Research Journal of Chemistry and Environment__________________________________________Vol. 22 (8) August (2018) Res. J. Chem. Environ.
Application of ZnO Nanoparticles in enhancing shelf life of cut flowers with special reference to Gerbera jamesonii Gupta Deepshikha*, Verma A.L. and Tyagi Monika Amity Institute of Applied Sciences; Amity University, Sector 125, Noida-201303, INDIA *
[email protected]
The use of chemical capping agents to control the particle size or prevention from coagulation is well established fact14. Some of the frequently used capping and stabilizing agents are triethyl amine, thioglycerol, polyethylene glycol, polyethylene phthalate, poly-vinyl alcohol, poly-vinyl pyrrolidone and many more. These chemical capping agents are found to be toxic. Despite inherent toxicity, organic and polymeric capping agents are majorly contributing in environmental pollution7. Many methods are known where polymeric chain is used to stabilize NPs in solution even though the poly-dispersed NPs undergo agglomeration to some extent due to lack of surface passivation.
Abstract In this work, we report unique application of zinc oxide nanoparticles in enhancing the shelf life of cut flowers (Gerbera jamesonii). ZnO nanoparticles were prepared using various plant based capping agents like citric acid, starch, xylan, chitosan and synthetic polymers like polyvinylpyrrolidone (PVP) and polyethyleneglycol (PEG). Starch capped ZnO nanoparticles showed best antibacterial action by Resazurin based Microtiter Dilution Assay (RMDA) with minimum inhibitory concentration (MIC) of 0.078mg/ml against Staphylococcous aureus (NCTC 6571).
In our study, starch, xylan and chitosan were chosen as stabilizing/capping agents due to their potential for multidentate action via its hydroxyl groups (amino group in chitosan) that might be able to interact with ZnO surface charges and as such act as a stabilizing template for nanoparticle synthesis. The structure of chitosan is very similar to that of cellulose; it consists of β (1-4)-linked Dglucosamine residue with the 2-hydroxyl group being substituted by an amino or acetylated amino group1. All of them are of natural origin, water-dispersible, biodegradable, inexpensive and commercially available and may provide a scaffold for further (bio) functionalization. Membrane related mechanism for nanoparticle was suggested for toxicity toward microbes10.
The capped ZnO nanoparticles showed significant changes in their morphology upon change of the capping agents. The enhancement of shelf life of cut flowers is observed due to potential antimicrobial action of the above mentioned synthesized nanoparticles. We suggest a membrane-damage mechanism of antibacterial action where membrane dysfunction is brought about by the interaction of ZnO nanoparticles with the cell membrane. The shelf life was found to be enhanced upto three times as compared to control under similar conditions.
The area under cut flower crops (with stems) has grown in recent years in India and the other countries. Main flowers in this category are rose, gladiolus, tuberose, carnation, orchids, liliums, gerbera, chrysanthemum and gypsophila but due to poor keeping quality floriculture industry faces 30-40% loss of total farm value. Therefore, to enhance the vase life of cut flower, effect of holding solution containing 8-HQC, sucrose, along with other floral preservatives in different combinations (like STS, Cobalt chloride, salicylic acid, aluminum sulphate, sodium nitroprusside, citric acid, Zinc and nano silver) were studied by various group of scientist11,12,16.
Keywords: ZnO, resazurin, antibacterial, cut flowers, Gerbera.
Introduction Zinc oxide (ZnO) in nano form is the most versatile metal oxide nanoparticles (NPs) that may be used in biomedical, gas sensors, drug delivery systems, biosensors, cosmetics, agriculture, waste water management, textile, medicine, degradation of harmful dyes and a number of other applications15. Apart from the technological significance of ZnO nanostructures, their quasi-one-dimensional structure with diameters in the range of nanometers makes them interesting from a scientific point of view.
When the cut flowers are kept in a vase, cellulose around the cut starts breaking down as bacteria starts colonizing at the open ends of the cut portion of the stems and block the channels through which water enters and reach the flower. This results in shortening of the vase life of cut flowers. In this regard, use of capped NPs of suitable size and quantity as antibacterial agents can become a stepping stone for overcoming post-harvest losses in the field of floriculture industry. ZnO nanoparticles showed bactericidal effects on
In the 5 to 100 nm size range, they are expected to possess interesting physical properties and pronounced reactivity due to available higher surface area quite different from their bulk counterparts. In order to enhance its properties, doping in ZnO is practiced with Ag, Cr, Mn, Fe, Co, Ni and Cu13.
