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ISSN 1021-4437, Russian Journal of Plant Physiology, 2024, Vol. 71:112. © Pleiades Publishing, Ltd., 2024.
Leaf Micromorphological Assessment, Chemical Composition
and Anatomical Responses of Trachyandra ciliata (L.F) Kunth
to Different Degrees of Salinity
S. Ngxabia, M. O. Jimoha, b, C. P. Laubschera, and L. Kambizia, *
a Department of Horticultural Sciences, Faculty of Applied Sciences, Cape Peninsula University of Technology,
P.O. Box 1905, Bellville, 7535 South Africa
b Department of Plant Science, Olabisi Onabanjo University, PMB 2002, Ago-Iwoye, 120107 Nigeria
*e-mail: KambiziL@cput.ac.za
Received December 29, 2023; revised March 14, 2024; accepted March 19, 2024
Abstract—Many studies have examined the morphological and micromorphological responses of different
halophytes to determine their salt tolerance mechanisms. However, few studies have focused on the South
African edible halophytes. This study examined the leaf micromorphology, elemental composition, and ana-
tomical responses using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy
(EDX) to examine salt tolerance levels in Trachyandra ciliata (L.F) Kunth. The treatments included varying
sodium chloride (NaCl) concentrations: 50 mM, 100 mM, 150 mM and 200 mM, while control (0 mM) was
watered with nutrient solution only. From the SEM micrographs, salt glands were observed protruding from
the epidermis along the vascular system under low salinity and salt crystals appeared under higher concentra-
tions, which makes this plant maintain cellular homeostasis under high salinity, and the plant can be classi-
fied as a recretohalophyte. Stomatal distribution, stomatal density and the number of open stomata decreased
wi th in creas ing sal init y. EDX r evea led t he p resen ce of some i mport ant e lement s su ch as potas sium, magne siu m,
phosphorus, calcium and more in the leaves. The results showed that increased salinity led to a decrease in the
percentage composition of P, K and Ca2+, while Mg2+ was high under the control and low salinity (50 mM),
decreased under 100 mM and increased again with increasing salinity. On the contrary, increasing salinity
caused an increase in Na+ and Cl- in a stable manner. These findings reveal that T. ciliata acquires salt toler-
ance through changes to its leaf surface properties, osmotic adjustment, and the regulation of Na+ uptake and
distribution in the leaves.
Keywords: Asphodelaceae, recretohalophytes, micromorphology, salt glands, Trachyandra ciliata, wild cab-
bage, salt stress
DOI: 10.1134/S1021443723603695
INTRODUCTION
One of the main abiotic factors in agriculture that
promotes deficiency symptoms, physiological abnor-
malities, and lower output of field crops around the
world is salinity, along with drought [1]. However,
halophytes have developed several adaptations to
withstand seawater and increased salinity concentra-
tions, such as accumulation of suitable organic solutes,
succulence, salt-secreting glands and bladders, ion
compartmentalization in cell vacuoles, and adjust-
ment of their internal water relations [2, 3]. It is stated
that the sensitivity of different plants to soil salinity
varies. Hence it is important to study anatomical
responses to salinity for different halophytes [4].
Many studies have been conducted to examine the
morphological and micromorphological responses of
different halophytes to determine their salt tolerance
mechanisms [5, 6]. However, few studies have focused
on the South African edible halophytes. Trachyandra
ciliata (L.f.) Kunth, sometimes known as wild cabbage
or Veldkool (Afrikaans), is a halophytic plant in the
Asphodelaceae family, endemic to the coastal winter
rainfall southwestern Cape in South Africa [7, 8]. The
therapeutic characteristics of the Asphodelaceae fam-
ily have been thoroughly investigated throughout the
years, and they are frequently employed in the bever-
age and pharmaceutical sectors [9, 10]. However, the
Trachyandra genus is underexploited with little to no
literature [11]. Ngxabi et al. reported that the inf lores-
cence of T. ciliata is edible and was used as a vegetable
by the native people that lived in the area before colo-
nization and removal of people from the area [8]. They
further studied its salt tolerance and phytochemicals in
response in response to salinity stress [12]. As such,
there is a need to further examine its anatomical and
micromorphological responses to understand the
RESEARCH PAPERS
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NGXABI et al.
internal mechanisms it employs to tolerate high levels
of salinity.
