E. coli cultures expressing a synthetic sequence of ptz gene (stz) promoted in vitro direct organogenesis in Nicotiana tabacum L.
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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.
KeywordsSynthetic biology Ptz Escherichia coli Cytokinins In vitro organogenesis Tobacco
Murashige and Skoog medium
Plant growth-promoting rhizobacteria
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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).
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.
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