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Recent Patents on Food, Nutrition & Agriculture, 2013, 5, 000-000

ZnO Nanofertilizer and He Ne Laser Irradiation for Promoting Growth


and Yield of Sweet Basil Plant
Mohammed A. El-Kereti1, Souad A. El-feky*,1, Mohammed S. Khater1, Yasser A. Osman2 and
El-sayed A. El-sherbini1
1

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

Sweet basil (Ocimum basilicum L.) is a popular annual


herb of the Lamiaceae (or Labiatae) family. Basil is a tropical species native to India and East Africa [1]. Essential oils
are extracted from basil by steam distillation from the leaves
and flowering tops, and used for food flavoring, dental and
oral products, fragrances and in traditional rituals and medicines [2]. The essential oils have also been shown to contain
biologically active constituents with insecticidal [3], antimicrobial [4] and antifungal [5] properties. These properties
can be attributed to Predominant essential oil constituents,
such as methyl chavicol, eugenol, linalool, camphor, and
methyl cinnamate [6]. Two minor components, juvocimene I
and II have been reported as potent juvenile hormone analogs [7]. Several studies have 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 accumulation in mint. Carbon dioxide and
glucose are precursors of monoterpene biosynthesis [8].
*Address correspondence to this author at the Department of Laser Applications in Metrology, Photochemistry and Agriculture. National Institute of
Laser Enhanced Science (NILES), Cairo University, Egypt. 12613; Tel:
(+447578617765); Fax: ?????????; E-mail: sae21@bath.ac.uk; Alternative
E-mail: dr_souad_elfeky@niles.edu.eg

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].

2013 Bentham Science Publishers

2 Recent Patents on Food, Nutrition & Agriculture, 2013, Vol. 5, No. 3

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).

In this study, we applied the zinc oxide nanoparticle


strategy alone and combined with pre-sowing irradiation of
He Ne laser to examine the possible improvement of sweet
basil growth and yield. We also examined the effects of laser
irradiation and ZnO NPs on the levels of the active constituents, with the aim of developing a technical approach for the
agricultural application of economical nanomaterials.

2.4. Seed Treatment

2. MATERIALS AND METHODS

2.5. Field Cultivation

2.1. Synthesis of ZnO NPs

A total of 1500 Ocimum basilicum seeds was used for the


study. The seeds were divided into three main groups for
field cultivation; the first group was considered the control,
the second group for ZnO NPs foliar spray application and
the last group for ZnO NPs foliar spray with pre-sowing of
He Ne laser irradiation.

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

Fig. (1). Set up of He Ne laser irradiation of Ocimum basilicum seeds.

2.3. He Ne Laser Irradiation


He Ne laser of power 3mW was used for the red-light
irradiation of Ocimum basilicum seeds. The spot diameter of
He Ne laser was 2 mm, thus it is suitable for direct irradiation to basil seeds without any divergence. The power density was calculated as (95 mW/cm2). The 2mm spot diameter
was enough for one-seed exposure in each time Fig. (1).

Ocimum basilicum seeds were treated before cultivation


with fungicides (Topsin M 70% w.p./vitavax 300% w.p.) at
2.5 g/kg. This treatment is generally used to prevent any
fungal damage during cultivation [22]. All treatment experiments were repeated three times.

Ocimum basilicum seeds were first sown on foam trays


(84 cells) on February 15th. The media for seed germination
contained peat moss: vermiculite (1:3 by volume). On April
1st uniform seedlings of approximately 15 cm in height were
transplanted to an open field. The uniform seedlings were
transplanted into plots 3x3.5 M on rows with 30 cm between
the seedlings.
Morphological characteristics such as the plant height
(cm), number of branches, number of leaves per plant, and
both fresh and dry matter of the herb were recorded.

