Elevating tolerance of drought stress in Ocimum basilicum using pollen grains extract

Drought is a major abiotic stress that limits performance of sweet basil (Ocimum basilicum L.), which is an
important aromatic plant for flavor foods and traditional medicines. The influence of palm pollen grains
extract (PGE) on growth, physio-biochemical attributes, water use efficiency (WUE), anatomical characteristics
and antioxidant defense system components in basil plants was investigated under normal and water
deficit stress conditions

1. Elevating tolerance of drought stress inOcimum basilicumusing pollen
grains extract; a natural biostimulant by regulation of plant performance
and antioxidant defense system
R.S. Taha
a
, H.F. Alharby
b
, A.A. Bamagoos
b
, R.A. Medani
a
, M.M. Rady
a
*
a
Botany Department, Faculty of Agriculture, Fayoum University, 63514 Fayoum, Egypt
b
Department of Biological Sciences, Faculty of Science, King Abdulaziz University, 21589 Jeddah, Saudi Arabia
ARTICLE INFO
Article History:
Received 22 December 2018
Revised 22 August 2019
Accepted 12 September 2019
Available online 12 November 2019
Edited by DK Tripathi
ABSTRACT
Drought is a major abiotic stress that limits performance of sweet basil (Ocimum basilicumL.), which is an
important aromatic plant forflavor foods and traditional medicines. The influence of palm pollen grains
extract (PGE) on growth, physio-biochemical attributes, water use efficiency (WUE), anatomical characteris-
tics and antioxidant defense system components in basil plants was investigated under normal and water
deficit stress conditions. The PGE was exogenously applied as a foliar spray at a rate of 1.0 g L
‒1
to plants
under full (70% of soil water-holding capacity; SWHC) or deficit irrigation (DI; 50% of SWHC) in a pot experi-
ment repeated three times. Exposing basil plants to drought stress significantly disorganized their perfor-
mance including oil yield and anatomical features, but increased their antioxidant defense system
components. However, the application of PGE to DI-stressed plants significantly improved growth character-
istics and the contents of essential oil, leaf photosynthetic pigments, soluble sugars, free proline, and ascorbic
acid. Antioxidant enzyme activities, relative water content (RWC), WUE, and anatomical characteristics were
also improved, while electrolyte leakage was significantly diminished compared to the corresponding con-
trol. Therefore, results obtained in this study recommend using PGE at 1.0 g L
‒1
as a foliar application strategy
forOcimum basilicumplants grown under DI stress to maintain higher RWC and WUE, and lower EL, and
improve plant growth and essential oil productivity by limiting the inhibitory effects of DI.
© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
Keywords:
Basil
Deficit irrigation
Biostimulants
Growth and productivity
Antioxidative defense system
1. Introduction
Basil (Ocimum basilicumL.) is one of the most important aromatic
plants belonging to the Lamiaceae family. It is very important forfla-
vor foods and traditional medicines (Yusuf et al., 1994). It has been
concluded that growth and essential oil production of aromatic plants
are influenced by various environmental stresses such as water defi-
cit (drought) stress (Forouzandeh et al., 2012;Moinuddin et al., 2012;
García-Caparr‒os et al., 2019).
Water deficit stress is one of the most dangerous stresses in plant
environment. In arid and semi-arid regions, in particular, it limits the
performance of plants (Moinuddin et al., 2012;Abd El-Mageed et al.,
2016;García-Caparr‒os et al., 2019). It overproduces the reactive oxy-
gen species (ROS; O
2
‒‒,H2O2,OH
‒
, and
1
O2) in chloroplasts, peroxi-
somes, and mitochondria. These overproduced ROS lead to
physiological disorders, negatively affecting photosynthesis, stomatal
conductance, transpiration and growth (Batra et al., 2014). Prolong-
ing the water stress period causes considerable damage and thus the
death of the plant cells because the production of excess ROS will
inhibit the scavenging action the antioxidant defense system compo-
nents (Cruz de Carvalho, 2008). Where available water for the agri-
cultural sector is reduced worldwide due to some reasons such as
rapid population growth, the greater impacts of climate change and
various human activities (World Bank, 2006), it is necessary to invent
some of the strategies applied to plants to support their growth
under stress such as antioxidants, including proline (Rady et al.,
2016;Merwad et al., 2018,2019b), salicylic acid (Abd El-Mageed et
al., 2016), sequenced ascorbate-proline-glutathione (Rady and Hem-
ida, 2016;Semida et al., 2018), and plant extracts that are rich sour-
ces in bioactive stimuli including antioxidants such as extracts of
Moringa oleiferaleaves, maize grains, and licorice roots (Rady et al.,
2013;Semida and Rady, 2014;Rady and Mohamed, 2015;Desoky et
al., 2018;Rehman et al., 2018;Rady et al., 2019a).
Pollens are the male reproductive cells offlowers and are pro-
duced by most uncultivated plants (Hassan, 2011). Palm pollen
grains (PPG) are a good source, rich in sugars, proteins, minerals,
vitamins, carotenoids, enzymes, phytohormones, amino acids, fatty
acids and antioxidants (Hassan, 2011;Bishr and Desoukey, 2012;
Basuny et al., 2013;Farouk et al., 2015). Therefore, PPG can be used
*Corresponding author.
