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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
  • 61 Downloads

Abstract

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.

Keywords

Synthetic biology Ptz Escherichia coli Cytokinins In vitro organogenesis Tobacco 

Abbreviations

ADP

Adenosine-5′-diphosphate

AMP

Adenosine-5′-monophosphate

ATP

Adenosine-5′-triphosphate

BAP

6-Benzylaminopurine

CKs

Cytokinins

HMBDP

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

IAA

Indole-3-acetic acid

iP

Isopentenyl adenine

IPT

Isopentenyl transferase

IPTG

Isopropyl β-D-1-thiogalactopyranoside

LB

Lysogeny broth

MS

Murashige and Skoog medium

PAA

Phenylacetic acid

PGPR

Plant growth-promoting rhizobacteria

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SE

Standard error

tZ

Trans-zeatin

stZ

Synthesized tZ

stz

Synthetic IPT gene

UPLC

Ultra performance liquid chromatography

Notes

Acknowledgements

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

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Supplementary material 1 (DOCX 134 KB)
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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

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