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Transient hairpin RNAi-induced silencing in floral tissues of Dendrobium Sonia ‘Earsakul’ by agroinfiltration for rapid assay of flower colour modification

Plant Cell, Tissue and Organ Culture (PCTOC) volume 120, pages 643–654 (2015)Cite this article

Abstract

Agroinfiltration is an Agrobacterium-mediated transient assay for the analysis of gene function and genetic modification in leaves, flowers and fruit tissues of various plants, and transient RNAi-induced silencing by agroinfiltration has been developed in leaves and fruits of several plant species. Here we report the establishment of a transient hairpin RNAi-induced silencing system for colour modification assay in floral tissues of Dendrobium Sonia ‘Earsakul’, a tropical hybrid orchid with purple and white bi-coloured flowers. Portions of the D. Sonia ‘Earsakul’ anthocyanin-related genes chalcone synthase (DseCHS-B) and dihydroflavonol 4-reductase (DseDFR) which are required for anthocyanin synthesis, were cloned into the hairpin-based RNAi vectors, pSTARGATE and pWATERGATE, under the control of the maize ubiquitin and Arabidopsis Rubisco small subunit (ARbcS) promoters, respectively. Characterisation of DseCHS-B and DseDFR expression profiles revealed that their expression was developmentally regulated in the flower and associated with flower pigmentation. Transient RNA silencing of DseCHS-B and DseDFR in the floral tissues of D. Sonia ‘Earsakul’ was induced by delivering 436-bp DseCHS-B and 470-bp DseDFR hairpin RNAs (hpRNAs) into the sepals and petals of flower buds at early developmental stages using agroinfiltration. Impaired anthocyanin accumulation and reduction of endogenous mRNAs of the corresponding targets were observed in the infiltrated areas of the sepals and petals. Silencing of the endogenous DseCHS-B and DseDFR mRNAs was highly effective at the early stages of mRNA synthesis and anthocyanin accumulation. This transient silencing system is a prototype for modification of the anthocyanin biosynthetic pathway in D. Sonia Earsakul and other orchids through gene suppression.

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Acknowledgments

This work was supported by Grants (P-10-11171) from the National Science and Technology Development Agency (NSTDA), Ministry of Science and Technology, and from Naresuan University, Thailand. We would like to give our sincere thanks to Dr. Orwan Chatchawankanphanich (National Center for Genetic Engineering and Biotechnology, Thailand) for providing A. tumefaciens strain EHA105. We are also grateful to Professor Dr. Duncan R. Smith (Institute of Molecular Biosciences, Mahidol University, Thailand) for English editing and scientific proofreading of this manuscript.

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Authors and Affiliations

  1. Department of Agricultural Science, Faculty of Agriculture, Natural Resources and Environment, Naresuan University, Phitsanulok, 65000, Thailand

    Kumrop Ratanasut & Chaiwat Monmai

  2. Center for Agricultural Biotechnology, Naresuan University, Phitsanulok, Thailand

    Kumrop Ratanasut, Chaiwat Monmai & Pornnapa Piluk

  3. Department of Biology, Faculty of Sciences, Naresuan University, Phitsanulok, Thailand

    Pornnapa Piluk

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  1. Kumrop Ratanasut
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  2. Chaiwat Monmai
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Correspondence to Kumrop Ratanasut.

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Key message: The work describes the establishment of a transient hairpin RNAi-induced silencing system for colour modification assay using agroinfiltration in floral tissues of Dendrobium Sonia ‘Earsakul’.

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Ratanasut, K., Monmai, C. & Piluk, P. Transient hairpin RNAi-induced silencing in floral tissues of Dendrobium Sonia ‘Earsakul’ by agroinfiltration for rapid assay of flower colour modification. Plant Cell Tiss Organ Cult 120, 643–654 (2015). https://doi.org/10.1007/s11240-014-0632-z

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Keyword

  • Agroinfiltration
  • Anthocyanin
  • Dendrobium
  • Flower colour
  • Gene silencing
  • Transient RNAi