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

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.     

Overexpression of the acidic dehydrin WCOR410 improves freezing tolerance in transgenic strawberry leaves

Progress in freezing tolerance (FT) improvement through plant breeding approaches has met with little success in the last 50 years. Engineering plants for greater FT through plant transformation is one possible way to reduce the damage caused by freezing. Here, we report an improvement of the selection procedure and the transfer of the wheat Wcor410a acidic dehydrin gene in strawberry. The encoded protein has previously been shown to be associated with the plasma membrane, and its level of accumulation has been correlated with the degree of FT in different wheat genotypes. The WCOR410 protein was expressed in transgenic strawberry at a level comparable with that in cold-acclimated wheat. Freezing tests showed that cold-acclimated transgenic strawberry leaves had a 5 degrees C improvement of FT over wild-type or transformed leaves not expressing the WCOR410 protein. However, no difference in FT was found between the different plants under non-acclimated conditions, suggesting that the WCOR410 protein needs to be activated by another factor induced during cold acclimation. These data demonstrate that the WCOR410 protein prevents membrane injury and greatly improves FT in leaves of transgenic strawberry. A better understanding of the limiting factors allowing its activation may open up the way for engineering FT in different plant organs, and may find applications for the cryopreservation of human tissues and organs.     

Engineered magnetic core shell nanoprobes:Synthesis and applications to cancer imaging and therapeutics

Magnetic core shell nanoparticles are composed of a highly magnetic core material surrounded by a thin shell of desired drug, polymer or metal oxide. These magnetic core shell nanoparticles have a wide range of applications in biomedical research, more specifically in tissue imaging, drug delivery and therapeutics. The present review discusses the up-to-date knowledge on the various procedures for synthesis of magnetic core shell nanoparticles along with their applications in cancer imaging, drug delivery and hyperthermia or cancer therapeutics. Literature in this area shows that magnetic core shell nanoparticle-based imaging, drug targeting and therapy through hyperthermia can potentially be a powerful tool for the advanced diagnosis and treatment of various cancers.     

Accumulation of magnetic nanoparticles in plants grown on soils of Apsheron peninsula

The disclosure of magnetic nanoparticles in five plant species growing in Apsheron peninsula have been detected by the EPR method. The EPR spectra of these nanoparticles proved to be similar to those of synthesized magnetic nanoparticles. The result demonstrated that plants are capable of absorbing magnetic nanoparticles from the soil. The accumulation of nanoparticles in plants is confirmed by the presence of a broad EPR signal whose maximum position of the low-field component changes from g = 2.38 and halfwidth of the signal of 32 mT at room temperature to g = 2.71 and 50-55 mT at 80 K. The intensity of the broad EPR signal for plants grown in radioactively contaminated areas (170-220 mkR per h) was substantially lower compared with plants grown on clean soil. The parameters of the broad EPR signal and its dependence on the temperature of recording were identical with those for synthetic magnetic nanoparticles. The photosynthetic activity and changes in the genome of irradiated plants by the analysis of PCR products were studied.     

Accumulation of magnetic nanoparticles in plants grown on soils of Apsheron peninsula

Magnetic nanoparticles in five plant species growing on the Apsheron peninsula have been detected by the EPR method. The EPR spectra of these nanoparticles proved to be similar to those of synthesized magnetic nanoparticles. The result demonstrated that plants are capable of absorbing magnetic nanoparticles from the soil. The accumulation of nanoparticles in plants is confirmed by the presence of a broad EPR signal whose maximum position of the low-field component changes from g = 2.38 and half-width of the signal of 32 mT at room temperature to g = 2.71 and 50–55 mT at 80 K. The intensity of the broad EPR signal for plants grown in radioactively contaminated areas (170‐220 μR/h) was substantially lower compared with plants grown on clean soil. The parameters of the broad EPR signal and its dependence on the temperature of recording were identical with those for synthetic magnetic nanoparticles. The photosynthetic activity and changes in the genome of irradiated plants by the analysis of PCR products were studied.     