E-mail address:[email protected](M.M. Rady).
https://doi.org/10.1016/j.sajb.2019.09.014
0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
South African Journal of Botany 128 (2020) 42fi53
Contents lists available atScienceDirect
South African Journal of Botany
journal homepage:www.elsevier.com/locate/sajb

2. after extraction as a bioactive stimulant for plants to improve their
tolerance to stress such as drought. This study was designed to
assess potential influences of palm pollen grains extract (PGE) on
increasing drought tolerance in basil plants grown under drought
stress (deficit irrigation at 50% of SWHC) through assessing basil
plant performance and status of plant antioxidant defensive sys-
tems and tissue health (assessed in terms of relative water content
and electrolyte leakage). Water use efficiency and oil content of
plants were also assessed.
2. Material and methods
2.1. Material of plant and growing conditions
At Fayoum University, Faculty of Agriculture (29° 17
0
N; 30° 53
0
E), a
pot experiment was performed and repeated three times during the
period from the end of March to mid-August 2018. Seeds of sweet basil
(Ocimum basilicumL.) were brought from the Center of the Agricultural
Research, Egypt. They were surface-sterilized for 2 min with a solution
(1%) of NaClO, thoroughly washed several times with distilled water,
and were then left to air-dry. Thereafter, seeds were sown in the nurs-
ery at the end of March. The uniform seedlings were selected, gently
removed and transplanted immediately into pots at the end of May.
Plastic pots (40 cm in diameter, 50 cm depth),filledby12kgsoilfor
each, were used and arranged for growing transplants in an open
greenhouse (mean day/night temperatures were 29°§5°C/14°§3°C,
respectively, rainfall ranged between 5 and 10 mm, relative humidity
ranged from 65.4% to 72.2%, and day-length varied in 12‒14 h).
Soil analyses were conducted as detailed inBlack et al. (1965)and
Jackson (1973)procedures. Texture of soil was sandy clay loam [clay%
(w/v) was 31.0, silt% (w/v) was 20.1, and sand% (w/v) was 48.9]. The
other main characteristics of the soil used for experiments were as
follows: pH [at a soil: water (w/v) ratio of 1:2.5] was 7.64, EC (dS m
fi1
;
soilfipaste extract) was 3.80, organic matter% (w/v) was 1.02, CaCO
3%
(w/v) was 4.96, and totalN% (w/v) was 0.081. In addition, some of the
available nutrient elements contents were as follows: P (mg kg
fi1
soil) was 9.67, K (mg kg
fi1
soil) was 196, Fe (mg kg
fi1
soil) was 5.82,
Mn (mg kg
fi1
soil) was 3.12, Zn (mg kg
fi1
soil) was 0.98, and Cu
(mg kg
fi1
soil) was 0.54.
Before transplanting, 1.5 g phosphorus fertilizer (calcium super-
phosphate, 15.5% P
2O
5) per pot was added, and then soil in all pots
was saturated with water. After transplanting, 3.0 g nitrogen (ammo-
nium sulfate, 21%N) and 1.5 g potassium (potassium sulfate, 48%
K
2O) fertilizers were added, each in two split doses. Thefirst dose
was added after one month from transplanting and the second was
added 15 days later.
2.2. Irrigation, PGE treatments, and experimental design
Maximum amount of water can held by soil is known as soil
water-holding capacity (SWHC), which was calculated from the fol-
lowing equation:
SWHC%ðÞ¼totalporosity%fiairspace%? :
Two irrigation treatments; 70% (calculated as optimum irrigation
water for optimum growth of basil in sandy clay loam) and 50% of
SWHC were used during the whole growing season after transplant-
ing, however, all pots were well-watered before transplanting. The
well irrigation treatment (70% of SWHC) was considered as a con-
trol, and the second treatment (50% of SWHC) was considered as a
deficit irrigation treatment. Water deficiency in 40 and 30% of
SWHC was also examined in a small preliminary study (data not
shown), however, they extremely reduced basil growth and oil yield
even with treatment by palm pollen grains extract (PGE). Deficit
irrigation (DI) was given to basil plants as a stress treatment begin-
ning from thefirst irrigation after transplanting. Moisture contents
of pots were daily monitored by HH2 moisture meter Version 4.0
(Delta-T Devices Ltd., U.K.) and maintained through water applica-
tion if required. Pots of each one of the two irrigation groups
(DI-stressed plants; 50% of SWHC and unstressed plants; 70% of
SWHC) were divided into two subgroups for PGE foliar application.
Therefore, the experiment consisted of four treatments as follows:
(1) irrigation with 70% of SWHC + foliar spray with distilled water
(control), (2) irrigation with 70% of SWHC + foliar spray with PGE
application, (3) irrigation with 50% of SWHC + foliar spray with dis-
tilled water, and (4) irrigation with 50% of SWHC + foliar spray with
PGE. The PGE was applied at 1.0 g L
‒1
for plants three times; at 30,
45, 60 days after transplanting to run off. Few drops of Tween-20
were used as a surfactant. The PGE was selected at a level of 1.0 g L
‒1
because it was generated highly effectiveness among all levels (i.e.,
0.5, 1.0 and 1.5 g L
‒1
) based on a preliminary study conducted with
small pots (data not shown). All pot experiments were organized in
a completely randomized design (CRD) with 20 replications/pots
per treatment.
2.3. The extraction of PPGE
Pollen grains were collected at the end of March from Egyptian
date palm;Phoenix dactyliferaL., Fayoum, Egypt. The palm pollen
grains were extracted as detailed inNikkon et al. (2003)method with
a modification. Each 10 g of pollen grain powder was well mixed
with 100 ml ethanol and kept aside for 72 h with occasional stirring.
After stirring process, solution wasfiltered twice using Whatman No.
