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Recent Patents on Food, Nutrition & Agriculture, 2013, 5, 000-000
Department of Laser Applications in Metrology, Photochemistry and Agriculture. National Institute of Laser Enhanced
Science (NILES), Cairo University, Egypt. 12613; 2Medicinal and Aromatic Plants Department, Desert Research Center
(DRC), Egypt
Received: August 19, 2013; Accepted: September 30, 2013; Revised September 19, 2013
Abstract: This study was conducted to evaluate the effectiveness of zinc nanofertilizer strategy on sweet basil yield,
through alone application or combined with pre-sowing laser irradiation. Furthermore, evaluate the growth of plant and
the level of active essential oil constituents. Zinc oxide (ZnO) nanoparticles (NPs) were synthesized, and transmission
electron microscope revealed particle size of approximately 10.5-15.5 nm. ZnO NPs were applied to sweet basil plants by
foliar spray at varying concentrations (10, 20 and 30 mg/L); He Ne laser of power 3mW was used for red light irradiation
of sweet basil seeds for 2 min. exposure time. Total chlorophyll, total carbohydrate, essential oil levels, zinc content, plant
height, branches/plant and fresh weight were measured.
In general, the combined foliar spray application of ZnO nanofertilizer with pre-sowing He Ne laser irradiation showed
more effectiveness than ZnO nanofertilizer alone and 20mg/L concentration gave the highest results of all measured traits.
Statistical analysis (t-test) showed significant differences among the effects of the various concentrations of zinc oxide
NPs on these attributes. The results showed an inverse relationship between the total carbohydrate content and the percentage of essential oil in the leaves. Together these findings support the usefulness and effectiveness of zinc oxide nanofertilizer and laser irradiation treatment to enhance the growth and yield of sweet basil plants.
Keywords: Basil, carbohydrate, chlorophyll, essential oil, He Ne laser, yields, ZnO nanofertilizer.
1. INTRODUCTION
2212-7984/13 $100.00+.00
Nano-agriculture involves the employment of nanoparticles in agriculture, with the particles imparting specific beneficial effects to the crops [9]. The success of nanoparticles
may be due to, a greater density in reactive areas, or increased reactivity of these areas on the particle surfaces.
Nanotechnology is a potential solution for increasing the
value of agricultural products and environmental problems.
For example, with the use of nanoparticles and nanopowders,
researchers can produce controlled or delayed release fertilizers [10]. In addition, several studies have shown that
nanoparticles can have a beneficial effect on seedling growth
and development [11].
Zinc is an essential component of a large number of enzymes participating in the synthesis and degradation of carbohydrates, lipids, proteins, and nucleic acids as well as in
the metabolism of other micronutrients and plays an important role in the production of biomass [12]. Therefore, soil
Zn deficiency can cause significant reductions in yield and
induces changes in plant metabolic processes such as cell
division, photosynthesis, and protein synthesis [13]. Several
studies have been shown that a small amount of Zn applied
by foliar spray can significantly increase the yield of crops
[14]. In addition, foliar nutrition is an option when nutrient
deficiencies cannot be corrected by applications of nutrients
to the soil [15].
El-Kereti et al.
Laser irradiation is considered as a new branch of agriculture, especially in the medicinal plants such as its effect
on seed germination and growth rate of plants. The results
showed improvements in sowing qualities, shortened the
phases of plant development, produced more vigorous
plants, increased the yields of both stems and seeds to a considerable extent and increasing the germination by 10-15%
[16, 17]. The stimulating effect of radiation was also observed on plant height, stalk thickness, height of the first ear
formation, number of leaves and leaf area size [18, 19]. It
was reported that the mutation frequency of plants derived
from wet seeds being higher than that of plants derived from
irradiated dry seeds [20, 21].
an electron acceleration voltage of 60 kV. The TEM samples were prepared by placing a few drops of the solution
on a carbon-coated copper grid (Okenshoji Co., Ltd., microgrid B).
Zinc acetate [Zn (H3 COO) 2.], NaOH and Isopropyl alcohol (2-propanol) with the reagent grade of 99.5% were obtained from Sigma Aldrich (99.9%).