For morphological microanalysis of plant tissues,
scanning electron microscopy (SEM) has been uti-
lized in the past [13, 14]. Great magnifications are
used in SEM, which results in highly precise images
with great resolution [4]. Several studies have been
conducted to examine salt gland shapes, stomatal dis-
tribution and density in different halophytes [6, 15].
However, there have not been many of these studies
regarding South African halophytes and T. c ili a ta is no
exception [12]. Furthermore, with methods like
energy-dispersive X-ray spectroscopy, the difficulty of
getting precise biological information on the classical
ultrastructure of plants has been substantially reduced
in recent years [5, 6]. Energy dispersive X-ray spectros-
copy, a chemical microanalysis, has been used to ascer-
tain the elemental composition of plant tissue. Addi-
tionally, silica body quantity and presence in the foliar
epidermis of various plant species have been evaluated
using EDX [4, 16]. The main aim of this study was to
examine leaf micromorphology, elemental compart-
mentalization and anatomical responses using SEM
and energy dispersive X-ray spectroscopy (EDX) to
clarify salt tolerance mechanisms in T. ciliata.
MATERIALS AND METHODS
Plant material and experimental design. This study
was conducted at the Bellville campus of the Cape
Peninsula University of Technology in Cape Town,
South Africa, at coordinates 33.55048.800 S and
18.38032.700 E. With the aid of environmental con-
trol, the study’s experimental greenhouse was main-
tained at a constant temperature of between 12 and
18°C at night and between 21 and 26°C during the day.
60% was the average relative humidity.
Healthy stalks of T. ciliata were acquired from a
local nursery. Due to the presence of rhizomes, the
plant material was propagated through the division
technique. A total of 150 plants of uniform size were
then transplanted into pots (12.5 cm height × 12.5 cm
length × 12.5 cm width) containing a mixture of Peat:
perlite: vermiculite (PPV) at (1 : 1 : 1). Cuttings were
obtained from the same mother stock population to
ensure they are as genetically identical as possible. The
plantlets were divided into 5 treatments of 30 replicates
each in a block arrangement. At the beginning of the
greenhouse experiment, for four weeks plants were
watered with only NUTRIFEED™ complete water-
soluble fertilizer manufactured by (STARKE AYRES
Pty. Ltd. Hartebeesfontein Farm, Bredell Rd, Kaal-
fontein, Kempton Park, Gauteng, South Africa, 1619)
dissolved in municipal water at 10 g per 5 L ratio for
plants to acclimatize in the experimental setup. The
aqueous solution contained the following ingredients:
N (65 mg/kg), P (27 mg/kg), K (130 mg/kg),
Ca (70 mg/kg), Cu (20 mg/kg), Fe (1500 mg/kg),
Mo (10 mg/kg), Mg (22 mg/kg), Mn (240 mg/kg),
S (75 mg/kg), B (240 mg/kg), and Zn (240 mg/kg).
Using sodium chloride (NaCl) in the nutrient solu-
tions, various salt concentrations were adjusted. Four
salt concentrations (50 mM, 100 mM, 150 mM, and
200 mM of NaCl) were tested in this experiment,
while the control was watered only with the nutrient
solution. Plants were equally watered at a three-day
interval with 300 mL of the nutrient solution with
and/or without NaCl. To ascertain that proper salinity
levels were maintained, drain water from every pot was
collected, and the electrical conductivity was mea-
sured. The pH of the solution was maintained at 6.0
with the aid of a calibrated hand-held digital pH meter
(Eurotech®TM pH 2 pen). Potassium hydroxide was
used to elevate pH, while phosphoric acid was used to
decrease the pH of the nutrient solution [12]. A cali-
brated hand-held digital EC meter (Hanna instru-
ments®TM HI 98312) was utilized to closely monitor
the electric conductivity of the nutrient solution. The
plants were harvested after 15 weeks of salinity treat-
ments to conduct the micromorphological assessments.
Sample preparation for SEM and EDX analyses. A
minora blade was used to cut the samples into 1 cm by
1 cm squares, and they were then immediately sub-
merged in 2.4% glutaraldehyde (GLA) for four h. Fol-
lowing the fixing procedure, the samples were dehy-
drated using 50, 70, 90% EtOH for 15 s, followed by
100% EtOH for 2 s on each sample. The samples were
mounted on aluminium stubs with double-sided car-
bon tape. The samples were subsequently covered in a
thin (10 nm) and thick (15 nm) layer of carbon using
the Quorum Q150TE carbon coater to make the sam-
ple surface electrically conductive to avoid electron
build-up on the sample surface, which may cause elec-
tron charge. This procedure was followed as suggested
by Sogoni et al. [6].
