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Optimizing the transient Fast Agro-mediated Seedling Transformation (FAST) method in Catharanthus roseus seedlings

Abstract

Key message

An Agro-mediated transformation method has been adapted in Catharanthus roseus seedlings for transient overexpression. Our results suggest that Agro-mediated methods may induce defense-related genes, which should be considered in its application.

Abstract

The Fast Agro-mediated Seedling Transformation (FAST) method, which involves the co-cultivation and transient transformation of young seedlings with Agrobacterium, was adapted and optimized in Catharanthus roseus. We investigated the optimal conditions for Gus expression by varying the Agrobacterium density (OD600 = 0.29 and 0.50), A. rhizogenes strain (15834 and R1000), and co-cultivation time in liquid (2, 12, or 24 h) followed by incubation time on solid media (1 or 2 days). Transformation efficiency was assessed quantitatively in terms of average GUS intensity per cotyledon surface area and percentage of cotyledons transformed. GUS staining was observed in 100 % of cotyledons co-cultivated with A. rhizogenes (OD600 = 0.50) co-transformed with the Mas promoter-driven Gus and pSoup helper plasmids, in the presence of 0.01 % v/v Silwet L-77 for 24 h in liquid followed by 2-days on solid media. In addition, we observed that co-cultivation with Agrobacterium strongly induced Zct1 and Orca3, two transcription factors known to regulate defense-related alkaloid biosynthesis in C. roseus. Homologous transcription factors regulate defense responses in many plant species. Therefore, possible induction of defense-related genes by Agro-mediated transformation should be a consideration in experimental design.

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Acknowledgments

This work was supported by the National Science Foundation (NSF-CAREER, Grant No. BES-0134511; NSF-CBET Grant No. 1033889). We would like to thank Dr. Jian-Feng Li and Dr. Andreas Nebenführ (University of Tennessee, Knoxville, Tennessee) for providing the pVKH-NLS-YFP-GUS plasmid and plasmid map and Dr. Hans-Joerg Jacobsen (Leibnitz University, Hannover, Germany) for providing the pGII0229TR-GUS-CP-LUC and pSoup plasmids.

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Correspondence to Carolyn W. T. Lee-Parsons.

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Communicated by K. Kamo.

J. Weaver and S. Goklany contributed equally.

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Weaver, J., Goklany, S., Rizvi, N. et al. Optimizing the transient Fast Agro-mediated Seedling Transformation (FAST) method in Catharanthus roseus seedlings. Plant Cell Rep 33, 89–97 (2014). https://doi.org/10.1007/s00299-013-1514-2

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  • DOI: https://doi.org/10.1007/s00299-013-1514-2

Keywords

  • Catharanthus roseus
  • Agrobacterium-mediated transient transformation
  • Orca APETALA2/ETHYLENE Response Factor (AP2/ERF) family
  • Zct zinc finger protein family
  • Transcription factor
  • Plant defense response