* Author for Correspondence 1
Research Journal of Chemistry and Environment__________________________________________Vol. 22 (8) August (2018) Res. J. Chem. Environ. gram-positive and gram-negative bacteria as well as the spores which are resistant to high temperature and high pressure1,13.
XRD instrument was operated at 40 kV and 30mA at room temperature. Infrared Spectra in KBr pellet were recorded using Perkin Elmer PE-Rx1 FTIR Spectrophotometer in the 400-4000 cm-1 range. Optical absorption spectra were measured using Shimadzu UV-1800 model in the 200-800 nm range by suspending and sonicating the synthesized samples in isopropanol. Scanning electron microscopy (SEM) Zeiss (MA EVO -18 Special Edition) was used to observe the surface morphology of the synthesized NPs. Analysis was done after gold coating on carbon tape.
In this study, we report the synthesis of ZnO NPs by wet chemical route using various capping agents of natural origin to produce sterically stabilized NPs. We shall compare the various NPs on the basis of their morphology and optical properties and their possible application and relative effectiveness in enhancing shelf life of cut flower (Gerbera species). Zinc compounds have been reported to decrease oxidative stress in cut flowers11. We have also verified the antibacterial properties of ZnO10 by in vitro microtiter plate method.
Determination of Antibacterial Activity by RMDA: Antibacterial activity of the synthesized NPs was determined using Resazurin based Microtiter Dilution Assay (RMDA)14. The indicator used is a blue colored dye Resazurin (7Hydroxy-3H-phenoxazin-3-one 10-oxide) which itself is feebly fluorescent. It is irreversibly reduced to the pink shade and profoundly red fluorescent Resorufin. It is utilized as an oxidation-reduction indicator in cell suitability tests for microorganisms and mammalian cells. Under aseptic conditions, 96 well microtitre plate was used for RMDA.
Material and Methods Materials: Materials used for the synthesis of ZnO NPs were: Zinc acetate dihydrate [Zn(CH3COO)2.2H2O], distilled water, resazurin , nutrient broth, agar, sodium hydroxide (NaOH) and different capping agents (Citric acid, Starch, Chitosan, Xylan (HIMEDIA), Poly-vinylpyrrolidone PVP (MW- 400,000) and Poly-ethyleneglycol PEG (MW 6000). The gram positive bacterial strains used were: Staphylococcus aureus (NCTC 6571), Bacillus subtilis (3610), Micrococcus luteus and gram-negative stains Escherichia coli (ATCC 25922) and Psuedomonas aeruginosa. All chemicals used were of analytical grade. Ampicillin trihydrate (AMP) and Chloramphenicol were used as positive control for antimicrobial activity. A 96 well microtitre plate (Falcon) was used for RMDA. Elico LI 120 digital pH meter was used to measure the pH of solution.
The first row of microtiter plate was filled with 100 μl of metal oxide solution of 1mg/ml concentration in sterile water. All the wells of microtiter plates were filled with 100 μl of nutrient broth. Two-fold serial dilution (throughout the column) was achieved by transferring 100 μl test sample from first row to the subsequent wells in the next row of the same column. Finally, volume of 10 μl was taken from bacterial suspension (0.5 McFarland turbidity containing 5×106 CFU/mL) and added to each well. To avoid the dehydration of bacterial culture, each plate was covered and wrapped loosely with cling film. Each microtiter plate had a set of 3 controls: (a) a column with chloramphenicol (0.01g in 10 ml autoclaved water w/v) as positive control diluted serially (b) a column with ampicillin (0.01g in 10 ml autoclaved water w/v) as positive control, (c) a column with all solutions with the exception of nanoparticle sample (as negative control).
The flowers chosen for the purpose of finding application of ZnO NPs in enhancing shelf life were red Gerbera species (Gerbera jamesonii) as it is a very delicate flower that shows stem bending, colour fading and other degradations as sign of flower senescence. Preparation of differently capped nanoparticles: Wet chemical route was followed for preparation of various ZnO NPs. 0.1 M aqueous solution of zinc acetate di-hydrate [Zn(CH3COO)2.2H2O] was mixed with equal volume of 1% solution of capping agent and 0.5 M sodium hydroxide solution was added dropwise on a REMI magnetic stirrer for 30 min at 75ºC to achieve a cloudy solution. NaOH was added dropwise so as to increase pH of the solution up to 10 monitored by digital pH meter. White precipitate of zinc hydroxide was obtained upon centrifugation. The supernatant was discarded and precipitate washed several times with distilled water to remove all the adhering impurities. It was further annealed at 300˚C for one hour to obtain ZnO in nano form which was characterized by different techniques.