ZnO Nanofertilizer and He Ne Laser for Promoting Growth of Basil

2.6. Preparing ZnO NPs Solution for Foliar Spray


In order to prepare the spraying solution, 2 g of ZnO NPs
was dissolved in distilled water, and then the solution was
completed to 1000 ml as a stock solution for the preparation
of different concentrations of ZnO NPs (10, 20, and 30
mg/L) by dilution. An ultrasound instrument was used to
homogenize the solution. The foliar spray application was
done by using a calibrated pressurized backpack sprayer (capacity 20 L).
2.7. Leaf anatomy Study
Leaf samples of Ocimum basilicum L. were collected
from the mature leaves (fourth node on the shoot from the
terminal end) during September 2012. The specimens were
taken from the leaf between the mid vein and the leaf margin
and then fixed in formalin-acetic acid-alcohol (FAA)) using
70% ethanol. The specimens were gradually dehydrated in a
tert-butyl alcohol (TBA) series [23], and embedded in paraffin wax (m.p. 56C). Sections were cut by a rotary microtome at a thickness of 8-10 m (Model RM2245, Leica Microsystems). Paraffin was removed with xylol and slides
were stained with safranin FCF methanol and fast green, and
then mounted in Canada balsam [23]. The selected sections
were examined and photographed using a light microscope
(Model BX51; Olympus Optical).
2.8. Essential Oil Extraction
Oil was extracted from the plants by hydrodistillation. A
laboratory distillation of essential oil from plant herb is often
necessary in order to evaluate the raw material to be used in
large-scale distillation. A sufficient quantity of the plant (25
g) and water was added in a short neck round bottom 250 ml
flask. The proper essential oil trap and condenser were attached to the flask, and water was added to fill the trap. The
flask was placed in an oil bath and electrically heated to approximately 130C. The temperature of the bath was adjusted
so that a condensate of about one drop per second was obtained. The distillation was continued until collection of oil
ceased (approximately three hours). When the distillation
was complete, the oil was left undisturbed so that good separation was obtained. The volume obtained was determined
and the yield was expressed as a volume/weight percentage,
i.e., volume of oil per 100 g of plant herb. The crude oil was
dried over pure anhydrous sodium sulfate (120150 g/L of
oil). The mixture was stored at 4oC and kept in closed bottles
in the dark to prevent light and oxygen exposure [24].
2.9. Determination of Carbohydrates
The percentage of carbohydrate content was determined
by 3, 5-dinitrosalicylic acid (DNS) [25]. A mixture of 1 g
dinitrosalicylic acid, 200 mg crystalline phenol and 50 mg
sodium sulphite in 100 ml 1% NaOH were dissolved by stirring, and stored at 4C. Leaf samples (100 mg) were extracted with 80% hot ethanol two times (5 ml each time).
The supernatant was collected and evaporated by incubating
the sample in the 80C water bath. Water (10 ml) was added
to dissolve the sugars, and three samples (each 3 ml) were
elected in three separate tubes, together with an additional 3
ml of water in each tube. DNS reagent (3 ml) was added, and

Recent Patents on Food, Nutrition & Agriculture, 2013, Vol. 5, No. 3

the samples were heated in a boiling-water bath for 5 min.


