ArticlePDF Available

Abstract and Figures

Lycium schweinfurthii is a Mediterranean wild shrub rich in plant secondary metabolites. In vitro propagation of this plant may support the production of valuable dietary supplements for humanity, introduction of it to the world market, and opportunities for further studies. The presented study aimed to introduce an efficient and reproducible protocol for in vitro micropropagation of L. schweinfurthii and assess the genetic stability of micropropagated plants (MiPs) as well as to estimate phenolic, flavonoid, ferulic acid contents, and the antioxidant activity in leaves of micropropagated plants. Two DNA-based techniques, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR), and one biochemical technique, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), were used to assess the genetic stability in MiPs. Spectrophotometric analysis was performed to estimate total phenolic and flavonoid contents and antioxidant activity of MiPs leaves, while ferulic acid content was estimated using high-performance thin-layer chromatography (HPTLC). Sufficient shoot proliferation was achieved at MS (Murashige and Skoog) medium supplemented with 0.4 mg L−1 kinetin and rooted successfully on half-strength MS medium fortified with 0.4 mg L−1 Indole-3-butyric acid (IBA). The Jaccard’s similarity coefficients detected in MiPs reached 52%, 55%, and 82% in the RAPD, ISSR, and SDS-PAGE analyses, respectively. In the dried leaves of MiPs, the phenolic, flavonoid, and ferulic acid contents of 11.53 mg gallic acid equivalent, 12.99 mg catechin equivalent, and 45.52 mg were estimated per gram, respectively. However, an IC50 of 0.43, and 1.99 mg mL−1 of MiP dried leaves’ methanolic extract was required to scavenge half of the DPPH, and ABTS free radicals, respectively. The study presented a successful protocol for in vitro propagation of a valued promising plant source of phenolic compounds.
Content may be subject to copyright.
plants
Article
Genetic Stability, Phenolic, Flavonoid, Ferulic Acid Contents,
and Antioxidant Activity of Micropropagated
Lycium schweinfurthii Plants
Diaa Mamdouh 1, 2, * , Hany A. M. Mahgoub 2, Ahmed M. M. Gabr 3, Emad A. Ewais 2and Iryna Smetanska 1, *


Citation: Mamdouh, D.; Mahgoub,
H.A.M.; Gabr, A.M.M.; Ewais, E.A.;
Smetanska, I. Genetic Stability,
Phenolic, Flavonoid, Ferulic Acid
Contents, and Antioxidant Activity of
Micropropagated Lycium
schweinfurthii Plants. Plants 2021,10,
2089. https://doi.org/10.3390/
plants10102089
Academic Editors: Laura Pistelli and
Kalina Danova
Received: 7 September 2021
Accepted: 28 September 2021
Published: 1 October 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Department of Plant Food Processing, Agricultural Faculty, University of Applied Sciences
Weihensteph-an-Triesdorf, Markgrafenstr 16, 91746 Weidenbach, Germany
2Botany & Microbiology Department, Faculty of Science, Al-Azhar University, Nasr City, Cairo 11884, Egypt;
h.mahgoub@azhar.edu.eg (H.A.M.M.); ewais_e@yahoo.com (E.A.E.)
3Department of Plant Biotechnology, Genetic Engineering and Biotechnology Research Division,
National Research Centre (NRC), Cairo 12622, Egypt; ahmedgabr01@gmail.com
*Correspondence: diaa.mamdouh@azhar.edu.eg (D.M.); iryna.smetanska@hswt.de (I.S.)
Abstract:
Lycium schweinfurthii is a Mediterranean wild shrub rich in plant secondary metabolites.
In vitro
propagation of this plant may support the production of valuable dietary supplements for
humanity, introduction of it to the world market, and opportunities for further studies. The presented
study aimed to introduce an efficient and reproducible protocol for
in vitro
micropropagation of
L. schweinfurthii and assess the genetic stability of micropropagated plants (MiPs) as well as to estimate
phenolic, flavonoid, ferulic acid contents, and the antioxidant activity in leaves of micropropagated
plants. Two DNA-based techniques, random amplified polymorphic DNA (RAPD) and inter-simple
sequence repeats (ISSR), and one biochemical technique, sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), were used to assess the genetic stability in MiPs. Spectrophotometric
analysis was performed to estimate total phenolic and flavonoid contents and antioxidant activity of
MiPs leaves, while ferulic acid content was estimated using high-performance thin-layer chromatog-
raphy (HPTLC). Sufficient shoot proliferation was achieved at MS (Murashige and Skoog) medium
supplemented with 0.4 mg L
1
kinetin and rooted successfully on half-strength MS medium fortified
with 0.4 mg L
1
Indole-3-butyric acid (IBA). The Jaccard’s similarity coefficients detected in MiPs
reached 52%, 55%, and 82% in the RAPD, ISSR, and SDS-PAGE analyses, respectively. In the dried
leaves of MiPs, the phenolic, flavonoid, and ferulic acid contents of 11.53 mg gallic acid equivalent,
12.99 mg catechin equivalent, and 45.52 mg were estimated per gram, respectively. However, an IC
50
of 0.43, and 1.99 mg mL
1
of MiP dried leaves’ methanolic extract was required to scavenge half
of the DPPH, and ABTS free radicals, respectively. The study presented a successful protocol for
in vitro propagation of a valued promising plant source of phenolic compounds.
Keywords:
Lycium schweinfurthii; micropropagation; genetic stability; ISSR-PCR; RAPD-PCR; SDS-
PAGE; HPTLC; DPPH; ABTS
1. Introduction
One member of the Solanaceae (the nightshade) family is the genus Lycium, com-
prising more than 70 species and which has a disjunctive distribution in temperate to
subtropical regions in South America, North America, Africa, Eurasia, and Australia [
1
].
Within buckthorns (Lycium), Lycium schweinfurthii is grouped according to phylogenetic
studies in a clade with other Old World species of the genus. Within this clade, this species
is closely related to L. acutifolium,L. eenii,L. shawii,L. bosciifolium,L. hirsutum, and L. villo-
sum. The species is sometimes put to L. intricatum [
2
]. L. schweinfurthii grows in temperate
climates and is well spread throughout the southern Mediterranean region as well as in
Egypt, Algeria, Tunisia, and Libya [
3
]. L. schweinfurthii is distributed in Egypt in the great
Plants 2021,10, 2089. https://doi.org/10.3390/plants10102089 https://www.mdpi.com/journal/plants
Plants 2021,10, 2089 2 of 16
south-western desert, northern coastal region [
4
], and islands of Lake Burullus [
3
]. The
plant is a 2–3 m high, rigid, upright shrub with a spiny stem. Its leaves are succulent
and hairless that are 12–20 mm long and 2–4 mm wide and arranged in alternate patterns
(one leaf per node) while its flowers are hermaphrodite. The fruit is a black, spherical,
sometimes egg-shaped berry that measures 4–5 mm in diameter [
5
]. L. schweinfurthii suffers
from different types of threats that affect its distribution, whether natural or caused by
human activities, i.e., soil fragmentation, cutting, grazing, and firing [3].
It is difficult in many seasons to obtain seeds or crops from wild plants, especially
with their small number and wide geographical distribution, as in L. schweinfurthii. Hence,
it is imperative to micropropagate plants
in vitro
to maintain the explant source at all times
of the year. For decades, the micropropagation of plants was the only technique that main-
tained and promoted the economic value of many agricultural species [
6
]. Furthermore, it is
an efficient technique for
in vitro
multiplication of endangered species, e.g., Magnolia sirind-
horniae [
7
], as well as for producing secondary metabolites, e.g., Eryngium alpinum L. [
8
].
Although no reports were found on the micropropagation of L. schweinfurthii, it is well
studied in other species of the Lycium genus. Multiple shoots and adventitious buds of
L. ruthenicum were developed
in vitro
not only from stems but also from leaf explants [
9
].
Moreover, the best shoot proliferation of L. depressum was achieved at a low concentration
of BA (6-benzyl adenine) and rooted in full-strength MS medium (Murashige and Skoog
medium) supplemented with IBA (indole-3-butyric acid) with a high survival rate [
10
].
Micropropagation protocols were also developed in L. barbarum [11] and L. chinense [12].
To maintain the effectiveness of
in vitro
propagation, genetic stability must be ensured,
especially with successive generations. Diverse techniques are used to determine the
genetic stability of regenerated plants in terms of plant genomes or transcribed proteins.
One of these is the random amplified polymorphic DNA (RAPD) PCR technique, which
is a rapid, inexpensive, and simple method for detecting genetic differences as it does
not require any previous information about the plant genome [
13
]. RAPD-PCR was
used to determine the genetic stability in micropropagated plants of Prunus salicina [
14
],
Echinacea purpurea [
15
], Dendrobium fimbriatum [
16
], and Rhynchostylis retusa [
17
]. A more
specific technique than RAPD is the inter-simple sequence repeats (ISSR) PCR technique.
It is an efficient, quick, and reproducible technique in which the targets are the DNA
fragments located between adjacent microsatellite regions, while the RAPD-PCR targets
are random [
18
]. Wójcik et al. [
19
] used ISSR primers to observe the genetic stability of
regenerated plants of Ribes grossularia L. Both techniques are used together to obtain more
realistic and accurate results [2022].
Otherwise, the differences in the protein profile of the regenerated plants also reflect
the extent of genetic stability at the level of gene expression. Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) is a technique used to show the differences
in the transcribed polypeptides in the micropropagated plants concerning the mother
plant [
23
,
24
]. The SDS-PAGE technique was assessed to check the genetic stability of
in vitro
micropropagated plants of Pilosocereus robinii [
24
], Musa spp. [
25
], and Phoenix
dactylifera L. [
26
]. At both levels, the DNA genome and the transcribed proteins are
essential for recognizing the genetic stability in the regenerated plants of L. schweinfurthii.
The functional effect of certain plant species and their use in folk medicine depend
mainly on their active secondary metabolites [
27
,
28
]. Plants of L. schweinfurthii have been
reported to contain a high level of phenolic compounds, particularly flavonoids [
29
]. These
secondary metabolites play a major role in adapting the plant to the environment and main-
taining its survival [
30
]. Flavonoids are naturally produced phenolic compounds in plants
and play an important role in the protection against unfavorable environmental conditions
such as drought [
31
], high concentrations of aluminum in soil [
32
], UV-irradiation [
33
], and
defense plants against herbivores, bacteria, and fungi [
34
]. Phenolic compounds have a role
in modern human therapy, e.g., controlling hyperglycemia associated with type 2 diabetes
at early stages when included in the human diet [
35
]. Moreover, flavonoids are reported to
protect humans against numerous diseases due to the fact of their strong anti-oxidative [
36
],
Plants 2021,10, 2089 3 of 16
anti-inflammatory [
37
], anticarcinogenic [
38
], antiviral [
39
], and antibacterial [
40
] activi-
ties as well as a direct cytoprotective effect on several human systems (i.e., coronary and
vascular systems) and organs (i.e., liver and pancreas) [
41
,
42
]. These features put them
among the most attractive natural substances available for enhancing the options of the
previously mentioned therapy [
43
]. The leaves of L. schweinfurthii contain large quantities
of flavonoids compared to roots, stems, and flowers [
44
]. The main phenolics found in
leaves are quercetin, kaempferol, gallic acid, ferulic acid, and apigenin [
29
]. Six glucosides
have been isolated from L. schweinfurthii. Four of them showed a potent inhibitory activity
that could decrease postprandial hyperglycemia in diabetic patients [45].
Although many plants contain high-value phenolic compounds, it is difficult to
cultivate at a large-scale due to the specific ecological conditions. Corresponding plant
in vitro
cultures, particularly plant cell cultures, provide an attractive alternative source of
phenolics that can overcome the limitations of extracting useful metabolites from limited
natural resources [
46
]. Obtaining phenolic compounds from plant’s
in vitro
cultures is one
of the more interesting research areas in recent decades due to the fact of their benefits.
Phenolic content can be elevated in culture medium such as in Zingiber officinale Rosc. [
47
],
Sequoia sempervirens [48], Rosa damascene Mill [49], and grape [50].
It is worth searching for alternative plant sources to meet the nutritional needs of
humans and to protect them from diseases resulting from malnutrition and a lack of
functional substances in the future. Thus, the present study is the first attempt to optimize
a protocol for direct
in vitro
plant regeneration in L. schweinfurthii as well as to evaluate its
phenolic, flavonoid, ferulic acid contents, and antioxidant activity of
in vitro
leaves’ extract.