The plates were incubated in temperature-controlled incubator at 37° C for 18 h for the growth of bacteria. 20 μl of resazurin solution as indicator was added in each well and plates were further incubated for 2-3 hours. The color change in the well was then observed visually. Any color change observed from purple to pink or colorless was taken as positive result. The lowest concentration of metal oxide nanoparticle at which color change induced was recorded as the minimum inhibitory concentration (MIC) value. All the experiments were performed in triplicate. The average values were calculated for the MIC of test material. The dye underwent change in coloration from deep purple to flourescent pink / colourless upon reduction due to cellular activity (fig. 1).
Characterization of synthesized nanoparticles: Structural details of the synthesized nanomatreials were obtained using a Philips X’Pert Pro multipurpose X-ray diffractometer which uses Cu- Kα line having wavelength of 1.5418 A. The
Determination of Shelf life of Cut flowers: Eight bottles containing 200 ml of sterilized water were marked and added
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Research Journal of Chemistry and Environment__________________________________________Vol. 22 (8) August (2018) Res. J. Chem. Environ. with differently capped ZnO NPs so as to get final concentration of 0.25 mg/ml each. Each bottle was introduced with two stems of red Gerbera sp. flower with about 20 cm stalk length with similar flower maturity. The bottles were kept in a thermostatic room at temperature 25±2°C with 12 hrs illumination and about 80% humidity. The first bottle was made as negative control containing only sterilized water. The flowers were observed for a period of 12 days by observing the physical parameters like shelf life, color change or fading of flower, turgidity, stem bending and color change of hydrating solution.
table 1. The band gap energy (Eg nano) was determined by effective mass approximation equation as equation: Eg nano = Eg bulk + 1.6 / r2 where r is the radius of the crystal as calculated from XRD. The Eg bulk corresponds to the energy band gap of bulk ZnO (3.37 eV). The band gap energy, Eg nano, calculated for the synthesized NPs ranged from 3.385 to 3.428 eV.
Fig. 2: XRD patterns of various ZnO nanoparticles capped with different capping agents: (a) uncapped, (b) Citric acid capped, (c) Starch capped, (d) Chitosan capped, (e) Xylan capped, (f) PVP capped, (g) PEG capped, (h) ZnO Sigma
Fig. 1: Structure of Resazurin and their different forms
IR Spectrum of synthesized ZnO nanoparticles capped with different capping agents: FTIR spectra (fig. 3) were measured for those prepared ZnO particles which showed good XRD pattern. We observe peaks in the IR spectra from 400 cm-1 to 500 cm-1 due to Zn–O stretching vibrations of ZnO NPs. The peaks from 1500 cm-1 to 1650 cm-1 can be ascribed due to deformation mode of H2O and the broad peak from 3200 cm-1 to 3650 cm-1 arises from the O–H stretching vibration of water. Additional bands due to traces of capping agents are not significant indicating that there is no covalent bond formation between the capping agent and synthesized ZnO NPs. The IR spectra showed rather broad features in other spectral regions making it difficult to draw much meaningful conclusions.
Results and Discussion X-ray Diffraction analysis and band gap calculation: The result obtained from room temperature X-ray diffraction pattern (XRD) of the capped ZnO nanoparticle showed sharp and intense peaks matching with JCPDS data card no. 792205 indicated the hexagonal wurtzite phase of prepared ZnO NPs with 2θ values close to 31.74°, 34.21°, 35.97°, 47.19°, 56.33°, 62.59°, 66.16°, 67.72°, 68.92°, 72.35°, 76.85° corresponding to (100), (002), (101), (102), (110), (103), (200), (112), (201), (004) and (202) diffraction planes. Fig. 2 shows a comparison of XRD peaks of differently capped ZnO. The sharpness of peaks clearly suggests the crystallinity in the structure. The uncapped, chitosan and PVP capped samples were showing additional impurity peaks indicating that they are not pure and do not form wellstructured crystallites.
UV-Visible absorption and calculation of band gap energy: UV-visible absorption spectroscopy is widely being used technique to examine the optical properties of nanosized particles. The absorption spectrum of ZnO NPs is shown in fig. 4 and λmax values were tabulated in table 2 The UV-Visible absorption spectra exhibit a strong absorption band in the 354-371 nm range.
