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Optimization of Agrobacterium-mediated transient gene expression and endogenous gene silencing in Piper colubrinum Link. by vacuum infiltration

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Abstract

The present study describes the successful development of vacuum infiltration method in the oomycete-resistant wild Piper sp., Piper colubrinum, as a rapid transient method for expression of GUS (β-Glucuronidase) reporter gene and introduction of hairpin vector for endogenous gene silencing. The GUS reporter gene construct pCAMBIA 1305.2 was used as a positive control to test the efficiency of vacuum infiltration strategy. Agrobacteria (EHA 105) harbouring GUS binary vector were vacuum-infiltrated into young detached in vitro leaf explants, which showed detectable GUS gene activity within 4 days of infiltration. This paper also reports for the first time the application of transient gene silencing in P. colubrinum by the delivery of in vitro synthesized hairpin vector construct (pHELLSGATE) containing endogenous serine threonine protein kinase (STPK) gene homologue into in vitro shoots. Introduction of hairpin vectors for the STPK gene into in vitro plantlets by vacuum infiltration resulted in significant reduction in transcript accumulation of the endogenous gene. The results indicate that transient gene silencing could be used as a rapid, preliminary high-throughput tool for P. colubrinum functional genomics.

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Abbreviations

BA:

6-Benzyladenine

GUS:

β-Glucuronidase

MMLV-RT:

Moloney murine leukemia virus reverse transcriptase

MS:

Murashige and Skoog

PCR:

Polymerase chain reaction

STPK:

Serine threonine protein kinase

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Acknowledgments

T.M. would like to acknowledge the Council for Scientific and Industrial Research, New Delhi, Government of India, for CSIR–Junior Research Fellowship, and M.S. gratefully acknowledges the Department of Biotechnology, Government of India, for financial support in the form of research grant. The authors also wish to acknowledge CSIRO, Australia for providing the pHELLSGATE vector.

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Authors and Affiliations

  1. Plant Molecular Biology Division, Rajiv Gandhi Centre for Biotechnology, Poojappura, Trivandrum, Kerala, 695014, India

    Tomson Mani & S. Manjula

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  1. Tomson Mani
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  2. S. Manjula
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Correspondence to S. Manjula.

Electronic supplementary material

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PCR amplification of P.colubrinum STPK target sequence (120 bp) for silencing experiments (JPEG 16 KB)

11240_2010_9836_MOESM2_ESM.jpg

Result of control agroinfiltration experiment showing GUS expression in leaves of Nicotiana tabacum under identical experimental conditions (JPEG 1389 KB)

11240_2010_9836_MOESM3_ESM.jpg

A diagrammatic representation of P.colubrinum STPK gene indicating the target sequence for silencing and the corresponding primer positions (JPEG 52 KB)

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Mani, T., Manjula, S. Optimization of Agrobacterium-mediated transient gene expression and endogenous gene silencing in Piper colubrinum Link. by vacuum infiltration. Plant Cell Tiss Organ Cult 105, 113–119 (2011). https://doi.org/10.1007/s11240-010-9836-z

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  • DOI: https://doi.org/10.1007/s11240-010-9836-z

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