Strengthening desert plant biotechnology research in the United Arab Emirates: a viewpoint

The biotechnology of desert plants is a vast subject. The main applications in this broad field of study comprises of plant tissue culture, genetic engineering, molecular markers and others. Biotechnology applications have the potential to address biodiversity conservation as well as agricultural, medicinal, and environmental issues. There is a need to increase our knowledge of the genetic diversity through the use of molecular genetics and biotechnological approaches in desert plants in the Arabian Gulf region including those in the United Arab Emirates (UAE). This article provides a prospective research for the study of UAE desert plant diversity through DNA fingerprinting as well as understanding the mechanisms of both abiotic stress resistance (including salinity, drought and heat stresses) and biotic stress resistance (including disease and insect resistance). Special attention is given to the desert halophytes and their utilization to alleviate the salinity stress, which is one of the major challenges in agriculture. In addition, symbioses with microorganisms are thought to be hypothesized as important components of desert plant survival under stressful environmental conditions. Thus, factors shaping the diversity and functionality of plant microbiomes in desert ecosystems are also emphasized in this article. It is important to establish a critical mass for biotechnology research and applications while strengthening the channels for collaboration among research/academic institutions in the area of desert plant biotechnology.     

Some effects of applied sodium and potassium chloride on yellow rust in winter wheat

In glasshouse experiments with plants in pots, applications of potassium chloride to the soil at 0.5 g/plant, 4 days before inoculation with Puccinia striiformis, decreased the severity of yellow rust on several winter wheat cultivars in comparison with untreated plants. Conversely, yellow rust was encouraged by applications of sodium nitrate. Sodium chloride in solution (8.6 g/l) reduced yellow rust when applied to the soil at the rate of 20 ml/plant but not when it was sprayed on to the leaves. In small-plot field experiments, sodium and potassium chlorides applied to the soil as dry powders in the spring at rates of 376, 1130 or 2260 kg/ha, significantly decreased the severity of yellow rust on most of the winter wheat cvs examined at each rate. The chlorides at these rates did not adversely affect the overall growth or yield in the absence of yellow rust.     

小麦遗传转化研究进展

小麦作为最重要的3大禾谷作物之一,其离体培养具有很强的惰性,再生频率与水稻、玉米相比要低一些,目前大多通过对基因型和外植体的选择来达到植株的高频再生分化,因此其遗传转化就远远滞后于水稻和玉米,更不用说与其它双子叶植物相比了.重点综述了小麦转基因技术和外源基因在小麦中的遗传转化研究现状,其内容包括几种主要的小麦转基因方法和以基因枪法为主的各种转化技术对品质基因、抗除草剂基因和抗病等基因在小麦中的遗传转化研究进展,并对存在的一些问题进行了简要的论述.     

Impact of bio-nanogold on seed germination and seedling growth in Pennisetum glaucum

Nanotechnology is leading towards the development of low cost applications to improve the cultivation and growth of plants. The use of nanotechnology in agriculture will leads to a significant effect on food industry along with opening a new area of research in agroecosystem. In this paper gold nanoparticles were biosynthesized with Cassia auriculata leaf extract at room temperature and characterized by UV–vis spectroscopy, X-ray diffraction and transmission electron microscopy. The objective of this study was to investigate effect of synthesized bio-nanogold on an important food and biofuel producing plant Pennisetum glaucum. Positive effects were observed on percentage of seed germination and growth of seedlings. Improved germination and increased plant biomass have high economic importance in production of biofuel or raw materials, agriculture and horticulture. Although the impact of nanoparticles on plants depends on concentration, size and shape. The biological synthesized AuNPs can replace the chemically synthesized AuNPs used in gene transfer method. The study gives brief insight on nanoparticles effects on plants, brings attention on both positive and negative side of nanomaterial which can resolve phytopathological infections by stimulating nutrition and growth.     

Geminivirus-based shuttle vectors capable of replication in Escherichia coli and monocotyledonous plant cells

Shuttle vectors have been constructed that are able to replicate in either Escherichia coli or plant cells. They contain the ColE1 origin of replication and parts of the wheat dwarf virus genome, a geminivirus infecting a variety of species of monocotyledonous plants. Such plasmids are able to replicate in E. coli and wheat cells. The plasmids can be rescued in E. coli and show no changes during their passage through plant cells. Such an E. coli/plant cell shuttle vector system could be used for the amplification of foreign genes in plant cells, for studies on DNA rearrangement or the isolation of plant transposons.     

Variation and inheritance of carbon isotope discrimination in wheat (Triticum aestivum)

Significant small variations exist in the proportion of the heavy isotope (¹³C) in organic and inorganic materials. The amounts of variations depend on the discrimination against or (in favor of) the heavy isotope through equilibrium reactions or kinetic processes particularly during CO₂ fixation in plants. These variations have also paved the way to measure carbon isotope discrimination in plant materials as a surrogate for water-use efficiency. Thus looking for variation and understanding the inheritance of this character is a prerequisite for improving wheat genotypes showing high water use efficiency and tolerance to drought. The main objectives of this review is to provide an overview of various aspects in which inheritance of ? is documented and of variation for carbon isotope discrimination reported in wheat under different circumstances. The relationship between carbon isotope discrimination and drought tolerance is also discussed exclusively in wheat.     

