Nanoparticles as smart treatment-delivery systems in plants: assessment of different techniques of microscopy for their visualization in plant tissues

Abstract

Background and aims: The great potential of using nanodevices as delivery systems to specific targets in living organisms was first explored for medical uses. In plants, the same principles can be applied for a broad range of uses, in particular to tackle infections. Nanoparticles tagged to agrochemicals or other substances could reduce the damage to other plant tissues and the amount of chemicals released into the environment. To explore the benefits of applying nanotechnology to agriculture, the first stage is to work out the correct penetration and transport of the nanoparticles into plants. This research is aimed (a) to put forward a number of tools for the detection and analysis of core-shell magnetic nanoparticles introduced into plants and (b) to assess the use of such magnetic nanoparticles for their concentration in selected plant tissues by magnetic field gradients.

Methods: Cucurbita pepo plants were cultivated in vitro and treated with carbon-coated Fe nanoparticles. Different microscopy techniques were used for the detection and analysis of these magnetic nanoparticles, ranging from conventional light microscopy to confocal and electron microscopy.

Key results: Penetration and translocation of magnetic nanoparticles in whole living plants and into plant cells were determined. The magnetic character allowed nanoparticles to be positioned in the desired plant tissue by applying a magnetic field gradient there; also the graphitic shell made good visualization possible using different microscopy techniques.

Conclusions: The results open a wide range of possibilities for using magnetic nanoparticles in general plant research and agronomy. The nanoparticles can be charged with different substances, introduced within the plants and, if necessary, concentrated into localized areas by using magnets. Also simple or more complex microscopical techniques can be used in localization studies.

Fig. 1.

(A) Pumpkin plant growing in the polyethylene bag system. Red circles indicate where magnets were placed. (B) Detail showing the point of application of the bioferrofluid (black arrow) and further expected movement of nanoparticles through the vascular system (red arrows). (C) Schematic representation of a transverse section of the pumpkin stem. ep, Epidermis; cx, cortex; fb, fibres; vc, xylem vessels; ih, internal hollow.

Fig. 2.

Hand-cut sections of petioles (A–C) and roots (D–F) of pumpkin plants treated with bioferrofluid. (A) Detail of vascular tissues at the application point. Dark coloration indicates accumulation of bioferrofluid. (B) Detail of vascular tissues adjacent to a magnet. Bioferrofluid is concentrated in xylem vessels. (C) Detail of vascular tissues opposite a magnet placement. No bioferrofluid accumulation is observed. (D) Detail of root vascular tissue preceding magnet localization. Bioferrofluid appears distributed through the xylem vessels as a dark staining. (E) As (D) but at the point of the magnet placement. Again strong presence of bioferrofluid is observed. (F) As (D) but after the point of the magnet placement. No bioferrofluid is observed, indicating its flux was mainly stopped at the magnet placement.

Fig. 3.

Techniques for the detection of nanoparticles injected into plants at the resolution of the light microscope. (A–C) Projection of 3-D confocal stacks from a cell of the stem, after 72 h at the position of the magnet. Aggregates of nanoparticles (arrow) are detected by Nomarski (A) and reflection (B) techniques. The overlay between (A) and (B) is shown in (C) with an almost complete colocalization. (D–G) Detection of nanoparticles on 7-μm sections of paraffin-embedded plant tissues. The particles, identified as dark dots under bright field (arrows in D and F) correspond to non-fluorescent areas (arrows in E and G) within the autofluorescent cell wall of xylem cells (xy) under an epi-fluorescence microscope. (H–K) Detection of nanoparticles on 1- to 2-μm sections from specimens embedded in Epon resin, after 24 h at the site of injection. The nanoparticles can be observed on a light photomicroscope as a punctuate pattern of dark or refringent areas (arrows) under phase contrast (H), bright field (I) and dark field (J). (K) shows a portion of the stem with a xylem vessel from a control plant without any signal, under phase contrast. Scale bar =10 µm.

Fig. 4.

Transmission electron microscopy. (A) A similar area of the cortex to the one shown in Fig. 3K, with a section of a xylem cell (xy). No particles are detected. (B–E) Nanoparticles detected on correlated light and electron microscopy imaging of the same specimen after 24 h at the injection site. (B) The nanoparticles (arrows) are seen as beads on a string along the cells on the outer side of the cell wall at a groove of the epidermis by phase contrast on a light microscope. (C) The same area as in (B) imaged on the transmission electron microscope where electron-dense areas can be seen (asterisks): (D, E) higher magnification of the areas marked in (C) with one and two asterisks, respectively, in which aggregates of nanoparticles of different sizes can clearly been recognized in the extracellular area. Scale bars: A = 10 µm; B = 50 µm; C = 5 µm; D, E = 0·1 µm.

Fig. 5.

Size distribution of nanoparticles found in the cells and the extracellular space after injection.