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Summer Research Fellowship Programme of India's Science Academies

The effect of Carbon Nanotubes (CNTs) on seed germination and seedling growth of Cyamopsis tetragonoloba (Guar)

Prerna Joshi

G. B. Pant University of Agriculture & Technology, Pantnagar

Professor S. L. Kothari

Amity University, Jaipur

Abstract

Carbon Nanotubes (CNTs) are engineered nanomaterials which are allotropes of carbon. They are categorized into single-walled CNTs (SWCNTs) and multi-walled CNTs (MWCNTs) based on the number of concentric layer of rolled graphene sheets. Due to their structure, they have exceptional properties i.e., they have the unique combination of rigidity, elasticity and strength compared with other fibrous material. CNTs have been recently introduced in the field of agriculture for the improvement of seed germination and production. CNTs can change the morphological and physiological characteristics of plant cells, but they can show either enhancing or toxic effect depending on type of CNTs, its concentration or the plant species etc. So, there is a need to evaluate the toxic effect of CNTs. Laboratory studies were conducted to study the effect of CNTs on seed germination and seedling growth of Cyamopsis tetragonoloba (L.) Taub. (cluster beans also known as guar) because of its diversified industrial applications viz., textile, paper, printing, food, cosmetics, mining, natural gas, petroleum, well drilling, oil industries, pharmaceuticals, etc. The guar endosperm contains galactomanan gum known as guar gum which has several diversified industrial uses. India is a major exporter of guar gum. Guar seeds were placed in four concentrations of COOH functionalised MWCNTs (10, 20, 30 mg/L) and was subjected to sonication for 30 minutes. After this, the seeds were germinated in autoclaved distilled water in Petri dishes which were covered and sealed with parafilm and were incubated in the dark at 37ºC temperature under laboratory conditions and observed after every 24 hours for 10 days. To evaluate the effect of CNTs on seed germination and seedling growth, germination percentage (GP), germination energy (GE), germination index (GI), relative germination rate (RGR), mean germination time (MGT), coefficient of velocity of germination (CVG), relative growth of seedling and seed vigor index (SVI) were calculated. The control showed the highest GP (93.33%) under laboratory conditions. The seedling growth was reduced in the CNTs treated seeds. However the differences were not significant. The control also showed highest GE, GI and SVI indicating that CNTs have some toxic effect on seed germination and seedling growth of guar though it was not significant.

Keywords: seed germination, seedling growth, guar, carbon nanotubes, nanomaterial, toxic effect

Abbreviations

Abbreviations
CNTsCarbon Nanotubes
MWCNTs Muti-walled Carbon Nanotubes 
SWCNTsSingle-walled Carbon Nanotubes
APEDA The Agricultural and Processed Food Products Export Development Authority  
GP  Germination Percentage
GE  Germination Enerngy
RGR  Relative Germination Rate
GI  Germination Index
SVI Seed Vigor Index
MGT Mean Germination Time
CVG Coefficient of Velocity of Germination
SEStandard Error 
MT Metric Ton (1000 kilograms)
PABS Poly-3-aminobenzenesulphonicacid
TEM Transmission Electron Microscopy
SEM Scanning Electron Microscopy
NA Not Applicable

INTRODUCTION

Background/Rationale​

In the past years, there is an increase in research and studies of nanomaterials, nanosciences and nanotechnology because of the small size (nanoscale) of nanomaterials that can be used for different biological and medical applications including gene and drug delivery, biosensing, diagnostic, and tissue engineering was widely documented during the last several years [Panyam & Labhasetwar, 2003; Oberdörster et al., 2005; Shi Kam et al., 2005; Borm et al., 2006; Zanello et al., 2006; Harrison & Atala, 2007; Aqel et al., 2012]. To improve the quality of life, nanotechnology has been used in consumer products ranging from health care to agriculture, and concerns about the possible side effects of this technology in ecosystems, human health, and agricultural industries has been addressed [Fiorito et al., 2006]. Nanomaterials are used for the improvement of seed germination and production. Carbon nanotubes (CNTs) are recently introduced in this field and there has been great interest in the use of carbon nanotubes (CNTs) in agriculture. However, the existing literature reveals mixed effects from CNT exposure on plants, ranging from enhanced crop yield to acute cytotoxicity and genetic alteration [Mukherjee et al., 2016]. So, there is a need to evaluate its effect (enhancing/toxic) on seed germination and production.

Scope and Objective

In the present study Cyamopsis tetragonoloba (Cluster beans, also known as guar) is used to study the effect of CNTs on its seed germination. Cyamopsis tetragonoloba (Guar) is a very important legume and industrial crop in arid areas of India. It has diversified industrial applications, viz., textile, printing, paper, food, cosmetics, mining, petroleum, natural gas, well drilling, oil industries, pharmaceuticals, etc. [Senapati et al., 2006; Pathak et al., 2009; Bhatt et al., 2016]. The guar endosperm contains natural hydrocolloid galactomanan gum known as guar gum, which has several diversified industrial uses (used as a thickening agent) [Mudgil et al., 2011]. India earns several thousand crores of rupees as foreign exchange from export of guar gum and its derivatives [Arora & Pahuja, 2008]. Additionaly, cluster beans have been reported to have beneficial effects when cultivated as an intercrop [Bhatt et al., 2016]. The vegetative parts of the plant and the cluster bean meal obtained from seed coat and germ cell are rich in protein are an excellent feed for monogastric animals and are used as fodder for cattles [Bhatt et al., 2016]. Guar gum has emerged as the most important agro-chemical. It is non-toxic, eco-friendly, and generally recognized as safe (GRAS) by FDA. In addition, it is used as vegetable (fruit part), fodder (shoot and fruit), and green manure (whole plant). The crop is grown as a cash crop across the world [Pathak et al., 2010].