Laboratory SEM and EDX examination of leaves.
SEM and EDX examination were conducted follow-
ing a procedure as described by Sogoni et al. [6]. The
back Scattered Electron images of treated samples
were examined in A Zeiss 5-diode Back Scattered
Electron (BSE) Detector (Zeiss NTS BSD) and Zeiss
Smart SEM software at different magnifications to
provide surface topography and morphological infor-
mation of the sample. The samples’ chemical compo-
sition was established by using semi-quantitative/full
quantitative energy dispersive X-ray spectrometry
using an Oxford Instruments® X-Max 20 mm2 detec-
tor and Oxford Aztec software/INCA Oxford soft-
ware. Beam conditions included a 20 kV accelerating
voltage, 1.4 nA probe current, a working distance of
9.5 mm, and a beam current of 11 nA for the quantita-
tive analysis and backscattered electron image analysis
on the Zeiss MERLIN. The counting time was 10 s
live-time/ Zeiss EVO MA15 accelerating voltage
20 kV, IProbe 1.1 nA, and specimen current of 19 nA
and 8.5 mm working distance. Stomata density, dis-
tance between two stomata, and percentage of open
and closed stomata were all considered as quantitative
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 71:112 2024
LEAF MICROMORPHOLOGICAL ASSESSMENT, CHEMICAL COMPOSITION Page 3 of 9 112
parameters. The qualitative parameters measured were
stomata distribution, salt glands, and salt crystals.
Statistical analysis. At the end of the experiments,
the data obtained from the treatments was expressed as
means ± SE of two replicates, and Fisher’s least signif-
icant difference (LSD) was used to compare the signif-
icant differences between treatment means at P ≤ 0.05.
A MINITAB 17 statistical package was used to deter-
mine the statistical significance of means of different
treatments for EDX chemical composition, stomata
density, and percentage of open and closed stomata.
RESULTS
Electron Microscopy Analysis of Leaf Surfaces:
Stomatal Density and Distribution
Based on stomatal distribution observed in SEM
micrographs under 200× magnification across all
treatments, the leaves are characterized by sunken,
anomocytic stomata that lack subsidiary cells (Fig. 1).
Stomatal density was calculated as the stomatal count
per unit area [17]. Results gathered from the experi-
ment reveal that salinity treatments significantly
influenced stomatal density and distribution. The
highest stomatal density mean value was recorded
under low salinity treatment (50 mM) followed by
moderate salinity treatment (100 mM), while control
and 150 mM recorded equivalent stomatal density
(Table 1). In contrast, a significantly low stomatal
density mean value was recorded under the highest
salinity treatment (200 mM). Stomatal distribution
was extracted from SEM micrographs under 200×
magnification across all treatments (Fig. 1).
Open and Closed Stomata
A microscopic analysis demonstrated clear changes
between the leaf surfaces of the five treatments due to
increasing salinity including the number of open and
closed stomata. Statistical analysis revealed that salin-
ity significantly inf luenced the percentage of open and
closed stomata among the treatments. Plants culti-
vated under control (0 mM) and low salinity (50 mM)
treatment recorded significantly high percentages of
open stomata compared to all other treatments, with
control recording the highest percentage (71%) (Fig. 2).
As salinity increased, the percentage of open stomata
reduced significantly, with the lowest percentage (28%)
recorded under the highest salt treatment (200 mM).
On the other hand, the highest percentage of closed
stomata (72%) was observed under the highest salt
treatment followed by 150 mM, which recorded
61.67% (Fig. 2). The lowest percentages of closed sto-
mata were observed under control treatment followed
by low salinity levels, which recorded 29 and 34.33%,
respectively. Under moderate salinity (100 mM) the
percentage of closed stomata was higher than that of
open stomata, but the numbers were statistically dif-
ferent.
Salt glands were observed protruding from the epi-
dermis along the vascular system under control, low
(50 mM), and moderate salinity levels (100 mM). The
oval-shaped salt glands are smaller under control, big-
ger and well-defined under low salinity, and smaller
again under moderate salinity (Fig. 3). Salt crystals
were observed on the leaf surface of wild cabbage under
higher salinity concentrations (150 mM and 200 mM).