2. Materials and Methods
2.1. Plant Material and Culture Conditions
Fruits of L. schweinfurthii were collected during March 2016 from Jazirat Al-Kawm
Al-Akhdar (the green islet) located in Burullus Lake (northern Nile Delta), Egypt. The
fruits were air-dried for approximately 120 h, and then their envelopes were removed
to obtain their seeds. The plant seeds were washed with 70% ethanol for 30 s, and then
they were surface sterilized by soaking in 30% commercial Clorox for 10 min. Seeds were
washed with sterilized distilled water 4 times to remove the remaining bleach.
After the sterilization process, seeds were cultured in 300 mL jars containing 30 mL
basal MS medium, including vitamins (Caisson Labs, Smithfield, UT, USA), with 3% sucrose
and solidified using 7% agar (ROTH Company, Carlsruhe, Germany) and incubated at
23 ±2C under a 16 h photoperiod of 2500 lux by cool fluorescent lamps.
2.2. In Vitro Micropropagation
For vegetative propagation, nodal segments were cut and cultivated on full-strength
MS media including vitamins supplemented with BA (0.4, 0.8, 1.6, or 3.2 mg L
1
), kinetin
(0.4, 0.8, 1.6, or 3.2 mg L
1
), BA + Kin (0.2 + 0.2, 0.4 + 0.4, 0.8 + 0.8, or 1.6 + 1.6 mg L
1
),
or BA + Kin + NAA (0.2 + 0.2 + 0.2 or 0.4 + 0.4 + 0.4 mg L
1
) and on basal MS medium
as a control. Seven nodal explants were used for shoot formation in each treatment.
Regenerated shootlets were then transferred to basal full-strength MS, half-strength MS,
half-strength MS medium fortified with NAA (0.4, 0.8, or 1.6 mg L
1
) or IBA (0.4, 0.8, or
1.6 mg L
1
). To determine the rooting capacity and the most suitable rooting medium,
eight shootlets were used in each treatment.
2.3. DNA Extraction and PCR Amplification Conditions
Total DNA was extracted from leaves of two
in vitro
mother plants and their micro-
propagated plantlets for three generations using the E.Z.N.A. kit (VWR, Darmstadt, Ger-
many). Twelve primers (i.e., 7 RAPD and 5 ISSR) out of a total of twenty primers (Thermo
Fisher, Frankfurt, Germany) were selected to amplify DNA fragments. The protocol for
RAPD and ISSR analysis was adapted from Martins et al. [
51
] and Williams et al. [
52
]. PCR
was performed in a volume of 20
µ
L using Invitrogen
Platinum
master mix (Thermo
Plants 2021,10, 2089 4 of 16
Fisher, Frankfurt, Germany). The amplification reaction consisted of an initial denaturation
step at 94
C for 5 min, followed by 43 cycles of 1 min at 92
C, 1 min at a specific annealing
temperature (Table 1), and 2 min at 72
C; there was one last extension step of 7 min
at 72
C. Amplifications were performed in a Bio-Rad T100
thermal cycler (Bio-Rad
Laboratories, Hercules, CA, USA) for both RAPD and ISSR. DNA amplification fragments
were separated with 1.5% agarose gel using 1x TBE buffer and stained with Red-Safe
nucleic acid staining solution. Gels were then analyzed with CAMAG
®
TLC Visualizer 2
(CAMAG, Muttenz, Switzerland).
Table 1. Sequences and annealing temperatures of primers used for the RAPD and ISSR analysis of L. schweinfurthii.
Primer Name Sequence Annealing Temperature (C)
RAPD primers
OPA-10 50-GTGATCGCAG-3040.5
OPAJ-01 50-ACGGGTCAGA-3043
OPAK-06 50-TCACGTCCCT-3042
OPAK-20 50-TGATGGCGTC-3041
OPAQ-20 50-GTGAACGCTC-3040.5
OPB-18 50-CCACAGCAGT-3042
OPR-09 50-TGAGCACGAG-3042
ISSR primers
HB11 50-GTGTGTGTGTGTCC-3054
HB12 50-CACCACCACGC-3050.9
HB13 50- GAGGAGGAGGC-3048
HB14 50-CTCCTCCTCGC-3048
HB15 50-GTGGTGGTGGC-3050.9
2.4. Protein Extraction and SDS-PAGE
Total protein was extracted from the healthy leaves of two
in vitro
mother plants
and their micropropagated plantlets for three generations. Ten milligrams of ground, fine
powder were homogenized thoroughly with a 400
µ
L extraction buffer (0.6 g Tris base, 0.2 g
sodium dodecyl sulfate (SDS), 30 g of urea, and 1 mL
β
-mercaptoethanol in 100 mL double-
distilled water) using vortex. The mixture was centrifuged at 13,000 rpm for 10 min at room
temperature after keeping overnight at 4
C. Twenty microliters of the extracted protein
samples were boiled in a water bath for 3–5 min before loading them on the gel. SDS-PAGE
was performed according to Laemmli [
53
] using 12.5% resolving gel, 4% stacking gel, and
bromophenol blue as a tracking dye. After carrying out the electrophoresis at 150 volts
and 25 milliamperes, the gel was de-stained in a methanol:glacial acetic acid:water (4:1:5)
mixture. Then, it was kept overnight in Coomassie Brilliant Blue buffer for staining. The
gel was photographed, and the molecular weights of the polypeptide bands were estimated
against protein molecular weight marker.
2.5. Secondary Metabolites
2.5.1. Sample Preparation and Extraction
Leaves of micropropagated plants were randomly collected, freeze-dried, and ground.
One gram was collected and Soxhlet extracted with 200 mL of 80% aqueous methanol
for 24 h. The extract was concentrated with a rotary evaporator to a concentration of
50 mg mL
1
which was then subjected to estimate the phenolic and flavonoid contents
as well as the antioxidant activity. More diluted leaves’ extract of 10 mg mL
1
was
used to quantify the ferulic acid content through HPTLC (high-performance thin layer
chromatography) analysis.
2.5.2. Total Phenolic Assay
The total phenolic content of the leaves was determined using the Folin–Ciocalteu
assay as described by Marinova et al. [
54
] with some modifications. An aliquot (200
µ
L)
of extracts or gallic acid (Sigma–Aldrich, St. Louis, MO, USA) standard solution (10, 20,
30, 40, 50, and 100 mg L
1
) was added to a 5 mL Eppendorf tube containing 1.8 mL
Plants 2021,10, 2089 5 of 16
distilled deionized water. Two hundred microliters of Folin-Ciocalteu’s reagent (Merck,
Schnelldorf, Germany) were added to the mixture and shaken. After 5 min, 2 mL of
7% sodium carbonate (VWR chemicals, Darmstadt, Germany) solution was added and
mixed thoroughly. The mixture was diluted to 5 mL with distilled water and incubated for
90 min in the dark at room temperature. The absorbance against the reagent blank was
determined at 750 nm with an Analytic Jena Specord
®
250 Plus UV-Vis spectrophotometer.
Total phenolic content is expressed as mg GAE g
1
DW (mg gallic acid equivalents/g dry
weight) and calculated as follows: T = CV/M, where T is the total phenolic content, C is
the concentration of gallic acid estimated in mg mL
1
, V is the volume of extract solution
in mL, and M is the weight of extract in g.
2.5.3. Total Flavonoid Assay
Total flavonoid content was measured using the aluminum chloride assay as described
by Marinova et al. [
54
] with some modifications. An aliquot (500
µ
L) of extracts or catechin
standard (Sigma–Aldrich, St. Louis, MO, USA) solution (10, 20, 30, 40, 50, and 100 mg L
1
)
was added to a 5 mL Eppendorf tube, containing 2 mL distilled water. To the diluted
sample, 150
µ
L of 5% sodium nitrite (AppliChem, Darmstadt, Germany) was added. After
5 min, 150
µ
L of 10% aluminum chloride (Carl-Roth, Carlsruhe, Germany) was added. At
the sixth min, 1 mL of 1 M sodium hydroxide was added, and the total volume was diluted
to 5 mL using distilled water. The absorbance was measured against reagent blank at
510 nm, and total flavonoids were expressed as mg CE g
1
DW (mg catechin equivalent/g
dry weight) and calculated by the equation: T = CV/M, where T is the total flavonoid
content, C is the concentration of catechin estimated in mg mL
1
, V is the volume of extract
solution in ml, and M is the weight of extract in g.
2.5.4. HPTLC Conditions
The high-performance thin-layer chromatography (HPTLC) system (Camag, Muttenz,
Switzerland) consisted of a Limomat 5 connected to compressed air, an Automatic Devel-
oping Chamber 2 (ADC 2), and a TLC Visualizer 2 supported with visionCATS software.
An analytical grade of ferulic acid (Merck, Germany) was used to prepare 400
µ
g ml
1
in
methanol as a calibration standard against dry leaves’ extracts of micropropagated plants.
TLC silica gel 60 F
254
aluminum plates (10
×
20 cm, Merck, Darmstadt, Germany) were
used for the TLC analysis. Standard and samples were applied to plates as 8 mm bands,
8 mm from the bottom edge of the layer, using Linomat 5. A ferulic acid standard solution
of 400
µ
g ml
1
of a volume of 2–9
µ
L was applied against 2, 4, 6, 8, 10, 12, and 14
µ
L of
dry leaves’ extract. A mixture of ethyl acetate/methanol/water (100:13.5:10, v/v/v) was
used as the mobile phase. Plates were developed at room temperature and 60% humidity
in an ADC2 automated development chamber. The migration distance of the mobile phase
was 70 mm with a development time of 9 min. After development, the chromatogram was
visualized and photographed by Visualizer 2 at 254 and 366 nm. The ferulic acid content in
the samples was expressed as mg g1DW.
2.6. Antioxidant Capacity
The antioxidant capacity of the micropropagated leaves’ extract was measured using
the DPPH (diphenyl-1-picryl-hydrazyl) assay according to Olalere et al. [
55
] and the ABTS
(2,2
0
-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) assay according to Gabr et al. [
56
].
2.6.1. DPPH Free Radical Scavenging
DPPH is highly sensitive in detecting small differences in antioxidant activities. It
is a stable free radical that can accept a hydrogen radical or an electron to convert to a
stable molecule. The stock solution of DPPH reagent (1 mM) was prepared and stored at
–20
C until use. The working solution (0.06 mM) was prepared to obtain an absorbance
value of 0.8
±
0.04 at 515 nm. Ten different extract concentrations of micropropagated
leaves (between 0.25 and 0.7 mg mL
1
) were prepared. The absorbance at 515 nm (A
1
)
Plants 2021,10, 2089 6 of 16
was measured for a mixture of 0.5 mL of each extract concentration with 2.5 mL DPPH
working solution after incubation in the dark at room temperature for 30 min. Ethanol was
used instead of extract to obtain the absorbance of the control reaction (A
0
). The DPPH
radical scavenging activity percentage was calculated as follows:
((A0A1)/A0)×
100.
The inhibition percentage was plotted against the different concentrations of the leaves’
extracts to generate a straight-line equation. The extract concentration required to scavenge
half of the DPPH radicals (IC50) was then determined.
2.6.2. ABTS Free Radical Scavenging
A 7 mM ABTS solution was reacted with 2.4 mM potassium persulphate solution at
a ratio of 1:1 (v/v). The solution was incubated in the dark at room temperature for 16 h.
One milliliter of the prepared ABTS
+
solution was diluted with 60 mL methanol resulting
in a working solution with an absorbance of 0.60
±
0.01 at 728 nm. Fourteen different
extract concentrations of micropropagated leaves (between 1.0 and 5.5 mg mL
1
) were
prepared. The absorbance at 734 nm (A
1
) was measured for a mixture of 40
µ
L of each
extract concentration with 1.96 mL blue-green ABTS
+
working solution after incubation in
the dark at 37
C for 10 min. The control reaction (A
0
), which contains all reagents except
the test compound, was run identically. The ABTS
+
radical scavenging activity percentage
was calculated as follows:
((A0A1)/A0)×
100. The inhibition percentage was plotted
against the different concentrations of the dry leaves’ extracts to generate a straight-line
equation. The concentration of extract required to scavenge half of the ABTS
+
radicals
(IC50) was then determined.