Guar with high gum content (>32%) and viscosity (4000-5000 cps) are more preferred for export [Bhatt et al., 2015]. India accounts for 90% of the world guar produce, of which 72% comes from Rajasthan. About 90% of guar gum processes in India is exported. India is a major exporter of guar gum to the world; it exports various form of guar products to a large number of countries (major export destinations in 2018-19 are USA, China, Russia, Norway and Germany). As per data released by APEDA the export of guar gum from India has increased by 57.8% in four years. The country has exported 5, 13, 211.91 MT of guar gum to the world for the worth of Rs. 4,707.10 crores/676.48 USD millions during the year 2018-19 [​APEDA 2019​].

Overall Objective

The overall objective of the study is to evaluate the effect of CNTs on seed germination and seedling growth of Cyamopsis tetragonoloba (guar).

LITERATURE REVIEW

Carbon Nanotubes

Carbon nanotubes (CNTs) are type of carbon nanomaterials (CNMs) which are class of engineered nanomaterials (ENMs) having number of applications because of their exceptional optical, electrical, mechanical and thermal properties [Hurt et al., 2006; Bennett et al., 2013; Srivastava et al., 2015]. The discovery and synthesis of Buckminster fullerene (C60) in 1985 was followed by CNTs in 1991 and the graphene family in 2004 which have large number of applications till now [Bergmann & Machado, 2015; Hong et al., 2015].

CNTs are a carbon allotrope, cylindrical in structure with open closed ends and are further categorized into single-walled carbon nanotubes (SWCNTs) (one graphene cylinder) and multi-walled carbon nanotubes (MWCNTs) (2-50 graphene cylinders with a common long axis) depending on the number of concentric layers of rolled graphene sheets [​Tan et al., 2009​; ​Yang et al., 2010​; ​De Volder et al., 2013​]. The outer diameter of CNTs is typically 1 to 3 nm for SWCNTs with a length of few micrometers and MWCNTs have a diameter of 5 to 40 nm and a length around 10 μm. Recently synthesis of CNTs with a length of even 550 mm has been reported [​Zhang et al., 2013​]. The structure of CNTs lead to exceptional properties with a unique combination of rigidity, strength and elasticity compared with other fibrous materials. For instance, CNTs exhibit considerably higher aspect ratios (length to diameter ratios) than other materials and larger aspect ratios for SWCNTs as compared with MWCNTs due to their smaller diameter [​Zaytseva & Neumann, 2016​ ]. The CNTs are one of the most promising nanomaterials in nanotechnology due to their unique physico-chemical, electronic, and mechanical properties and the large number of potential applications [​Tan et al., 2009​].

A wide variety of carbon nanotubes can be further expanded by so-called chemical functionalisation. They are often functionalized by linking certain molecules to the nanoparticle surface, in order to modify the physical and chemical properties of the particles [Hirsch & Vostrowsky, 2005], which in turn greatly expands the field of applications [Hirsch & Vostrowsky, 2005; Hernández-Fernández et al., 2010]. One example for functionalisation of carbon-based nanoparticles is an oxidation of CNTs. This process comprises an ultrasonic treatment of nanotubes in a mixture of acids, leading to attachment of carboxylic functional groups (–COOH) on the sidewalls of the nanotubes. Oxidized CNTs acquire solubility in aqueous solutions, but retain their mechanical and electrical properties. Moreover, carboxylic groups attached to the nanotube surface can serve as sites for further functionalisation [Zaytseva & Neumann, 2016].

In the present study we have used COOH functionalised MWCNT that have carboxyl (COOH) functional groups bonded to the ends and sidewalls of the CNTs.

Agricultural Applications of Carbon Nanotubes

It is important in the present time to increase and optimize the agriculture production on limited area of arable land sustainably with a continuously increasing world population. This can be done by the use of modern nanotechnology in the following ways [Khot et al., 2012; Hong et al., 2013]:

1. Use of plant stimulators and new fertilizers based on nanomaterials. A range of studies have reported a positive impact of carbon-based nanomaterials on plant growth stimulating research on nano-carbon containing fertilizers. The majority of these fertilizers are based on amendments of mineral and organic fertilizers with nano-carbon, which in most cases acts as a fertilizer synergist with the aim of improving plant nutrient availability, reducing nutrient losses and stimulating plant growth [Zaytseva & Neumann, 2016].

