Observación de la práctica de la lectura de los estudiantes

As discussed earlier, nanomaterials face numerous challenges and hurdles during the course of gaining entry into the plant systems. Some may arise due to intrinsic limitations of the NMs with respect to size, charge, shape, etc., but majority of obstacles are posed during the application and contact of NMs with the microen-vironment of the target exposure tissues. Even when the NMs are successful in gaining entry into the system, either by force or by active/passive diffusion, still, gaining access to the conductive tissues of the plants viz., xylem and phloem remains a challenge. One solution, taken from thefield of medicine, is the age-old tradition of direct injection of substances into the tissues. Though, when applied to the plant systems, this technique provides direct access to the desired tissues bypassing the discussed barriers, it remains a matter of debate when considering the limitations of large-scale application.

Pumpkin plants were selected by Gonzalez-Melendi et al. (2008) due to their large-sized vessels, which are expected to facilitate efficient transport of NPs through the vascular system. Seeds were germinated in Petri dishes on moistened filter paper, and 15-day-old seedlings with a radicle length of about 4–5 cm were transferred to a bag, with Hoagland nutrient solution. The bags were suspended vertically in a controlled environment chamber. Carbon-coated iron NPs were dispersed by sonication in gelafundin, a commercial succinated gel to obtain a biocompatible magneticfluid. The aim of this work was to analyze the capacity of a magnetic field to retain the particles in specific parts of the plant in addition to studying their penetration and movement in plant cells. The bioferrofluid was directly injected inside the internal hollow of the leaf petiole to deliver instant access to the nanoparticles to the vascular system for faster and efficient translo-cation and distribution in the plant body (Fig.4.5). The experimental setup con-sisted of small magnets placed on the petiole of the leaf opposite to the injection point and on some of the roots. It was observed how the bioferrofluid can be concentrated in the desired areas by using magnets. No particles greater than 50 nm were detected inside the tissues, implying a possible size-based selection mecha-nism, probably involving a barrier of cell walls and waxes.

Corredor et al. (2009) performed similar experiments to observe the subcellular localization of carbon-coated nanoparticles under the influence of external magnets.

The employability of magnetic nanoparticles in a large-scale scenario is somewhat a debatable prospect as placing magnets in annual extensive crops (e.g., cereals) is impractical. However, the possibility of using such a system under greenhouse/

controlled conditions would be possible for specific treatments in fruit trees (e.g.,

Table4.3ExamplesofhydroponicallyculturedplantexposuretoNPs NanoparticlePlantMethodObjectiveReference TiO2NPsOnionandtobaccoGerminatedonionbulbsandfourth-leafstage tobaccoplantletswereexposedtoTiO2NPsGenotoxicityGhoshetal.(2010) ZnONPsVelvetmesquiteSeedlingswereexposedtoNPsolutionZnaccumulationintissuesand physiologicalevaluationHernandez-Viezcas etal.(2011) CdSe/ZnS QDsArabidopsisSeedlingsexposedtomediasupplementedQD solutionUptakemechanismNavarroetal. (2012) AgNPsBrahmiplantWeekoldseedlingswereexposedtohydroponic mediumsupplementedwithAgNPsPlantgrowthmetabolismand biochemicalparametersKrishnarajetal. (2012) AuNPsTobaccoandwheat30daysgerminatedseedlingsoftobaccoand 7daysgerminatedseedlingsofwheatwere exposedtoAuNPs Bioaccumulation/Uptakeof differentsizeandsurfacecoatedAu NPs Judyetal.(2012) AuNPsRice,radish, pumpkin,and perennialryegrass

ExposureofseedlingstoAuNPsEffectofsurfacechargeonthe uptakeanddistributionofAuNPsZhuetal.(2012) AuNPsRice9-day-oldgerminatedseedlingswereexposedto NPsdispersedinMilliQwaterTissueleveluptakeandspatial distributionofAuNPsinriceroots andshoots Koelmeletal. (2013) Magnetite NPsSoybeanSeedswereexposedtonutrientmedia containingdifferentconc.ofNPsUptake,translocation,andeffecton chlorophyllcontentinsoybean, underhydroponicconditions

Ghafariyanetal. (2013) CeO2NPsWheatand pumpkinNPswereaddedtothehydroponicculture mediumInfluenceoforganicmatteronNP– plantinteractionSchwabeetal. (2013) ZnONPsMustardSeedsweregerminatedunderahydroponic conditionwithvaryingconcentrationsofZnO NPs

Estimationofplantbiomass, biochemicalparameters,and bioaccumulationofZnO RaoandShekhawat (2014) (continued)