1filter paper, and then with non-absorbent cotton. Thefiltered solu-
tion was evaporated with help of the vacuum rotary evaporator. The
crude extract obtained from 10 g pollen grains was made as an
extract at a concentration of 1.0 g L
‒1
by dissolving in 10 L distilled
water with the help of ethanol and then ethanol was eliminated from
the extract by evaporation. The extract was used immediately or
stored in the refrigerator at‒20 °C until use.
The crude extract was analyzed and its chemical constituents (on
a dry weight basis) are shown inTable 1.
Table 1
Chemical constituents of palm pollen grains extract (PPGE; on a dry
weight basis).
Component Unit Value
Moisture g 100 g
‒1
28.9§1.64
Ash 6.2 §0.42
Protein 31.0 §1.52
Total free amino acids 30.2 §1.16
Free proline 0.34 §0.01
Soluble sugars 14.2 §0.31
Phosphorus (P) g kg
‒1
9.04§0.54
Calcium (Ca) 2.49 §0.13
Magnesium (Mg) 3.42 §0.18
Potassium (K) 8.63 §0.49
Sulfur (S) 6.16 §0.32
Molybdenum (Mo) 2.94 §0.16
Boron (B) 2.98 §0.14
Iron (Fe) 4.55 §0.20
Manganese (Mn) 2.92 §0.17
Zinc (Zn) 2.72 §0.12
Copper (Cu) 3.03 §0.16
Sodium (Na) 0.52 §0.02
Soluble phenols 0.72 §0.03
Totalflavonoids 0.61 §0.02
Total carotenoids 14.2 §0.24
Vitamin C (ascorbic acid) 1.06 §0.01
Vitamin A IU kg
‒1
747§12.4
Vitamin E 335 §5.42
DPPH (antioxidant activity) % 85.2 §1.38
Phytohormones Indole-3-acetic acid mg kg
‒1
4.92§0.03
Gibberellins 6.74 §0.05
Cytokinins 7.87 §0.06
Values are means (n=5)§SE.
R.S. Taha et al. / South African Journal of Botany 128 (2020) 42fi53 43

3. 2.4. Measurements of basil growth, essential oil content and water use
efficiency (WUE)
At 75 days from transplanting, roots and shoots of 9 basil plants
obtained in each time from each treatment were separated carefully
and were then washed in distilled water. By a meter scale, root and
shoot lengths were measured and were then weighed (fresh weight;
FW). Numbers of leaves, branches, and inflorescences were counted.
Leaf area per plant was measured by using a graph sheet. Thereafter,
roots and shoots were dried at 70 °C until a constant weight was
obtained (dry weight; DW). Essential oil content was determined (ml
per 100 g fresh leaves) following the method ofBritish Pharmaco-
poeia (1963). In a gram of dry matter of plant per L of water applied,
values of WUE were calculated at 75 days after transplanting accord-
ing to the following equation (Jensen, 1983):
WUE¼basilyield gplant
fi1
flfi
waterapplied Lplant
fi1
flfi
2.5. Leaf photosynthetic pigments determination
Chlorophylls and carotenoids contents were assessed and were
then calculated as detailed inArnon (1949)method. A sample (0.2 g)
of leaf was homogenized in 50 ml of 80% (v/v) acetone. Thereafter,
centrifugation was practiced at 10,000£g for 10 min at room tem-
perature (22§2 °C). Afterfiltration, absorbance was read at 665, 649
and 440 nm.
2.6. Relative water content (RWC) and electrolyte leakage (EL)
assessments
Using fresh fully-expanded leaf after excluding the midrib, assess-
ment of RWC following the method detailed inWeatherly (1950)and
modified inOsman and Rady (2014)method was performed, as well
as EL in leaf with the same specification was assessed as followed in
Sullivan and Ross (1979)method.
2.7. Determination of osmoprotectants (free proline and soluble sugar)
and low molecular weight antioxidants (ascorbic acid and glutathione)
contents
2.7.1. Determination of free proline
Leaf free proline contents (mg g
fi1
DW) were measured (n=9)using
the rapid colorimetric method, as suggested byBates et al. (1973).Pro-
line was extracted from 0.5 g of each leaf sample by grinding in 10 ml
3% (v/v) sulphosalicylic acid and the mixture was then centrifuged at
10,000£g for 10 min. Two ml of the supernatant was added to a test
tube, to which 2 ml of a freshly prepared acid-ninhydrin solution was
then added. The tubes were incubated in a water bath at 90 °C for
30 min and the reaction was terminated in an ice bath. Each reaction
mixture was extracted with 5 ml toluene and vortex-mixed for 15 s.
The tubes were allowed to stand for at least 20 min in the dark at room
temperature to allow separation of the toluene and aqueous phases.
Each toluene phase was then carefully collected into a clean test tube
and its absorbance was read at 520 nm. The free proline content in the
sample was determined from a standard curve prepared using analyti-
cal grade proline, and calculated on a % DW basis.
2.7.2. Determination of total soluble sugars
Dried ground leaves (0.5 g,n= 9) were homogenized in 80% etha-
nol for 10 min and centrifuged at 600 g. The supernatant was col-
lected. The extraction procedure was repeated twice to ensure the
complete extraction of sugars. The collected supernatant was
completely evaporated on a china dish in a hot water bath. The resi-
due was re-dissolved in 1fi3 mL distilled water and then passed
through 0.45-
mmfilterfilm for assay of contents of sugars. Total
soluble sugars were measured by the anthrone reagents method (Iri-
goyen et al., 1992). Five mL anthrone sulphuric acid solution (75% v:
v) were added to 0.1 mL supernatant. The mixture was boiled in 90 °C
for 15 min after it was refrigerated in a cool water bath (0 °C). The
absorbance was read at 620 nm, using a spectrophotometer and com-
pared with the calibration curve, using pure glucose (Sigma).