2. Application of nanomaterial-based plant protection products [Gogos et al., 2012] including pesticides [Suresh Kumar et al., 2013; Sarlak et al., 2014] and herbicides [Periera et al., 2014];

3. Application of nanotechnology in precision farming [Auernhammer, 2001].

According to Gogos et al. [Gogos et al., 2012], 40 % of all contributions of nanotechnology to agriculture will be provided by carbon-based nanomaterials acting as additives as well as active components. The majority of such applications are still in the developmental stage.

A number of studies were done to find out the influence of CNTs on seed germination of various plant species. The following table shows the influence of CNTs on seed germ ofination some plant species.

Influence of CNTs on seed germination
Plant species Type of CNTs Size of CNTs Concentration (mg/L) Germination medium Exposure duration Effects Reference 
MaizeSWCNTs Diameter 1-2 nm, Length 30 μm20 MS medium 72 hoursNo effect on seed germination Yan et al., 2013
Raddish, rape, ryegrass, lettuce, maize, cucumber MWCNTs Diameter 10-20 nm, Length 1-2 μm 2000Distilled water  5 daysNo effect on seed germination Lin & Xing, 2007
Alfaalfa, wheatMWCNTsDiameter 3±4 nm 40-2560 Agar medium 4 daysNo effect on seed germination Miralles et al., 2012
Zucchini MWCNTsDiameter 13-16 nm, Length 1-10 μm 1000 Hoagland medium 12 daysNo effect on seed germinationStampoulis et al., 2009
Mustard, black lentilMWCNTsDiameter 110-170 nm, Length 5-9 μm 10, 20, 40Distilled water  7 daysNo effect on seed germinationGhodake et al., 2010
WheatOxidised MWCNTsDiameter 6-13 nm, Length 2.5-0 μm 10, 20, 40, 80, 160 Distilled water 7 daysNo effect on seed germinationWang et al., 2012
Wheat, maize, garlic bulb, peanutWater soluble CNTsDiameter 10-20 nm, Length 10-30 μm 20, 50 Distilled water 5-10 daysEnhanced germinationSrivastava & Rao, 2014
Barley, maize, soybeanMWCNTs NA 50, 100, 200 MS medium 10 daysAccelerated germinationLahiani et al., 2013
Barley, maize, soybeanMWCNTs NA 25, 50, 100 (spray) Water 20 daysAt 25 mg/L no effect, at 100 mg/L increased germination rateLahiani et al., 2013
TomatoMWCNTs NA 10, 20, 40 MS medium 20 daysIncreased seed moisture content, accelerated seed germination, improvrd germination rateKhodakovskaya et al., 2009
RiceCNTsDiameter 8 nm, Length 30 μm 50, 100, 150 MS medium 6 daysEnhanced germination speed and rate Jiang et al., 2012
RiceCNTsNA NA Basal growth medium NAIncreased seed water content, germination rateNair et al., 2012
Tomato, onion, turnip, raddishCNTsDiameter 8-15 nm, Length >10 μm 10, 20, 40 Ultra-pure water 12 days Improved germination of tomato and onionHaghighi & Teixeira da Silva, 2014

Effect of CNTs in Plants

CNTs have recently gained interest due to their possible applications in regulating plant growth [Khot et al., 2012]. Many studies on plant interactions with the various types of carbon nanotubes are available, describing effects on seed germination, early plant growth, cell culture, gene expression and various physiological processes. Concerning toxicity aspects, due to smaller size, SWCNTs seem to be more toxic than MWCNTs, and toxicity is further increased by functionalization of nanotubes [Cañas et al., 2008]. Since CNTs exhibit great tensile strength, mechanical damage of tissues may be induced by piercing effects. Importantly, the literature shows both positive and negative effects on terrestrial plant species, depending upon the type of CNTs and their concentration, growth conditions, and plant species.

Impact of SWCNT on seed germination

A few studies report effects of SWCNTs on germination rate. Stimulation of seed germination in response to SWCNTs treatments (10–40 mg L −1), potentially induced by perforation of the seed coat, has been reported for salvia (Salvia macrosiphon), pepper (Capsicum annuum), and tall fescue (Festuca arundinacea) [​Pourkhaloee et al., 2011​]. Among the tested treatments, the highest germination rates were obtained by applying moderate SWCNT concentrations. e.g., 10 mg L −1 SWCNTs for pepper (C. annuum) and 30 mg L−1 of for salvia (S. macrosiphon) and tall fescue (F. arundinacea). However, a similar concentration of SWCNTs (20 mg L −1) did not affect the germination of maize (Z. mays) seeds [​Yan et al., 2013​]. We have used COOH functionalised MWCNT in our study so we will discuss about impact of MWCNT in plants more.