Average crystallite size, D, has been obtained from the highest diffraction peaks along (100) and (101) planes using the Debye-Scherrer formula (D = 0.9 λ / β cosθ) where λ is the wavelength of the CuKα radiation at wavelength of 1.54178 A and β is Full Width of Half Maxima (FWHM obtained in degree)17. The average crystallite sizes were determined along the (101) diffraction plane and were found to lie between 20.4 – 23.8 nm for samples (A-G) given in
The band gap increases along with the decrease of the radius due to the radial confinement in comparison to bulk material
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Research Journal of Chemistry and Environment__________________________________________Vol. 22 (8) August (2018) Res. J. Chem. Environ. (Eg bulk of ZnO = 3.37 eV). The direct band gap in ZnO is due to electronic transitions from valence band to conduction band (O2p to Zn3d). The UV-visible spectrum shows a strong exciton absorption peak in the range of 354-371 nm for differently capped ZnO NPs and blue shift relative to the bulk exciton absorption (388 nm) was observed as a result of the quantum size effect18. The energy band gap of the NPs has been calculated using the relation (Eg = 1243/λmax)4.
Scanning Electron Microscopy for size and morphological Properties: It was observed that both the morphology and the size of the NPs got improved on using a capping agent. The prepared samples showed a change in morphology with changing capping agents although the method of preparation was kept the same. As evident in fig. 5, very fine spherical structures of nanomaterials were obtained on capping with citric acid, well defined elliptical structures with starch and elongated rod like structure with PVP. Starch is a good example of “green” capping agent as it adopts right handed helical conformation in aqueous solution in which the extensive number of hydroxyl groups can facilitate the complexation of metal ions to the molecular matrix.
According to the quantum confinement theory8, the electrons in the conduction band and holes in the valance band are confined spatially by the potential barrier of the surface18. Because of the confinement of electrons and holes, the optical transition energy from the top of valence to the bottom of conduction band increases and the absorption maximum shifts to the shorter wavelength region. The stronger exciton effect is an important characteristic of quantum confinement in nano-semiconductor, in which the electrons, holes and excitons have limited space to move and their motion is possible for definite values of energies.
Synthetic organic polymer like PVP and PEG are water soluble and biocompatible which may generate sterically stabilized NPs in solution. PVP capped ZnO NPs ranged from spherical to elongated rods. PEG capping did not improve the morphology to a large extent. Both the uncapped ZnO NPs and the PEG capped NPs were irregular in shape. Xylan capped ZnO NPs were also spherical in shape and capping improved the morphology and particle size. Antimicrobial Assay of prepared ZnO nanoparticles: The results obtained by evaluating the antimicrobial potential of synthesized NPs using RMDA are given in fig. 6. Resazurin shows change in coloration due to reduction in its structure upon bacterial growth. The change in coloration of the dye from blue to pink (Resorufin) or colorless (dihydroresorufin) (structure given in fig. 1) indicates cellular activity or growth of bacteria. The cells that do not show change in coloration show bactericidal concentrations. MICs of synthesized NPs were calculated against the following bacteria strains in mg/ml. The structure of dye is given in figure 5. MICs of synthesized NPs were calculated against the selected bacteria strains in mg/ml as given in table 3.
Fig. 3: IR Spectrum of synthesized ZnO nanoparticles: (a) ZnO sigma, (b) PVP capped, (c) Citric acid capped, (d) Xylan capped (e) Starch capped
Effect on Shelf life of cut flowers (Gerbera jamesonii): For comparing the influence of differently capped nanomaterials on shelf life of cut flowers, the flower samples were maintained at similar conditions and observed up to 12th day of experiment. The details are shown in fig. 7. The keeping quality of flowers was assessed by flower size, color, stem stiffness, yellowing of hydrating solution and fading of flowers. The flowers in bottle 1(control) showed wilting of petals on 3rd day, drooping on 4th day onwards with yellowing of hydrating solution indicating bacterial growth. Bending of stalk more than 90˚ took place with wilting of flower petals and turbidity in solution indicating microbial growth. ZnO NPs have antibacterial applications and proved them to be bacteriostatic in nature. Hence, no turbidity was observed in any of the bottles containing synthesized NP up to 10 days.