2.7.3. Determination of ascorbic acid
Ascorbic acid (AsA) contents in basil plants were determined
using the method ofMukherjee and Choudhuri (1983). Each fully-
expanded leaf sample (0.5 g,n= 9) was extracted with 10 ml of 6%
(w/v) TCA. The extract was mixed with 2 ml of 2% (w/v) dinitrophe-
nylhydrazine, followed by the addition of one drop of 10% (w/v) thio-
urea in 70% (v/v) ethanol. The mixture was then boiled for 15 min in
a water bath and, after cooling to room temperature, 5 ml of 80%
(v/v) H
2SO4was added at 0 °C. The absorbance was recorded at
530 nm. The concentration of AsA was calculated from a standard
curve plotted using known concentrations of AsA. AsA concentrations
were calculated on a dry weight basis.
2.7.4. Determination of glutathione
Glutathione (GSH) contents were determined using the method
described byGriffth (1980). Fresh, fully-expanded leaf tissue (50 mg)
was homogenized in 2 ml of 2% (v/v) metaphosphoric acid and centri-
fuged at 17,000£g for 10 min. An aliquot (0.9 ml) of the supernatant
(n= 9) was neutralized by adding 0.6 ml of 10% (w/v) sodium citrate.
Each 1.0 ml assay contained 700
ml of 0.3 mM NADPH, 100mlof
6 mM 5,5
0
-dithio-bis-2-nitrobenzoic acid, 100ml distilled water, and
100
ml of extract and was stabilized at 25 °Cfor 3fi4 min. Ten mlof50
Units ml
fi1
GSH reductase was added and the absorbance was
recorded at 412 nm. GSH contents were calculated from a standard
curve and expressed on a dry weight basis.
2.8. Preparation of leaf extract for protein and enzyme activity assays
Using potassium phosphate buffer (100 mM) with a pre-chilled
pestle and mortar, leaf sample was homogenized. After supernatant
centrifugation at 15,000 g for 20 min, protein content (Lowry et al.,
1951) and antioxidant enzyme activities [Putter (1974)for guaiacol
peroxidase; GPOX,Aebi (1984)for catalase; CAT andKono (1978)for
superoxide dismutase; SOD] were assessed.
Activity of GPOX (EC 1.11.1.7) was assessed in a reaction mixture
consisted of phosphate buffer (3 ml), guaiacol solution (50
ml), enzyme
extract (100
ml) and H
2O
2solution (30ml). A rate of oxidized guaiacol
formed was spectrophotometrically read at 436 nm. Activity of CAT (EC
1.11.1.6) was assessed in a reaction mixture consisted of potassium
phosphate buffer (50 mM, 1.5 ml), H
2O2(150 mM, 1.2 ml) and enzyme
extract (30
ml). The absorbance change was spectrophotometrically
read at 240 nm. Activity of SOD (EC 1.15.1.1) was assessed in a reaction
mixture (sodium carbonate buffer (1.8 ml) + nitroblue tetrazolium
(NBT; 750
ml) + Triton X-100 (150ml) initiated by addition of hydroxyl-
amine hydrochloride (150
ml). After incubation for 2 min, an enzyme
extract (70
ml) was added, and an inhibition in the rate of NBT reduc-
tion was spectrophotometrically read at 540 nm.
The UV/VIS Absorption spectrophotometer (Specord M-40, Jena,
Germany), and the UV-160A UVfivisible recording spectrometer (Shi-
madzu, Kyoto, Japan) were used for all the previous assessments.
2.9. Leaf and stem anatomy
Leaf and stem anatomy was conducted using samples from two
cuts (at 50 and 75 days after transplanting). To identify the anatomi-
cal features of basil leaf and main stem, 1 cm length samples were
brought from the middle of thefifth leaf from the apex and from the
fifth internode, respectively. After killing andfixing leaf and stem
samples in FAA solution composed of 95% ethyl alcohol (50 ml),
44 R.S. Taha et al. / South African Journal of Botany 128 (2020) 42fi53

4. formalin (10 ml), glacial acetic acid (5 ml) and distilled water (35 ml)
for 48 h, they were washed in 50% ethyl alcohol. Leaf and stem sam-
ples were then dehydrated and cleared in tertiary butyl alcohol
series. Thereafter, samples were embedded in paraffin wax of
54 °Cfi56 °C m.p., and were then cut to cross-sections, 20
mthick
using a rotary microtome. Sections of leaves and stems were adhered
with Haupt's adhesive and stained with the crystal vio-
letfierythrosine combination (Sass, 1961). Sections were then cleared
in carbol xylene and mounted in Canada balsam. Using an upright
light microscope (AxioPlan, Zeiss, Jena, Germany), sections were
spotted and authenticated, and measurements were taken using a
micrometer eyepiece and an average of 9 readings was calculated.
Fig. 1.Effect of 1.0 g L
‒1
palm pollen grains extract (PGE) application on growth traits ofOcimum basilicumplants grown under normal and deficit irrigation.
Values are means§SE (n= 9), and means in the same row for each trait with the same letter are not significantly different atP0.05. *SWHC means soil water-holding capacity.