Impact of MWCNT on seed germination

In contrast to SWCNTs, stimulatory effects of MWCNTs have been reported for a wider range of different crops. Improved germination rates were described for tomato (S. lycopersicum) [Khodakovskaya et al., 2009] and rice (O. sativa) [Nair et al., 2012], while germination speed was accelerated in barley (Hordeum vulgare), soybean (G. max), maize (Z. mays) [Lahiani et al., 2013; Mondal et al., 2011] and mustard seeds (B. juncea) [Mondal et al., 2011]. One of the most frequently encountered theories to explain beneficial effects on germination is associated with improved water uptake, demonstrated for tomato (S. lycopersicum) [Khodakovskaya et al., 2009], rice (O. sativa) [Nair et al., 2012], and mustard (B. juncea) [Mondal et al., 2011]. Accelerated water flow into the seeds has been related with the ability of CNTs to perforate the seed coat [Khodakovskaya et al., 2009]. Later, a concentration-dependent effect of MWCNTs on the expression of aquaporin genes has been reported for germinating seeds of soybean (G. max), barley (H. vulgare) and maize (Z. mays) [Lahiani et al., 2013]. Due to a central role of aquaporins in germination, it has been speculated that the beneficial effects of MWCNTs on seed water uptake and germination may be mediated by the described aquaporin effect. However, this assumption is still speculative since additionally to water uptake, aquaporins are involved in many physiological processes including stress responses also induced by CNTs and therefore, more detailed investigation of the involved aquaporin genes is necessary.

Apart from positive effects of MWCNTs on germination, there are also numerous reports claiming the absence of any MWCNT effect in a wide range of different plant species, including radish (R. sativus), rape (B. napus), ryegrass (Lolium perenne), lettuce (L. sativa), maize (Z. mays), cucumber (C. sativus) [Lin & Xing, 2007], wheat (T. aestivum) [Wang et al., 2012], mustard (B. juncea), black lentil (P. mungo), and zucchini (C. pepo) [Ghodake et al., 2010]. On the one hand, this discrepancy may be attributed to genotypic differences or variability in seed lot quality of the tested seed material but it may also be the test conditions.

Uptake of MWCNTs

Despite the fact, that the diameter and length of MWCNTs are frequently greater than the size of fullerenes and SWCNTs, plant uptake and internal translocation has been reported also for MWCNTs. The majority of such studies have been carried out in hydroponics or agar-like growth media. Multiwalled nanotubes can penetrate not only cells of developing seedlings, but even rigid seed coats by perforation and creation of new pores [Khodakovskaya et al., 2009]. For instance, MWCNTs with a diameter range of 15–40 nm were detected in germinating seeds of barley (H. vulgare), soybean (G. max) and maize (Z. mays) [Lahiani et al., 2013]. Once nanotubes have passed the seed coat, contact with the radicle and other seedling organs is possible. Accordingly, small diameter MWCNTs (<13 nm), present in the germination medium, could penetrate cell walls and were later detected in the roots of wheat (T. aestivum) [Wang et al., 2012] and red spinach (A. tricolor) seedlings [Begum & Fugetsu, 2012]. Wild and Jones [Wild & Jones, 2009] demonstrated that MWCNTs with a diameter of 110–170 nm could pierce the epidermal cell wall and thus penetrate up to 4 μm into the cytoplasm of wheat (T. aestivum) root hairs. MWCNTs taken up by plant roots were even detected in the xylem and in phloem cells [Zhai et al., 2015]. A root to shoot translocation of MWCNTs is most probably driven by transpiration [Chen et al., 2015] as demonstrated for wheat (T. aestivum) and rapeseed (B. napus) [Larue et al., 2012]. In soil-grown tomato plants MWCNTs have been detected in vegetative shoot organs and even in the flowers [Khodakovskaya et al., 2013].

The following tables show both positive and negative effects of CNTs based on different studies done in various plant species.

Positive effects of CNTs in plant
Plant species Type of CNT Treatment Effect References
 Lolium perenne (ryegrass) MWCNT 2000 mg/L Increased root length (~17%)Lin & Xing, 2007
Brassica juncea (mustard) Oxidized-MWCNT and Pristine (diameter ~30 mn) 2.3-46.0 μg Enhanced germination, increased root and shoot growthMondal et al., 2011
 Wheat Oxidized-MWCNT 40, 80, and 160 mg/L for 3 and 7 days Increse in root length of seedlingsWang et al., 2012
 Barley, soybean and corn. MWCNT 10-11 days at 50, 100 and 200 mg/L 50% (in barley and soybean) and 90% (in corn) increase in germination. In soybean, the root length increased upto 26%. In corn, shoot and leaf length were enlarged by 40% and more  than threefold, respectively. Internalisation was visualised by both Raman spectroscopy and TEM.Lahiani et al., 2013
Cucumis sativus (cucumber) and Allium sepa (onion). Uncoated and PABS coated SWCNTs Coated (0, 160, 900 and 5000 mg/L) and uncoated-CNTs (0, 104, 315 and 1750 mg/L) for 24 hours and 48 hours. Uncoated-CNTs increased the root length in onion and cucumber as compared to the coated-CNTs. Cañas et al., 2008
Cicer arietinum (gram) Citrate coated water-soluble CNTs 10 days exposure to 6 mg/mL Visualise internalisation of citrate coated water-soluble CNTs by SEM and TEM.Tripathi et al., 2011
Negative effects of CNTs in plant
Plant species  Type of CNTTreatment Effect References 
 ZucchiniMWCNT  15 days exposure to 1000 mg/L60% reduction in biomass reduction  Stampoulis et al., 2009
Mustard Oxidised MWCNT Hydroponic mustard, 6.9 mg/L Reduced germination and dry biomass Mondal et al., 2011

Considering the above-described effects of CNTs on plants, several tentative conclusions can be formulated: The cases of positive effects on seed germination seem to be related to seed coat perforation by nanotubes and improved seed water uptake. The effects of CNTs on cell cultures are negative as well as positive, but in both cases a contact of CNTs and cells was observed. Many studies show plant responses to CNT treatments comparable with reactions induced by various biotic and abiotic stress factors. Generally, the interaction of CNTs (and other CNMs) with entire plants appears to be a highly complex process, in which three components (plant, CNMs and growth medium) are closely interlinked. Therefore, a variation in one of these components can completely change the expression profile of responses to CNT-plant interactions, as a main source of contradictions and variability in different studies [Zaytseva & Neumann, 2016] .