It was observed that the plant keeps salt in salt glands
under low salt concentrations, while glands get rup-
tured by the emergence of salt crystals under higher
salt concentrations (Fig. 3) as the plant excludes salts
to avoid cell damage by sodium ions. It was also
observed that as the salt glands get ruptured by salt
crystals, the surface becomes flaccid demonstrating
loss of water and cell damage under high salt concen-
tration (Fig. 3e) compared to the turgid surface
observed in control (Fig. 3a).
Energy Dispersive Spectroscopy (EDX) Analysis
of Leaf Surface
The percentage of chemical atomic composition in
the leaf epidermal layer of T. ciliata was subsequently
determined by using energy dispersive spectroscopy.
The EDX analysis confirmed that increasing salinity
modulated significantly the distribution of chemical
elements on the leaf surface of T. ciliata as presented in
Table 2 and Fig. 4. The concentration of sodium (Na)
increased in direct proportion with increasing salinity
levels, with the lowest concentration (0.6 ± 0.07%)
detected under the control treatment and the highest
concentration (3.9 ± 0.22%) detected under the high-
est salt concentration. Control and low salinity con-
centrations produced the highest levels of magnesium
(Mg) with 0.65 ± 0.01 and 0.59 ± 0.04% respectively,
while the lowest atomic mass (0.12 ± 0.02%) was
found under moderate salinity level (100 mM) and
surprisingly increasing again under 150 mM salinity.
An equally higher amount of phosphorus (P) was
found under control and 50 mM salinity, decreased
under moderate (100 mM) salinity, and surprisingly
increased again as salinity increased. Likewise with
potassium (K), an equally high composition (3.9%)
Table 1. Effect of salinity treatments on the stomatal density
Mean values that do not share the same letters are statistically different at P ≤ 0.001.
NaCl treatments Control 50 mM 100 mM 150 mM 200 mM
Stomatal density, mm211.2 ± 0.86bc 17.9 ± 1.29a 13.9 ± 1.08b 11.2 ± 1.29bc 7.9 ± 0.9c
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NGXABI et al.
was detected under control and 50 mM salinity levels
exponentially declined with increasing salinity. Chlo-
rine (Cl) composition was directly proportional to the
increasing salinity as described for sodium. However,
calcium (Ca) composition was significantly higher
(3.56 ± 0.21%) under 50 mM salinity, followed by
control (2.65 ± 0.06%), and then gradually decreased
as salinity increased.
DISCUSSION
This study examined the leaf micromorphology,
elemental composition and anatomical responses
using SEM and EDX to clarify salt tolerance mecha-
nisms in T. ciliata. The results revealed that salinity
induced stomatal density and the ratio of open and
closed stomata, which are important factors in regu-
lating gaseous exchange and water loss. The highest
stomatal density was recorded under low salinity
(50 mM) followed by moderate salinity (100 mM),
while control had the same stomatal density as 150 mM,
and the lowest stomatal density was recorded under
high salinity treatment (200 mM). Control and low
salinity treatment had a significantly higher ratio of
open stomata, while high salinity treatment resulted in
a significantly low percentage of open stomata. The
Fig. 1. Control (a), 50 mM NaCl (b), 100 mM NaCl (c), 150 mM NaCl (d), and 200 mM (e) SEM images demonstrating stomatal
density and distribution on the leaf surface of wild cabbage.
(a) (b)
(c) (d)
(e)
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LEAF MICROMORPHOLOGICAL ASSESSMENT, CHEMICAL COMPOSITION Page 5 of 9 112
reduction in stomatal density and reduced percentage of
open stomata under salt stress has also been recorded in
other halophytic species such as Chenopodium quinoa,
Tetragonia decumbens, and Chenopodium album [6, 18].
These findings suggest that halophytes can regulate
stomatal number in response to salinity stress. It has
also been reported in another study that halophytes
typically utilize this strategy to prevent dehydration
and perhaps slow down the rate at which transpiration
water fluxes supply salt to the leaves [19].