R.S. Taha et al. / South African Journal of Botany 128 (2020) 42fi53 45

5. 2.10. Analysis of data
After applying the homogeneity test for error variances following
Gomez and Gomez (1984)procedure, data were subjected to one-
way ANOVA for a CRD. Combined analysis of 3-replicate experiment
and Tukey’s test were practiced to evaluate the LSD at a probability
level of 95% (P0.05).
3. Results
3.1. Response of growth traits of water deficit-stressed plants to palm
pollen grain extract (PPGE)
Treatment of basil plants with PGE under normal irrigation (70%
of SWHC) significantly (P0.05) increased all assessed growth
characteristics compared to the corresponding control as shown in
Fig. 1.Deficit irrigation water (DI; 50% of SWHC) treatment signifi-
cantly reduced all evaluated growth characteristics compared to
appropriate irrigation (70% of SWHC). However, PGE application sig-
nificantly increased all assessed growth characteristics of DI-
stressed plants compared to corresponding controls. Plant height,
root length, leaf area, shoot and root fresh weights, and shoot and
root dry weights were increased by 22.6, 49.1, 77.4, 43.8, 37.7, 76.1,
and 38.6%, respectively. In addition, numbers of leaves per main
stem, branches per plant, and inflorescences per plant were
increased by 73.3, 56.7, and 56.1%, respectively compared to the
stressed controls. The positive effect of PGE application was more
pronounced under DI than its application under normal irrigation
and awarded no significant differences between all tested parame-
ters of full-irrigated plants and those of DI-stressed plants treated
with PGE.
3.2. Response of photosynthetic pigments and oil contents, tissue health
and water use efficiency (WUE) of water deficit-stressed plants to palm
pollen grain extract (PPGE)
Under normal irrigation (70% of SWHC), application of basil plants
with PGE significantly increased leaf photosynthetic pigments and oil
contents, relative water content (RWC) and WUE. On the other hand,
electrolyte leakage (EL) was not affected compared to the corre-
sponding control;Figs. 2 and 3). DI (50% of SWHC) treatment signifi-
cantly decreased all abovementioned parameters, while significantly
increased El compared to 70% of SWHC. However, PGE treatment sig-
nificantly increased all aforementioned parameters, while signifi-
cantly reduced El of DI-stressed plants compared to the
corresponding controls. The increases were 18.3, 40.9, 29.2, 19.0,
38.5, 68.4, and 18.8% for chlorophyll“a”, chlorophyll“b”, total chloro-
phylls, total carotenoids, essential oil, WUE, and RWC, respectively,
while, the reduction in El was 12.1%. The positive effect of PGE appli-
cation was more pronounced under DI than under the appropriate
Fig. 2.Effect of 1.0 g L
‒1
palm pollen grains extract (PGE) application on leaf photosynthetic pigment contents (mg g
‒1
FW) ofOcimum basilicumplants grown under normal and def-
icit irrigation.
Values are means§SE (n= 9), and means (bars) with different letters are significantly different atP0.05. *SWHC means soil water-holding capacity.
46 R.S. Taha et al. / South African Journal of Botany 128 (2020) 42fi53

6. irrigation. It conferred, generally, no significant differences between
all tested parameters of well-irrigated plants and those of DI-stressed
plants treated with PGE.
3.3. Response of antioxidant defense systems of water deficit-stressed
plants to palm pollen grain extract (PPGE)
Data shown inFigs. 4 and 5show that spraying of basil plants with
PGE under normal irrigation (70% of SWHC) significantly increased
osmoprotectants and non-enzymatic antioxidants contents and
enzymatic antioxidants activities. On the other hand, protein content
was not affected compared to the corresponding control. Exposing
plants to 50% of SWHC significantly increased the contents of osmo-
protectants and non-enzymatic antioxidants. In addition, the activi-
ties of enzymatic antioxidants were significantly increased, while the
content of proteins was significantly reduced compared with expos-
ing to 70% of SWHC. PGE treatment further increased all osmoprotec-
tants and non-enzymatic antioxidants contents and enzymatic
antioxidants activities in DI-stressed plants. The elevations were
23.8, 37.2, 29.8, 19.6, 21.9, and 47.2% for proline, soluble sugars,
ascorbate, glutathione, and protein contents. Activities of superoxide
dismutase, catalase, and guaiacol peroxidase were also increased by
47.2, 39.5, and 35.1%, respectively. The positive effect of PGE applica-
tion was more evidenced under DI than under well irrigation (Figs. 4
and 5).
3.4. Response of leaf and stem anatomy of water deficit-stressed plants
to palm pollen grain extract (PPGE)
Data shown inTables 2and3;Figs. 6 and 7reveal that application
of basil plants with PGE under normal irrigation (70% of SWHC) sig-
nificantly improved, in general, stem and leaf anatomical features
compared to corresponding controls. DI (50% of SWHC) negatively
affected stem and leaf anatomical features compared to well irriga-
tion (70% of SWHC). However, PGE treatment significantly improved,
in general, stem and leaf anatomical features of DI-stressed plants
compared to the corresponding controls. The improvements
recorded for stem anatomical features were 8.3, 13.4, 14.3, 7.5,7.1,
18.7, and 10.0% for section length, section width, cortex thickness,
vascular cylinder thickness, xylem vessels thickness, pith diameter,
and pith layers infirst cut. In addition, they were 8.8, 16.0, 20.0, 14.6,
and 10.5% for section length, cortex thickness, xylem vessels thick-
ness, pith diameter, and pith layers in second cut. The improvements
recorded for leaf anatomical features were 11.6, 8.3, 25.0, 18.2, and
7.5% for midvein length, midvein width, number of xylem rows per
vascular bundle, spongy tissue thickness, and vascular bundle diame-
ter infirst cut. In addition, they were 5.6, 9.1, 11.1, 46.2, and 20.0% for
midvein width, number of xylem rows per vascular bundle, palisade
tissue thickness, spongy tissue thickness, and vascular bundle diame-
ter in second cut compared to the corresponding controls. The posi-
tive effect of PGE application was more evidenced under DI than
Fig. 3.Effect of 1.0 g L
‒1
palm pollen grains extract (PGE) application on oil content, water use efficiency (WUE), relative water content (RWC%), and electrolyte leakage (EL%) ofOci-
mum basilicumplants grown under normal and deficit irrigation.