METHODOLOGY

The study was conducted in the plant biotechnology lab of Amity University, Jaipur. The materials and equipments etc. used were, COOH functionalised MWCNTs, cluster bean (guar) seeds, ultra pure water, sterile Petri dishes, sterile tissue paper, sterile filter paper, sterile forceps, sonicator bath, vortex mixer, laminar flow cabinet, measuring scale etc.

Objective

To evaluate the toxic effect of CNTs on seed germination and seedling growth of Cyamopsis tetragonoloba.

Concepts

In order to evaluate the effect of CNTs on seed germination and seedling growth of guar the parameters used are: germination percentage (GP), germination energy (GE), germination index (GI), relative germination rate (RGR), relative salt injury (RGR), mean germination time (MGT), coefficient of velocity of germination (CVG), relative growth of seedling and seed vigor index (SVI). The formulae for these parameters are as follows:

Germination percentage

GP = (number of seeds germinated x 100)/ total number of seeds kept for germination.

Germination energy

GE = total number of germinated seeds in a particular treatment in 3 days/seed germination percentage in that particular treatment.

Germination index

GI = total number of seed germinated in total days/number of corresponding germination days.

Relative germination rate

RGR = germination percentage in a treatment/germination percentage in control.

Mean germination time

MGT = Σ ni di /n

Coefficient of velocity of germination

CVG = G1 + G2 +…Gn/(1 x G1) + (2 x G2) +…(n x Gn)

Where n is the total number of seeds germinated during study period; ni is number of seed germinated on day di; di is day during germination period; and G is number of germinated seeds on nth day.

Relative growth of seedling

Relative growth of seedling = seedling growth in a treatment/seedling growth in a control

Seed vigor index

SVI = germination percentage x seedling growth

Methods

The following procedures were performed in the experiment.

  • COOH functionalized multi walled carbon nanotubes were used in this experiment. Different doses of CNTs were prepared i.e. suspension of 10, 20, 30 mg/L in distilled water (total volume 10 ml) using a vortex mixer. A control i.e. with no CNTs is also taken.
  • 30 seeds were placed in each suspension of CNTS (10, 20, 30 mg/L) and the control.
  • All the three suspensions containing 30 seeds each were undergone sonication for 30 minutes in a sonicator bath.
  • The following steps were done in the laminar cabinet:
  • One piece of sterile filter paper was placed on 150mm X 15mm sterile Petri dish above the filter paper, one piece of tissue paper was placed. 10 ml of autoclaved distilled water was added in it.
  • Seeds undergone sonication with different concentration of CNTs and seeds with no treatment of CNTs were transferred onto the tissue paper, with 5 seeds per dish.

5. Petri dishes were covered and sealed with parafilm tape and were incubated in the dark at 37 degree celsius temperature.

6. The observations of germination and seedling growth progress were noted every day after every 24 hours. This was done for 10 days (Fig. 1).

0.jpg
    Seed germination and seedling growth in untreated and CNT treated (10, 20, 30 mg/L) guar seeds after 10 days.

    Data Analysis

    Each experiment was performed in triplicates and repeated three times. Data were analysed through one way ANOVA using SIGMA PLOT 13.0 and SPSS version 10.0 statistical package to determine the significance among mean values of control and treatments. Data was presented as mean ± SE.

    RESUTS & DISCUSSION

    The results of the experiment of the effect of CNTs on seed germination of Cyamopsis tetragonoloba are below.

    GP.png
    Germination percentage
      GR.png
      Germination rate
        GI.png
        Germination index
          MGT.png
          Mean germination time
            GE.png
            Germination energy
              CVG.png
              Coefficient of velocity of grmination
                R G S.png
                Relative growth of seedling
                  SVI.png
                  Seed vigor index
                    Graphs showing the results of the experiment.

                    Seed germination potential and recovery of seed germination in C. tetragonoloba. Seed germination in C. tetragonoloba treated with 10, 20 and 30 mg/L CNTs were compared to control. The germination percentage were less in the CNT treated seeds, however the difference was very low, 83.3% for 20 mg/L CNT treated seeds, 76.6% for 10 mg/L treated seeds and 73.3% for 30 mg/L treated seeds [Fig. 2 (a)]. The germination percentage for control was the highest (93.3%).

                    Similarly, germination energy, germination rate and germination index decreased in the CNT treated seeds [Fig. 2 (e), (b) & (c)], however, the difference was not significant.

