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Adventitious Root Induction in Arabidopsis thaliana as a Model for In Vitro Root Organogenesis

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Plant Organogenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 959))

Abstract

Adventitious root formation, the development of roots on non-root tissue (e.g. leaves, hypocotyls and stems) is a critical step during micropropagation. Although root induction treatments are routinely used for a large number of species micropropagated in vitro as well as for in vivo cuttings, the mechanisms controlling adventitious rooting are still poorly understood. Researchers attempt to gain better insight into the molecular aspects by studying adventitious rooting in Arabidopsis thaliana. The existing assay involves etiolation of seedlings and measurements of de novo formed roots on the elongated hypocotyl. The etiolated hypocotyls express a novel auxin-controlled signal transduction pathway in which auxin response factors (ARFs), microRNAs and environmental conditions that drive adventitious rooting are integrated. An alternative assay makes use of so-called thin cell layers (TCL), excised strips of cells from the inflorescence stem of Arabidopsis thaliana. However, both the etiolated seedling system and the TCL assay are only distantly related to industrial rooting processes in which roots are induced on adult stem tissue. Here, we describe an adventitious root induction system that uses segments of the inflorescence stems of Arabidopsis thaliana, which have a histological structure similar to cuttings or in vitro micropropagated shoots. The system allows multiple treatments with chemicals as well as the evaluation of different environmental conditions on a large number of explants. It is therefore suitable for high throughput chemical screenings and experiments that require numerous data points for statistical analysis. Using this assay, the adventitious root induction capacity of classical auxins was evaluated and a differential response to the different auxins could be demonstrated. NAA, IBA and IAA stimulated adventitious rooting on the stem segment, whereas 2,4-D and picloram did not. Light conditions profoundly influenced the root induction capacity of the auxins. Additionally to the environmental control of adventitious root formation, we also investigated the spatial and temporal aspects of stem-based adventitious root organogenesis. To determine the cells involved in de novo root initiation on the adult stems, we adopted scanning electron microscopy, which allows the visualization of the auxin responsive stem tissue. Using this technique, direct (without callus interface) and indirect (with intermediate callus phase) organogenesis was readily distinguished. The described micro-stem segment system is also suitable for other non-woody species and it is a valuable tool to perform fast evaluations of different treatments to study adventitious root induction.

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Acknowledgments

This research was supported by the IWT (The Flemish institute for Innovation by Science and Technology). We are grateful to Marjolein Couvreur (Faculty of Sciences, Department Biology) for the critical point drying of the samples and to Dr. Daniel Van Damme for the introduction of the SEM.

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Authors and Affiliations

  1. Department of Plant Production, Faculty of bioscience engineering, Ghent University, Ghent, Belgium

    Inge Verstraeten & Danny Geelen

  2. Department of Plant Systems Biology, VIB (Flanders Institute for Biotechnology), Ghent, Belgium

    Inge Verstraeten

  3. Department of Plant Systems Biology, VIB, Ghent, Belgium

    Tom Beeckman

  4. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

    Tom Beeckman

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  1. Inge Verstraeten
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  2. Tom Beeckman
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  3. Danny Geelen
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Correspondence to Danny Geelen .

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  1. School of Biosciences, University of Nottingham, Loughborough, LE12 5RD, United Kingdom

    Ive De Smet

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Verstraeten, I., Beeckman, T., Geelen, D. (2013). Adventitious Root Induction in Arabidopsis thaliana as a Model for In Vitro Root Organogenesis. In: De Smet, I. (eds) Plant Organogenesis. Methods in Molecular Biology, vol 959. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-221-6_10

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  • DOI: https://doi.org/10.1007/978-1-62703-221-6_10

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