From microscopic analysis, the oval-shaped salt
glands were observed protruding from the epidermis
along the vascular system. These glands enable plants
to store and secrete excess salts to maintain osmotic
balance and prevent damage to the cells [20, 21]. The
salt glands were observed under control where they
were relatively small, 50 mM where they appeared big-
ger and well defined, and at 100 mM where they
appeared small again, while there was no sign of the
glands under higher salt concentrations. Salt crystals
were observed under 150 and 200 mM, while none
were observed under lower concentrations. This sug-
gests that salt glands may have been ruptured by the
emergence of salt cr ystals which appeared at high con-
centrations as observed in Sporobolus virginicus [22].
The disappearance of salt glands and observation of
salt crystals on the leaf surface of Wild cabbage under
higher concentrations is a clear indication of ion
secretion, which implies that the plant can be catego-
rized under halophytes that are salt secretors (exo-rec-
retohalophytes) [21, 23, 24]. This is supported by
reports suggesting that certain halophytes (recretoha-
lophytes) could excrete excess salt as a liquid that crys-
tallizes and appears apparent on the surface of plant
Fig. 2. The influence of different NaCl treatments on the stomatal opening.
Open Stomata, % Closed Stomata, %
90
70
80
60
50
40
30
20
10
0Control 50 mM 100 mM 150 mM 200 mM
Stomatal opening, %
NaCl treatments
a
a
a
ab
ccc
bbc
b
Table 2. Main chemical components detected on the surface and subsurface of leaves using energy dispersive spectroscopic
analysis
Values (Mean ± SE) followed by different letters are statistically different from each other as determined by Fisher’s least significant dif-
ference at P ≤ 0.05 (*) and P ≤ 0.001 (**) probability levels.
Treatments Na, % Mg, % P, % K, % Cl, % Ca, %
0 mM 0.6 ± 0.07d 0.65 ± 0.01a 0.35 ± 0.03a 3.9 ± 0.09a 0.2 ± 0.01e 2.65 ± 0.06b
50 mM 2.2 ± 0.08c 0.59 ± 0.04a 0.35 ± 0.03a 3.9 ± 0.03a 1.97 ± 0.08d 3.56 ± 0.21a
100 mM 3.3 ± 0.06b 0.12 ± 0.02d 0.21 ± 0.03b 2.8 ± 0.11b 4 ± 0.15c 1.59 ± 0.01c
150 mM 3.5 ± 0.12ab 0.4 ± 0.03b 0.17 ± 0.03b 2.7 ± 0.05b 5.6 ± 0.16b 1.44 ± 0.06c
200 mM 3.9 ± 0.22a 0.3 ± 0.01c 0.18 ± 0.04b 1.7 ± 0.07c 6.09 ± 0.23a 1.3 ± 0.06c
F-statistics
F-Value 155.9** 74.3** 7.6* 44.1** 288** 85.9**
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NGXABI et al.
leaves when exposed to air [2, 4, 25]. It was also
observed at the highest concentration (200 mM) that
the surface became flaccid, which demonstrates water
loss and cell damage. This is consistent with the
reports that extreme salinity levels decrease leaf
growth due to the osmotic and ionic stress caused by
inadequate water absorption, which causes cell shrink-
ing [26, 27].
The percentage of elemental composition of T. cil-
iata varied with regard to the nutrient ions acquired
from various salinity treatments, as revealed by EDX
spectroscopy of the leaf epidermis. As anticipated, a
significantly high amount of carbon was due to carbon
coating. [5] also reported high gold content due to gold
coating. The EDX analysis illustrated the increase of
Na+ and Cl– in direct proportion to increasing salin-
ity, with the lowest amounts found at 0 mM and the
highest amounts detected under high salinity treat-
ments. However, the increase in Na+ was not statisti-
cally different between 100, 150 and 200 mM salinity
treatments, which shows the ability of T. c i l iat a to
maintain chemical balance under high salinity [27].
Fig. 3. Control (a), 50 mM NaCl (b), 100 mM NaCl (c), 150 mM NaCl (d), and 200 mM (e) SEM images demonstrating the
appearance of salt glands and salt crystals on the leaf surface of Wild cabbage under different levels of salinity. SG = salt glands;
SC = salt crystals; FS = f laccid surface.