With the contents of the tubes still warm, 1 ml of 40% Rochelle salt solution was added. After cooling, the intensity of
the dark-red color absorption was read at a wavelength of
510 nm on a spectrophotometer.
2.10. Measurement of Chlorophyll Content
Measurement of chlorophyll content was performed using the SPAD-502 meter (Konica-Minolta, Japan). The instrument measures the transmission of red light at 650 nm, in
which chlorophyll absorbs light, and transmission of infrared
light at 940 nm, at which no absorption occurs [26].
2.11. Determination of Plant Macronutrient Content
A half gram of each plant sample was digested using the
H2SO4 and H2O2 [27]. The extracted samples were used to
determine the nitrogen content in the digested solution by the
modified microkjeldahl method [28]. Crude phosphorus content was determined colorimetrically [29]. Potassium and
sodium content were determined against a standard using the
flame photometer [30].
2.12. Determination of Plant Micronutrient Content
Each ashed sample was dissolved in 0.1N HCl and the
volumes were completed to the mark (100ml). This solution
was used for the quantitative determination of Fe, Zn and Cu
by atomic absorption spectrophotometer (Thermo Jarrell Ash
Model AA SCAN1). All the determinations were carried out
with an air-acetylene gas mixture at a rate of 5 L/min according to the method described by Reuter [30].
2.13. T-test Statistical Analysis
Average values and standard deviations were calculated;
t-test was applied to assess the statistical significance of the
differences between the control and ZnO nanoparticle treated
plants using OriginPro.
3. RESULTS
3.1. Characterization of ZnO NPs
Physicochemical properties of ZnO nanoparticles were
characterized via TEM imaging Fig. (2). The images of the
synthesized ZnO nanoparticles reveal a spherical shape and
an average particle size of 10.5 to 15.5 nm.
3.2. The Effect ZnO NPs Fertilization on the Zn Content
of Basil Shoots
Figure (3) shows that the application of ZnO NPs foliar
spray combined with pre-sowing laser irradiation at 10, 20
and 30 mg/L significantly increased shoot zinc level by approximately 4.5-, 4.7- and 5.6-fold, respectively, compared
to the controls. ZnO NPs foliar spray alone at the same concentrations significantly increased shoot zinc level by approximately 4.0-, 4.2- and 4.9-fold, respectively, compared
to the controls.
Table 1 shows the foliar application of ZnO NPs results
in significantly increased plant N, P and K, Fe, Zn and Cu
through the combined application of pre-sowing laser

4 Recent Patents on Food, Nutrition & Agriculture, 2013, Vol. 5, No. 3

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)

ZnO NPs Control.

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

ZnO NPs + He Ne laser Control.

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

ZnO Nanofertilizer and He Ne Laser for Promoting Growth of Basil

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)

Recent Patents on Food, Nutrition & Agriculture, 2013, Vol. 5, No. 3

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.

6 Recent Patents on Food, Nutrition & Agriculture, 2013, Vol. 5, No. 3

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,

fresh weight of 268 (control) to 588 gm, dry matter from


79.3 (control) to 81.3%, number of branches/plant from 45
(control) to 80 and the number of leaves/plant from 744
(control) to 1154.
In case of ZnO NPs treatment alone, at 20 mg/L ZnO
NPs increases the plant height of 93 (control) to 133 cm,
fresh weight of 268 (control) to 655 gm, dry matter from
79.3 (control) to 82.8%, number of branches/plant from 45
(control) to 91 and the number of leaves/plant from 744
(control) to 1290.
3.7. Statistical Analysis
The results presented in Table 3 are statistically significant. These results suggest that zinc NPs treatment and He
Ne laser irradiation in these samples induced an extreme and
unusual significantly increase in the essential oil, mineral
content, chlorophyll content, carbohydrate content and morphological structures of plants compared to zinc NPs treatment alone and control samples.

ZnO Nanofertilizer and He Ne Laser for Promoting Growth of Basil

Table 2.

Recent Patents on Food, Nutrition & Agriculture, 2013, Vol. 5, No. 3

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

Plant Height (cm)

Fresh Weight (g)

Dry Matter %

Branches /plant

Leaves/plant

ZnO NPs Control

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

ZnO NPs + He Ne laser Control

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.

Statistical Analysis of Results (M: Mean, SD: Standard Deviation, P: P Value)


M

Treatment

SD

ZnO NPs + He Ne laser

ZnO NPs

ZnO NPs + He Ne laser

ZnO NPs

ZnO NPs + He Ne laser

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,

which should increase the dissolution rate of fertilizers with


low solubility in water such as zinc oxide (ZnO) [32].
The mobility of the nanoparticles is known to be a very
high, which ensures the phloem transport and ensures the
nutrient to reach all parts of the plant. The presence of
nanoparticles both in the extracellular space and within
some cells in the living plant, Cucurbita pepo that was reported [33].
The application of Pre-sowing laser irradiation combined
with ZnO NPs at (30 mg/L), increased tissue zinc content to
level 5.6 times higher than control. ZnO NPs foliar spray
treatment alone at 10, 20 and 30 mg/L ZnO NPs significantly, leaf Zn content increased whenever Zn supply in-