Fig. 4: UV-Visible spectrum of ZnO nanoparticles capped with different capping agents: (a) PVP capped, (b) Strach capped, (c) PEG capped, (d) ZnO sigma, (e) Citric acid capped, (f) Xylan capped, (g) Chitosan capped, (h) uncapped
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Research Journal of Chemistry and Environment__________________________________________Vol. 22 (8) August (2018) Res. J. Chem. Environ. On 10th day, slight turbidity was observed in chitosan capped ZnO NPs. Both the two flowers of bottle 3 and bottle 5 showed bending and less solution uptake on 7th day and got completely dried on 10th day. On the other hand, flowers in the rest of the bottles except control were quite hydrated and fresh till 7 days, but slight drying begins to occur on the 10th day of observation, except the flowers in bottle 4 (starch capped ZnO NPs solution), which were still hydrated and most alive on the 12th day of observation. We may conclude from the above experiments that among the synthesized samples, starch capped and xylan capped ZnO NPs were of great interest as they showed a well-defined antimicrobial property which in turn prevented the cut flowers from being attacked by microbes resulting in increase in the shelf life of flowers greatly as compared to control, uncapped, citric acid, PVP, PEG and chitosan capped ZnO NPs. Mechanism of antibacterial activity of capped ZnO nanomaterials: The mechanisms of NP toxicity depend on composition, surface modification, intrinsic properties, and the bacterial species 6. There are mainly two mechanisms discussed in literature for understanding the antibacterial activities of ZnO and capped ZnO nanomaterials. These have both bacteriostatic and bactericidal activities which inhibits the growth of microbes and helps keeping the flower healthy for longer duration. The interesting characteristics of ZnO NPs are due to their large UV band gap (~3.37ev), large
exciton binding energy (~ 60 meV) and presence of intrinsic point defects in the crystal structure. Light irradiation of ZnO NPs induces a charge separation generating a hole (h+) in the valence band and an electron (e-) in the conduction band. ZnO (in presence of light)→ e– + h+ (on the surface of ZnO particles) At the surface of the excited ZnO NPs, the holes abstract electrons from water and / or hydroxyl ions, generating hydroxyl radicals (OH•). Moreover, electrons can reduce O2 to produce the superoxide anion O2.–. In an alternative mechanism, ZnO nanomaterials can adhere to the bacterial cell wall surface and eventually pierce into cell leading to lipid peroxidation in membranes causing bacterial death1. The highly reactive free radicals produce lethal H2O2 that damage the cells of bacteria and its death. H2O2 generation mainly occurs on the surface of ZnO NPs to yield additional active molecules which also actively participate in the antibacterial activities. The capping of ZnO NPs with starch, PVP, xylan is effective in controlling the particle size as well as act as stabilizers of ZnO NPs in water. Starch capping of the NPs appeared to provide greater protection from bacteria possibly due to the OH-related quenching of positive charges on the ZnO nanoparticle surface.
Fig. 5: SEM micrographs of various ZnO nanoparticles synthesized using various capping agents showing spherical, elongated to irregular shapes; A: Uncapped, B: Citric acid capped, C: Starch capped, D: Chitosan capped, E: Xylan capped, F: PVP capped. 5
Research Journal of Chemistry and Environment__________________________________________Vol. 22 (8) August (2018) Res. J. Chem. Environ. Conc. (mg/ml)
2.5 1.25 0.625 0.312 0.156 0.078 0.039 0.019 2.5 1.25 0.625 0.312 0.156
0.078 0.039 0.019
Fig. 6: Results showing Resazurin based Microtiter Dilution Assay (RMDA) on a 96 well microtiter plate with initial sample concentration of 2.5 mg/ml diluted serially in columns loaded with various samples A-G (A-uncapped, BCitric acid capped, C-Starch capped, D-Chitosan capped, E-Xylan capped, F-PVP capped, G-PEG capped); AcAntibiotic Chloramphenicol; AA-Antibiotic Ampicillin; C: Control
Fig. 7: This figure depicts the results obtained till 12 th day upon treating the cut flowers with synthesized ZnO nanomaterials: I-VII in water. Bottle number 1: Control (autoclaved water), 2: Uncapped ZnO, 3: Citric acid capped, 4: Starch capped, 5: Chitosan capped, 6: Xylan capped, 7: PVP