(a) (b)
(c) (d)
(e)
SG SG
SG
SC
SC
FS
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LEAF MICROMORPHOLOGICAL ASSESSMENT, CHEMICAL COMPOSITION Page 7 of 9 112
These findings are consistence with previous studies,
which reported the capacity of halophytes to maintain
osmotic balance in cells by storing excess salts in salt
glands [4, 28, 29]. Furthermore, other studies reported
the presence of salt crystals at high concentrations as a
clear indication of ion secretion for the maintenance
of cellular ion homeostasis [25, 26, 30].
Results from EDX analysis revealed an equally high
composition of K+ in 0 and 50 mM salinity levels,
which then significantly decreased with increasing
salinity. Potassium ions function as a prerequisite fea-
ture to build a salt-tolerance and play a critical role in
mitigating the effects of salinity stress on plants by
recasting essential plant processes [27]. In addition,
Fig. 4. Control (a), 50 mM NaCl (b), 100 mM NaCl (c), 150 mM NaCl (d), and 200 mM (e) randomly picked plates demon-
strating the main elemental components on the leaf epidermis of T. ciliata by energy dispersive spectroscopic analysis.
cps/eV
30
20
10
002468
(c)
cps/eV
30
20
10
002468
(b)
cps/eV
30
50
40
20
10
002468
(a)
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NGXABI et al.
salinity negatively affected the composition of Mg2+,
which is primarily required for the formation of chlo-
rophyll, its formation, movement, and use of photo-
assimilates, as well as the activation of enzymes and
proteins [27]. Interestingly, 50 mM recorded a signifi-
cantly higher accumulation of Ca2+ than the control,
while there was no significant difference from 100 to
200 mM. Ca2+ is reported to be the requirement for
structural functions in the membranes and cell walls,
and in the vacuole as a counter-cation for both inor-
ganic and organic anions [27]. The high accumulation
of Ca2+ under low salinity and its stability under high
salinity may be related to the reports stating that T. c i l -
iata achieves maximum growth and chemical balance
under low salinity and the secretion of Na+ under high
salinity [2, 12]. A similar trend was found in a halo-
phyte, Tet r a go nia d e c u mben s where salinity did not
have a significant influence on the accumulation of
Ca2+ [6]. Similarly, leaves under control and 50 mM
treatments accumulated an equally high amount of
Phosphorus (P), which lowered under 100 mM and
increased in direct proportion to increasing salinity.
Phosphorus plays a role in many plant processes
which include the transfer of energy, photosynthesis,
the processing of sugars and starches, the f low of
nutrients throughout the plant, and the genetic trans-
fer [31]. The stability of P in this plant may be related
to its ability to maintain ion homeostasis and osmotic
adjustment under high salinity.
CONCLUSIONS
For the first time, the leaf surface and cross-sec-
tional properties of T. ciliata were examined using
SEM and EDX in this study. The results obtained from
the analyses confirmed that salinity had a significant
influence on the leaf surface characteristics and per-
centage chemical composition of Wild cabbage. The
observation of salt glands protruding from the epider-
mis along the vascular system under low salinity and
the emergence of salt crystals under higher concentra-
tions makes this plant superior compared to other
plants in the maintenance of cellular homeostasis
under high salinity, and the plant can be classified as
recretohalophytes. Increased salinity led to a decrease
in the percentage composition of some important
chemical elements such as P, K and Ca2+, while Na+
and Cl– increased in a stable manner. The results of
this study demonstrate the complexity of Wild cab-
bage’s reaction to salinity, which includes a variety of
physiological mechanisms such as ion exchange and
excretion in a form of salt crystals through salt glands,
a decrease in stomatal density, and the distribution of
Na+ and Cl– inside the plant. The findings from this
study confirm that T. ciliata achieves salt tolerance by
modifying leaf surface characteristics, osmotic adjust-
ment, and the regulation of Na+ uptake and distribu-
tion in the leaves, and the results may be used to
update the existing taxonomic information on leaf
ultrastructure of the species. Further studies are rec-
ommended to unravel the mechanism of ion exchange
and determine candidate genes controlling salt toler-
ance in T. ciliata.
ABBREVIATIONS AND NOTATION
FUNDING
This research was supported by the South African Med-
ical Research Council (SAMRC) and National Research
Foundation (grant no. MND210624615237).
ETHICS APPROVAL
AND CONSENT TO PARTICIPATE
This work does not contain any studies involving ani-
mals or human participants as objects of research.
CONFLICT OF INTEREST
The authors of this work declare that they have no con-
flicts of interest.
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