8 Recent Patents on Food, Nutrition & Agriculture, 2013, Vol. 5, No. 3

creased compared with control. These results were in close


agreement with the findings of Grawel [34] who reported
that the foliar application of zinc increased the zinc content
in the leaves. These results were in agreement with those
reported by El-Gazzar [35], Foregoni [36] on chlorotic
Grapevines and Marschner [13] who reported that Zn content
in cereal grain increase with an increase in Zn fertilizer additions. This increase in leaves content may be due to its
maximum absorption from Zn source and less translocation
to the other parts of the plant [37] and may be attributed to
the plant enzymes activated by Zn are involved in carbohydrate metabolism, maintenance of the integrity of cellular
membranes, protein synthesis, and regulation of auxin synthesis [13].
The data of this work revealed that ZnO NPs foliar application treatments significantly increased leaves nutrients
content, i.e. N, P, K, Fe, Zn and Cu compared to the control
leaves. These results of enhancing leaf nutrient contents may
be due to that Zn is essential for sugar regulation and enzymes that control plant growth [38]. The obtained results
are in conformity with those of El-Khayat [39], (Gomaa [40]
on Antholyza aethiopica, Yadav [41] on tuberose and Samia
and Mahmoud [42] on Tritonia crocata.
In this work, the foliar spray application of ZnO NPs
increased tissue iron contents. These results were in close
agreement with the findings of Aref [43] who reported that
zinc foliar spray aided Zn in affecting the increase of Fe concentration in the leaf.
The data of this work showed that Zn foliar application significantly increased leaves nutrients content, i.e.
(N, P, K, Fe, Zn and Cu) whenever Zn supply increased
then they reached their maximum values at (20 mg/L) and
turned to decreasing at (30 mg/L), this may be due to the
antagonistic relationship between Zn and other micronutrients [43]. This indicated that sweet basil plant in this
study was intolerant of excess Zn at (30 mg/L). These
results match with Aref [43] who found that the excess Zn
at (10 mg /L) reduced leaf chlorophyll content in wheat.
Our results also concur with Fontes and Cox [44] who
reported reduced growth and leaf chlorosis in soybean
plants grown in culture solutions containing 40 M of Zn.
The visual symptoms were similar to those observed in a
0 M of Fe treatment, leading Fontes and Cox [45] to
conclude that Fe deficiency is one of the factors responsible for reduced growth in plants exposed to Zn toxicity
(excess Zn). The mechanism of Zn induced chlorosis is
not clear. One possible explanation is that Zn may compete with Fe for a Fe-requiring step in the chlorophyll
biosynthesis [46]. Kaya [46] found that Fe addition could
remedy chlorosis induced by excess Zn.
The toxicity limit for Zn depends on plant species, genotype, and growth stage. Toxicity limits generally range between (100-500g/g) [47].
Our study showed that the supply of Zn at (30 mg /L)
increased Zn level in sweet basil leaves to toxic level this
due to the well-known antagonism between Fe and Zn. Previous studies have shown that Zn interfered with Fe uptake
and translocation, whereas Fe interfered with Zn translocation, but only when Zn concentrations were high [48].

El-Kereti et al.

There are three possible mechanisms for this antagonism.