Values are means§SE (n= 9), and means (bars) with different letters are significantly different atP0.05. *SWHC means soil water-holding capacity.
R.S. Taha et al. / South African Journal of Botany 128 (2020) 42fi53 47

7. under normal irrigation. It awarded, in general, no significant differ-
ences between all tested anatomical features of well-irrigated plants
and those of DI-stressed plants treated with PGE.
4. Discussion
This study shows that palm pollen grains extract (PGE) is rich in
phytohormones (i.e., cytokinins, gibberellins, and indole-3-acetic
acid), mineral nutrients (i.e., P, Ca
2+
,Mg
2+
,K
+
, S, Mo, Bo, Fe, Mn, Zn
and Cu), soluble sugars, free proline, and antioxidants and vitamins
(i.e., AsA, GSH, vitamin A, and vitamin E) (Table 1). These core ingre-
dients make PGE a vigorous growth bio-stimulant. Therefore, it was
used as a foliar application strategy for deficit irrigation (DI)-stressed
basil plants. It has also a high value of DPPH-radical scavenging activ-
ity (85.2%), which has been considered as an important mechanism
functioned by antioxidants to inhibit lipid peroxidation (Hatano et
al., 1989;Silva et al., 2012). Therefore, PGE has some crucial mecha-
nisms for DI-stressed plants to increase their tolerance to drought
stress. Under DI (50% of SWHC), in this study, basil plants were per-
formed well with PGE application and awarded growth characteris-
tics and oil content similar to those of the well-watered plants. So,
PGE seems to be a good, inexpensive mean to replace commercially
synthesized phytohormones, antioxidants, vitamins, mineral
nutrients or osmoprotectants, which are expensive and unsafe. In
addition, PGE is easy to prepare as a natural bioactive stimulant for
environmentally stressful plants.
The PGE has succeeded as a growth bio-stimulant when applied to
basil plants grown under DI stress. Here, we conclude that the effec-
tive ingredients of PGE have been penetrated through the stomata,
providing plants with stimuli to overcome stress conditions. The
major stimuli of PGE are phytohormones and antioxidants (Table 1).
Results of this study suggest PGE involvement in beneficial roles dur-
ing basil growth under stress by improving plant metabolism. This
may be due to the richness of PGE in phytohormones (gibberellins,
auxin, and cytokinins), antioxidants (AsA, GSH, free proline, and vita-
mins) and other bio-stimulants. These PGE stimuli can act in DI-
stressed basil as mechanisms to promote cell division and elongation
and restore the plant's nutritional status due to nutrients in PGE
(Table 1), increasing basil growth characteristics (Fig. 1). PGE contains
many antioxidants that acquired it a protective effect, increasing pho-
tosynthetic pigments in stressed-basil (Fig. 2). This contributed to
improve photosynthesis and metabolic processes, maintaining tissue
health and turgidity in terms of increased RWC and decreased EL
(Fig. 3). This result contributed also to maintain vigorous basil growth
under DI stress (Fig. 1). In this study, a strong correlation was
observed between the elevated levels of proline and soluble sugars
(Fig. 4) and the capacity to survive stress effects.Sairam and Tyagi
(2004)have reported that proline has some mechanisms in stressful
plants. It acts as a scavenger of free radicals, an enzyme protectant,
and/or a compatible solute, which contributes to osmotic adjustment.
In addition,Hassanein et al. (2009)have reported that soluble sugars
are a massive category of compatible solutes, which play a crucial
Fig. 4.Effect of 1.0 g L
‒1
palm pollen grains extract (PGE) application on the contents of free proline, total soluble sugar, ascorbic acid (AsA) and glutathione (GSH) ofOcimum basili-
cumplants grown under normal and deficit irrigation.
Values are means§SE (n= 9), and means (bars) with different letters are significantly different atP0.05. *SWHC means soil water-holding capacity.
48 R.S. Taha et al. / South African Journal of Botany 128 (2020) 42fi53

8. role in mitigating the adverse effects of stress either by osmotic
adjustment or by awarding a desiccation resistance to plant cells.
Therefore, these increased levels of osmoprotectants (soluble sugars
and proline), due to PGE application, played an important role in
increasing cellular RWC and alleviating DI stress effects in basil
plants.