                    CVG was recorded highest and equal in both 10, 20 mg/L CNT treated seeds followed by 30 mg/L CNT treated seeds, which was approximately equal to the control [Fig. 2 (f)].

                    Seed vigor index was recorded significantly highest in control thereafter it decreased significantly with decrease in concentration of CNT treated seeds [Fig. 2 (h)].

                    Mean germination time was found the highest for 10 mg/L CNT treated seeds, followed by the control, and was equal for both 20 and 30 mg/L CNT treated seeds [Fig. 2 (d)].

                    The seedling growth also decreased in the CNT treated seeds with decrease in concentration of CNT treatment [Fig. 2 (g)].

                    CONCLUSION

                    The results of the experiment showed that the germination percentage, germination energy, germination rate and germination index decreased in the CNT treated seeds. But that effect was not significant. CNTs are recently introduced in this field to enhance germination and production and studies in many plant species proved that CNTs have enhancing effect in germination and production. In the present study, we did not get significant results, that maybe because it is difficult to make CNT suspension in water as it is hydrophobic in nature.

                    However, CNTs showed negative effect in germination and seedling growth regardless of CNT concentration, but that was lower in comparision to control and the difference in the result between the control and CNT treatment was almost insignificant.

                    Future studies should be directed to phytotoxicity mechanisms, for example, size distribution of CNTs in solution and its effect on phytotoxicity, possible uptake and translocation of nanoparticles by plants, and physical and chemical properties of CNTs.

                    ACKNOWLEDGEMENT

                    It was an honor to work under the guidance of your group members and collaborators and these summer research programme are inspirational and innovative. I would like to pay my gratitude to Professor D. N. Rao of IISC, Bangalore who visited our University (GBPUAT, Pantnagar) in an alumni meet and let us know about the Summer Research Fellowships 2019 organized by Indian Academy of Sciences, Bangalore; Indian National Academy of Sciences, New Delhi and The National Academy of Sciences India, Allahabad.

                    I found myself blessed to get selected for the fellowship under the guidance of Professor S. L. Kothari, who gave me this opportunity, which certainly added values and knowledge to my life. I am thankful to Dr. Vinod Singh Gour, for his supportive actions and his guidance towards the work, which was the backbone of this entire project. I would like to thank all the members of AMITY UNIVERSITY, JAIPUR for their encouraging and caring behavior that always let me feel like a family here. It was a great experience here, which introduced me to the process required for any research. This will always be helpful for future. It is very important to have courage and prerequisite knowledge to begin something new, and this was possible because of the support of my academic advisor, Dr. Vandana A. Kumar, and my parents. Above all I am thankful to God who always gives me strength and inspire me towards the righteous wisdom.

                    References

                    • Panyam, J. & Labhasetwar, V. (2003). Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Advanced Drug Delivery Reviews, 55 (3), 329-347.

                    • Oberdörster, G., Oberdörster, E. & Oberdörster, J. (2005). Nanotoxicology: An Emerging Discipline Evolving from Studies of Ultrafine Particles. Environmental Health Perspectives, 113 (7), 823-839.

                    • Shi Kam, N. W., O'Connell, M., Wisdom, J. A. & Dai, H. (2005). Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction. Proceedings of the National Academy of Sciences, 102 (33), 11600-11605.

                    • Borm, P. J. A., Robbins, D., Haubold, S., Kuhlbusch, T., Fissan, H., Donaldson, K., et al. (2006). The potential risks of nanomaterials: a review carried out for ECETOC. Particle and Fibre Toxicology, 3 (1), 11.

                    • Zanello, L. P., Zhao, B., Hu, H. & Haddon, R. C. (2006). Bone Cell Proliferation on Carbon Nanotubes. Nano Letters, 6 (3), 562-567.

                    • Harrison, B. S. & Atala , A. (2007). Carbon nanotube applications for tissue engineering. Biomaterials, 28 (2), 344-353.

                    • Aqel, A., Abou El-Nour, K. M. M., Ammar, R. A. A. & Al-Warthan, A. (2012). Carbon nanotubes, science and technology part (I) structure, synthesis and characterisation. Arabian Journal of Chemistry, 5 (1),1-23.

                    • Fiorito, S., Serafino, A., Andreola, F., Togna, A. & Togna, G. (2006). Toxicity and Biocompatibility of Carbon Nanoparticles. Journal of Nanoscience and Nanotechnology, 6 (3), 591-599.

                    • Mukherjee, A., Majumdar, S., Servin, A. D., Pagano, L., Dhankher, O. P. & White, J. C. (2016). Carbon Nanomaterials in Agriculture: A Critical Review. Frontiers in Plant Science, 7:172.

                    • Pandit, J. K., Senapati, M. K. & Srinatha, A. (2006). In vitrorelease characteristics of matrix tablets: Study of Karaya gum and Guar gum as release modulators. Indian Journal of Pharmaceutical Sciences, 68 (6), 824.

                    • Pathak, R., Singh, M. & Henry, A. (2009). Genetic divergence in cluster bean (Cyamopsis tetragonoloba) for seed yield and gum content under rainfed conditions. Indian J. Agric. Sci., 79:559–561.