2.7. Recording Data and Statistical Analysis
The number of plantlets, leaves, distinct nodes, and shootlet length were estimated and
recorded after five weeks of cultivation. Recorded data were subjected to statistical analysis
of variance (ANOVA) using SigmaPlot v.12.5. The Shapiro–Wilk normality test failed for
all data and also for the transformations in the number of plantlets, nodes, roots, and root
length. Then, the power of the performed test decreased from 0.50 to 0.001. The normality
test passed in shootlet length and passed in the number of leaves after transformation into
the square root. The Holm–Sidak method was applied for pairwise comparisons.
RAPD, ISSR, and SDS-PAGE data were scored for presence (1) and absence (0). Three
matrices were generated, one for each analysis type. The genetic similarities were calculated
according to Jaccard’s index. A dendrogram showing the genetic stability between the three
generations’ individuals and the mother plant was constructed using UPGMA (unweighted
pair group method with arithmetic average) through CAP 1.2 software [57].
3. Results and Discussion
3.1. In Vitro Propagation
During the study of
in vitro
seed germination of L. schweinfurthii, the percentage of
microbial contamination based on the method of sterilization used in culture media was
16.67%, and the maximum percentage of germination in non-contaminated cultures was
30% (Figure 1). Although due to the plant’s relatively low germination percentage, it was
noticed that the germinated plants had plenty of leaves, convergent nodes, and elongated
stems. The low growth rate reflected the low spread of the plant across large areas in the
wildlife, which means that the in vitro multiplication of the plant is of great significance.
In an attempt for intensive plant micropropagation, nodal segments of sterilized
germinated seedlings were cut and transferred into full-strength MS medium fortified with
different concentrations of BA, Kin, and NAA as explained in Table 2. Strong variability
was obtained in the number of leaves and shoot length of regenerated plantlets after
5 weeks of culture.
Plants 2021,10, 2089 7 of 16
Plants 2021, 10, x FOR PEER REVIEW 8 of 17
a total concentration the same as the concentration of only one of them. This allows saying
that shoot formation in L. schweinfurthii may depend more on the concentration of the
hormone than its type. These results are different from results obtained in the microprop-
agation of Magnolia sirindhorniae, Eryngium alpinum, and Argania spinosa. Shoots of M.
sirindhorniae were optimally induced in a half-strength MS medium supplemented with a
combination of BA, NAA, and gibberellic acid (GA3) with higher concentrations, i.e. 2.0 +
0.1 + 2.0 mgL1, respectively [7]. A solid MS medium combined with BA, IAA, and GA3
was successful in shoot proliferation of E. alpinum [8]. Moreover, the highest adventitious
shoots of the endangered plant, A. spinosa, were observed on MS medium containing 1
mg L1 BA and 2 mg L1 GA3 [59].
For completing in vitro micropropagation of the studied species, shootlets of the
plant were transferred firstly to full- and half-strength MS media without growth regula-
tors for root initiation. It was noticed that the number and length of roots that emerged in
1/2 MS were better than in the full-strength MS. Therefore, the experiment was repeated
with the same treatments in addition to adding NAA and IBA to half-strength MS for
rooting enhancement. It was found that increasing NAA concentration to medium re-
versely affected rooting production. Otherwise, the addition of 0.4 mg L1 IBA enhanced
the number of roots and the root length but with non-considerable significance with other
treatments according to pairwise comparison using the Holm–Sidak method (Figure 1).
Although the highest mean of the number of roots emerged per plant and long roots ob-
tained in IBA treatments, not all eight shootlets showed a rooting response to the treat-
ment. This led to a high standard error in several treatments and hid the significant dif-
ferences between the different IBA concentrations used (Figure 2). Ho wever, the IBA treat-
ments showed significantly better root formation and enhancement than NAA treatments.
Figure 1. In vitro plant micropropagation protocol of L. schweinfurthii: (a) aseptic seedling; (b) shoot formation after five
weeks of culture on MS medium supplemented with 0.4 mg L1 Kinetin; (c) roots formed on MS medium fortified with 0.4
mg L1 IBA (indole-3-butyric acid).
b
a c
Figure 1. In vitro
plant micropropagation protocol of L. schweinfurthii: (
a
) aseptic seedling; (
b
) shoot
formation after five weeks of culture on MS medium supplemented with 0.4 mg L
1
Kinetin; (
c
) roots
formed on MS medium fortified with 0.4 mg L1IBA (indole-3-butyric acid).
Table 2.
Effect of different concentrations of BA, Kin, and NAA on micropropagation of L. schweinfurthii from nodal cuttings.
Treatment (mg L1)Number of
Plantlets
Number of
Distinct Nodes
Number of
Leaves
Shootlet Length
(cm)
BA Kin NAA
---1.00 ±0.00 abc 1.00 ±0.00 abc 4.14 ±0.46 c0.56 ±0.03 b
0.4 - - 1.43 ±0.48 abc 3.29 ±0.89 ab 20.71 ±3.66 ad 1.24 ±0.40 b
0.8 - - 1.00 ±0.22 abc 1.71 ±0.47 abc 5.86 ±2.19 cd 0.83 ±0.17 b
1.6 - - 1.14 ±0.14 abc 1.29 ±0.18 abc 7.14 ±2.44 bcd 0.71 ±0.19 b
3.2 - - 0.29 ±0.18 c0.43 ±0.30 b1.86 ±1.70 c0.20 ±0.13 b
- 0.4 - 1.86 ±0.46 ab 5.86 ±0.91 a26.00 ±4.34 a2.83 ±0.39 a
- 0.8 - 0.86 ±0.14 abc 1.71 ±0.52 abc 8.00 ±2.17 bcd 0.93 ±0.25 b
- 1.6 - 0.14 ±0.14 c0.14 ±0.14 b0.43 ±0.43 c0.07 ±0.07 b
- 3.2 - 0.86 ±0.14 abc 1.00 ±0.22 abc 8.14 ±1.97 bcd 0.64 ±0.13 b
0.2 0.2 - 2.00 ±0.44 a3.86 ±1.12 a22.14 ±4.49 ab 1.23 ±0.20 b
0.4 0.4 - 1.29 ±0.18 abc 2.14 ±0.55 abc 12.71 ±4.20 a1.11 ±0.24 b
0.8 0.8 - 1.43 ±0.30 abc 2.14 ±0.46 abc 12.43 ±3.61 a1.14 ±0.31 b
1.6 1.6 - 1.00 ±0.22 abc 1.71 ±0.61 abc 8.29 ±2.73 bcd 0.91 ±0.26 b
0.2 0.2 0.2 0.57 ±0.37 c1.00 ±0.66 abc 6.00 ±3.93 cd 0.51 ±0.34 b
0.4 0.4 0.4 0.43 ±0.20 c1.29 ±0.97 abc 4.43 ±2.41 c0.63 ±0.38 b
Pairwise comparison was conducted according to the Holm–Sidak method at p
0.05. Seven replicates were used for each treatment;
BA, 6-benzyl adenine; Kin, kinetin; NAA, naphthalene acetic acid. The letters a, b, c, and d represent the pairwise comparison and the
significance between treatments.
The highest significant results of shootlet length were observed in plantlets produced
in MS medium with 0.4 mg L
1
kinetin, while 0.4 BA, 0.4 kinetin, and 0.2 BA + 0.2 Kin
(in mg L
1
) were recorded as highly significant in the number of distinct nodes (Figure 1).
Although 0.4 mg L
1
kinetin was non-significant in other variables, with most treatments
used it was the best in terms of the average number of leaves at approximately 26 leaves
per regenerated plant. Moreover, it was second (1.86 plantlets/nodal segment) after
0.2 + 0.2 mg L
1
Kin + BA in terms of the number of plantlets regenerated per inoculated
cut (2 plantlets/ nodal segment).
In vitro
propagation of plants depends mainly on the addition of cytokinins to culture
media and, sometimes, in addition to a lower concentration of auxins [
58
]. Two cytokinins
Plants 2021,10, 2089 8 of 16
(BA and Kin) and one auxin (NAA) were used for multiple shoot formations from nodal
segments of L. schweinfurthii. The lower concentrations of cytokinins (BA or Kin) were
the best in all determined variables, such as the number of plantlets, nodes, leaves, and
shoot length. In the present study, a reduction in shoot proliferation by increasing benzyl-
adenine or kinetin in the culture medium was noticed. Furthermore, similar results were
observed when combinations between both growth regulators were added but with a total
concentration the same as the concentration of only one of them. This allows saying that
shoot formation in L. schweinfurthii may depend more on the concentration of the hormone
than its type. These results are different from results obtained in the micropropagation of
Magnolia sirindhorniae, Eryngium alpinum, and Argania spinosa. Shoots of M. sirindhorniae
were optimally induced in a half-strength MS medium supplemented with a combination of
BA, NAA, and gibberellic acid (GA
3
) with higher concentrations, i.e. 2.0 + 0.1 + 2.0 mgL
1
,
respectively [
7
]. A solid MS medium combined with BA, IAA, and GA
3
was successful
in shoot proliferation of E. alpinum [
8
]. Moreover, the highest adventitious shoots of the
endangered plant, A. spinosa, were observed on MS medium containing 1 mg L
1
BA and
2 mg L1GA3[59].
For completing
in vitro
micropropagation of the studied species, shootlets of the plant
were transferred firstly to full- and half-strength MS media without growth regulators for
root initiation. It was noticed that the number and length of roots that emerged in 1/2 MS
were better than in the full-strength MS. Therefore, the experiment was repeated with the
same treatments in addition to adding NAA and IBA to half-strength MS for rooting en-
hancement. It was found that increasing NAA concentration to medium reversely affected
rooting production. Otherwise, the addition of 0.4 mg L
1
IBA enhanced the number of
roots and the root length but with non-considerable significance with other treatments
according to pairwise comparison using the Holm–Sidak method (Figure 1). Although the
highest mean of the number of roots emerged per plant and long roots obtained in IBA
treatments, not all eight shootlets showed a rooting response to the treatment. This led
to a high standard error in several treatments and hid the significant differences between
the different IBA concentrations used (Figure 2). However, the IBA treatments showed
significantly better root formation and enhancement than NAA treatments.
Plants 2021, 10, x FOR PEER REVIEW 9 of 17
Figure 2. In vitro rooting of L. schweinfurthii shoots on MS medium fortified with different auxins. Pairwise comparison
showed no significant differences between treatments at p 0.05 using the Holm–Sidak method. Eight replicates were
used in each treatment. PGR, plant growth regulators; MS, full-strength MS salts; 1/2MS, half MS salts; IBA, indole-3-
butyric acid; NAA, naphthalene acetic acid.
In this study, half-strength MS medium with NAA and IBA were used for root stim-
ulation, and IBA was the best for root formation enhancement. The results were consistent
with other studies where IBA stimulated sufficient root induction in several species in-
cluding Cardiospermum halicacabum [60], Dorem ammoniacum [61], Achyranthes aspera [62],
and Prunus armeniaca L. [63].
3.2. Genetic Stability of Micropropagated Plantlets
For determining the genetic stability in the suggested micropropagation protocol,
RAPD, ISSR, and SDS-PAGE analyses were performed to compare between the in vitro
mother plant and its micropropagated plantlets, which resulted from using MS medium
fortified with 0.4 mg L1 BA for three generations and two individuals from each genera-
tion. Among the 20 primers screened (10 RAPD and 10 ISSR), only 12 primers produced
clear and detectable amplified DNA fragments and were used in further PCR analysis.
With seven RAPD primers, 29 DNA fragments (a total of 137 scorable bands) were
amplified in the mother plant and its three generations plantlets. Jaccard’s similarity co-
efficient, ranging between 0.36 and 0.56, was obtained. The second and third generations
showed a similarity of 0.48 and 0.52 to the mother plant, respectively. The highest poly-
morphism of 100% was observed in fragments amplified with OPA10 and OPAJ01 pri-
mers, while the lowest of 50% was in the amplified fragments using OPB18 primer. Only
eight monomorphic fragments out of 29 DNA fragments were recorded. Furthermore, 28
DNA fragments (a total of 107 scorable bands) were amplified using five ISSR primers,
while a similarity of 0.33-0.70 was recorded. The highest similarities to the mother plant
were in the first (0.66) and third (0.55) generations (Figure 3). A higher polymorphism was
observed over RAPD, where the lowest was 75% in the HB13 and HB14 primers and the
highest was 100% in the HB11 primer. Out of 28 DNA fragments amplified with five ISSR
primers, only four fragments were monomorphic.