First, there could be competition between Zn2+ and Fe2+ during uptake [47]. Second, there could be interference in the
chelation process during Fe uptake and translocation [47].
Third, there could be competitive inhibition between Zn and
Fe during unloading in the xylem [48].
The combined treatment of pre-sowing laser irradiation
with ZnO NPs foliar spray results in significantly increasing
of zinc content in treated leaves than the content of control
leaves (untreated). This can be explained by the plant chemical contents that are related to the other organic compounds
and morphological characteristics of the plant. Any change
in plant proteins will cause changes in nitrogen percentage,
which is a part of these compounds. The laser treatment increased the nitrogen content that led to increases in protein
content, which needs to cover the increase of plant organs
(branches and number of umbels). Increasing of the cell
number of laser treated seeds led to an increase in nucleic
acids and phospholipids membranes and significant increased phosphorus content of laser treated fruits more than
control one. The laser effect on gibberellic acid resulted in
cell elongation and an increase in the cell vacuoles that led to
potassium increase because it is found in the cellular sap
vacuoles, which controls the cell osmotic pressure and electrical balance [49].
The results showed that laser irradiation results in significantly increasing of plant micronutrients such as Fe, Zn
and they increased with laser irradiation used than the content of the control. Our results were in close agreement with
the findings of Truchliski [50] whom reported that the
Triticale seeds of Gabo and Migo cv. That were irradiated by
laser (red light) caused an increase of mineral content, notably that of Zn and Fe.
The foliar spray application of ZnO NPs increased nitrogen uptake. These results were in close agreement with the
findings of Alloway [51] who reported that the increase of
total nitrogen uptake induced by zinc application corroborates the thesis of its primary effect on main physiological
processes, related to nutrient uptake. Increase of plant height
is due to enhancement of auxin biosynthesis and synergistic
relation between iron and nitrogen. Zinc plays the important
role in the biosynthesis of tryptophan, which is a precursor
of auxin. Zinc also found in phosphoenolpyruvate carboxylase structure [52].
The data of this work revealed that the protein content of
treated leaves increased whenever Zn supply increasedcompared to controls leaves and reached its maximum value at
(20 mg/L). These results agreed with the results of Zakaria
[53] on cotton. In addition, Cakmak [54] stated that under a
condition of zinc deficiency protein content decreases due to
zinc is an important element in enzyme structure involved in
amino acid biosynthesis and thus amino acids are the base of
protein synthesis. Protein content decreased at (30 mg/L) of
ZnO NPs, as mentioned above that; basil leaves intolerant of
excess Zn which, reduces leaf chlorophyll content, as a result
of Zn may compete with Fe for a Fe-requiring step in the
chlorophyll biosynthesis [55] which, decreases the metabolic
activity, nutrient absorption, photosynthesis and consequently, protein synthesis.

ZnO Nanofertilizer and He Ne Laser for Promoting Growth of Basil

The Pre-sowing He Ne laser irradiation combined with


ZnO NPs foliar spray resulted in an extreme increase in nitrogen content compared to ZnO NPs treatment alone. Fennel Feoniculum vulgare Mill seeds irradiated by laser increased the nitrogen content, which led to increase in protein,
that needs to cover the increase of plant organs of fennel and
coriander plants [56]. Perennial ryegrass seeds irradiated by
red laser caused the increase of some macroelements: phosphorus, potassium, magnesium [57].
Weber [58] stated that Laser primarily resulted in the
perforation of the plant cell wall and consequently, facilitated the internalization of nutrients essential for plant
growth and development; which is likely the apparent reason
for the increased growth in the treated plants [59]. The Presowing laser irradiation combined with ZnO NPs foliar spray
resulted in an extreme increase in chlorophyll content compared to ZnO NPs treatment alone at (10, 20 and 30 mg/L).
Chlorophyll content increased whenever Zn supply increased
and reached its maximum value at (20 mg/L). But it decreased at (30 mg/L), as we mentioned above that; basil
leaves intolerant of excess Zn (Zn toxicity) at (30 mg/L)
which, reduced leaf iron content and plant growth that due to
zinc may compete with Fe for a Fe-requiring step in chlorophyll biosynthesis resulting in chlorophyll content decreased.
These results were in close covenant to the findings of
Kumar [60], who reported that regarding the beneficial effect
of Zn on photosynthetic pigments, it might be due to its role
in the increase rate of phytochemical reduction regarding the
beneficial effect of Zn mentioned from many investigators
Nahed [61] on Salvia farinacea and Massoud [62] on pea
plants. They reported that Zn increased chlorophyll content.
Zn plays an important role in many biochemical reactions
within the plants like formation of chlorophyll and carbohydrates [63, 64]. Regarding the beneficial effect of Zn on photosynthetic pigments, may be due to its role in increasing the
rates of photochemical reduction [60], chloroplast structure,
photosynthetic electron transfer as well as photosynthesis
[65].
Chlorophyll content of sweet basil herb significantly increased through pre-sowing laser irradiation. It well known
that, there are many enzymes and hormones control the plant
growth as gibberellic acid (GA3) and cytokinin. The endogenous content of GA1, the main biologically active GA in
lettuce seeds increases after red light treatment [66]. This
means that, the red light, which induces GA3 hydroxylase
gene (S3 h1 expression), promotes the complex cycle of GA
formation. This expression is inhibited by far red light, this
means that, monochromatic light is the only possible way to
promote GA3 hydroxylase gene (S3 h1 expression). Therefore, the red light laser can induce this effect but not the
polychromatic light (sunlight). The GA3 mainly induces cell
elongation and many other effects, i.e. weakens the cell wall
[67], formation of proteolytic enzymes [68], increase of
auxin content [69], the hydrolysis of starch, which increases
the concentration of sugars and raising the osmotic pressure
in the cell sap so that water enters the cell and tends to
stretch it [70]. The cell elongation causes an increase of plant
height in plants treated with helium-neon than untreated
ones, so the shoot internodes increase, which gave a chance
for growing more branches as well as umbels. There is a

