312
C. Marquina
also observed inside the xylem vessels. After 168 h there were almost no nanoparticles, neither in the pit cavity, nor in the tissues of the stem near the point of application. TEM analysis of samples collected 48 h after the administration, close to the
magnets placed far from the point of injection, revealed the presence of individual
nanoparticles in the cytoplasm of cells close to the vascular system. Nanoparticles
were also observed inside the xylem vessels, suggesting that the nanoparticles use
them for travelling long distances inside the plant. The microscopy study carried
out in the different samples allowed also to analyze the structure of the cytoplasm.
A dense cytoplasm with starch-containing organelles was observed in those cells
collected 24 h after injection, with nanoparticle aggregates in the cytosol. However
these starch structures were neither detected in adjacent cells without nanoparticles,
nor in the cytoplasm of cells in tissues collected far from the application point 48 h
later, containing individual nanoparticles. This fact suggests that these changes in
their subcellular organization could be a plant response to the presence of a high
density of nanoparticles. Cytotoxicity has been associated with the dose of nanoparticles [65], in particular with those in a magnetic ferrofluid [66–68]. However, only
with our experiments it is not possible to assess if the observed reaction to the Fe@C
nanoparticles is specific of the plant cells, or is a common cell reaction to high
nanoparticle concentrations in the cytoplasm. Moreover, an effect of the calcium in
the gelafundine in which nanoparticles are suspended cannot be completely excluded.
13.2.2 Nanoparticle Application by Spray
This methodology was chosen because is more similar to that used by agronomists
in cultivated plants, for example for phytosanitary control. The experiments were as
well carried out on living pumpkin plants and with the Fe@C nanoparticles suspended
in gelafundine [52]. Drops of nanoparticle suspension were sprayed of the surface
of leaves close to the insertion of the petiole. Tissue samples (close and far from
the application point) were also collected 24, 48 and 168 h after spraying. In this
case, nanoparticles were only observed in samples taken 168 h after application.
It was not possible to distinguish any nanoparticle aggregates by light microscopy,
and the nanoparticles were only visible in TEM images of cells from the epidermis
of the petiole close to the application point. Moreover, there were no nanoparticles
in cells beyond the first epidermal layer. In these cells the nanoparticles appeared
isolated, not in aggregates. There was no difference between the intracellular structure density observed in those cells with nanoparticles and in the neighboring cells
without nanoparticles in their cytoplasm. As the epidermic outer cell wall has a
considerable thickness and is covered by protective waxes, it is quite likely that the
nanoparticles penetrate through the stomata and the subestomatic chambers. In fact,
this is a route used by pathogens of different species [69, 70]. The fact that nanoparticles passed through the epidermal cell wall opens up the possible application of these
nanotechnology tools for agronomical purposes. However, these results should be
taken as preliminary. For example, the fact that the amount of nanoparticles found in
C. Marquina
also observed inside the xylem vessels. After 168 h there were almost no nanoparticles, neither in the pit cavity, nor in the tissues of the stem near the point of application. TEM analysis of samples collected 48 h after the administration, close to the
magnets placed far from the point of injection, revealed the presence of individual
nanoparticles in the cytoplasm of cells close to the vascular system. Nanoparticles
were also observed inside the xylem vessels, suggesting that the nanoparticles use
them for travelling long distances inside the plant. The microscopy study carried
out in the different samples allowed also to analyze the structure of the cytoplasm.
A dense cytoplasm with starch-containing organelles was observed in those cells
collected 24 h after injection, with nanoparticle aggregates in the cytosol. However
these starch structures were neither detected in adjacent cells without nanoparticles,
nor in the cytoplasm of cells in tissues collected far from the application point 48 h
later, containing individual nanoparticles. This fact suggests that these changes in
their subcellular organization could be a plant response to the presence of a high
density of nanoparticles. Cytotoxicity has been associated with the dose of nanoparticles [65], in particular with those in a magnetic ferrofluid [66–68]. However, only
with our experiments it is not possible to assess if the observed reaction to the Fe@C
nanoparticles is specific of the plant cells, or is a common cell reaction to high
nanoparticle concentrations in the cytoplasm. Moreover, an effect of the calcium in
the gelafundine in which nanoparticles are suspended cannot be completely excluded.
13.2.2 Nanoparticle Application by Spray
This methodology was chosen because is more similar to that used by agronomists
in cultivated plants, for example for phytosanitary control. The experiments were as
well carried out on living pumpkin plants and with the Fe@C nanoparticles suspended
in gelafundine [52]. Drops of nanoparticle suspension were sprayed of the surface
of leaves close to the insertion of the petiole. Tissue samples (close and far from
the application point) were also collected 24, 48 and 168 h after spraying. In this
case, nanoparticles were only observed in samples taken 168 h after application.
It was not possible to distinguish any nanoparticle aggregates by light microscopy,
and the nanoparticles were only visible in TEM images of cells from the epidermis
of the petiole close to the application point. Moreover, there were no nanoparticles
in cells beyond the first epidermal layer. In these cells the nanoparticles appeared
isolated, not in aggregates. There was no difference between the intracellular structure density observed in those cells with nanoparticles and in the neighboring cells
without nanoparticles in their cytoplasm. As the epidermic outer cell wall has a
considerable thickness and is covered by protective waxes, it is quite likely that the
nanoparticles penetrate through the stomata and the subestomatic chambers. In fact,
this is a route used by pathogens of different species [69, 70]. The fact that nanoparticles passed through the epidermal cell wall opens up the possible application of these
nanotechnology tools for agronomical purposes. However, these results should be
taken as preliminary. For example, the fact that the amount of nanoparticles found in