In SDS-PAGE analysis, sixteen polypeptides were separated with a similarity be-
tween 0.54 and 0.82. The first and second generations showed high similarity to the
mother plant of 0.68 and 0.74, respectively (Figure 4). Half of the separated polypeptides
were monomorphic, as they were found in all protein extracts. It was noticed also that
there were two unique polypeptides of 82 and 108 KDa that were separated only in a plant
in the third generation (3rd_1). The expressed protein showed uniformity between the
012345678
MS
1/2 MS
1/2MS+NAA0.4
1/2MS+NAA0.8
1/2MS+NAA1.6
1/2MS+IBA0.4
1/2MS+IBA0.8
1/2MS+IBA1.6
PGR concentration (mgL-1)
Root length (cm) No. of Emerged roots/Plant
Figure 2. In vitro
rooting of L. schweinfurthii shoots on MS medium fortified with different auxins.
Pairwise comparison showed no significant differences between treatments at p
0.05 using the
Holm–Sidak method. Eight replicates were used in each treatment. PGR, plant growth regulators;
MS, full-strength MS salts; 1/2MS, half MS salts; IBA, indole-3-butyric acid; NAA, naphthalene
acetic acid.
Plants 2021,10, 2089 9 of 16
In this study, half-strength MS medium with NAA and IBA were used for root stimu-
lation, and IBA was the best for root formation enhancement. The results were consistent
with other studies where IBA stimulated sufficient root induction in several species includ-
ing Cardiospermum halicacabum [
60
], Dorem ammoniacum [
61
], Achyranthes aspera [
62
], and
Prunus armeniaca L. [63].
3.2. Genetic Stability of Micropropagated Plantlets
For determining the genetic stability in the suggested micropropagation protocol,
RAPD, ISSR, and SDS-PAGE analyses were performed to compare between the
in vitro
mother plant and its micropropagated plantlets, which resulted from using MS medium
fortified with 0.4 mg L
1
BA for three generations and two individuals from each genera-
tion. Among the 20 primers screened (10 RAPD and 10 ISSR), only 12 primers produced
clear and detectable amplified DNA fragments and were used in further PCR analysis.
With seven RAPD primers, 29 DNA fragments (a total of 137 scorable bands) were
amplified in the mother plant and its three generations plantlets. Jaccard’s similarity coef-
ficient, ranging between 0.36 and 0.56, was obtained. The second and third generations
showed a similarity of 0.48 and 0.52 to the mother plant, respectively. The highest poly-
morphism of 100% was observed in fragments amplified with OPA10 and OPAJ01 primers,
while the lowest of 50% was in the amplified fragments using OPB18 primer. Only eight
monomorphic fragments out of 29 DNA fragments were recorded. Furthermore, 28 DNA
fragments (a total of 107 scorable bands) were amplified using five ISSR primers, while a
similarity of 0.33-0.70 was recorded. The highest similarities to the mother plant were in the
first (0.66) and third (0.55) generations (Figure 3). A higher polymorphism was observed
over RAPD, where the lowest was 75% in the HB13 and HB14 primers and the highest was
100% in the HB11 primer. Out of 28 DNA fragments amplified with five ISSR primers, only
four fragments were monomorphic.
Plants 2021, 10, x FOR PEER REVIEW 10 of 17
mother plant and most of the plant individuals studied. On the contrary, only eight poly-
peptides were separated from L. schweinfurthii seed proteins in the study by El-Ghamry et
al. [64].
OPA-10
OPAJ-01 OPAK-06
OPAK-20
OPAQ-20
OPB-18
OPR-09
HB11
HB12
HB13 HB14
HB15
Figure 3. RAPD and ISSR profiles with primers mentioned in Table 1 of three micropropagated generations of L. schwein-
furthii compared to the mother plant. MP, mother plant; 1st, first-generation plantlets; 2nd, second-generation plantlets;
3rd, third-generation plantlets.
Figure 4. SDS-PAGE analysis of total protein bans extracted from three micropropagated generations of L. schweinfurthii
compared to the mother plant. M, marker; MP, mother plant; 1st, first-generation plantlets; 2nd, second-generation plant-
lets; 3rd, third-generation plantlets showing three of the monomorphic polypeptides detected.
Figure 3.
RAPD and ISSR profiles with primers mentioned in Table 1of three micropropagated
generations of L. schweinfurthii compared to the mother plant. MP, mother plant; 1st, first-generation
plantlets; 2nd, second-generation plantlets; 3rd, third-generation plantlets.
In SDS-PAGE analysis, sixteen polypeptides were separated with a similarity between
0.54 and 0.82. The first and second generations showed high similarity to the mother
plant of 0.68 and 0.74, respectively (Figure 4). Half of the separated polypeptides were
monomorphic, as they were found in all protein extracts. It was noticed also that there
were two unique polypeptides of 82 and 108 KDa that were separated only in a plant in the
third generation (3rd_1). The expressed protein showed uniformity between the mother
Plants 2021,10, 2089 10 of 16
plant and most of the plant individuals studied. On the contrary, only eight polypeptides
were separated from L. schweinfurthii seed proteins in the study by El-Ghamry et al. [64].
Plants 2021, 10, x FOR PEER REVIEW 10 of 17
mother plant and most of the plant individuals studied. On the contrary, only eight poly-
peptides were separated from L. schweinfurthii seed proteins in the study by El-Ghamry et
al. [64].
OPA-10
OPAJ-01 OPAK-06
OPAK-20
OPAQ-20
OPB-18
OPR-09
HB11
HB12
HB13 HB14
HB15
Figure 3. RAPD and ISSR profiles with primers mentioned in Table 1 of three micropropagated generations of L. schwein-
furthii compared to the mother plant. MP, mother plant; 1st, first-generation plantlets; 2nd, second-generation plantlets;
3rd, third-generation plantlets.
Figure 4. SDS-PAGE analysis of total protein bans extracted from three micropropagated generations of L. schweinfurthii
compared to the mother plant. M, marker; MP, mother plant; 1st, first-generation plantlets; 2nd, second-generation plant-
lets; 3rd, third-generation plantlets showing three of the monomorphic polypeptides detected.
Figure 4.
SDS-PAGE analysis of total protein bans extracted from three micropropagated generations
of L. schweinfurthii compared to the mother plant. M, marker; MP, mother plant; 1st, first-generation
plantlets; 2nd, second-generation plantlets; 3rd, third-generation plantlets showing three of the
monomorphic polypeptides detected.
Three matrices of RAPD, ISSR, and SDS-PAGE were merged and analyzed to show
the clonal fidelity of the DNA and protein levels together. The dendrogram of genetic dis-
tances among the
in vitro
and micropropagated plants based on amplified DNA fragments
generated by RAPD and ISSR primers and polypeptides separated in SDS-PAGE is shown
in Figure 5. The distances in the dendrogram revealed that the first and third generations
of the first plant individuals (1st_1 and 3rd_1) were more similar than the second genera-
tion (2nd_1). Furthermore, the second generation of the second individual (2nd_2) was
more similar to the mother plant than the first (1st_2) and third (3rd_2) generations. The
results showed that the generation that was more similar to the
in vitro
plants had the
higher Jaccard’s similarity coefficient which ranged between 0 (completely different) and
1 (identical). The first micropropagated generation showed a higher similarity coefficient to
the mother
in vitro
plants of 0.56–0.58. On the other hand, the second generation showed
a similarity coefficient of 0.44–0.61, while the third one showed a similarity coefficient of
0.52–0.56 (Table 3). It was also obtained that the conditions of propagation in this study
lowered the tendency of the plants to be genetically stable.
It is necessary after micropropagation to check the genetic uniformity of microprop-
agated plantlets [
65
]. Two PCR-based techniques (RAPD and ISSR) and a biochemical
marker technique (SDS-PAGE) were used in the present study to test the genetic stability
and polypeptide content because of their rapidity, simplicity, and effectiveness as well as
the fact that they do not need prior information about the DNA sequence [
66
]. Moreover,
the use of different markers in parallel provides better opportunities for genetic alteration
identification between different clones [
67
]. The molecular markers were not affected
by external environmental factors which, consequently, accurately detected the genetic
variability among the plant clones [
68
]. The advantage of using both biochemical and
molecular markers is the ability to give an account of the expression stability level of the
DNA regarding the variability that occurred in the plant genome. In the present investi-
gation, it was concluded that molecular and biochemical markers are equally important
for genetic analysis and for the evaluation of the amount of genetic variability among
Plants 2021,10, 2089 11 of 16
the different micropropagated plantlets of L. schweinfurthii. In addition, Osman et al. [
69
]
determined the genetic relationship between several species of Zea mays and Sorghum using
SDS-PAGE of seed protein as well as RAPD-PCR markers.
Plants 2021, 10, x FOR PEER REVIEW 11 of 17
Three matrices of RAPD, ISSR, and SDS-PAGE were merged and analyzed to show
the clonal fidelity of the DNA and protein levels together. The dendrogram of genetic
distances among the in vitro and micropropagated plants based on amplified DNA frag-
ments generated by RAPD and ISSR primers and polypeptides separated in SDS-PAGE is
shown in Figure 5. The distances in the dendrogram revealed that the first and third gen-
erations of the first plant individuals (1st_1 and 3rd_1) were more similar than the second
generation (2nd_1). Furthermore, the second generation of the second individual (2nd_2)
was more similar to the mother plant than the first (1st_2) and third (3rd_2) generations.
The results showed that the generation that was more similar to the in vitro plants had
the higher Jaccard’s similarity coefficient which ranged between 0 (completely different)
and 1 (identical). The first micropropagated generation showed a higher similarity coeffi-
cient to the mother in vitro plants of 0.56–0.58. On the other hand, the second generation
showed a similarity coefficient of 0.44–0.61, while the third one showed a similarity coef-
ficient of 0.52–0.56 (Table 3). It was also obtained that the conditions of propagation in this
study lowered the tendency of the plants to be genetically stable.
Figure 5. UPGMA dendrogram based on data generated from biochemical and molecular markers, showing the genetic
linkage distance among the different micropropagated plantlets in different generations of L. schweinfurthii. MP, mother
plant; 1st, first-generation plantlets; 2nd, second-generation plantlets; 3rd, third-generation plantlets.
Table 3. Jaccard’s similarity coefficient concerning similarities in DNA fragments generated in RAPD and ISSR analyses
and protein polypeptides through SDS-PAGE.
Mother Plant 1st_1 1st_2 2nd_1 2nd_2 3rd_1 3rd_2
Mother Plant
1st_1 0.5806
1st_2 0.5593 0.5738
2nd_1 0.4364 0.4821 0.5714
2nd_2 0.614 0.6271 0.5 0.54
3rd_1 0.5625 0.6508 0.5556 0.4912 0.6333
Figure 5.
UPGMA dendrogram based on data generated from biochemical and molecular markers,
showing the genetic linkage distance among the different micropropagated plantlets in different gen-
erations of L. schweinfurthii. MP, mother plant; 1st, first-generation plantlets; 2nd, second-generation
plantlets; 3rd, third-generation plantlets.
Table 3.
Jaccard’s similarity coefficient concerning similarities in DNA fragments generated in RAPD
and ISSR analyses and protein polypeptides through SDS-PAGE.
Mother Plant 1st_1 1st_2 2nd_1 2nd_2 3rd_1 3rd_2
Mother Plant
1st_1 0.5806
1st_2 0.5593 0.5738
2nd_1 0.4364 0.4821 0.5714
2nd_2 0.614 0.6271 0.5 0.54
3rd_1 0.5625 0.6508 0.5556 0.4912 0.6333
3rd_2 0.5246 0.5645 0.5424 0.4717 0.569 0.7368
In the present analysis, SDS-PAGE revealed the high stability of expressed proteins
in the micropropagated plantlets compared to the amplified DNA fragments assessed by
RAPD- and ISSR-PCR techniques. This indicates that it was supposed to have modifications
in plantlet DNA, especially in the non-coding region. This effect may be related to the PGR
used in micropropagation, as it was noticed that 6-benzyl adenine affects DNA and causes
mutations [
70
]. In a study by Alizadeh and Singh [
71
], the similarity coefficient was 1 (in
both RAPD and ISSR) in most clones, although there were low coefficients of 0.53 (RAPD)
and 0.63 (ISSR) recorded in some clones of Vitis spp. micropropagated plantlets. This also
raises the idea of the effects of PGRs and the cultivation conditions on the genetic stability
of cloned plants.