Table4.3(continued) NanoparticlePlantMethodObjectiveReference ZnONPsMaize7-day-oldseedlingswereexposedtoNPsinthe hydroponicculturemediumRootmetabolicactivities, accumulationinroots, biotransformation,phytotoxicity Lvetal.(2015) CeONPsKidneybeanPlantswereexposedtosuspensions of8±1nmnCeO2for15daysin hydroponicconditions

Primaryindicatorsofstress, chlorophyllcontents, bioaccumulation,translocation,and cellularhomeostasis

Majumdaretal. (2014) NaYF4:Yb,Er upconversion nanocrystals

Pumpkin6-day-oldseedlingsinhydroponicculturewere exposedtoNPsKineticsoftheuptakeandthe translocationofNPsNordmannetal. (2015) Fe(nZVI) NPsArabidopsisPlantswereexposedto½strengthMSmedia fortifiedwithNPsPlasmamembraneH+ -ATPase activity—stomatalopeningand geneexpression

Kimetal.(2015) ZnONPsAquaticplant mesocosm— Schoenoplectus tabernaemontani

PlantexposuretoNPsaugmentedhydroponic nutrientmediumPhytotoxicityandbioaccumulationZhangetal.(2015) CuNPsLettuceandalfalfa10-to15-day-oldhydroponicallygrownplants exposedtonCu,bulkCu,nCuO,bulkCuO,Cu (OH)2(CuPRO2005,Kocide3000),andCuCl2

Phytotoxicity-rootlength,catalase andascorbateperoxidase measurement,nutrientcontent

Hongetal.(2015)

olive (Olea europaea) trees) or high-input crops. Still, the major scope of using this model, as of now, would be for laboratory-scale research applications since it allows a very precise localization of particles.

4.5 Spraying

Currently, most of the studies on plants involve exposure of NPs through roots, either directly to established roots or during the germination stage. However, it has to be noted that plants also interact with atmospheric NPs through the leaves, foliar pathway, and knowledge on their response to this contact is limited. Significant effects of NPs on plant foliage are inevitable due to deposition of atmospheric particles or application of purpose-made NPs.

Corredor et al. (2009) apart from studying the localization of carbon-coated NPs by direct injection also employed spray technique to analyze the progressive pen-etration of NPs in the plant tissues. This methodology closely resembles that employed by agronomist and breeders in field applications. Droplets of ferrofluid were placed on the leaf surface, close to the insertion point of the petiole, in an Fig. 4.5 aPumpkin plant growing in the polyethylene bag system. Red circles indicate positions of magnets. b Detail showing the point of application of the bioferrofluid (black arrow) and further expected movement of NPs through the vascular system (red arrows). Reprinted from Gonzalez-Melendi et al. (2008), with permission from Oxford University Press

attempt to emulate spraying of a nanoparticle solution onto a cultivated plant. The method used in this work is similar to the large-scale and hands-on spraying pro-cedures, which are used by breeders and coordinators of phyto-sanitary control.

This spray technique seemed to be a more practical approach from an agronomic perspective, as large-scale and hands-on modules currently exist for the spraying of pesticides and chemical fertilizers.

To control the pathogenic fungi in green zucchini plants infected with powdery mildew, Park et al. (2006) tested nanosized silica-silver (nano-silver combined with silica molecules and water-soluble polymer) by uniformly spraying the nanocom-posite onto green squash plants infected with powdery mildew at 0.3 ppm con-centration. After the nanosized silica-silver had been applied, the progress of powdery mildew was observed for three weeks. In addition to the anti-fungal assay, the possible chemical injuries to plants due to the application of nanosized silica-silver were assessed with the spray of undiluted and 10, 100 and 1000 times diluted solutions of the nanocomposite on the surface of squash leaves including new leaves of cucumber and pansy (Viola tricolor). After three days, chemical injuries on plants were observed.

Birbaum et al. (2010) focused on the quantitative investigation of uptake and translocation of ceria NPs into maize plants using ICP-MS. Various scenarios were simulated, wherein ceria NPs were introduced to leaves as airborne aerosols and aqueous suspensions. The NP exposure to plants was conducted under artificial daylight, to facilitate stomatal opening, and under dark conditions, where stomata are closed. A 10lg/mL of CeO2 NP suspensions were diluted from freshly pre-pared stock suspension. Three- to five-week-old, greenhouse-grown maize plants (Birko) were used for NP exposure studies in an in-house-built glove box of 2.25 m³, wherein the NPs were produced (Fig.4.6). The exposition unit consisted of NP production unit and an exposure chamber, hosting the plants. Once the NP production was initiated, a total exposure of 0.4 g of NPs was achieved in 1 min.

A fan was used to disperse the NPs homogeneously over the plants for an exposure time of 20 min. During harvesting, the leaves were separately rinsed with deionized water and abraded with a glove, simulating a possible naturally occurring washing procedure by rain and the wind. Post-exposure, a batch of NP exposed plants was returned to the greenhouse for a further 12 weeks to determine translocation of NPs into newly developed leaves.