一个新的小麦黄化突变体的遗传研究

摘要: 通过对冬小麦“西农1718” 自然黄化突变体多年连续自交、与突变亲本回交以及与其他基因型进行正反交, 研究突变性状的遗传规律。观察统计发现, 突变体中金黄株发育至抽穗－开花期前后死亡, 黄-绿株自交后代表现为1金黄株︰2黄-绿株︰1绿株, 绿株自交后代不再分离; 黄-绿株与突变亲本回交及与其他基因型正反交, 后代均表现为1黄-绿株︰1绿株。遗传分析表明, 该小麦黄化突变体是由一对核基因控制的不完全显性遗传, 其中, 金黄株是纯合体(au/au), 表现为致死, 黄-绿株为杂合体(Au/au), 绿株为纯合体(Au/Au)。     

Carbon-iron magnetic nanoparticles for agronomic use in plants: Promising but still a long way to go

In the recent years, multiple ways of interaction between the fields of nanotechnology and biology have been opened, mainly in the biomedical research, with the development of tools for diagnosis and controlled delivery of substances.^1,2 On the other hand, in the field of plant biology, the interaction between both disciplines has been less frequent. Most of the published work on this field has focus in the environmental impact of nanoparticles on crop growth and development;^3,4 and also on the bio production of nanoparticles using plant extracts (reviewed in refs. ^5–8). Much less attention has taken other possible aspects of the interrelationship between nanotechnology and plant biology, such as the development of nanodevices for controlled delivery of drugs or different kind of substances,^9,10 in a similar way to that already developed in the medical research.Key words: plant nanobiotechnology, phytosanitaries, nanoparticle uptake, electron microscopy, nanoparticle subcellular localizationRecently, our group has developed an approach for the application of carbon-coated iron nanoparticles to pumpkin plants. The goal of this project was the development of tools for the treatment of pathologies affecting specific areas or organs of the plants. To achieve that, nanoparticles carrying the phyto-remedy will be applied to the affected plants, and magnetic fields will be applied to the organs of the plant affected by the pathology. In this way, the nanoparticles will be retained in the affected area, and the effect of the active compound linked to the nanoparticle will be concentrated in a restricted area.In a previous report, we described the capability of different microscopic methodologies to identify and locate nanoparticles in plant tissue samples, including both electron microscopy and light microscopy approaches.¹¹ Using this knowledge, we used a correlative microscopy approach, identifying first the presence of nanoparticles in sections of resin-embedded tissue by light microscopy (bright field, phase contrast and dark field), followed by a further analysis of consecutive sections from the same block, this time by transmission electron microscopy. This approach allowed us to unveil many different aspects of the behavior of the nanoparticles in the living tissue. First of all, movement of the nanoparticles was detected at different levels: chains of nanoparticle-aggregates carrying cells were apparent close to the application point, when such application was made by ‘injection’ of the nanoparticle suspension into the pith cavity of the stem, suggesting the flux of nanoparticles from one cell to another. Also, after the same kind of application, nanoparticles were detected in an area close to the vascular core, but appearing as isolated particles in the cytoplasm of the cell. Furthermore, after application of the nanoparticle by ‘spray’ (that is, application of a drop of the solution over the surface of the leaf, close to the petiole insertion point), isolated nanoparticles were also detected close to the application point (Fig. 1). This last data is particularly relevant, as this application method attempted to emulate that of breeders and coordinators of phytosanitary control. The fact that the nanoparticles are capable of penetrating through the leaf cuticule and into the cell cytoplasm opens the possibility for the use of this approach in phytosanitary applications.Open in a separate windowFigure 1Isolated nanoparticles localized close to the epidermis after spray application. (A) Low amplification image of the area were the nanoparticle is (squared area): SEC, sub estomatal cavity; GC, guard cell; E, exterior; LP, lagunar parenchyma; EP, epidermis. (B) Detail of the region squared in (A), showing an isolated nanoparticle (arrow). Bars in (A): 5 µm, (B): 200 nm.A second question of special interest in our analysis was the differential response to the presence of the nanoparticles shown by the cell cytoplasm when they appear in the form of aggregates when compared with cells carrying non-clustered nanoparticles. In fact, a dense cytoplasm with starch-containing organelles was observed concomitantly with nanoparticle aggregates in the cytosol (Fig. 2), suggesting that plant cells could respond to the presence of a high density of nanoparticles by changing their subcellular organization. On the other hand, no response was observed in those cells in which only isolated nanoparticles were detected (Fig. 1). The change on the cytoplasm of the cells was accompanied by the fact that the cell-to-cell movement of the particles in regions with a high density of aggregates seems to direct them to the exterior of the organism, what points to a physiological response from the plant to the intracellular presence of nanoparticle aggregates.Open in a separate windowFigure 2Different behavior between cell cytoplasm