Ascorbic acid (AsA) and glutathione (GSH) antioxidants are major
components in PGE. Their endogenous levels were significantly
increased by treating basil plants by PGE under normal or DI stress
conditions (Fig. 4). As reported byCruz de Carvalho (2008), both AsA
and GSH antioxidants play crucial roles to increase plant tolerance to
stress conditions through improving the scavenging activities to
eliminate reactive oxygen species (ROS). These antioxidants act as
mechanisms to mitigate and repair damages generated by ROS and
enable plants to evolve complex defense systems to raise the cellular
defense strategies against drought-induced oxidative stress. Ascor-
bate is a redox active molecule that reacts with
1
O
2,H
2O
2,O
2
‒‒,OH
‒
and lipid hydroperoxides. It also implicates in various biological
activities in plants related to resistance to oxidative stress (Conklin,
2001). In addition, GSH is also a redox active molecule that quenches
ROS and involves in the ascorbate-glutathione cycle, eliminating the
damaging peroxides (Galant et al., 2011). Therefore, PGE as a rich
source in AsA and GSH along with its high DPPH-scavenging activity
(85.2%) enabled basil plants to perform well, conferring high oil con-
tent under DI stress (Fig. 3).
Tissue RWC was increased and EL was reduced with PGE applica-
tion in DI-stressed basil plants (Fig. 3). PGE repaired damages
occurred in cell membranes under DI stress. This may be due to that
PGE as an effective bio-stimulant contributed to maintain membrane
integrity that is agreed withHammad and Ali (2014). The RWC is a
convenient measure of water status in plant as a physiological out-
come of tissue water deficit, whilst water potential is a measure of
plant water transport in the soil-plant-atmosphere continuum (Dar-
vishan et al., 2013). Osmotic adjustment is an important mechanism
included in plant adaptations to various stresses, including drought.
The elevated contents of proline and soluble sugars by PGE applica-
tion (Fig. 4) improved cellular osmotic adjustment and contributed to
maintain higher RWC and lower EL in DI- stressed plants. The
improved plant water status (RWC) by PGE application is highly cor-
related with the increase in water use efficiency (WUE) in basil plants
(Fig. 3). This helped to maintain healthy metabolic processes in basil
leaves as an effective mechanism generated in DI-stressed plants.
Significant increases in the activities of antioxidant enzymes by
PGE foliar spray, especially under DI stress were obtained (Fig. 5).
This may be attributed to crucial roles of amino acids and phytohor-
mones found in PGE (Table 1), maintaining higher antioxidant
defense system activities to cope with the deleterious effects of DI.
Therefore, phytohormones and antioxidants are mechanisms by
which PGE application enabled basil plants to mitigate the dangerous
effects of DI stress. In addition, high DPPH-radical scavenging activity
of PGE (85.2%) increased the efficiency of the antioxidant defense sys-
tem components in DI-stressed plants. In this concern,Cao et al.
(2005)have reported that the elevation in antioxidant (enzymatic
and non-enzymatic) activities, on the basis of molecular, physiologi-
cal and genetic approaches, is the outcome of enhanced expression of
DET2gene to enhance resistance to oxidative stress inArabidopsis
thaliana. Due to that PGE is rich source in cytokinins (CKs), gibberel-
lins (GAs) and auxins, its foliar application to basil plants improved
Fig. 5.Effect of 1.0 g L
‒1
palm pollen grains extract (PGE) application on the activities of enzymatic antioxidants (superoxide dismutase; SOD, catalase; CAT, and guaiacol peroxidase;
GPOX) ofOcimum basilicumplants grown under normal and deficit irrigation.
Values are means§SE (n= 9), and means (bars) with different letters are significantly different atP0.05. *SWHC means soil water-holding capacity.
R.S. Taha et al. / South African Journal of Botany 128 (2020) 42fi53 49

9. plant defense against DI stress. CKs antagonize ABA to reduce its con-
centration in stressed plants for many developmental processes. More
specifically, zeatin-type CK acts as a direct free radical scavenger or it
may involve in antioxidant mechanism related to the protection of
purine breakdown (Chakrabarti and Mukherji, 2003). In addition, in
response to environmental stresses, many changes appear in the tran-
script of cytokinin genes inArabidopsis(Argueso et al., 2009). Cross-
talking between phytohormones is occurred at the molecular level
such as auxin and ethylene signaling act as a mechanism to modulate
the GAs-promoted destabilization ofDELLAproteins (Achard et al.,
2006). GAs are accumulated rapidly when plants are exposed to abiotic
stress to reduce stomatal resistance and enhance plant water use (Leh-
mann et al., 1995).Gonai et al. (2004)have concluded that free radicals
induced lipid peroxidations are inhibited by GAs, which also catabolize
ABA. Therefore, hormonal homeostasis might be a possible mechanism
of plant tolerance to stress (Iqbal and Ashraf, 2013). In this regard, PGE,
a rich source of plant hormones, can contribute to the hormonal bal-
ance in DI-stressed basil plants to withstand the effects of drought
stress effectively.
All improved growth characteristics, water relations of plant, and
antioxidant defense system components in response to foliar
application of PGE under DI were correlated with improved anatomi-
cal characteristics of basil leaf and stem (Tables 2and3; Pictures 1
and 2). These improved anatomical characteristics by PGE awarded
an improvement in nutrient translocation along with other assimi-
lates into leaf cells for use in different metabolic processes. Therefore,
vigorous plant growth and high oil productivity were obtained under
the adverse conditions of DI.
Finally, bioactive stimuli found in PGE (Table 1) showed some
potential mechanisms to increase basil plant tolerance to DI stress.
Osmoprotectant compounds (e.g., free amino acids, free proline, and
soluble sugars), which significantly increased in stressed plants by
PGE application award a potential mechanism to prevent water loss
from plant shoot under stress. This elevates RWC and WUE and
reduces ion leakage (EL) to maintain membrane stability. Another
potential mechanism is the improvement of enzymatic and non-
enzymatic antioxidants activities by PGE treatment to strength the
antioxidant defense system to increase resistance to stress in plants.