To find an effective solution against the soilborne Oomycete Pythium aphani-dermatum, the causal agent of one of the most serious threats of rhizome rot disease to turmeric (Curcuma longa) crops, Anusuya and Sathiyabama (2015) developed b-D-Glucan nanoparticles (GNPs) with b-Glucan isolated from P. aphanidermatum mycelium. Rhizomes of turmeric were thoroughly cleaned under running tap water and surface-sterilized. Two–three rhizomes each with three nodes were planted in earthen pots containing soil and manure and maintained under glass house condi-tion. A foliar spray of GNPs (0.1 %, w/v) was applied to 30-day-old plants (5 mL/plant) at intervals of 30 days till 210 days after which the leaves were excised and rhizomes left for another 30 days for harvest.

In their study, Hong et al. (2014) aerially treated hydroponically grown cucumber plants with nanoceria powder (nCeO2). Fifteen days after treatment, the test plants were assayed for Ce uptake by using inductively coupled plasma optical emission spectrometry (ICP-OES) and transmission electron microscopy (TEM).

Post-surface sterilization and soaking in deionized water for 24 h, the seeds were placed on the edge of wet germination paper towels, rolled, and supplemented with 10 drops of antimycotic/antibiotic solution, and kept in Mason jars with distilled water at the bottom, set in the dark for four days, and, after that, exposed to light for one day. Seven-day-old plants were transferred to hydroponic jars with 300 mL of a modified Hoagland solution. After two weeks of growth, the young plants were transferred to separate chambers and treated with 0, 0.98 and 2.94 g/m³NP con-centrations. CeO₂NPs were kept on a tray in front of a fan (120 V, 0.25 A, 60 Hz) inside the chamber, and blowing times werefixed at 15 and 45 min. In another set of experiments, leaves of 2-week-old plants were sprayed with CeO₂suspended in distilled water at concentrations 0, 40, 80, 160, and 320 mg/L, with a handheld sprayer bottle (Fig.4.7). A total of 100 mL of respective concentrations were sprayed every 4 h. At defined time points after treatment, samples of leaves, stems, and roots were collected and prepared for analysis.

Fig. 4.6 Cerium dioxide nanoparticles were exposed as in situ-prepared aerosol. Aerosol exposure was performed in a setup developed for application at the air–liquid interface. a The nanoparticles production unit. b The ventilator for the well-characterized and homogeneous particles deposition and c the maize plants within the totally enclosed setup. Reprinted with permission from Birbaum et al. (2010). Copyright (2010) American Chemical Society

The uptake of Ag NPs in lettuce after foliar exposure and possible NP bio-transformation and phytotoxic effects were studied by Larue et al. (2014). Lettuce was chosen as model species because of its large foliar surface and cosmopolitan occurrence in gardens or farmlands making it an ideal model to assess the foliar transfer of NPs. Young lettuce plantlets were exposed to 1, 10, or 100lg Ag NPs.

Plants were harvested after a 7-day exposure.

4.6 Biolistics

Torney et al. (2007) coated DNA onto Type-II MSNs for endocytosis experiments by incubating 1 mg of purified plasmid DNA with 10 mg MSN in 50 ml water for 2 h. The MSNs were thoroughly washed with W5 media prior to incubation with isolated protoplasts. Bio-Rad Biolistic PDS-1000/He particle delivery system was used for MSN delivery into plant cells. To coat DNA onto gold-capped MSNs, a standard protocol was followed similar for biolistic gun with minor modifications.

The particle bombardment parameters for tobacco plantlets were 650 p.s.i. rupture disk, 10-cm gap distance, and 10-cm target distance. A sterile 150-mm mesh was used between the macro-carrier and the target tissue. Bombardment of maize immature embryos was performed using the standard laboratory procedure. After bombardment, the embryos were placed on N6-30 medium for 10 days to avoid Fig. 4.7 a Hydroponic setup for cucumber plants treated with CeO₂ NPs. b Nanoparticle application as powder and c suspension. The plants were cultivated for 15 days before NP treatment and harvested 15 days after treatment. Reprinted with permission from Hong et al.

(2014). Copyright (2014) American Chemical Society

callus autofluorescence interfering with the GFP expression evaluation. Plants were germinated and grown in Y-segmented Petri dishes. Plants were grown for 48 h after bombardment before being subjected to evaluation.