3.3. Phenolic and Flavonoid Content Estimation
The phenolic and flavonoid contents of the micropropagated plant leaves’ extract
were estimated spectrophotometrically in terms of gallic acid and catechin equivalence
(GAE: gallic acid equivalent; CE: catechin equivalent) at 750 and 510 nm, respectively.
Plants 2021,10, 2089 12 of 16
Three replicates of different concentrations of gallic acid and catechin (10, 20, 30, 40, 50,
100, 150, 200, and 300
µ
g ml
1
) were used to deduce the standard curves for determination
of phenolic and flavonoid content, respectively. The generated equation for the gallic
acid standard curve was
y=
0.0043
x+
0.0019
R2=0.9995
. Furthermore, the generated
one for the catechin standard curve was
y=
0.0034
x
0.0039
R2=0.9993
. The result
obtained from the total phenolic content estimation of the
in vitro
leaves’ extracts was
11.53 mg GAE g
1
DW. However, the total flavonoid content was estimated as 12.99 mg
CE g1DW.
From the rich plant sources of phenolics, Acacia nilotica,Acacia catechu, and Albizia
lebbeck contain 80.63, 78.12, and 66.23 mg GAE, respectively [
72
]. Moreover, higher phenolic
contents were estimated in the fruits of Solanum indicum and S. surattense of 250.4–289.5 mg
GAE g
1
DW [
73
]. Despite the relatively lower total phenolics detected in this study,
the global problem of food shortage necessitates the search for nutritional alternatives as
well as nutritional supplements that preserve human health and vitality. On the other
hand, the production of the active substance
in vitro
will remain the most appropriate
solution that saves time and effort, especially due to the decline of global cultivated land
and climate risks.
3.4. HPTLC Analysis
During the estimation of ferulic acid in dry leaves’ extract, the retardation factor (Rf)
of the 400
µ
g ml
1
standard was 0.62 (Figure 6). The eight reference volumes (2–9
µ
L) of
the standard were used to generate a linear calibration curve. The linear equation obtained
was y= 5.601
×
10
8
x where R = 95.21%, and the coefficient of variation (CV) was 11.77%.
Only four of the seven different volumes of dry leaves’ extract samples (2, 4, 6, and 8
µ
L)
were detected in the calibration range (Figure S5). The final results showed that the mean
of ferulic acid content in the three samples within the calibration range was 45.52 mg g
1
DW where the CV = 1.19% (Table 4). The HPTLC method was simple, reproducible, and
sensitive in the separation and determination of ferulic acid. It was used to estimate ferulic
acid in Lycopodium clavatum [74], Setaria italica [75], and Ricinus communis Linn. [76].
g
g
y
g
Figure 6.
HPTLC chromatogram of L. schweinfurthii micropropagated leaves’ extract against ferulic
acid standard captured at 366 nm. Tracks 1–8: ferulic acid, 400
µ
g ml
1
of volume 2–9
µ
L; Tracks
9–15: dry leaves’ extract of volume 2, 4, 6, 8, 10, 12, and 14 µL.
Table 4.
A summary of the results of the total phenolic content, total flavonoid content, ferulic acid
content, and antioxidant activity of micropropagated L. schweinfurthii dried leaves.
Contents and Antioxidant Capacity Obtained Results
Total phenolic content 11.53 GAE g1DW
Total flavonoid content 12.99 CE g1DW
Ferulic acid content 45.52 mg g1DW
IC50 with DPPH analysis 0.43 mg mL1
IC50 with ABTS+analysis 1.99 mg mL1
Plants 2021,10, 2089 13 of 16
3.5. Antioxidant Activities
The results obtained from the antioxidant assay revealed that 0.43 mg mL
1
of the
in vitro
leaves’ extract were required to scavenge half of the DPPH stable radicals (IC
50
).
However, 1.99 mg mL
1
of the leaves’ extract were required to scavenge half of the stable
ABTS free radicals (Table 4). According to plotting the inhibitory effect, the sensitivity and
efficiency of the DPPH assay were higher than the ABTS assay. On the other hand, only
107.57 and 94.71
µ
g ml
1
of black pepper extracts were required to scavenge half of the
DPPH and ABTS stable radicals, respectively [55].
4. Conclusions
In this study, we successfully established a suitable, rapid, and efficient protocol for
in vitro
micropropagation of L. schweinfurthii from nodal segments. Reproducible genetic
and biochemical techniques were performed to determine the stability of plant genome and
expressed proteins in regenerated
in vitro
plants. The importance of the leaves’ extract was
proven through the content and activity. This protocol should be useful in future studies
for in vitro secondary metabolite production from this plant.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10
.3390/plants10102089/s1, Figure S1: Jazirat Al-Kawm Al-Akhdar (the green islet) which is located in
Burullus Lake (northern of Nile Delta) in Egypt showing the populations of Lycium schweinfurthii.
Figure S2: The blooming of L. schweinfurthii during the spring season. Figure S3: L.schweinfurthii
branch showing leaves and immature fruits. Figure S4: Ripe fruits of L. schweinfurthii. Figure S5:
Calibration range of the HPTLC analysis of micropropagated dry leaves’ extract samples against a
reference of ferulic acid 400 µg ml1.
Author Contributions:
Conceptualization, D.M. and H.A.M.M.; Methodology, D.M., A.M.M.G.
and I.S.; Validation, H.A.M.M., E.A.E. and I.S.; Investigation, Funding Acquisition, and Software,
D.M. and I.S.; Data Curation, D.M., E.A.E. and I.S.; Statistical Analysis, Writing—Original Draft
Preparation, D.M.; Writing—Review and Editing, D.M., H.A.M.M., A.M.M.G. and I.S.; Visualization,
A.M.M.G., E.A.E. and I.S.; Resources, Supervision, and Project Administration, I.S. All authors have
read and agreed to the published version of the manuscript.
Funding:
D.M. was funded by a full scholarship (No. 308923) from the Ministry of Higher Education
of the Arab Republic of Egypt. This Article is funded by the Open Access Publication Fund of
Weihenstephan-Triesdorf University of Applied Sciences.
Acknowledgments:
The authors thank Mahmoud H. Sultan, Ramadan Bedair, and Osama Gamal
for their help in collecting the plant material and identification of the plant species.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Yao, R.; Heinrich, M.; Weckerle, C.S. The Genus Lycium as Food and Medicine: A Botanical, Ethnobotanical and Historical
Review. J. Ethnopharmacol. 2018,212, 50–66. [CrossRef] [PubMed]
2.
Stearn, W.T. Lycium. In Flora Europaea; Tutin, T.G., Heywood, V.H., Burges, N.A., Moore, D.M., Valentine, D.H., Walters, S.M.,
Webb, D.A., Eds.; Cambridge University Press: Cambridge, UK, 1972; p. 194.
3.
Shaltout, K.H.; El-Din, A.S.; El-Fahar, R.A.; Beshara, H.M. Associated Species and Threats upon Lycium Schweinfurthii var.
Schweinfurthii in the Deltaic Mediterranean Coast, Egypt. Taeckholmia 2018,38, 107–122.
4.
El-Amier, Y.; El-Halawany, E.; Abdullah, T. Composition and Diversity of Plant Communities in Sand Formations Along the
Northern Coast of the Nile Delta in Egypt. Res. J. Pharm. Biol. Chem. Sci. 2014,5, 826–847.
5.
Khafagi, A.; El- Ghamery, A.; Ghaly, O.; Ragab, O. Fruit and Seed Morphology of Some Species of Solanaceae. Taeckholmia
2018
,
38, 123–140. [CrossRef]
6.
Cardoso, J.C.; Sheng Gerald, L.T.; Teixeira da Silva, J.A. Micropropagation in the Twenty-First Century. Methods Mol. Biol.
2018
,
1815, 17–46. [CrossRef] [PubMed]
7.
Cui, Y.; Deng, Y.; Zheng, K.; Hu, X.; Zhu, M.; Deng, X.; Xi, R. An Efficient Micropropagation Protocol for an Endangered
Ornamental Tree Species (Magnolia sirindhorniae Noot. & Chalermglin) and Assessment of Genetic Uniformity through DNA
Markers. Sci. Rep. 2019,9, 1–10. [CrossRef]
Plants 2021,10, 2089 14 of 16
8.
Kikowska, M.; Thiem, B.; Szopa, A.; Ekiert, H. Accumulation of Valuable Secondary Metabolites: Phenolic Acids and Flavonoids
in Different
in vitro
Systems of Shoot Cultures of the Endangered Plant Species—Eryngium alpinum L. Plant Cell Tissue Organ Cult.
2020,141, 381–391. [CrossRef]
9.
Gao, Y.; Wang, Q.-M.; An, Q.; Cui, J.; Zhou, Y.; Qi, X.; Zhang, L.; Li, L. A Novel Micropropagation of Lycium ruthenicum and
Epigenetic Fidelity Assessment of Three Types of Micropropagated Plants
in vitro
and ex vitro. PLoS ONE
2021
,16, e0247666.
[CrossRef]
10.
Samiei, L.; Mirshahi, H.; Pahnehkolayi, M.D.; Tehranifar, A. Micropropagation of Lycium depressum, a Promising Native Shrub for
Urban landscape. Iran. J. Hortic. Sci. 2020,51, 741–751. [CrossRef]
11.
Silvestri, C.; Sabbatini, G.; Marangelli, F.; Rugini, E.; Cristofori, V. Micropropagation and ex vitro Rooting of Wolfberry. HortScience
2018,53, 1494–1499. [CrossRef]
12.
Jung, W.-S.; Chung, I.-M.; Kim, S.-H.; Chi, H.-Y.; Yu, C.Y.; Ghimire, B.K. Direct Shoot Organogenesis from Lycium chinense Miller
Leaf Explants and Assessment of Genetic Stability Using ISSR Markers. Agronomy 2021,11, 503. [CrossRef]
13.
Samaha, G.M.; Ahmed, M.A.; Abd El-Hameid, A.R. Assessment of Growth and Productivity of Five Peanut Cultivars and Genetic
Diversity Using RAPD Markers. Bull. Natl. Res. Cent. 2019,43, 1–11. [CrossRef]
14.
Thakur, M.; Soni, M.; Sharma, D.P.; Vivek, M.; Sharma, V.
In vitro
Propagation of Plum (Prunus salicina) cv. ‘Santa Rosa’ and
Assessment of Genetic Stability Using RAPD Markers. Indian J. Plant Physiol. 2018,23, 161–168. [CrossRef]
15.
Lema-Rumi´nska, J.; Kulus, D.; Tymoszuk, A.; Varejão, J.M.T.B.; Bahcevandziev, K. Profile of Secondary Metabolites and Genetic
Stability Analysis in New Lines of Echinacea purpurea (L.) Moench Micropropagated via Somatic Embryogenesis. Ind. Crops Prod.
2019,142, 111851. [CrossRef]
16.
Tikendra, L.; Potshangbam, A.M.; Dey, A.; Devi, T.R.; Sahoo, M.R.; Nongdam, P. RAPD, ISSR, and SCoT Markers Based Genetic
Stability Assessment of Micropropagated Dendrobium fimbriatum Lindl. Var. Oculatum Hk. f.-an Important Endangered Orchid.
Physiol. Mol. Biol. Plants 2021,27, 341–357. [CrossRef]
17.
Oliya, B.K.; Chand, K.; Thakuri, L.S.; Baniya, M.K.; Sah, A.K.; Pant, B. Assessment of Genetic Stability of Micropropagated Plants
of Rhynchostylis retusa (L.) Using RAPD Markers. Sci. Hortic. 2021,281, 110008. [CrossRef]
18.
Patel, P.; Rajkumar, B.K.; Parmar, P.; Shah, R.; Krishnamurthy, R. Assessment of Genetic Diversity in Colletotrichum falcatum Went
Accessions Based on RAPD and ISSR Markers. J. Genet. Eng. Bio-Technol. 2018,16, 153–159. [CrossRef]
19.
Wójcik, D.; Trzewik, A.; Kucharska, D. Field Performance and Genetic Stability of Micropropagated Gooseberry Plants (Ribes
grossularia L.). Agronomy 2021,11, 45. [CrossRef]
20.