0.073 mmol Zn (OAc)2. 2H2O was dissolved at 60oC in
50 ml 2-propanol under stirring. In a second flask 1.5 mmol
NaOH was dissolved under vigorous stirring in 25 ml 2propanol at 60oC. NaOH solution was added drop wise under
stirring to the acetate solution. The product was stirred for an
hour at 60oC and then cooled to room temperature. The precipitate was washed twice with 2-propanol and centrifuged at
4500 rpm for 30 min.
2.2. Characterization of ZnO NPs
The size and shape of ZnO nanoparticles were observed
directly by transmission electron microscope (TEM) using
El-Kereti et al.
Fig. (2). TEM imaging of the prepared ZnO nanoparticles revealed a spherical shape of the particles, with an average size of around (10.515.5 nm).
Fig. (3). The effect of different concentrations of ZnO NPs foliar spray on Zn content (ppm) of Ocimum basilicum plants with and without
pre-sowing He Ne laser irradiation.
Table 1.
The Effect of Different Concentrations from ZnO NPs Foliar Spray on N, P, K, Fe, Zn and Cu % of Ocimum basilicum
plants Cultivated from Seeds Treated with and without Pre-sowing He Ne Laser Irradiation
Treatment
N (%)
P (%)
K (%)
Cu (ppm)
Fe (ppm)
1.22
0.108
2.21
14
690
10mg/L
1.59
0.108
3.22
18
788
20mg/L
3.41
0.175
4.89
26
1108
30mg/L
3.01
0.226
3.73
22
811
1.22
0.108
2.21
14
690
10mg/L
2.06
0.318
4.24
25
866
20mg/L
3.80
0.411
5.19
32
1178
30mg/L
3.43
0.348
4.56
27
989
irradiation with ZnO NPs than ZnO NPs treatment alone and
their levels increased whenever ZnO NPs concentration increased.
3.3. Study the Effect of ZnO NPs Treatment on the Anatomical Structure of Sweet Basil Leaf
As shown in Fig. (4a) the epidermis cells of the control
were similar in their shape and size, while the epidermal
cells of the ZnO NPs-treated leaves became larger in size
and reached to their maximum size at 20 mg/L (Fig. 4b). In
addition, the thickness of mesophyll tissue, which is specialized photosynthetic tissue that contains chloroplasts in the
palisade and spongy parenchyma tissue was greater with
ZnO-treated leaves compared to control leaves. The presowing laser irradiation combined with ZnO NPs more effective than ZnO NPs treatment alone (Fig. 4c), the air spaces
were largest in ZnO sprayed leaves cultivated from laser
irradiated seeds. Therefore, laser irradiation combined with
ZnO NPs treatment was the most effective treatment, followed by ZnO NPs treatment alone and control leaves.
3.4. Study the Effect of ZnO NPs Treatment on Chlorophyll Content
As shown in Fig. (5). The foliar treatment of basil of ZnO
NPs alone at 10, 20 and 30 mg/L significantly increased the
4a)
4b)
chlorophyll content in the experimental plants than the control ones. While the application of ZnO NPs at 20 mg/L for
the plants that cultivated from laser irradiated seeds had a
marked enhancement of the total chlorophyll content compared to the plants that treated by ZnO NPs alone and control
plants.
The results were 43 SPAD units for plants cultivated
from the combined application of pre-sowing laser irradiation with ZnO NPs vs. 39 SPAD units for the plants cultivated from ZnO NPs alone and 25 SPAD units for control
plants.
3.5. Effect of ZnO NPs on Total Carbohydrate and Essential Oil
(Figures 6 and 7) showed that plants treated with (20
mg/L) ZnO NPs had the lowest total carbohydrate levels
(16.3% and 21.4%), for plants that cultivated from the combined application of pre-sowing laser irradiation with ZnO
NPs and plants treated with ZnO NPs alone, respectively.
While the same plants had the highest essential oil content
(0.95% and 0.51%) respectively, that due to the consumption
of carbohydrate in the oil production process, which confirmed our proposed relationship between carbohydrate content and oil production.
4c)
Fig. (4). 4a): Leaf cross section in Ocimum basilicum control plants. 4b): Leaf cross section of Ocimum basilicum treated with 20 mg/L ZnO
NPs by foliar spray application. 4c): Leaf cross section of Ocimum basilicum cultivated from pre-sowing He Ne laser irradiation and treated
with ZnO NPs foliar spray (20 mg /L).
Fig. (5). Effect of different concentrations of ZnO NPs foliar spray on leaves total chlorophyll (SPAD units) of Ocimum basilicum plants
cultivated from seeds treated with and without pre-sowing He Ne laser irradiation.
