to thank Yolanda Rodríguez for her technical assistance with the HPLC analysis and to I.Q. Enrique Ramírez for his technical assistance in the GC-MS study. We are grateful to Collen A. Beard and Carolyn Smith, Peace Corps Response Volunteers, for the English language edition of this manuscript. The present investigation was financialy supported by Fondo de Innovación Tecnológica del Estado de Guanajuato, México (CFINN0173).

Author contributions

Conceived and designed the experiments: AM-A, AC-R, and AJ-G. Performed the experiments: AC-R, RL-R,AJ-R, FC-M, AH-O, and JM-T. Analyzed the data: AC-R, RL-R, and AJ-G. Contributed reagents/materials/analysis tools: AM-A, AJ-G, and JM-T. Contributed to the writing of the manuscript: AC-R and AM-A. All the authors read and approved the manuscript.

Supplementary material

11240_2018_1554_MOESM1_ESM.docx (134 kb)
Supplementary material 1 (DOCX 134 KB)
11240_2018_1554_MOESM2_ESM.pptx (92 kb)
Supplementary material 2 (PPTX 92 KB)
11240_2018_1554_MOESM3_ESM.pptx (370 kb)
Supplementary material 3 (PPTX 370 KB)
11240_2018_1554_MOESM4_ESM.pptx (1.7 mb)
Supplementary material 4 (PPTX 1784 KB)
11240_2018_1554_MOESM5_ESM.pptx (115 kb)
Supplementary material 5 (PPTX 114 KB)