Recent Patents on Food, Nutrition & Agriculture, 2013, Vol. 5, No. 3

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-

10 Recent Patents on Food, Nutrition & Agriculture, 2013, Vol. 5, No. 3

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,

ZnO Nanofertilizer and He Ne Laser for Promoting Growth of Basil

iron content, and plant growth characteristics (branches and


leaves number/plant, fresh and dry weight) and consequently, will increase the total yield of sweet basil. The results of this work show strong evidence for the high efficiency of this new nanofertilizer on plant growth enhancement. These powerful and inexpensive nanofertilizers could
replace traditional methods of plant growth enhancement.
Furthermore ZnO nanofertilizers development could have
large-scale economic implications and multiple benefits for
consumers, producers, farmers. This work confirmed that
ZnO nanofertilizers using is eco-friendly, safe and produce
healthier and more resistant to disease plants at appropriate
concentrations but must avoid the high ZnO NPs concentrations.
Additionally, using cheap laser sources for seed irradiation such as, He Ne could significantly enhance the growth,
the active constituents of sweet basil to a higher extent. He
Ne treatment did not cause any damages to plant tissues, but
it improves seed germination rate. It is well known that the
traditional methods depended on expensive materials like the
application of external growth hormones. Our technique using soft laser did this effect by enhancing the growth and the
essential oil yield of sweet basil plant.
CURRENT AND FUTURE DEVELOPMENTS
Zinc oxide nanofertilizer can be used as a foliar spray on
leaves and as a seed coating. Nanotechnology can present a
solution to increasing the value of agricultural products and
environmental problems that makes nanofertilizers a promising technology in the future. These powerful and inexpensive nanofertilizers could replace traditional methods of
plant growth enhancement. By using of nano-particles and
nano-powders, we can produce controlled or delayed releasing fertilizers. Nano-particles have high reactivity because of
the more specific surface area, more density of reactive areas, or increased reactivity of these areas of the particle surfaces and improvement physical properties of soil. These
features simplify the absorption of fertilizers and pesticides
that produced at nano scale.

Recent Patents on Food, Nutrition & Agriculture, 2013, Vol. 5, No. 3

[5]

[6]

[7]
[8]
[9]
[10]

[11]
[12]
[13]
[14]
[15]
[16]

[17]

[18]
[19]

CONFLICT OF INTEREST

[20]

The authors confirm that this article content has no conflict of interest.

[21]

ACKNOWLEDGEMENTS

[22]

This work was supported by Cairo University, National


Institute of Laser Enhanced Sciences, Department of Laser
Application in Metrology, Photochemistry and Agriculture,
Cairo, Egypt.

[23]
[24]

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