depending of the content in nanoparticle aggregates. (A) low amplification image showing unmodified cells (UMC) without nanoparticles, next to a nanoparticle carrying cell (NCC), whose cytoplasm is full of organelles. (B) Detail of the area squared in (A), indicating the presence of nanoparticle aggregates (asterisks). (C) Detail of a nanoparticle aggregate. Bars in (A): 10 µm, (B): 2 µm, (C): 200 nm.Despite the obvious advance that supposes the possibility of the application of nanoparticles on agronomical applications, it is also clear that there are many aspects in the protocols for detection of the nanoparticles and for their infiltration into the plant than can be improved. As shown in our work, the detection of isolated nanoparticles is almost impossible to achieve just by the resolution of the conventional optical microscopy, and their detection by direct scanning of ultrathin sections by electron microscopy is a tedious and time consuming task, especially if there is no clear evidence of the presence or not of nanoparticles in a certain part of the plant. Therefore, it is convenient to develop protocols with auxiliary methodologies to increase the sensibility of the microscopy techniques, that could allow directing the analysis by TEM to samples in which presence of nanoparticles has been previously assessed with certainty. Several methodologies have been employed for the detection of metallic nanoparticles in large tissue samples or in living tissues, with a special development in the field of therapy and diagnosis of diseases in the central nervous system. In this area magnetic resonance imaging,^12,13 phototermal interference contrast¹⁴ and conventional iron staining¹³ approaches have been successfully applied, although the resolution level is still low, with ranges between 1 µm and the cell size. A suitable solution could be the optimization of current methods for iron staining (reviewed in ref. ¹⁵), provided that lesser equipment requirements are needed than for the other approaches. In this line of action, a protocol should be optimized for their use to detect our particles in plant cells using as a starting point previous reports of iron detection in plant tissues, as the one used by Green¹⁶ for detection of inorganic ferric iron in Arabidopsis thaliana. Also, as a suitable system for the detection of global uptake of magnetic nanoparticles into plant organs, a vibrating sample magnetometer has been used successfully to measure the amount of nanoparticles taken by different organs of Cucurbita maxima plants,¹⁷ what could be a good option for the selection of samples of interest to be analyzed with microscopy in future experiments. Last, but not least, an even more straight forward approach is the attachment of colored, fluorescent or chemically detectable compounds to the nanoparticles, allowing an enhancement of the capacity of optical microscopy to detect those.¹⁸Our approach has been shown to allow the internalization of nanoparticles into plant cells in vivo, but the system as established initially is far from being functional, and several questions require improvement. First, the distribution of nanoparticles is still very limited, and most of the intracellular translocation of nanoparticles takes place near the application point, and much more efficiently when it is effectuated by injection. The most interesting way of application, the pulverization, has given only very limited results, with presence of intracellular nanoparticles but just in the first cellular layer, the epidermis. Also, long-range transport has still a low efficiency. Observations in samples equivalent to the ones that we have analyzed of fresh vibratome section, in which nanoparticles seemed to be present¹¹ may point to the possibility of loosing some NPs during the processing for EM analysis. Again, this possibility reinforces the need of a pre-scan of the samples to identify the presence of nanoparticles before a further analysis.Interestingly, it has been described recently the uptake of magnetite nanoparticles through the root system in Cucurbita maxima plants, as well as when applied by spraying, but the first method was tested only in plants growing in liquid media, and in the second only plants growing again in liquid media showed a significant uptake of particles.¹⁷ Also, in Arabidopsis and Phalaenopsis plants, it has been possible to perform live imaging of the uptake of other kind of nanoparticles (NaYF4:Yb,Er).¹⁹As stated previously, these experiments support the applicability of magnetic nanoparticles for use in agronomic purposes. But despite of the promising results, there is still a long way to go until they are suited for their use. This commentary has attempted to focus in some of the aspects to improve in the methodology employed.     

Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology

Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer-scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self-assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site-specific (spatially addressable) DNA 'staples'. This revolutionary approach has led to the creation of a multitude of two-dimensional and three-dimensional scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy.     