  1. Akiyoshi DE, Regier TDA, Gordon MP (1985) Cloning and nucleotide sequence of the tzs gene from Agrobacterium tumefaciens strain T37. Nucleic Acids Res 13(8):2773–2788CrossRefGoogle Scholar
  2. Akiyoshi DE, Regier TDA, Gordon MP (1987) Cytokinin production by Agrobacterium and Pseudomonas spp. J Bacteriol 169(9):4242–4248CrossRefGoogle Scholar
  3. Bertani G (1951) Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300Google Scholar
  4. Blunt JW, Calder VL, Fenwick GD, Lake RJ, McCombs JD, Munro MHG, Perry NB (1987) Reverse phase flash chromatography: a method for the rapid partitioning of natural products extracts. J Nat Prod 50(2):290–292CrossRefGoogle Scholar
  5. Chen X, Qu Y, Sheng L, Liu J, Huang H, Xu L (2014) A simple method suitable to study de novo root organogenesis. Front Plant Sci 5(208):1–6Google Scholar
  6. Cheng ZJ, Wang L, Sun W et al (2013) Pattern of auxin and cytokinin responses for shoot meristem induction results from the regulation of cytokinin biosynthesis by AUXIN RESPONSE FACTOR3. Plant Physiol 161:240–251CrossRefGoogle Scholar
  7. Dakha N, Kothari SL (2002) Phenylacetic acid improves bud elongation and in vitro regeneration efficiency in Helianthus annuus L. Plant Cell Rep 21:29–34CrossRefGoogle Scholar
  8. Drisch RC, Stahl Y (2015) Function and regulation of transcription factors involved in root apical meristem and stem cell maintenance. Front Plant Sci 6:505. CrossRefGoogle Scholar
  9. El-showk S, Ruonala R, Helariutta Y (2013) Crossing paths: cytokinins signalling and crosstalk. Development 140:1373–1383CrossRefGoogle Scholar
  10. Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual. 4th edn. In Kielkopf CL, Bauer W, Urbatsch IL (eds) Expression of cloned genes in E. coli using IPTG-inducible promoters. Vol 3 Part 4: Gene expression, Chap 19. Cold Spring Harbor Laboratory Press, New York, p 1508Google Scholar
  11. Hecht S, Eisenreich W, Adam P, Amslinger S, Kis K, Bacher A, Arigoni D, Rohdich F (2001) Studies on the nonmevalonate pathway to terpenes: the role of the GcpE (IspG) protein. PNAS 98(26):14837–14842CrossRefGoogle Scholar
  12. Hussain S, Jain A, Kothari SL (1999) Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Capsicum annuum L. Plant Cell Rep 19:64–68CrossRefGoogle Scholar
  13. Idris EE, Iglesias DJ, Talon M, Borris R (2007) Tryptophan-dependent production of indole-3-acetic acid (IAA) affects level of plant growth promotion by Bacillus amyloliquefaciens FZB42. MPMI 20(6):619–626CrossRefGoogle Scholar
  14. Kakimoto T (2001) Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate: ATP/ADP isopentenyl transferases. Plant Cell Physiol 42:677–685CrossRefGoogle Scholar
  15. Kakimoto T (2003) Cytokinin biosynthesis. J Plant Res 116:233–239CrossRefGoogle Scholar
  16. Kamada-Nobusada T, Sakakibara H (2009) Molecular basis for cytokinin biosynthesis. Phytochemistry 70:444–449CrossRefGoogle Scholar
  17. Krall L, Raschke M, Zenk MH, Baron C (2002) The Tzs protein from Agrobacterium tumefaciens C58 produces zeatin riboside 5′-phosphate from 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate and AMP. FEBS Lett 527:315–318CrossRefGoogle Scholar
  18. Laloue M, Terrine C, Guern J (1977) Cytokinins: metabolism and biological activity of N6-(∆2-Isopentenyl) adenosine and N6-(∆2-Isopentenyl) adenine in tobacco cells and callus. Plant Physiol 59:478–483CrossRefGoogle Scholar
  19. Liu J, Sheng L, Xu Y, Li J, Yang Z, Huang H, Xu L (2014) WOX11 and 12 are involved in the first-step cell fate transition during de novo root organogenesis in Arabidopsis. Plant Cell 26(3):1081–1093CrossRefGoogle Scholar
  20. MacDonald EM, Powell Gary K, Regier Dean A (1986) Secretion of zeatin, ribosylzeatin, and ribosyl-1″-methylzeatin by Pseudomonas savastanoi. Plant Physiol 82:742–747CrossRefGoogle Scholar
  21. Martens DA, Frankenberger WT Jr (1994) Assimilation of exogenous 2′-14C-indole-3-acetic acid and 3′-14C-tryptophan exposed to the roots of three wheat varieties. Plant Soil 166:281–290CrossRefGoogle Scholar
  22. Milborrow BV, Purse JG, Wightman F (1975) On the auxin activity of phenylacetic acid. Ann Bot 39:1143–1146Google Scholar
  23. Miyawaki K, Matsumoto-Kitano M, Kakimoto T (2004) Expression of cytokinin biosynthetic isopentenyl transferase genes in Arabidopsis: tissue specificity and regulation by auxin, cytokinin, and nitrate. Plant J 37:138CrossRefGoogle Scholar
  24. Molnár-Perl I, Katona ZF (2000) GC-MS of amino acids as their trimethylsilyl/t-butyldimethylsilyl derivatives: in model solutions III. Chromatographia 51:S228–S236CrossRefGoogle Scholar