Tikendra, L.; Amom, T.; Nongdam, P. Molecular Genetic Homogeneity Assessment of Micropropagated Dendrobium moschatum
Sw.—A Rare Medicinal Orchid, Using RAPD and ISSR Markers. Plant Gene 2019,19, 100196. [CrossRef]
21.
Chittora, M. Assessment of Genetic Fidelity of Long Term Micropropagated Shoot Cultures of Achras sapota L. Var. “Cricket Ball”
as Assessed by RAPD and ISSR Markers. Indian J. Biotechnol. 2018,17, 492–495.
22.
Ahmed, M.R.; Anis, M.; Alatar, A.A.; Faisal, M. In Vitro Clonal Propagation and Evaluation of Genetic Fidelity Using RAPD and
ISSR Marker in Micropropagated Plants of Cassia alata L.: A Potential Medicinal Plant. Agrofor. Syst.
2017
,91, 637–647. [CrossRef]
23.
Jong, L.W.; Thien, V.Y.; Yong, Y.S.; Rodrigues, K.F.; Yong, W.T.L. Micropropagation and Protein Profile Analysis by SDS-PAGE of
Gracilaria changii (Rhodophyta, Solieriaceae). Aquac. Rep. 2015,1, 10–14. [CrossRef]
24.
Khattab, S.; El Sherif, F.; El-Garhy, H.A.; Ahmed, S.; Ibrahim, A. Genetic and Phytochemical Analysis of the
in vitro
Regenerated
Pilosocereus robinii by ISSR, SDS-PAGE and HPLC. Gene 2014,533, 313–321. [CrossRef]
25.
Mahmoud, R.A.; Hassan, O.S.; Abou-Hashish, A.; Amin, A. Role of Trehalose during Recovery from Drought Stress in Micro-
propagated Banana (Musa Spp.) Transplants. Res. J. Pharm. Biol. Chem. Sci. 2017,8, 1335–1345.
26.
El-Mageid, I.S. Evaluation of Genetic Stability by Using Protein and ISSR Markers during Callus Development Stage of Some
Date Palm (Phoenix dactylifera L.) Cultivars under Effect of 2,4-D and Picloram. Middle East J. Appl. Sci. 2019,9, 483–493.
27.
Konarska, A. Microstructural and Histochemical Characteristics of Lycium barbarum L. Fruits Used in Folk Herbal Medicine and
as Functional Food. Protoplasma 2018,255, 1839–1854. [CrossRef]
28.
Wink, M. Modes of Action of Herbal Medicines and Plant Secondary Metabolites. Medicines
2015
,2, 251–286. [CrossRef] [PubMed]
29.
Ewais, E.A.; Abd El-Maboud, M.M.; Elhaw, M.H.; Haggag, M.I. Phytochemical Studies on Lycium schweinfurthii Var. Schweinfurthii
(Solanaceae) and Isolation of Five Flavonoids from Leaves. J. Med. Plants Stud. 2016,4, 288–300.
30.
Cushnie, T.P.T.; Lamb, A.J. Recent Advances in Understanding the Antibacterial Properties of Flavonoids. Int. J. Antimicrob.
Agents 2011,38, 99–107. [CrossRef]
31.
Tattini, M.; Galardi, C.; Pinelli, P.; Massai, R.; Remorini, D.; Agati, G. Differential Accumulation of Flavonoids and Hydrox-
ycinnamates in Leaves of Ligustrum vulgare under Excess Light and Drought Stress. New Phytol.
2004
,163, 547–561. [CrossRef]
[PubMed]
32.
Barceló, J.; Poschenrieder, C. Fast Root Growth Responses, Root Exudates, and Internal Detoxification as Clues to the Mechanisms
of Aluminium Toxicity and Resistance: A Review. Environ. Exp. Bot. 2002,48, 75–92. [CrossRef]
33.
Ryan, K.G.; Swinny, E.E.; Markham, K.R.; Winefield, C. Flavonoid Gene Expression and UV Photoprotection in Transgenic and
Mutant Petunia Leaves. Phytochemistry 2002,59, 23–32. [CrossRef]
34. Treutter, D. Significance of Flavonoids in Plant Resistance: A Review. Environ. Chem. Lett. 2006,4, 147–157. [CrossRef]
35.
Lin, D.; Xiao, M.; Zhao, J.; Li, Z.; Xing, B.; Li, X.; Kong, M.; Li, L.; Zhang, Q.; Liu, Y.; et al. An Overview of Plant Phenolic
Compounds and Their Importance in Human Nutrition and Management of Type 2 Diabetes. Molecules
2016
,21, 1374. [CrossRef]
Plants 2021,10, 2089 15 of 16
36. Pietta, P.G. Flavonoids as Antioxidants. J. Nat. Prod. 2000,63, 1035–1042. [CrossRef]
37. Maleki, S.J.; Crespo, J.F.; Cabanillas, B. Anti-Inflammatory Effects of Flavonoids. Food Chem. 2019,299, 125124. [CrossRef]
38.
Yamamoto, Y.; Gaynor, R.B. Therapeutic Potential of Inhibition of the NF-
κ
B Pathway in the Treatment of Inflammation and
Cancer. J. Clin. Invest. 2001,107, 135–142. [CrossRef]
39.
Lani, R.; Hassandarvish, P.; Shu, M.H.; Phoon, W.H.; Chu, J.J.H.; Higgs, S.; Vanlandingham, D.; Abu Bakar, S.; Zandi, K. Antiviral
Activity of Selected Flavonoids against Chikungunya Virus. Antiviral Res. 2016,133, 50–61. [CrossRef] [PubMed]
40.
Manner, S.; Skogman, M.; Goeres, D.; Vuorela, P.; Fallarero, A. Systematic Exploration of Natural and Synthetic Flavonoids for the
Inhibition of Staphylococcus aureus Biofilms. Int. J. Mol. Sci. 2013,14, 19434–19451. [CrossRef] [PubMed]
41.
Peterson, J.J.; Dwyer, J.T.; Jacques, P.F.; McCullough, M.L. Associations between Flavonoids and Cardiovascular Disease Incidence
or Mortality in European and US Populations. Nutr. Rev. 2012,70, 491–508. [CrossRef] [PubMed]
42. Rezende, B.A.; Pereira, A.; Cortes, S.; Lemos, V. Vascular Effects of Flavonoids. Curr. Med. Chem. 2016,23, 87–102. [CrossRef]
43.
Cazarolli, L.; Zanatta, L.; Alberton, E.; Bonorino Figueiredo, M.S.; Folador, P.; Damazio, R.; Pizzo-latti, M.; Barreto Silva, F.R.
Flavonoids: Prospective Drug Candidates. Mini-Rev. Med. Chem. 2008,8, 1429–1440. [CrossRef] [PubMed]
44.
Jamous, R.; Zaitoun, S.; Husein, A.; Qasem, I.; Ali-Shtayeh, M. Screening for Biological Activities of Medicinal Plants Used in
Traditional Arabic Palestinian Herbal Medicine. Eur. J. Med. Plants 2015,9, 1–13. [CrossRef]
45.
Elbermawi, A.; Halim, A.F.; Mansour, E.S.S.; Ahmad, K.F.; Ashour, A.; Amen, Y.; Shimizu, K. A New Glucoside with a Potent
α-Glucosidase Inhibitory Activity from Lycium schweinfurthii.Nat. Prod. Res. 2021,35, 976–983. [CrossRef]
46.
Smetanska, I. Sustainable Production of Polyphenols and Antioxidants by Plant
in vitro
Cultures. In Bioprocessing of Plant In Vitro
Systems; Pavlov, A., Bley, T., Eds.; Springer: Cham, Switzerland, 2018; pp. 225–269. ISBN 978-3-319-54599-8.
47.
Ali, A.M.A.; El-Nour, M.E.A.M.; Yagi, S.M. Total Phenolic and Flavonoid Contents and Antioxidant Activity of Ginger (Zingiber
officinale Rosc.) Rhizome, Callus and Callus Treated with Some Elicitors. J. Genet. Eng. Biotechnol. 2018,16, 677–682. [CrossRef]
48.
El-Hawary, S.S.; Abd El-Kader, E.M.; Rabeh, M.A.; Abdel Jaleel, G.A.; Arafat, M.A.; Schirmeister, T.; Abdelmohsen, U.R. Eliciting
Callus Culture for Production of Hepatoprotective Flavonoids and Phenolics from Sequoia sempervirens (D. Don Endl). Nat. Prod.
Res. 2020,34, 3125–3129. [CrossRef]
49.
Darwish, H.Y.; Ahmed, S.M. Elicitors Enhancing Phenolics Content and Related Gene Expression Variation in Petal—Derived
Calli of Rosa damascena Mill. Egypt. J. Bot. 2020,60, 71–79. [CrossRef]
50.
Çelik, M.; Keskin, N.; Özdemir, F.A. The Effects of UV Irradiation and Incubation Time on
in vitro
Phenolic Compound Production
in “Karaerik” Callus Culture. Kahramanmara¸s Sütçü ˙
Imam Üniversitesi Tarım Ve Do˘ga Derg. 2020,23, 1428–1434. [CrossRef]
51.
Martins, M.; Tenreiro, R.; Oliveira, M.M. Genetic Relatedness of Portuguese Almond Cultivars Assessed by RAPD and ISSR
Markers. Plant Cell Rep. 2003,22, 71–78. [CrossRef] [PubMed]
52.
Williams, J.G.K.; Kubelik, A.R.; Livak, K.J.; Rafalski, J.A.; Tingey, S.V. DNA Polymorphisms Amplified by Arbitrary Primers Are
Useful as Genetic Markers. Nucleic Acids Res. 1990,18, 6531–6535. [CrossRef]
53.
Laemmli, U.K. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature
1970
,227, 680–685.
[CrossRef] [PubMed]
54.
Marinova, D.; Ribarova, F.; Atanassova, M. Total Phenolics and Total Flavonoids in Bulgarian Fruits and Vegetables. J. Univ.
Chem. Technol. Metall. 2005,40, 255–260.
55.
Olalere, O.A.; Abdurahman, H.N.; Yunus, R.B.M.; Alara, O.R.; Ahmad, M.M.; Zaki, Y.H.; Abdlrhman, H.S.M. Parameter Study,
Antioxidant Activities, Morphological and Functional Characteristics in Microwave Extraction of Medicinal Oleoresins from
Black and White Pepper. J. Taibah Univ. Sci. 2018,12, 730–737. [CrossRef]
56.
Gabr, A.M.M.; Mabrok, H.B.; Ghanem, K.Z.; Blaut, M.; Smetanska, I. Lignan Accumulation in Callus and Agrobacterium Rhizogenes-
Mediated Hairy Root Cultures of Flax (Linum usitatissimum). Plant Cell Tissue Organ Cult. 2016,126, 255–267. [CrossRef]
57.
Sneath, P.H.A.; Sokal, R.R. Numerical Taxonomy: The Principles and Practice of Numerical Classification; W. H. Freeman: San Francisco,
CA, USA, 1973.
58.
Hasan, M.N.; Nigar, S.; Rabbi, M.A.K.; Mizan, S.B.; Rahman, M.S. Micropropagation Of Strawberry (Fragaria x Ananassa Duch.).
Int. J. Sustain. Crop. Prod. 2010,5, 36–41.
59.
Amghar, I.; Diria, G.; Boumlik, I.; Gaboun, F.; Iraqi, D.; Labhilili, M.; Mentag, R.; Meziani, R.; Mazri, M.A.; Ibriz, M.; et al. An
Efficient Regeneration Pathway through Adventitious Organogenesis for the Endangered Argania spinosa (L.) Skeels. Vegetos
2021
,
34, 355–367. [CrossRef]
60.
Thomas, T.D.; Maseena, E.A. Callus Induction and Plant Regeneration in Cardiospermum halicacabum Linn. an Important Medicinal
Plant. Sci. Hortic. 2006,108, 332–336. [CrossRef]
61.
Irvani, N.; Solouki, M.; Omidi, M.; Zare, A.R.; Shahnazi, S. Callus Induction and Plant Regeneration in Dorem ammoniacum D., an
Endangered Medicinal Plant. Plant Cell Tissue Organ Cult. 2010,100, 293–299. [CrossRef]
62.