4.7 Discussion

Any mode of nanoparticle application to plants, either under laboratory conditions or infield scenario, must take into account certain essential aspects as the acces-sibility of the nanoformulations to target tissues, minimum concentrations with maximum effect, scalability of the approach, and, of course, the economics of the whole process. Transportation of molecules across plant cells is more complicated due to the cell wall, which is mostly made up of cross-linked polysaccharides, and cell membrane, which pose a great challenge for nanoparticulate movement. This particular aspect is one of the main distinguishing factors between plant and animal cells, the main reason why nanotechnology has moved at a rapid pace in medicine and still at infancy in the agro-related field. The complexity to overcome these phyto-cellular barriers due to their complex architecture has limited the use of many mammalian cells-applicable nanomaterials in plants. Therefore, preprocessing of plant cells to pave the way for smoother nanomaterial access has been attempted.

Protoplasts, plant cells whose cell wall is removed enzymatically together with certain cell surface proteins, have been employed to show the internalization of certain nanomaterials such as silicon NPs and polystyrene nanospheres. However, uncertainties regarding the similarities between isolated protoplasts and intact cells raise questions on this mode of NP administration. The technique along with the more modern biolistic approach, though seem lucrative under in vitro settings, may not be feasible in the long run due to major limitations as high cost and restrictions in large-scale applications.

Foliar application of nanoparticles as powders dispersed manually or mechani-cally and aqueous suspension by spray technique are being widely practiced as plant leaves provide a wider canvas for the NPs to exercise their effects.

Nanoparticles applied on leaf surfaces are found to gain entry through stomatal openings, cuts, and wounds, or through the bases of trichomes and subsequently get translocated to various tissues (Eichert et al.2008; Navarro et al.2008; Fernandez and Eichert2009; Uzu et al.2010). Such a mode of aerial application of NPs on leaf and stem surface has not been found to alter essential physiological processes as photosynthesis or respiration in several groups of horticultural and crop plants.

Also, they have not been responsible for alteration of gene expressions in insect trachea and have been qualified for use as nanobiopesticides. One successful example is of amorphous silica, which is designated as safe for humans by World Health Organization and US Department of Agriculture (Barik et al.2008). Still, this cannot be taken as a general statement for all classes of nanoparticles as it is well known that the characteristics of elements vastly differ at the nanoscale

compared to the bulk and are dependent on the constitutional make of individual or composite NPs. There have been reports of foliar heating due to the accumulation of NPs on photosynthetic surface leading to alterations in gas exchange due to stomatal obstruction resulting in various physiological and cellular functional impairments in plants (Da Silva et al.2006).

Small size and huge surface energy of nanoparticles renders them susceptible to aggregation in aqueous media, which in turn may modulate their bioavailability.

Root tips and hairs are found to secrete large amounts of mucilage (Campbell1990) composed of highly hydrated polysaccharides, which might contribute to the aggregation of NPs causing clogging of pore channels and arresting NP entry.

Changes in zeta potentials of tested NPs also are indicators of the ability of root exudates to change the property and behavior of NPs.

One major emergingfield of plant cultivation is hydroponics. The hydroponic strategy has many advantages as plants can be grown anywhere and it uses only 1/20th of water compared to traditional (soil-based) gardening. This technique does not require the use of pesticides, fertilizers, and other chemicals, as the risk of soilborne pathogens is negated. However, care has to be taken when considering infections posed by air and waterborne diseases. As hydroponic system is a closed and controlled system, crops grow faster. The NP exposure to the plant system can be constantly monitored, and numerous extraneous interactions, which would otherwise hinder in the efficiency of the experiments, can be avoided. Apart from the usual hydroponic cultures, there have also been studies on the extension of shelf life of post-harvest products as in horticulture using hydro-treatment. One example is from the experiments conducted by Solgi et al. (2009) who utilized silver nanoparticles and essential oils as novel antimicrobial agents for extending the vase life of gerbera (Gerbera jamesonii cv. ‘Dune’) flowers. Flowers were harvested from the plants and incubated in solutions of silver nanoparticles with or without essential oils. Similarly, the vase life of Rose (Rosa hybrida L.), one of the major cut flower crops, was significantly improved by treatment with biologically syn-thesized Ag NPs (Hassana et al.2014).

4.8 Conclusion

The secure application of nanomaterials to agriculture and related crop cultivation has been extensively delayed when compared to the rapid strides in fields as electronics, energy harvesting, and medicine. Though one main reason for this scenario is the limited understanding of plant–NP interactions, as unlike the mammalian cell system, the growth and related phenotypic/genotypic expressions in plants are a lengthier process, one other limiting aspect is the choice of methods employed for the application of NPs. It does depend on factors as the running cost, the area of application, availability of related resources, disease, and pest man-agement, etc., still, a major emphasis has to be given to the select methods that

allow for ready access to plant tissues in consideration for optimal desired effects.

Though a lot of research has focused on this aspect, conclusions have varied.

A solid framework for the NP–plant interaction has to be designed for the devel-opment of fool proof and highly efficient nanoparticle administration to crops.

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