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Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

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Abstract

The insertion of foreign DNA in plants occurs through a complex interaction between Agrobacteria and host plant cells. The marker gene β-glucuronidase of Escherichia coli and cytological methods were used to characterize competent cells for Agrobacterium-mediated transformation, to study early cellular events of transformation, and to identify the potential host-cell barriers that limit transformation in Arabidopsis thaliana L. Heynh. In cotyledon and leaf explants, competent cells were mesophyll cells that were dedifferentiating, a process induced by wounding and-or phytohormones. The cells were located either at the cut surface or within the explant after phytohormone pretreatment. In root explants, competent cells were present in dedifferentiating pericycle, and were produced only after phytohormone pretreatment. Irrespective of their origin, the competent cells were small, isodiametric with thin primary cell walls, small and multiple vacuoles, prominent nuclei and dense cytoplasm. In both cotyledon and root explants, histological enumeration and β-glucuronidase assays showed that the number of putatively competent cells was increased by preculture treatment, indicating that cell activation and cell division following wounding were insufficient for transformation without phytohormone treatment. Exposure of explants for 48 h to A. tumefaciens produced no characteristic stress response nor any gradual loss of viability nor cell death. However, in the competent cell, association between the polysaccharide of the host cell wall and that of the bacterial filament was frequently observed, indicating that transformation required polysaccharide-to-polysaccharide contact. Flow cytofluorometry and histological analysis showed that abundant transformation required not only cell activation (an early state exhibiting an increase in nuclear protein) but also cell proliferation (which in cotyledon tissue occurred at many ploidy levels). Noncompetent cells could be made competent with the appropriate phytohormone treatments before bacterial infection: this should aid analysis of critical steps in transformation procedures and should facilitate developing new strategies to transform recalcitrant plants.

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Abbreviations

BAP:

6-benzylaminopurine

BM:

basal medium

2,4-D:

2,4-dichlorophenoxyacetic acid

GUS:

β-glucuronidase

K:

kinetin

KmR :

kanamycin resistant

NAA:

naphthaleneacetic acid

PAS:

periodic acid-Schiff's

35S-GUS:

GUS marker gene driven by the promoter of cauliflower mosaic virus

T-DNA:

transferred DNA

TR-GUS:

GUS marker gene driven by TR-promoter

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Authors and Affiliations

  1. Laboratoire Androgenèse et Biotechnologie, Université de Picardie Jules Verne, 33 Rue Saint-Leu Ilot des Poulies, F-80039, Amiens Cédex, France

    Rajbir S. Sangwan, Yvan Bourgeois, Gérard Vasseur & Brigitte Sangwan-Norreel

  2. Institut des Sciences Végétales, Service de Cytométrie, C.N.R.S., F-91198, Gif-sur-Yvette, France

    Spencer Brown

Authors
  1. Rajbir S. Sangwan
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  2. Yvan Bourgeois
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  3. Spencer Brown
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  4. Gérard Vasseur
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  5. Brigitte Sangwan-Norreel
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Dedicated to the memory of Professor H. Camefort

We thank Drs. M. Van Montagu (Laboratory of Genetics, State University, Gent, Belgium), J. Leemans (Plant Genetic systems, Gent, Belgium), D.P.S. Verma (Biotechnology Centre, Ohio State University, Columbus, USA) and L. Willmitzer (Institut für Gen-biologische Forschung, Berlin, FRG) for generous gifts of plas-mids; Drs. V. Raghavan (Department of Plant Biology, Ohio State University), E. Zyprian (Laboratoire AEB, Université de Picardie Jules Verne), B. Gronenborn and J. Leung (Institut des Sciences Végétales, CNRS) for discussion, and Dr. B. Ahloowalia (Kinsealy Research Center, Dublin, Ireland) for critical reading the manuscript. We also express thanks to D. Marie (Institut des Sciences Végétales, CNRS) for performing numerous flow-cytometric analyses and M. Poiret for technical assistance.

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Sangwan, R.S., Bourgeois, Y., Brown, S. et al. Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana . Planta 188, 439–456 (1992). https://doi.org/10.1007/BF00192812

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