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Research Journal of Chemistry and Environment__________________________________________Vol. 22 (8) August (2018) Res. J. Chem. Environ. Table 1 Calculated crystallite sizes and band gap energy of the sample A to G from XRD for (101) diffraction plane Sample Number
Capping Agent used
Observed 2
Intensity
B (2)
Calculated D
Egnano
H
K L
A B
Uncapped Citric acid capped
36.05 35.79
1519.4 13332.6
0.797 0.409
10.486 20.415
3.428 3.385
1 1
0 0
1 1
C
35.99
3519.5
0.390
21.422
3.384
1
0
1
D E F G
Starch capped Chitosan Xylan PVP PEG
35.90 35.90 35.99 35.87
2622.4 3064.37 3871.39 1389.97
0.390 0.401 0.350 0.399
21.415 20.827 23.871 20.884
3.384 3.385 3.381 3.385
1 1 1 1
0 0 0 0
1 1 1 1
H
Market
35.99
3777.42
0.401
20.833
3.385
1
0
1
Table 2 λmax (nm) and Band gap energy Eg (in eV) of various ZnO Nps calculated from UV-Vis Spectrum Sample A B C D E F G H
Capping agent λmax (nm) value used Uncapped 368 Citric acid 378 Starch 371 Chitosan 366 Xylan 371 PVP 371 PEG 370 Market sample 354
Band gap energy Eg (in eV) 3.369 3.28 3.342 3.387 3.342 3.342 3.351 3.502
Table 3 Table indicating the minimum inhibitory concentration (MIC) in mg/ml for various ZnO Nps (A to G) against four bacterial species: S. aureus, B. subtilis, E. coli and P. aeruginosa Sample code A B C D E F G
Capping agent used Uncapped Citric acid Starch Chitosan Xylan PVP PEG
S. aureus (MIC mg/ml) 0.625 0.625 0.078 0.625 0.078 0.625 1.25
B. subtilis (MIC mg/ml) 1.25 2.5 1.25 2.5 0.625 1.25 0.625
E. coli (MIC mg/ml) 0.625 0.625 0.156 0.625 0.312 0.312 1.25
P. aeruginosa (MIC mg/ml) 0.156 0.156 0.312 0.625 1.25 0.156 0.625
MIC –Minimum Inhibitory Concentration in mg/ml, Results shown are mean of triplicate measurement
Our results suggest a membrane-damage mechanism of antibacterial action more relevant in comparison to the reactive oxygen species (ROS) mechanism. The membrane dysfunction process is aided by the fact that ZnO particles containing zinc rich positively charged surface, would exhibit a strong electrostatic interaction with the negatively charged cell membrane. This mechanism is consistent with the observed increase in antibacterial activity with decreasing size of nanomaterials, because the smaller sized particles would be expected to have a higher surface charge due to the increased surface area per unit volume. Membrane
related mechanism was also suggested for toxicity of nanoparticles towards microbes10
Conclusion It can be concluded that starch capped ZnO NPs is best suited to increase the shelf life of cut flowers up to three times compared to that of the control. It is of natural origin, waterdispersible and biodegradable. Starch granules consist of two major molecular components, amylose and amylopectin, both of which are polymers of α-glucose units
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Research Journal of Chemistry and Environment__________________________________________Vol. 22 (8) August (2018) Res. J. Chem. Environ. in the 4 C 1 conformation 20. ZnO NPs with starch capping also promote sap action by preventing flowers from microbial attack and also provide food source to the cut flowers and hence proved to be the best among all.
10. Nair S., Sasidharan A., Divya Rani V.V., Menon D., Nair S., Manzoor K. and Raina S., Role of size scale of ZnO nanoparticles and microparticles on toxicity toward bacteria and osteoblast cancer cells, J Mater Sci Mater Med., 20(Suppl. 1), S235 (2009)
Acknowledgement
11. Saeed T., Hassan I., Jilani G. and Abbasi N.A., Zinc augments the growth and floral attributes of gladiolus and alleviates oxidative stress in cut flowers, Scientia Horticulturae, 164, 124-129 (2013)
Authors thank Amity University to provide necessary infrastructure and facilities to carry out this work. Authors specially thank Dr. P.K. Paul, Amity Institute of Biotechnology, Noida for providing necessary lab facilities to carry out flower shelf life experiments under controlled conditions.
12. Safa Z., Hashemabadi D., Kaviani B., Nikchi N. and Zarchini M., Studies on quality and vase life of cut Gerbera jamesonii cv. 'Balance' flowers by silver nanoparticles and chlorophenol, J Environ Biol, 36(2), 425-431 (2015) 13. Schelonka D., Tolasz J. and Štengl V., Doping of Zinc Oxide with Selected First Row Transition Metals for Photocatalytic Applications, Photochemistry and Photobiology, 91(5), 1071-1077 (2015)
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(Received 10th January 2018, accepted 13th March 2018)
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