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

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.

The Petunia tra1 gene controls cell elongation and plant development, and mediates responses to cytokinins

“…After dehydration by a graded ethanol series, the sample were embedded in Araldite, Technovit (LaboNord, Villeneuve d'Ascq, France) or LR White resin (TAAB Laboratories, Almerston, Reading, Berks, UK). For light microscopy, the semi-thin sections (1-5 I~m) were stained with Azure blue, PAS (periodic acid-Schiff's) and PAS-naphthol blue-black as described (Sangwan et al, 1992). PAS specifically stained polysaccharides, cell wall and starch red, while naphthol blue-black specifically revealed soluble proteins and reserve proteins by staining them blue-black.…”

“…The specimens were fixed in 4% glutaraldehyde as described above, and postfixed in 1% osmium tetroxide for 2 h. After dehydration in an ethanol series, the samples were embedded in Araldite as described (Sangwan et al, 1992). Ultra-thin sections (50-70 nm) were poststained with aqueous uranyl acetate followed by lead citrate and examined under an electron microscope (301 G; Philips, Eindhoven, The Netherlands).…”

Citrus biotechnology: Achievements, limitations and future directions

“…Pre-culturing of explants on co-cultivation medium rich in auxins prior to Agrobacterium inoculation has been shown to increase transformation efficiency in many woody plants such as plum (Mante et al, 1991), apricot (Laimer et al, 1992) and Arabidopsis (Sangwan et al, 1992) by increasing the number of competent cells at cut ends for transformation, but in case of citrus the reverse happened to be true as the transformation efficiency dropped to half after pre-culture treatment (Cervera et al,1998c;Costa et al, 2002).…”

Activity of antioxidant enzymes during induction of morphogenesis of Fritillaria meleagris in bulb scale culture

“…Injuries to plant tissue can be a trigger for cell division (Sangwan et al, 1992). Low temperature, which is necessary to overcome dormancy, also represents a kind of oxidative stress for the plant.…”