Interfacing carbon nanotubes CNT with plants en

Authors: D K Tiwari N DasguptaSchubert L M Villaseñor Cendejas J Villegas L Carreto Montoya S E Borjas García

Publish Date: 2013/06/02

Volume: 4, Issue: 5, Pages: 577-591

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Abstract

The application of nanobiotechnology to cropscience/agriculture ‘nanoagriculture’ is a recent development While carbon nanotubes CNTs have been shown to dramatically improve germination of some comestible plants deficiencies in consistency of behavior and reproducibility arise partially from the variability of the CNTs used In this work factorysynthesized multiwalledCNTs MWCNTs of qualitycontrolled specifications were seen to enhance the germinative growth of maize seedlings at low concentrations but depress it at higher concentrations Growth enhancement principally arose through improved water delivery by the MWCNT Polarized EDXRF spectrometry showed that MWCNTs affect mineral nutrient supply to the seedling through the action of the mutually opposing forces of inflow with water and retention in the medium by the ionCNT transientdipole interaction The effect varied with ion type and MWCNT concentration The differences of the Fe tissue concentrations when relatively high equimolar Fe2+ or Fe3+ was introduced implied that the ionCNT interaction might induce redox changes to the ion The tissue Ca2+ concentration manifested as the antipode of the Fe2+ concentration indicating a possible cationic exchange in the cell wall matrix SEM images showed that MWCNTs perforated the blacklayer seedcoat that could explain the enhanced water delivery The absence of perforations with the introduction of FeCl2/FeCl3 reinforces the idea of the modification of MWCNT functionality by the ionCNT interaction Overall in normal media low dose MWCNTs were seen to be beneficial improving water absorption plant biomass and the concentrations of the essential Ca Fe nutrients opening a potential for possible future commercial agricultural applicationsDuring the last decade noteworthy advances have been reported in the study of the fundamental characteristics of nanomaterials and their utilization for a multitude of applications In medicine and biology nanomaterials have been investigated for diverse enduses such as gene and drug delivery biosensing and diagnostic and tissue engineering Panyam and Labhatsevar 2003 Merkoçi 2009 Tiwari and Tiwari 2013 Lee et al 2010 Since their discovery Ijima 1991 carbon nanotubes CNT amongst all nano materials have attracted a major share of the interest because of their unique mechanical electrical thermal and chemical properties Dresselhaus et al 2004 Most studies of CNTs in the biosciences have focused on their influence on animal and human cells Donaldson et al 2006 Martinelli et al 2012 Relatively scant attention has been paid to the effect of CNTs on plant cells and the way they might influence the physiology and the development of plants Yet plants are fundamental to higher life on earth—they are the geophysicochemical transducers that produce food and oxygen that sustain life in the biosphere They are also endreceivers of environmental contaminants including the CNTbased ones Thus the plantCNT interaction needs to be thoroughly investigated from the cellular to the organismic level to understand its multifaceted complexity This knowledge will also serve to develop the technology of ‘nanoagriculture’—a nascent area of nanobiotechnology that holds promise for growth acceleration and higher biomass yields of crop plants Srinivasan and Saraswathi 2010At the cellular level there has been substantial interest in the possibility of CNTs penetrating the plant cell wall to work as a smart delivery system of chemicals to the cell The ability of singlewalledCNTs SWCNTs to penetrate the cell wall and membrane of tobacco cells Liu et al 2009 and of cupstacked cellulase impregnated CNTs to penetrate the cell wall and transport intracellularly through cellulaseinduced nanoholes Serag et al 2012 have been demonstrated At the whole plant level a number of researchers have reported the dramatic effects on seed germination and plant growth by multiwalled CNTs MWCNTs MWCNTs have been shown to penetrate the seedcoat and stimulate the growth of tomato seeds Khodakovskaya et al 2009 Villagarcia et al 2012 as well as the growth of mustard seeds Mondal et al 2011 while water soluble MWCNTs wsMWCNT have been shown to exhibit the same dramatic improvement of the growth of gram plants Tripathi et al 2011 More efficient water uptake induced by the CNT has been implied as the growth stimulator Khodakovskaya et al 2009 Villagarcia et al 2012 Mondal et al 2011 Tripathi et al 2011 On the other hand there exist contradictory reports that show the toxicity of CNTs to the growth of a number of different plants Begum et al 2012 Lin and Xing 2007 MWCNT reduced the root fresh weights FW of rice and cucumber seedlings while the root lengths RL varied in a nonorderly manner with MWCNT concentration rice or decreased cucumber Begum et al 2012 the germination rate of maize and rye grass decreased with 2000 mg/l of MWCNT but their RLs increased Lin and Xing 2007 MWCNT increased the FW and RLs of wheat seedlings but had no effect on seed germination and shoot lengths SL Wang et al 2012The contradictory observations are a result of the variability between the experimental entities employed in the studies The effect of the MWCNT will principally vary with the type of plant studied the type of growth medium and ambient growth conditions the type of MWCNT pristine or functioanlised its concentration and the ultrasonication and associated techniques used to obtain the aqueous dispersion of the MWCNT The dependence of seedling growth on the first three factors