                    • Bhatt, R. K., Jukanti, A. K. & Roy, M. M. (2016). Cluster bean [Cyamopsis tetragonoloba (L.) Taub.], an important industrial arid legume: A review. Legume Research-An International Journal, 40:207-214.

                    • Mudgil, D., Barak, S. & Khatkar, B. S. (2011). Guar gum: processing, properties and food applications—A Review. Journal of Food Science and Technology, 51 (3), 409-418.

                    • Arora, R. N. and Pahuja, S. K. (2008). Mutagenesis in guar [Cyamompsis tetragonoloba (L.) Taub] Plant Mutation Reports, 2:7-9

                    • Pathak, R., Singh, S. K., Singh, M. & Henry, A. (2010). Molecular assessment of genetic diversity in cluster bean (Cyamopsis tetragonoloba) genotypes. Journal of Genetics, 89 (2), 243-246.

                    • APEDA (2019). Guargum. http://www.apeda.gov.in/apedawebsite/SubHead_Products/Guargum.htm

                    • Hurt, R. H., Monthioux, M. & Kane, A. (2006). Toxicology of carbon nanomaterials: Status, trends, and perspectives on the special issue. Carbon, 44 (6),1028-1033.

                    • Bennett, S. W., Adeleye, A., Ji, Z. & Keller, A. A. (2013). Stability, metal leaching, photoactivity and toxicity in freshwater systems of commercial single wall carbon nanotubes. Water Research, 47 (12), 4074-4085.

                    • Srivastava, V., Gusain, D. & Sharma, Y. C. (2015). Critical Review on the Toxicity of Some Widely Used Engineered Nanoparticles. Industrial & Engineering Chemistry Research, 54 (24), 6209-6233.

                    • Bergmann, C. P. & Machado, F. (2015). Carbon Nanomaterials as Adsorbents for Environmental and Biological Applications. Berlin: Springer, 1–122.

                    • Hong, G., Diao, S. ,Antaris, A. L. & Dai, H. (2015). Carbon nanomaterials for biological imaging and nanomedicinal therapy. Chemical Reviews, 115, 10816–10906.

                    • Tan, X., Lin, C. & Fugetsu, B. (2009). Studies on toxicity of multi-walled carbon nanotubes on suspension rice cells. Carbon, 47 (15), 3479-3487.

                    • De Volder, M. F., Tawfick, S. H., Baughman, R. H. & Hart, A. J. (2013). Carbon nanotubes: Present and future commercial applications. Science, 339, 535–539.

                    • Zhang, R., Zhang, Y., Zhang, Q., Xie, H., Qian, W. & Wei, F. (2013). Growth of Half-Meter Long Carbon Nanotubes Based on Schulz–Flory Distribution. ACS Nano, 7 (7), 6156-616.

                    • Zaytseva, O. & Neumann, G. (2016). Carbon nanomaterials: production, impact on plant development, agricultural and environmental applications. Chemical and Biological Technologies in Agriculture, 3:17.

                    • Hirsch, A., Vostrowsky, O. (2005). Functionalization of carbon nanotubes. Top Curr Chem, 245:193–237.

                    • Hernández-Fernández, P., Montiel, M., Ocón, P., de la Fuente, J. L. G., García- Rodríguez, S., Rojas, S. & Fierro, J. L. (2010). Functionalization of multi-walled carbon nanotubes and application as supports for electrocatalysts in protonexchange membrane fuel cell. Appl. Catal. B., 99:343–52.

                    • Khot, L. R., Sankaran, S., Maja, J. M., Ehsani, R. & Schuster, E. W. (2012). Applications of nanomaterials in agricultural production and crop protection: a review. Crop Prot., 35:64–70. 74.

                    • Hong, J., Peralta-Videa, J. R. & Gardea-Torresdey, J. L. (2013). Nanomaterials in agricultural production: benefits and possible threats? In: Shamim, N.& Sharma, V. K., editors. Sustainable nanotechnology and the environment: advances and achievements. Washington, D.C: American Chemical Society, 73–90.

                    • Gogos, A., Knauer, K. & Bucheli, T. D. (2012). Nanomaterials in plant protection and fertilization: current state, foreseen applications, and research priorities. J. Agric. Food Chem., 60:9781–92.

                    • Suresh Kumar, R. S., Shiny, P. J., Anjali, C. H., Jerobin, J., Goshen, K. M., Magdassi, S., et al. (2013). Distinctive effects of nanosized permethrin in the environment. Environ. Sci. Pollut. Res. Int., 20:2593–602.

                    • Sarlak, N., Taherifar, A. & Salehi, F. (2014). Synthesis of nanopesticides by encapsulating pesticide nanoparticles using functionalized carbon nanotubes and application of new nanocomposite for plant disease treatment. J. Agric. Food Chem., 62:4833–8.

                    • Auernhammer, H. (2001). Precision farming—the environmental challenge. Comput. Electron. Agric., 30:31–43.

                    • Yan, S., Zhao, L., Li, H., Zhang, Q., Tan, J., Huang, M., He, S. & Li, L. (2013). Single-walled carbon nanotubes selectively influence maize root tissue development accompanied by the change in the related gene expression. J. Hazard Mater., 246–247:110–8.