  25. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  26. Nautiyal CS, Rehman A, Chauhan PS (2010) Environmental Escherichia coli occur as natural plant growth-promoting soil bacterium. Arch Microbiol 192:185–193CrossRefGoogle Scholar
  27. Nordström A, Tarkowski P, Tarkowska D, Norbaek R, Astot C, Dolezal K, Sandberg G (2004) Auxin regulation of cytokinin biosynthesis in Arabidopsis thaliana: a factor of potential importance for auxin-cytokinin-regulated development. PNAS 101:8039–8044CrossRefGoogle Scholar
  28. Parrot S, Jones S, Cooper RA (1987) 2-Phenylethylamine catabolism by Escherichia coli K12. J Gen Microbiol 133:347–351Google Scholar
  29. Pérez-Martínez I, Zhao Y, Murillo J, Sundin GW, Ramos C (2008) Global genomic analysis of Pseudomonas savastanoi pv. savastanoi plasmids. J Bacteriol 190(2):625–635CrossRefGoogle Scholar
  30. Powell GK, Morris RO (1986) Nucleotide sequence and expression of a Pseudomonas savastanoi cytokinin biosynthetic gene: homology with Agrobacterium tumefaciens tmr and tzs loci. Nucleic Acids Res 14:2555–2565CrossRefGoogle Scholar
  31. Riefler M, Novak O, Strnad M, Schmülling T (2006) Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism. Plant Cell 18:40–54CrossRefGoogle Scholar
  32. Romasi EF, Lee J (2013) Development of indole-3-acetic acid-producing Escherichia coli by functional expression of IpdC, AspC, and Iad1. J Microbiol Biotechnol 23(12):1726–1736CrossRefGoogle Scholar
  33. Sakakibara H (2005) Cytokinins biosynthesis and regulation. In: Litwack G (ed) Vitamins and hormones vol 72. Plant Hormones. Academic Press, Cambridge, pp 271–287Google Scholar
  34. Shetty RP, Endy D, Knight TF Jr (2008) Engineering biobrick vectors from Biobrick parts. J Biol Engin 2:5. CrossRefGoogle Scholar
  35. Skoog F, Miller CO (1957) Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol 54:118–130Google Scholar
  36. Skoog F, Hamzi HQ, Zweykowska A, Leonard NJ, Carraway KL, Fujii T, Helgeson JP, Loeppky RN (1967) Cytokinin: structure/activity relationships. Phytochemstry 6:1169–1192CrossRefGoogle Scholar
  37. Spaepen S, Vaderleyden J, Remans R (2007) Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiol Rev 31:425–448CrossRefGoogle Scholar
  38. Su YH, Liu YB, Zhang XS (2011) Auxin-cytokinin interaction regulates meristem development. Mol Plant 4(4):616–625CrossRefGoogle Scholar
  39. Sun J, Niu QW, Tarkowski P, Zheng B, Tarkowska D, Sandberg G, Chua NH, Zuo J (2003) The Arabidopsis AtIPT8/PGA22 gene encodes an isopentenyl transferase that is involved in de novo cytokinin biosynthesis. Plant Physiol 131:167–176CrossRefGoogle Scholar
  40. Taiz L, Zeiger E (2010) Cytokinin: regulators of cell division regulator. In: Plant Physiology, 5th edn. Sinauer Associates Inc., Sunderland, ISBN 978-0-87893-866-7Google Scholar
  41. Walker V, Bruto M, Bellvert F et al (2013) Unexpected phytostimulatory behavior for Escherichia coli. and Agrobacterium tumefaciens model strains. MPMI 26(5):495–502CrossRefGoogle Scholar
  42. Werner T, Motyka V, Strnad M, Schmulling T (2001) Regulation of plant growth by cytokinin. PNAS 98:10487–10492CrossRefGoogle Scholar
  43. Werner T, Motyka V, Laucou V, Smets R, van Onckelen H, Schmulling T (2003) Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15:2532–2550CrossRefGoogle Scholar
  44. Wightman F, Lighty DL (1982) Identification of phenyl acetic acid as a natural auxin in the shoots of higher plants. Physiol Plant 55(1):17–24CrossRefGoogle Scholar
  45. Yamada Y, Sekiya J, Koshimizu K (1972) Cytokinin-induced shoot formation. Phytochem 11:1019–1021CrossRefGoogle Scholar
  46. Ying-Hua S, Liu Y-B, Xia-Sheng Z (2011) Auxin-cytokinin interaction regulates meristem development. Mol Plant 4(4):616–625CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Alberto Camas-Reyes
    • 1
  • Ricardo Laguna-Ramírez
    • 1
  • Alba E. Jofre-Garfias
    • 2
  • Faviola Cardoso-Martínez
    • 1
  • Ana Lilia Hernández-Orihuela
    • 1
  • Jorge Molina-Torres
    • 3
  • Agustino Martínez-Antonio
    • 1
    Email author
  1. 1.Laboratorio de Ingeniería Biológica, Dpto. de Ingeniería GenéticaCentro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV Unidad Irapuato)IrapuatoMexico
  2. 2.Laboratorio de Cultivo de Tejidos y Transformación Genética de Plantas, Dpto. de Ingeniería GenéticaCentro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV Unidad Irapuato)IrapuatoMexico
  3. 3.Laboratorio de Fitobioquímica, Depto. de Biotecnología y BioquímicaCentro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV Unidad Irapuato)IrapuatoMexico

Personalised recommendations