is well known in plant physiology An assortment of media have been used in the aforesaid works—wet filter paper Mondal et al 2011 Lin and Xing 2007 Wang et al 2012 agar with MS nutrients Khodakovskaya et al 2009 Villagarcia et al 2012 and hydroponics with Hoagland’s nutrients Begum et al 2012 or without Tripathi et al 2011 Pristine MWCNTs are hydrophobic and form aggregates in aqueous media that reduce their aspect ratio and hence their effectiveness To disperse their CNTs the researchers in the aforesaid studies had functionalised their CNTs with different hydrophilic groups Khodakovskaya et al 2009 Villagarcia et al 2012 Mondal et al 2011 Tripathi et al 2011 or had used a surfactant PEG Villagarcia et al 2012 and had used different protocols for ultrasonication Khodakovskaya et al 2009 Villagarcia et al 2012 Mondal et al 2011 Tripathi et al 2011 Lin and Xing 2007 Wang et al 2012 Functionalization and micellar cage formation by a surfactant Vaisman et al 2006 alter the surface chemistry of the CNT which changes the way it interacts with the plant Villagarcia et al 2012 Furthermore different durations or intensities of ultrasonication will change the degree of dispersion of the CNT introducing its component to the variability Too vigorous an ultrasonication has been supposed damage the CNT Vaisman et al 2006 In yet another variation ultrasonication had been replaced by bead milling Begum et al 2012 Pristine MWCNTs where used were pure but were individualistic in type because they had been fabricated in the researchers’ own laboratories with different techniques resulting in MWCNTs with different morphologies or remnant functional groups Thus the substantial variability in the characteristics of the CNTs used is also the reason for the variable observationsTo define the basic response of the given plant to the MWCNT unambiguously a baseline study that can be easily replicated across independent laboratories becomes necessary Such a study would incorporate standard components of controlled specifications that are universally available utilize minimal processing and for the plant growth employ a simple wellestablished protocol Such a baseline could also serve as reference to more elaborate studies involving different processing techniques growth protocols and other features Also if CNTdriven nanoagriculture is to be developed as a largescale commercial venture consistency and reproducibility of results acquire added importance The general goal of the present work is a baselinetype study with addons as described further belowWe report the study of maize Zea mays seedlings grown in nutrient agar gel under controlled ambient conditions with the gel being treated by different concentrations of pristine MWCNT nonultrasonically distributed within it The MWCNT were obtained from an established chemicals company and possessed reliable qualitycontrolled specifications Maize is one of the species recommended by the USEPA US Environmental Protection Agency 1996 for the determination of the ecological effects of toxic substances Moreover it is an important food and agroindustrial crop such that an enhancement of its growth rate and biomass production if CNTs do indeed promote these would be highly beneficial Few studies of CNT affected seed germination have been conducted on maize The objective of the present study was to observe the effect of the MWCNT on the indices of growth and water absorption of the whole seedling as well as its morphological parts and to determine the mineral nutrient concentrations in the seedling Seedling growth in the initial phases of seed germination is principally driven by water imbibition MacDonald 2007 and Private Communication Feb 2013 If the mechanism of the CNTseed interaction is through the facilitation of water uptake then this is also expected to enhance mineral nutrient uptake and affect growth However given the fact that CNTs purportedly display a marked interaction with ions and polar molecules Miskovic 2008 Frolov et al 2012 it would be interesting to see how enhanced water delivery versus ionCNT interactions play out as far as seedling nutrient uptake is concerned The mechanism of ionic interactions with the CNT surface Miskovic 2008 implies that redox type changes of the nutrient ion of a given oxidation state might take place with the MWCNTs in the medium This is explored in the later part of this work with the introduction of the important mineral nutrient iron as FeII and FeIII chloride in agarose gel Agarose was chosen because it retains the gel platform of the previous study but is practically nutrient free so that the effects on iron will not be obscured by interference by other ions or polar species The initiation of germination is not affected by the lack of substrate nutrients because it is driven by the stored food in the seed endoderm/cotyledon MacDonald 2007 and Private Communication Feb 2013 The growth experiments were terminated after 7 days of growth because up till ~7 days the phases I–III of germination predominate where as stated before water imbibition is of paramount importanceMWCNTs were purchased from SigmaAldrich® St Louis MO USA purity 95  OD 6–9 nm L 5 μm Bacteriological agar BA Bioxon® was purchased from Becton–Dickinson BD de Mexico Mexico City Mexico and Agarose from SigmaAldrich® A9539 BA contains a certain level of mineral nutrients as ionic or polar species Difco™ and BBL™ manual 2009 but their levels in agarose are negligible The reagents FeCl2·4H2O 99  pure and FeCl3·6H2O ≥97  pure were purchased from SigmaAldrich® and Mallinckrodt Inc Phillipsburg NJ USA respectively Maize sweet corn seeds were purchased from a local seed supply company Hortaflor Rancho Los Molinos SA de CV Mexico

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