                    • Lin, D. & Xing, B. (2007). Phytotoxicity of nanoparticles: Inhibition of seed germination and root growth. Environmental Pollution, 150 (2), 243-250.

                    • Miralles, P., Johnson, E., Church, T. L. & Harris, A. T. (2012). Multiwalled carbon nanotubes in alfalfa and wheat: toxicology and uptake. J. R. Soc. Interface., 9:3514–27.

                    • Stampoulis, D., Sinha, S. K. & White, J. C. (2009). Assay-dependent phytotoxicity of nanoparticles to plants. Environ. Sci. Technol., 43:9473–9.

                    • Ghodake, G., Seo, Y. D., Park, D. & Lee, D. S. (2010). Phytotoxicity of carbon nanotubes assessed by Brassica juncea and Phaseolus mungo. J. Nanoelectron. Optoelectron., 5:157–60.

                    • Wang, X., Han, H., Liu, X., Gu, X., Chen, K. & Lu, D. (2012). Multi-walled carbon nanotubes can enhance root elongation of wheat (Triticum aestivum) plants. Journal of Nanoparticle Research, 14 (6).

                    • Srivastava, A. & Rao, D. P. (2014). Enhancement of seed germination and plant growth of wheat, maize, penut and garlic using multiwalled carbon nanotubes. Eur. Chem. Bull., 3:502–4.

                    • Lahiani, M. H., Dervishi, E., Chen, J., Nima, Z., Gaume, A., Biris, A. S., et al. (2013). Impact of Carbon Nanotube Exposure to Seeds of Valuable Crops. ACS Applied Materials & Interfaces, 5 (16), 7965-7973.

                    • Khodakovskaya, M., Dervishi, E., Mahmood, M., Xu, Y., Li, Z., Watanabe, F. & Biris, A. S. (2009). Carbon nanotubes are able to penetrate plant seed coat and dramatically affect seed germination and plant growth. ACS Nano., 3:3221–7.

                    • Jiang, Y., Hua, Z., Zhao, Y., Liu, Q., Wang, F. & Zhang, Q. (2012). The effect of carbon nanotubes on rice seed germination and root growth. In: Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012). Berlin: Springer, 1207-1212.

                    • Nair, R., Mohamed, M. S., Gao, W., Maekawa, T., Yoshida, Y., Ajayan, P., et al. (2012). Effect of carbon nanomaterials on the germination and growth of rice plants. J. Nanosci. Nanotechnol., 12:2212–20.

                    • Haghighi, M. & Teixeira da Silva, J. A. (2014). The effect of carbon nanotubes on the seed germination and seedling growth of four vegetable species. J Crop. Sci. Biotechnol., 17:201–8.

                    • Cañas, J. E., Long, M., Nations, S., Vadan, R., Dai, L., Luo, M., et al. (2008). Effects of functionalised and nonfunctionalised single-walled carbon nanotupes on root elongation of select crop species. Environmental Toxicology and Chemistry, 27 (9), 1922.

                    • Pourkhaloee, A., Haghighi, M., Saharkhiz, M. J., Jouzi, H. & Doroodmand, M. M. (2013). Carbon nanotubes can promote seed germination via seed coat penetration. Seed Technol., 33:155–69.

                    • Mondal, A., Basu, R., Das, S. & Nandy, P. (2011). Beneficial role of carbon nanotubes on mustard plant growth: an agricultural prospect. Journal of Nanoparticle Research, 13 (10) 4519-4528.

                    • Begum, P. & Fugetsu, B. (2012).  Phytotoxicity of multi-walled carbon nanotubes on red spinach (Amaranthus tricolor L.) and the role of ascorbic acid as an antioxidant. J. Hazard Mater., 243:212–22.

                    • Wild, E. & Jones, K. C. (2009). Novel Method for the Direct Visualization of in Vivo Nanomaterials and Chemical Interactions in Plants. Environmental Science & Technology, 43 (14), 5290-5294.

                    • Zhai, G., Gutowski, S. M., Walters, K. S., Yan, B. & Schnoor, J. L. (2015). Charge, size, and cellular selectivity for multiwall carbon nanotubes by maize and soybean. Environ. Sci. Technol., 49:7380–90.

                    • Chen, G., Qiu, J., Liu, Y., Jiang, R., Cai, S., Liu, Y.,et al. (2015). Carbon nanotubes act as contaminant carriers and translocate within plants. Sci. Rep., 5:15682.

                    • Larue, C., Pinault, M., Czarny, B., Georgin, D., Jaillard, D., Bendiab, N., et al. (2012). Quantitative evaluation of multi-walled carbon nanotube uptake in wheat and rapeseed. J. Hazard Mater., 155–63.

                    • Khodakovskaya, M. V, Kim, B., Kim, J. N., Alimohammadi, M., Dervishi, E., Mustafa, T., et al. (2013). Carbon nanotubes as plant growth regulators: effects on tomato growth, reproductive system, and soil microbial community. Small, 9:115–23.

                    • Tripathi, S., Sonkar, S. K. & Sarkar, S. (2011). Growth stimulation of gram (Cicer arietinum) plant by water soluble carbon nanotubes. Nanoscale, 3 (3), 1176.

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