Sen, M.K.; Nasrin, S.; Rahman, S.; Jamal, A.H.M. In Vitro Callus Induction and Plantlet Regeneration of Achyranthes aspera L., a
High Value Medicinal Plant. Asian Pac. J. Trop. Biomed. 2014,4, 40–46. [CrossRef]
63.
Ozdemir, F.A.; Gur, N. In Vitro Propagation of Cataloglu Apricot (Prunus armeniaca L.) Cultivar Using Apical Node As Explant.
Prog. Nutr. 2018,20, 176–181. [CrossRef]
64.
El-Ghamery, A.A.; Khafagi, A.A.F.; Ragab, O.G. Taxonomic Implication of Pollen Morphology and Seed Protein Electrophoresis
of Some Species of Solanaceae in Egypt. Azhar Bull. Sci. 2018,29, 43–54.
Plants 2021,10, 2089 16 of 16
65.
Khawale, R.N.; Singh, S.K.; Yerramilli, V.; Grover, M. Assessment of Clonal Fidelity of Micropropagated Grape (Vitis vinifera L.)
Plants by RAPD Analysis. Physiol. Mol. Biol. Plants 2006,12, 189–192.
66.
Lakshmanan, V.; Reddampalli Venkataramareddy, S.; Neelwarne, B. Molecular Analysis of Genetic Stability in Long-Term
Micropropagated Shoots of Banana Using RAPD and ISSR Markers. Electron. J. Biotechnol. 2007,10, 717–3458. [CrossRef]
67.
Martins, M.; Sarmento, D.; Oliveira, M.M. Genetic Stability of Micropropagated Almond Plantlets, as Assessed by RAPD and
ISSR Markers. Plant Cell Rep. 2004,23, 492–496. [CrossRef] [PubMed]
68.
Parida, R.; Mohanty, S.; Nayak, S. Molecular Characterization of Endangered Medicinal Plant Species Hedychium coronarium from
Eastern India. Int. J. Pharm. Pharm. Sci. 2017,9, 173–178. [CrossRef]
69.
Osman, G.; Munshi, A.; Altf, F.; Mutawie, H. Genetic Variation and Relationships of Zea mays and Sorghum Species Using
RAPD-PCR and SDS-PAGE of Seed Proteins. Afr. J. Biotechnol. 2013,12, 4269–4276. [CrossRef]
70.
Seesangboon, A.; Pokawattana, T.; Eungwanichayapant, P.D.; Tovaranonte, J.; Popluechai, S. Effects of 6-Benzyladenine on
Jatropha Gene Expression and Flower Development. Russ. J. Plant Physiol. 2018,65, 345–356. [CrossRef]
71.
Alizadeh, M.; Singh, S.K. Molecular Assessment of Clonal Fidelity in Micropropagated Grape (Vitis Spp.) Rootstock Genotypes
Using RAPD and ISSR Markers. Iran. J. Biotechnol. 2009,7, 17–44.
72.
Sulaiman, C.T.; Balachandran, I. Total phenolics and total flavonoids in selected Indian medicinal plants. Indian J. Pharm. Sci.
2012,74, 258–260. [CrossRef] [PubMed]
73.
Yasir, M.; Sultana, B.; Anwar, F. LC–ESI–MS/MS Based Characterization of Phenolic Components in Fruits of Two Species of
Solanaceae. J. Food Sci. Technol. 2018,55, 2370–2376. [CrossRef] [PubMed]
74.
Srivastava, S.; Singh, A.P.; Singh Rawat, A.K. A HPTLC Method for the Identification of Ferulic Acid from Lycopodium clavatum.
Asian Pac. J. Trop. Biomed. 2012,2, S12–S14. [CrossRef]
75.
Goudar, G.; Sathisha, G.J. Effect of Processing on Ferulic Acid Content in Foxtail Millet (Setaria italica) Grain Cultivars Evaluated
by HPTLC. Orient. J. Chem. 2016,32, 2251–2258. [CrossRef]
76.
Verma, S.C.; Rani, R.; Pant, P.; Padhi, M.M.; Jain, C.L.; Babu, R. Quantitative Determination of Ferulic Acid in Ricinus communis
Linn. Leaves and Its Geographical Variation Using HPTLC Finger-print. Chem. Sin. 2011,2, 127–135.
... Several reports have been conducted about the in vitro production of flavonoids using different biotechnological methods, including callus and cell suspension cultures [33]. The production of phenolic and flavonoid compounds was reported in micropropagated plants of L. schweinfurthii [34]. Obtaining phenolic and flavonoid compounds from the plant callus cultures is a common tool in recent decades, such as in Plantago ovata Forsk. ...
... The total phenolic content of callus and cell cultures was determined by using the Folin-Ciocalteu assay, as described by Mamdouh et al. [34]. Gallic acid (Sigma-Aldrich, St. Louis, MO, USA) was used for generating a standard curve using standard solutions of 10,20,30,40,50,100,150,200, and 300 µg ml −1 . ...
... Total flavonoid content was measured by aluminum chloride assay as described by Mamdouh et al. [34]. Catechin (Sigma-Aldrich, Louis, MO, USA) was used for generating a standard curve using standard solutions of 20, 40, 60, 80, 100, and 120 µg mL −1 . ...
Article
Full-text available
Lycium schweinfurthii is a traditional medicinal plant grown in the Mediterranean region. As it is used in folk medicine to treat stomach ulcers, it took more attention as a source of valuable secondary metabolites. The in vitro cultures of L. schweinfurthii could be a great tool to produce secondary metabolites at low costs. The presented study aimed to introduce and optimize a protocol for inducing callus and cell suspension cultures as well as estimating phenolic, flavonoid compounds, and antioxidant activity in the cultures of the studied species. Three plant growth regulators (PGRs) were supplemented to MS medium solely or in combination to induce callus from leaf explants. The combination between 2,4-dichlorophenoxy acetic acid (2,4-D) and 1-naphthyl acetic acid (NAA) induced callus in all explants regardless of the concentration. The highest fresh weight of callus (3.92 g) was obtained on MS medium fortified with 1 mg L−1 of both 2,4-D and NAA (DN1) after 7 weeks of culture. DN1 was the best medium for callus multiplication regarding the increase in fresh weight and size of callus. Otherwise, the highest phenolics, flavonoids, and antioxidant activity against DPPH free radicals were of callus on MS fortified with 2 mg L−1 NAA (N2). The cell suspension cultures were cultivated on a liquid N2 medium with different sucrose concentrations of 5–30 g L−1 to observe the possible effects on cells’ multiplication and secondary metabolite production. The highest fresh and viable biomass of 12.01 g was obtained on N2 containing 30 g L−1 sucrose. On the other hand, the cell cultures on N2 medium of 5 and 30 g L−1 sucrose produced phenolics and flavonoids, and revealed antioxidant activity against DPPH and ABTS+ free radicals more than other sucrose concentrations. The presented protocol should be useful in the large-scale production of phenolic and flavonoid compounds from callus and cell cultures of L. schweinfurthii.
... This may be explained by the fact that in vitro culture systems and PGRs enhance or accumulate several of the bioactive compounds. In vitro culture has been shown to increase the amounts of phytochemical compounds in many medicinal plants [15][16][17][42][43][44], which is consistent with our findings. In the Aloe arborescens, media supplemented with PGRs increased the quantity of total phenolics, tannins, and flavonoids compared to media without PGRs during micropropagation [45]. ...
Article
Full-text available
Efficient methods for callus induction and the high-frequency plant regeneration of Ruta chalepensis L. were established, and the phytochemical potential and antioxidant activity of a donor plant, ex-vitro-established micropropagated plants, and callus were also studied. Yellowish-green callus was induced with a frequency of 97.8% from internode shoot segments of the donor plant growing in soil in the botanical garden cultured on Murashige and Skoog (MS) medium containing 10 μM 2,4-D (2,4-dichlorophenoxyacetic acid) and 1 μM BA (6-benzyladenine). Adventitious shoots were regenerated from the yellowish-green callus on MS medium containing 5.0 μM (BA) and 1.0 μM 1-naphthaleneacetic acid (NAA), with a regeneration frequency of 98.4% and a maximum of 54.6 shoots with an average length of 4.5 cm after 8 weeks. The regenerated shoots were rooted in a medium containing 1.0 μM IBA (indole-3-butyric acid) and successfully transferred to ex vitro conditions in pots containing normal garden soil, with a 95% survival rate. The amounts of alkaloids, phenolics, flavonoids, tannins, and antioxidant activity of the ex-vitro-established micropropagated plants were higher than in the donor plant and callus. The highest contents of hesperidin and rutin (93.3 and 55.9 µg/mg, respectively) were found in the ex-vitro-established micropropagated plants compared to those obtained from the donor plant (91.4 and 31.0 µg/mg, respectively) and callus (59.1 and 21.6 µg/mg, respectively). The genetic uniformity of the ex-vitro-established micropropagated plants was appraised by the ISSR markers and compared with the donor plant. This is the first report describing the callus-mediated plant regeneration, as well as the production of phenolic compounds and antioxidant activities in R. chalepensis, which might be a potential alternative technique for the mass propagation and synthesis of bioactive compounds such as hesperidin and rutin.
... Different groups of metabolites have been examined in the biomass of shoots, roots or calluses grown in vitro in several plant species such as: shoot cultures of Ruta graveolens [22], a biomass of shoots and differentiating callus culture of Schisandra chinensis [23], the shoots and roots of Eryngium planum [24], biomass from Aronia melanocarpa shoots and callus cultures [25], the leaves and roots of Rehmannia glutinosa [26], leaves of micropropagated plants of Lycium schweinfurthii [27], leaves of Kaempferia parviflora from in vitro cultured plantlets [28], tissue cultures of six genotypes of Deschampsia Antarctica [29], biomass of in vitro shoots and roots of Eryngium species (E. campestre, E. maritimum, E. planum) [30] or leaves from micropropagated plantlets of Passiflora setacea cv BRS Pérola do Cerrado [31]. ...
Article
Full-text available
In vitro culture has become a dependable approach for the mass production of plant material as the market for innovative plant-derived medicinal approaches has grown significantly. Furthermore, because it permits manipulation of biosynthetic routes to boost the production and accumulation of certain compounds, this technology has enormous potential for the manufacture of natural bioactive chemicals. As a result, the goal of this study was to develop an efficient micropropagation system for biomass production and to investigate the accumulation of bioactive compounds from Vaccinium corymbosum L., Duke and Hortblue Petite cultivars. Two in vitro plant tissue culture systems were used for shoots production: a solid medium (5 g/L Plant agar) and liquid medium (Plantform bioreactor). The culture medium used was Woddy Plant Medium (WPM) supplemented with two growth regulators: 0.5 mg/L and 1 mg/L zeatina (Z) and 5 mg/L N6-(2-Isopentenyl) adenine (2iP). The content of phenolic compounds, carotenoids, and chlorophylls of the in vitro shoot extracts were examined via the HPLC-DAD-MS/MS technique. The results showed that cv. Hortblue Petite produced a higher amount of biomass compared with cv. Duke, on all variants of culture media in both systems (solid and liquid), while the shoots extract of the Duke variety in the liquid culture system (under all concentrations of growth regulators) had the highest content of total phenolic compounds (16,665.61 ± 424.93 μg/g). In the case of the lipophilic compounds analysed (chlorophylls and carotenoids), the solid medium reported the highest values, whereas media supplemented with 0.5 mg/L Z was proved to have the richest total content for both cultivars.
Article
Full-text available
An efficient in vitro propagation system through adventitious organogenesis is reported for argan (Argania spinosa (L.) Skeels). Seed germination of two argan genotypes, ‘Mejji’ and ‘R’zwa’, was evaluated under different treatments. Afterwards, the effects of different factors on adventitious shoot bud induction, shoot multiplication, elongation and rooting were evaluated. The findings of this study showed that soaking argan seeds in GA3 during 12 h speeds germination increases the germination percentage. Besides, the use of a sucrose-free medium, without ammonium and with a low nitrate concentration significantly increased the germination percentage. To induce organogenesis, different seedling-derived explants were used. However, epicotyl segments were the only explants capable of regenerating adventitious shoot buds. Highest organogenesis percentage (79.17%) was observed in genotype ‘Mejji’ on Murashige and Skoog (MS) medium containing 2 mg L−1 6-benzylaminopurine (BAP) under dark conditions. Adventitious shoot bud multiplication was performed on MS medium supplemented with different combinations of BAP and GA3. The highest number of adventitious shoot buds per explant (4.0) was observed on MS medium containing 1 mg L−1 BAP and 2 mg L−1 GA3. Regarding in vitro root induction, it was found that combining indole-3-butyric acid (IBA) and putrescine is necessary for rhizogenesis. The highest rooting percentage (56.66% in genotype ‘R’zwa’) was observed on MS medium supplemented with 1.5 mg L−1 IBA and 160 mg L−1 putrescine, with an average number of 2.74 roots per shoot and an average root length of 1.93 cm. The regenerated plantlets were successfully acclimatized and showed normal growth and development.