El-Kereti et al.
Fig. (6). Effect of different concentrations from ZnO NPs on total carbohydrate% of Ocimum basilicum plants cultivated from seeds treated
with and without pre-sowing He Ne laser irradiation.
Fig. (7). Effect of different concentrations from ZnO NPs on the essential oil % of Ocimum basilicum plants cultivated from seeds treated
with and without pre-sowing He Ne laser irradiation.
The total carbohydrate content of laser irradiation combined with ZnO NPs (16.3%) was less than the plants treated
with ZnO NPs alone (21.4%) (Fig. 6). This could be explained from the view of the higher essential oil in plants
cultivated from the combined application of laser irradiation
with ZnO NPs than the plants treated with ZnO NPs alone
(0.95% and 0.51%) respectively (Fig. 7).
3.6. Morphological Characterization and Yield
The data presented in Table 2 showed that pre-sowing
laser irradiation combined with ZnO NPs and ZnO NPs
treatment alone significantly, increased plant height, number
of leaves/plant and number of branches/plant, especially at
of 20 mg/L. Fresh and dry weight of herb followed the same
trend. In addition, the results presented in Table 2 show that
pre-sowing laser irradiation combined with ZnO NPs had an
observable positive effect higher than ZnO NPs treatment
alone, on all measurements.
Pre-sowing laser irradiation combined with ZnO NPs at
20 mg/L increased plant height of 93 (control) to 129 cm,
Table 2.
The Effect of Different Concentrations from ZnO NPs Foliar Spray on Morphological Parameters of Ocimum basilicum
Plants Cultivated from Seeds Treated with and without Pre-sowing He Ne Laser Irradiation
Treatment
Dry Matter %
Branches /plant
Leaves/plant
93
268
79.3
45
744
10mg/L
115
460
80.1
66
1109
20mg/L
129
588
81.3
80
1154
30mg/L
120
471
80.7
76
1112
93
268
79.3
45
744
10mg/L
122
520
81.7
81
1136
20mg/L
133
655
82.8
91
1290
30mg/L
130
536
82.1
87
1205
Table 3.
Treatment
SD
ZnO NPs
ZnO NPs
ZnO NPs
Plant height
119.50000
114.2500
18.26655
15.30523
0.000482
0.000326
Fresh weight
494.75000
446.75000
162.72139
132.49748
0.004464
0.003329
Dry matter
81.47500
80.35000
1.51959
.85440
0.000001
0.000000
Branches/plant
76.00000
66.75000
21.07131
15.64981
0.002746
0.001692
Leaves/plant
1093.7500
1029.750
241.52346
191.60441
0.001422
0.000861
Total chlorophyll
36.75000
34.00000
8.09835
6.21825
0.001413
0.000818
Total carbohydrate
21.32500
25.25000
4.53900
2.66145
0.001277
0.000160
Essential oil
.60250
.30500
.32877
.16258
0.017561
0.016535
Nitrogen
2.63025
2.31225
1.20036
1.06487
0.011002
0.011267
Phosphor
.29625
.18100
.13135
.05337
0.010178
0.003276
potassium
4.05000
3.51250
1.28859
1.11458
0.004065
0.004034
Iron content
930.75000
849.25000
205.49027
180.30229
0.001421
0.001267
Copper content
24.50000
20.00000
7.59386
5.16398
0.003774
0.002237
Zinc content
524.00000
470.25000
271.78791
230.56218
0.015408
0.013304
4. DISCUSSION
In our results, the images of the synthesized ZnO
nanoparticles revealed a spherical shape and an average particle size of 10.5 to 15.5 nm. This average means that
nanoparticles subsequently used on plants in this study suggests the particles have the ability to pass through biomembranes.
Longnecker and Robson [31] who suggested that zinc
efficiency depend on the supplied amount and the nature of
plant species. Particle size may affect the agronomic effectiveness of Zn fertilizers. The decreased particle size results
in the increased number of particles per unit weight of applied Zn and increases the specific surface area of a fertilizer,
El-Kereti et al.
possible reason for increasing essential oil plants due to helium-neon exposure, which is GA increases the concentration of sugars, which form essential oil through acetyl co-A
and mevalonic acid synthesis pathways.