Plant protein-based hydrophobic fine and ultrafine carrier particles in drug delivery systems

For thousands of years, plants and their products have been used as the mainstay of medicinal therapy. In recent years, besides attempts to isolate the active ingredients of medicinal plants, other new applications of plant products, such as their use to prepare drug delivery vehicles, have been discovered. Nanobiotechnology is a branch of pharmacology that can provide new approaches for drug delivery by the preparation of biocompatible carrier nanoparticles (NPs). In this article, we review recent studies with four important plant proteins that have been used as carriers for targeted delivery of drugs and genes. Zein is a water-insoluble protein from maize; Gliadin is a 70% alcohol-soluble protein from wheat and corn; legumin is a casein-like protein from leguminous seeds such as peas; lectins are glycoproteins naturally occurring in many plants that recognize specific carbohydrate residues. NPs formed from these proteins show good biocompatibility, possess the ability to enhance solubility, and provide sustained release of drugs and reduce their toxicity and side effects. The effects of preparation methods on the size and loading capacity of these NPs are also described in this review.     

Plant nanotoxicology

The anthropogenic release of nanoparticles (NPs) to the environment poses a potential hazard to human health and life. The interplay between NPs and biological processes is receiving increasing attention. Plants expose huge interfaces to the air and soil environment. Thus, NPs are adsorbed to the plant surfaces, taken up through nano- or micrometer-scale openings of plants and are translocated within the plant body. Persistent NPs associated with plants enter the human food chain. In this Opinion, we document the occurrence and character of NPs in the environment and evaluate the need for future research on toxicological effects. Plant nanotoxicology is introduced as a discipline that explores the effects and toxicity mechanisms of NPs in plants, including transport, surface interactions and material-specific responses.     

Bionanoscience at the plant virus-inorganic chemistry interface

Bionanoscience is an inter-disciplinary area of research that sits at the interface of chemistry, biology, materials science, engineering and medicine. During the past 10 years the suitability and applicability of using plant viruses as building blocks, synthons, scaffolds or templates in bionanoscience/technology have begun to be explored. This short review describes how the plant Cowpea mosaic virus can be functionalised on its outer surface to form electroactive nanoparticles, can be used to construct monolayers on solid surfaces and multilayer arrays by a bottom-up, layer-by-layer approach, and how it can template mineralization processes to give new routes to monodisperse nanoparticles. Potential applications of virus-derived nanoparticles include nanoelectronics, sensory devices, catalysis, photonics and medical applications from imaging to the targeting and delivery of therapeutic agents.     

植物释放N2O速率及施肥的影响

采用开放式箱法和乙炔抑制技术,研究了大豆、春小麦和谷子3种植物不同生育期的N₂O释放速率以及施肥对春小麦N₂O释放速率的影响.研究发现,3种植物N₂O释放速率不同,但都具有在生长发育的前期阶段逐渐增加,至开花期前后达到高峰,然后又迅速下降的相似变化规律.在相应生育期,大豆表现出比谷子和春小麦更高的N₂O释放速率.不同的施肥量造成春小麦不同的生长状况,其N₂O释放速率也随之不同,过量施肥引起N₂O释放速率增加.     

Plant-based green synthesis of silver nanoparticles and its effective role in abiotic stress tolerance in crop plants

The development of effective and environmentally friendly methods for the green synthesis of nanoparticles (NPs) is a critical stage in the field of nanotechnology. Silver nanoparticles (AgNPs) are significant due to their unique physical, chemical, and biological properties, as well as their numerous applications. Physical, chemical, and green synthesis approaches can all be used to produce AgNPs; however, synthesis using biological precursors, particularly plant-based green synthesis, has shown outstanding results. In recent years, owing to a combination of frequent droughts, unusual rainfall, salt-affected areas, and high temperatures, climate change has changed several ecosystems. Crop yields have decreased globally as a result of these changes in the environment. Green synthesized AgNPs role in boosting antioxidant defense mechanisms, methylglyoxal (MG) detoxification, and developing tolerance for abiotic stress-induced oxidative damage has been thoroughly described in plant species over the last decade. Although various studies on abiotic stress tolerance and metallic nanoparticles (NPs) in plants have been conducted, but the details of AgNPs mediated abiotic stress tolerance have not been well summarized. Therefore, the plant responses to abiotic stress need to be well understood and to apply the gained knowledge to increase stress tolerance by using AgNPs for crop plants. In this review, we outlined the green synthesis of AgNPs extracted from plant extract. We also have updates on the most important accomplishments through exogenous application of AgNPs to improve plant tolerance to drought, salinity, low and high-temperature stresses.