Article
Full-text available
An efficient in vitro direct shoot regeneration system has been described for Lycium chinense Miller using leaf explants. Influence of various parameters such as growth regulator concentration, explant type, effect of basal salt type, Murashige and Skoog (1962) medium (MS), Schenk and Hildebrandt (1972) medium (SH), Gamborg et al. (1968) medium (B5), and carbon sources (sucrose, maltose, and fructose) on the regenerating shoots has been studied. Micromorphological studies and genetic fidelity of regenerated shoots were assessed and compared with those of the donor plants. Among the different concentrations of plant growth regulator (PGRs) tested, MS supplemented with lower concentration of 6-benzylaminopurine (BAP) (0.5 mgL−1) and thidiazuron (TDZ) (0.5 mgL−1) increased the frequency of shoot. Comparatively, indole-3-butyric acid (IBA) was more effective in the regeneration and growth of the root system. A higher number of root formation (6.67 ± 1.25) was observed when the rooting medium comprised half-strength MS salts supplemented with 3% sucrose. The surviving plantlets were gradually transferred to the greenhouse and natural soil. More than 90% of the plantlets survived and matured within 85 days. Similarity in the band patterns produced by inter simple sequence repeat (ISSR) primers confirmed the genetic stability and uniformity between the regenerated and donor plants. The present optimized direct shoot regeneration system may be useful for mass propagation and improving the genetic traits in L. chinense.
Article
Full-text available
Lycium ruthenicum is an excellent eco-economic shrub. Numerous researches have been conducted for the function of its fruits but scarcely focused on the somaclonal variation and DNA methylation. An efficient micropropagation protocol from leaves and stems of L . ruthenicum was developed in this study, in which not only the leaf explants but also the stem explants of L . ruthenicum were dedifferentiated and produced adventitious buds/multiple shoots on one type of medium. Notably, the efficient indirect organogenesis of stem explants was independent of exogenous auxin, which is contrary to the common conclusion that induction and proliferation of calli is dependent on exogenous auxin. We proposed that sucrose supply might be the crucial regulator of stem callus induction and proliferation of L . ruthenicum . Furthermore, results of methylation-sensitive amplified polymorphism (MSAP) showed that DNA methylation somaclonal variation (MSV) of CNG decreased but that of CG increased after acclimatization. Three types of micropropagated plants (from leaf calli, stem calli and axillary buds) were epigenetically diverged more from each other after acclimatization and the ex vitro micropropagated plants should be selected to determine the fidelity. In summary, plants micropropagated from axillary buds and leaves of L . ruthenicum was more fidelity and might be suitable for preservation and propagation of elite germplasm. Also, leaf explants should be used in transformation. Meanwhile, plants from stem calli showed the highest MSV and might be used in somaclonal variation breeding. Moreover, one MSV hotspot was found based on biological replicates. The study not only provided foundations for molecular breeding, somaclonal variation breeding, preservation and propagation of elite germplasm, but also offered clues for further revealing novel mechanisms of both stem-explant dedifferentiation and MSV of L . ruthenicum .
Article
Full-text available
Gooseberry (Ribes grossularia L.) is a small fruit crop producing valuable fruits, which is constantly gaining importance. In vitro propagation of this species can significantly support the production of virus-free planting material and accelerate the introduction of new cultivars to the market. The aim of presented study was to assess field performance and genetic stability of micropropagated plants (MPs) of four gooseberry cultivars, “Captivator”, “Hinnonmaki Rot”, “Invicta”, and “Resika”. The growth vigor and yield of MPs and plants propagated by standard methods from softwood cuttings (ST) were evaluated in a field experiment. Microscopic observations of the number and length of the stomata of MP and ST plants were carried out. Two DNA-based techniques, amplified fragment length polymorphism (AFLP) and inter simple sequence repeat (ISSR), were used to assess genetic stability of MP plants. For analysis of genetic stability of ST plants, the ISSR technique was applied. For three cultivars, Captivator, Hinnonmaki Rot, and Invicta, the plants’ growth vigor and fruit yield were greater in MP plants than in ST plants. In the case of Resika, most of these parameters were higher in ST plants. Microscopic observations of the stomata indicated a lack of differences in the length between MP and ST plants, while the stomata frequency on leaves of MP plants was higher than that of ST plants. The genetic variability of MP plants, assessed by AFLP, ranged from 0.35% for Hinnonmaki Rot to 2.12% for Resika. The results of ISSR analysis of MP plants showed variability from 0% in the case of Hinnonmaki Rot and Resika to 4% and 8.69% for Captivator and Invicta, respectively. No polymorphism was detected among ST plants of all analyzed gooseberry cultivars.
Article
Full-text available
In this study, the effect of Ultraviolet (UV) irradiation on induction of individual and total phenolics production on callus cultures of 'Karaerik' grape cultivar was investigated. Callus tissues were obtained from the leaves of the cuttings grown in in vitro plants. As a culture medium, Gamborg B-5 was utilized with 0.1 mg L-1 NAA (Naphthaleneacetic acid) and 0.2 mg L-1 Kin (Kinetin). Callus tissues were sub-cultured twice with 21 days intervals. After the second subculture, 12-day-old callus tissues were exposed to 254 nm UV-C light at 10 cm distance from the source for 10 and 15 min by opening covers of the petri dishes in sterile cabin. After the treatment, callus tissues were incubated under dark conditions. Phenolic compounds were measured at 24 th , 48 th and 72 nd hours. Individual phenolic compounds were analyzed by HPLC (High Pressure Liquid Chromatography) and total phenolic compounds were measured by spectrophotometer. As a result of the study, it was found that UV irradiation was effective for induction the production of phenolic compounds in the callus tissues of 'Karaerik' grape cultivar and this effect was closely related to the application time.
Article
Full-text available
In vitro cultures give the opportunity to perform the phytochemical studies on the protected species without harvesting the plant material from the natural environment. Shoots of Eryngium alpinum L. were multiplied on Murashige and Skoog (MS) medium in various systems, namely on the solid media and in two liquid cultures—stationary and agitated, as well as via regeneration from callus. The biomass increments were closely correlated with the number of shoots arising from one explant, which was connected with the supplementation of the culture media with the studied plant growth regulators. The methanolic extracts from shoots grown in the tested systems were subjected to phenolic acids and flavonoids qualitative and quantitative analysis. Biomass from in vitro shoot cultures accumulated from 19.59 to 32.95 times more phenolic acids [the total content ranged from 272.52 to 458.38 mg/100 g dry weight (DW)] and from 3.02 to 4.43 times more flavonoids (the total content ranged from 100.03 to 146.98 mg/100 g DW), depending on the culture system, than the extracts from basal leaves from the intact plant (13.91 and 33.16 mg/100 g DW, respectively). The phenolics present in shoot cultures include seven phenolic acids—3,4-dihydroxyphenylacetic, caftaric, caffeic, neochlorogenic, chlorogenic, isochlorogenic, and rosmarinic acids, and three flavonoids—isoquercetin, quercitrin and robinin. The best system for shoot proliferation resulting in the highest biomass growth and phenolic acids and flavonoids accumulation was solid culture on MS medium with BAP, IAA, and GA3 (each 1.0 mg/l). The aim of this work was to check the effect of various culture systems (stationary and agitated, on solidified and in liquid media) on the production of phenolic compounds in E. alpinum shoots cultured in vitro.
Article
Full-text available
Background This study was conducted to evaluate the genetic diversity of five peanut cultivars grown under field conditions. A field experiment was conducted using five peanut cultivars (Giza-5, Giza-6, Ismailia-1, Gregory, and R92) in a randomized complete block design with five replications during two following seasons to estimate the performance of five peanut cultivars for vegetative growth, yield, and yield component traits as well as seed quality traits. Twenty RAPD primers were used to identify a unique fingerprint for each of five cultivars. Results Giza-6 cultivar surpassed all the tested peanut cultivars in the most vegetative growth traits and yield and its components traits, while the lowest values were observed in Giza-5 cultivar. The dendrogram constructed from RAPD analysis showed that Gregory and Giza-5 were the most distant among five peanut cultivars. Conclusions RAPD markers are useful in the detection of genetic diversity of peanut. The availability of genetic diversity is important for the genetic improvement of peanut.
Article
Full-text available
Echinacea purpurea (L.) Moench is a plant species important for the phytopharmaceutical industry and in horticulture. Currently, there is lack of standardized plant material with an increased content of secondary meta-bolites in purple coneflower. The following research meets the expectations of the industry, as new selected lines of purple coneflower were micropropagated by somatic embryogenesis. The plant lines were analyzed both in terms of the content of main secondary metabolites by High Performance Liquid Chromatography (HPLC), as well as the genetic stability within the line and the genetic distance between lines using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) genetic markers. Significant differences were found in the relative percentage composition of individual phenolic acids in the tested plant material. Among six selected lines of Echinacea purpurea, three were characterized by a higher content of cichoric acid in relation to the other lines studied. A higher mean polymorphism rate (> 90%) was found with the RAPD technique, with a total of 1427 scorable bands produced (142.7 products per one primer). Unlike the RAPD analysis, ISSRs detected mostly monomorphic loci (63.4%), followed by polymorphic ones (36.6%), while there were no specific loci present. Cluster analysis of both marker systems showed that the tested genotypes were grouped according to their respective lines.
Article
Rhynchostylis retusa (L.) is an epiphytic orchid having both medicinal and ornamental value. The present study aimed to develop a protocol for mass propagation of R. retusa and evaluate the genetic stability of in vitro regenerants. The immature seeds obtained from capsule of mother plants (wild type) were cultured on different strength of MS medium viz. full (FMS), half (HMS), quarter (QMS) supplemented with BAP (0.5 mg/L), NAA (0.5 mg/L), and 5% or 10 % coconut water (CW). The earliest germination and protocorm development were observed on HMS and QMS media. Mature protocorms produced higher number of shoots (12.8) and longest shoots (5.3 cm) on FMS supplemented with 10 % CW. The maximum number of roots (7.3) and root length (5.0 cm) were observed on FMS when supplemented with fungal elicitor CVS4 (extracted from the stem of Vanda cristata). Ten RAPD primers were used to analyze genetic stability among six samples (five in vitro and one mother plant) produced a total of 23 fragments ranging from 275bp to 1100bp. The polymorphic information content (PIC) values ranged from 0.28 to 0.50. The amplified bands of all the samples of in vitro plants were similar to bands of mother plant. The result demonstrated the genetic stability of the micropropagated plants of R. retusa. Research reported here is indicating the applicability of tissue culture for true-to-type plant production and conservation of R. retusa.
Article
Dendrobium fimbriatum is an ornamental and medicinal orchid listed in the Red data book of IUCN. Phytohormones' effect on the in vitro regeneration of the orchid was studied using Mitra medium supplemented with different growth regulators. KN produced effective shoot formation when present alone or in combination with IBA or NAA. The shooting was gradually increased when KN concentration was increased from 0.8 to 4.8 mg L-1 , but the opposite response was observed with BAP at higher concentration (4.8 mg L-1). IBA either in combination with BAP or KN promoted effective root development and multiplication. Micropropagated orchids grown in the basal medium devoid of any phytohormone showed 100% monomorphism, while low genetic polymorphism of 1.52% (RAPD-Random Amplification of Polymorphic DNA), 1.19% (ISSR-Inter Simple Sequence Repeat) and 3.97% (SCoT-Start Codon Targeted) was exhibited among the regenerants propagated in the hormone enriched medium. UPGMA (Unweighted pair group method using arithmetic averages) dendrograms showed the grouping of mother plant (MP) with the in vitro regenerants. The principal coordinate analysis (PCoA) further confirmed the clustering patterns as determined by the cluster analysis. The study reported for the first time the successful in vitro propagation of Dendrobium fimbriatum and their genetic stability assessment using molecular markers.