Laser significantly increased chlorophyll contents of
Ocimum basilicum herb and ZnO NPs. Chlorophylls are
very important for increasing oil production because the
chlorophyll concentration increase induces more photosynthesis in the plant and producing a large amount of carbohydrate that will be used partially for essential oil production.
In a similar study, cucumber seeds irradiated by He-Ne
Laser increased the quantity of the photosynthetic product
and the content of plastid pigments in leaves [71]. Suitable
doses of laser irradiation improved germination capacity of
plant seeds, enzymatic activities and the concentration of
chlorophyll [72]. Sebanek [73] found an increase of chlorophyll content in the leaves of pea plants, which were grown
from laser light irradiated seeds. In the case of laser treatment, the stimulation was due to the increasing energy supply of seeds. In that case, the photon energy of laser radiation was absorbed by chlorophyll and directly affected the
photosynthetic intensity.
There was a positive relationship between the amount of
carbohydrate in sweet basil herb and the percentage of essential oil that can be distilled from these herbs. This is because
the oil production in plant cells starts from the carbohydrate
stock of these cells. Our findings of the relationship between
the amount of carbohydrate in sweet basil herbs and the percentage of essential oil agree with previous work Dubey [74]
that proved that the secretory cells of the oil glands responsible for the essential oil biosynthesis from monoterpenes of
primary carbohydrate precursors [75]. This means that any
increase of total plant carbohydrate will be utilized in the
biosynthesis of essential oil, consequently increase the production of essential oil. The results confirmed that plants
treated with the ZnO NPs at (20 mg/L) had the lowest total
carbohydrate level consequently; they have the highest essential oil percentage, this is due to the consumption of carbohydrate in the oil production process, which confirmed our
proposed relationship between carbohydrate content and oil
production. Whenever Zn supply increased the essential oil
content reached its maximum value at (20 mg/L) but it decreased at (30 mg/L), this can be due to the intolerance of
sweet basil plant to excess Zn (Zn toxicity) that we have
discussed above which resulting in chlorophyll content decrease and consequently, carbohydrates and essential oil content drop.
Pre-sowing laser irradiation combined with ZnO NPs
resulted in an extreme increase in essential oil yield compared to ZnO NPs treatment alone. This may be due to the
previously discussed of chlorophyll content increase that
caused by laser impact on seeds and zinc impact on leaves,
this would result in a high photosynthesis rate and consequently, more production of carbohydrate precursors for essential oil synthesis. Our findings of the significant increase
of essential oil and carbohydrate content by nano zinc oxides
applied agree with previous work of Misra and Sharma [8]
whom reported that the requirement of Zn for Japanese mint
and its limitations imposed on photosynthetic carbon metabolism and translocation in relation to the essential oil ac-
cumulation in mind. Carbon dioxide and glucose are precursors of monoterpene biosynthesis. Saccarides are also a
source of energy and reducing power for terpenoid synthesis.
As zinc is involved in photosynthesis and saccaride metabolism, CO2 and glucose are the most likely sources of carbon
utilized in the terpene biosynthesis, the role of zinc in influencing the essential oil accumulation seems particularly important [76]. Either zinc is an essential micronutrient that
acts as a metal component of various enzymes or as a functional, structural, or regulatory cofactor associated with saccharide metabolism, photosynthesis, and protein synthesis
[13].
The stimulatory effect of zinc on carbohydrate content
may be attributed to its role in the activation of the enzymes
responsible for photosynthesis, biosynthesis and transformation of carbohydrates, regulation of sugars and starch formation [13]. Zn has an important role can be adjudged as it controls the synthesis of indole acetic acid (IAA), a phytohormone which dramatically regulates the plant growth. It is
also necessary for the chlorophyll synthesis and carbohydrate formation [77].
These results confirmed that Pre-sowing He Ne laser
irradiation combined with ZnO NPs foliar spray resulted in
an extreme increase in all parameters compared to ZnO NPs
foliar spray treatment alone. The results showed that the foliar application of nano zinc oxides, resulted in a significant
increase of plant morphological structures and they increased
whenever nano zinc oxides increased compared to the control plants. The morphological structures parameters reached
its maximum values at (20 mg/L), then they turned to decreasing at (30 mg/L), due to Zn toxicity as we mentioned
above. These results were in close agreement with the findings of Kobayashi and Mizutani [78] whom reported that the
plants that were treated with nanoscale ZnO recorded the
highest plant growth, due to extended inter-nodal length.