Plant hormones found in PGE may be penetrated with the spraying
solution into leaf tissue to award a potential mechanism to maintain
hormonal balance in stressed plants. This lead to stay greenness and
delay senescence of leaves, and improves chlorophyll content and
Fig. 6.Effect of palm pollen grains extract (PGE) on stem anatomy in two cuts of basil (Ocimum basilicum) plants grown under deficit irrigation. (A) 70% of SWHC* (first cut); (B) 50%
of SWHC + 1.0 g L
‒1
of PGE (first cut); (C) 70% of SWHC (second cut); (D) 50% of SWHC + 1.0 g L
‒1
of PGE (second cut); (cx = cortex, xv = xylem vessel and pi= pith). *SWHC means soil
water holding capacity.
50 R.S. Taha et al. / South African Journal of Botany 128 (2020) 42fi53

10. photosynthesis to maintain healthy growth of plants under stress.
Mineral nutrients found in PGE can also penetrate into leaf tissue to
show an additional mechanism. This maintains mineral balance for
healthy growth of plants under stress. Taken together, PGE compo-
nents helped plants to take up and retain more water to enhance the
water status of the upper parts of plants. This helps in limiting the
Fig. 7.Effect of palm pollen grains extract (PPGE) on leaf anatomy in two cuts of basil (Ocimum basilicum) plants grown under deficit irrigation. (A) 70% of SWHC* (first cut); (B) 50%
of SWHC + 1.0 g L
‒1
of PGE (first cut); (C) 70% of SWHC (second cut); (D) 50% of SWHC + 1.0 g L
‒1
of PGE (second cut); (mv = mid vein, xv = xylem vessel, pa= Palisade and sp= spongy
tissue). *SWHC means soil water holding capacity.
Table 2 Effect of 1.0 g L
‒1
palm pollen grains extract (PGE) application on stem anatomy in two cuts ofOcimum basilicumplants grown under normal and
deficit irrigation.
Treatments Parameters
Stem dimension (
mm) Cortex thickness Vascular cylinder Xylem vessels Pith diameter Pith layers
Length Width
(
mm) thickness ( mm) thickness (mm) (mm) ( mm)
First cut
70% of SWHC* 2275§57b 2325§58a 165§6b 435 §15a 230 §7a 1400 §29a 22 §1b
70% of SWHC+PGE 2350 §61a 2335§58a 200§8a 436 §14a 235 §7a 1425 §31a 27 §1a
50% of SWHC 2100 §51c 2050§52b 140§4c 400 §12b 210 §6b 1150 §24b 20 §0c
50% of SWHC+PGE 2275 §58b 2325§56a 160§5b 430 §14a 225 §7a 1365 §27a 22 §1b
Second cut
70% of SWHC* 2315§59b 2075§55b 295§12b 291 §9b 250 §8b 1400 §28a 21 §1a
70% of SWHC+PGE 3137 §66a 2925§60a 320§14a 400 §11a 325 §11a 1450 §30a 22 §1a
50% of SWHC 2125 §54c 2050§
53b 250§9c 270 §9b 200 §6c 1200 §24b 19 §0b
50% of SWHC+PGE 2312 §60b 2062§54b 290§11b 288 §9b 240 §8b 1375 §26a 21 §1a
Means (n= 9) in the same column for each trait with the same letter are not significantly different atP0.05.
* SWHC means soil water-holding capacity.
R.S. Taha et al. / South African Journal of Botany 128 (2020) 42fi53 51

11. oxidative damage induced by DI stress by the improvement of antiox-
idant defense components (carotenoids, free proline, AsA, GSH, SOD,
CAT, and GPOX).
5. Conclusions
Analysis of PGE (Table 1) indicated presence of osmoprotectants
(free amino acids, free proline, and soluble sugars) essential nutrients
(P, K, Ca, Mg, S, Mo, B, Fe, Mn, Zn, and Cu), antioxidants (soluble phe-
nols, carotenoids,flavonoids, vitamins; C, A, and E), and phytohor-
mones (IAA, GAs, and CKs). These different bioactive components
indicate that PGE can use as an effective bioactive stimulant to
improve basil plant growth and oil productivity. This due to that PGE
was effective in alleviating the physiological response of DI (drought)
stress damages. The positive effects of PGE were more pronounced
under DI (50% of SWHC) than under normal irrigation (70% of
SWHC). The DI-stressed basil plants applied with PGE maintained
higher RWC, osmoprotectants (soluble sugars and free proline), anti-
oxidants (AsA and GSH), and enzymatic activities, and lower EL.
Therefore, the bioactive components implicated in PGE played impor-
tant roles in plant development and metabolism and response to DI
stress due to their effective mechanisms that enabled plants to per-
form well under DI stress. Stressed basil plants treated with PGE
awarded growth characteristics and oil yield similar to those of plants
received normal irrigation. It could be concluded that PGE can be
used as an effective bioactive stimulant for growing plants under nor-
mal or drought stress conditions as an economic and natural source
rich in plant growth stimuli rather than synthetic growth regulators.
Declaration of Competing Interest
The authors have no conflict of interest.
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Treatments Parameters
Midvein (
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Spongy tissue
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50% of SWHC 820 §30a 850 §c22 §0c 80 §3b 130 §5b 400 §14c
50% of SWHC+PGE 825 §
31a 900 §b24 §1b 903 §a 190 §6a 480 §16b
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* SWHC means soil water-holding capacity.
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