Such increase can be ascribed to the higher precursor activity
of nanoscale zinc in auxin production. Similarly, nanoscale
ZnO produced early flowers compared to control and bulk
ZnSO4. Such effects can be due to the higher seedling vigour and early vegetative growth. Nanoscale ZnO increased
leaf chlorophyll content irrespective of concentrations compared to bulk ZnSO4 and control.
Moreover, zinc is a component of carbonic anhydrase, as
well as several dehydrogenases and auxin production, which
in turn enhanced the elongation processes, besides the function of zinc in CO2 assimilation. Consequently, the fresh and
dry weights of herbs could be increased [13].
This finding also confirmed by Kaya [46] and Cakmak
[12] found that micronutrients such as zinc have prominent
effects on dry matter, grain yield and straw yield in wheat.
Due to the metabolic role of Zn in synthesis of proteins, enzyme activation and metabolism of carbohydrate, utilization
of fertilizers containing this element increase qualitative and
quantitative performance of potato tubers [48].
Foliar spraying with zinc encouraged the vegetative
growth and increased the plant capacity for building metabolites. Such response may be due to that zinc is known to play
an activator of several enzymes in plants and is directly involved in the biosynthesis of growth substances such as
El-Kereti et al.
auxin, which produces more plant cells and more dry matter.
Similar results were obtained by Darwish [79], Tomar [80].
The important role of Zn comes from its apparent requirement for the synthesis of optimum tryptophan (Precursor of IAA) levels and for the activation of enzymes involved in the synthesis of IAA [81]. El-Khayat [39], mentioned that spraying Antholyza aethiopica with Zn had a
promoting effect on increasing the leaf number per plant.
The positive effects of the treatment were expressed in
increasing of the germination, plant height and weight of the
plant in comparison to the control. Aladjadjian and Svetleva
[82] confirmed that the laser irradiation could raise the activities of SOD, CAT and APX enzymes in plants. As a result, laser exposure accelerated plant physiological metabolism and increased plant growth Aladjadjiyan [83].
From the obtained results, we can interpret that the action
of laser light is quite similar to photosynthesis in plants. Photons of light from a laser penetrate deeply into tissue and
power the synthesis of adenosine tri-phosphate (ATP). ATP
is a molecule that is a major carrier of energy from one reaction site to another in all living cells. These increases in ATP
because of laser light increase the energy available to the cell
biochemical reaction. Tobgy [49] who stated that light irradiation enhanced and accelerated the steps of carbohydrate to
oil conversation. Laser exposure accelerated plant physiological metabolism and increased plant growth (laser
biostimulation effect). This effect started with the seeds and
continued in the herbs. So may laser enhances the centers of
hormone production in the cells of plant seeds and this enhancement continued with the meristematic and permanent
tissues of the plant after cultivation or the laser radiation
enhances the endogenous seeds hormones do certain action
in the cells of the seed embryo which had an effect on the
plant cells after seed cultivation.
The cell elongation causes an increase of plant height in
plants that cultivated from He Ne laser irradiated seeds than
untreated ones, so the shoot internodes increase which gives
a chance for growing more branches as well as umbels.
There is a possible reason for the increasing of plant essential oil due to He Ne laser exposure which is GA increases
the concentration of sugars which form essential oil through
acetyl co-A and mevalonic acid synthesis pathways [49]. In
addition, alfalfa, clover, burr reed irradiated by He Ne increasing fresh weight, proteins [84].
Our results are statistically significant and suggest that;
zinc NPs foliar spray only, or combined with pre-sowing
laser irradiation induced an extreme and unusual increase in
the essential oil of plants compared to the control. These
results confirmed the accomplishment of our main objective
of this study in enhancing the production of sweet basil oil.
Essential oil of basil is a good source of minerals and other
phytochemicals, which are biologically active substances
that have been potential for various therapeutic applications
[85].
5. CONCLUSION
ZnO NPs foliar spray only or combined with pre-sowing
He Ne laser irradiation had a significant enhancement on the
total chlorophyll, total carbohydrate, essential oil content,
[5]
[6]
[7]
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CONFLICT OF INTEREST
[20]
The authors confirm that this article content has no conflict of interest.
[21]
ACKNOWLEDGEMENTS
[22]
[23]
[24]
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