Introduction
Sorghum (Sorghum bicolor) is a tropical grass with high adaptation and productivity in hot and dry climate regions. One
of the Indonesian superior hybrid Numbu cv, is a sweet
sorghum hybrid from the SADC (South
African Development Community) IS23509 strain. The
sorghum hybrid Numbu cv is drought and heat tolerant, and rust disease
and leaf spot resistant. Furthermore, it has
an average height of 135 cm, immature panicle closed by leaf and opened when
mature, harvesting age of 100–110 days and a productivity of 7.695 ton/ha. Sorghum is an important plant
used for the production of food, feed, renewable
energy, and national food diversification (Pabendon et al. 2012). Therefore, the biotechnology is
an important technique for improving sorghum cultivars that requires
development of the tissue culture. However, tissue culture of sorghum is found an obstacle due to tissue
browning caused by a high level of phenolic compound production and recalcitrant
traits.
Somatic embryogenesis (SE) in tissue culture is an essential process for micropropagation starting with the formation of an embryo from a somatic cell, which then
develops into a complete plant (Ikeuchi et al. 2013). There are
several advantages such as bipolar
structure for shoot and root, plant
propagation improvement, as well as facilitation of
physiological and biochemical plant study. The SE process consists of 3
stages, namely callus induction, proliferation, and regeneration. Several studies explored the
embryogenic callus induction of sorghum using different explant sources or plant growth regulators (PGRs). Furthermore, some of the explants used for the induction include leaf whorl (Silva et al. 2020), immature embryo (Flinn et al. 2020), mature embryo (Avci 2019) and leaf (Amali et al. 2014), but they all have a low
regeneration rate. Combining PGRs
is expected to overcome this problem as well
as to improve the induction of embryogenic
callus and its regeneration into sorghum plantlets.
The PGRs have an essential role in the physiological and genetic process for the growth and development of plants. Recently, 1–6 mg L-1 of 2,4-D was used for embryogenic callus induction in sorghum. The 2,4-D is an auxin that is often used for callus induction from explant tissue in various
plants (Hu et al. 2000; Ikeuchi et al. 2013). The callus induction was achieved by adding of 2,4-D at different
concentration and combination with kinetin or supplements, such as
proline, glutamine, or casein hydrolysate (Pola et al. 2009; Wu et
al. 2014). The combination of proline (as the nitrogen source) and auxin improved sorghum callus induction and generated a large amount
of callus (Assem et al. 2014). The concentration of proline at 2 mg L-1 increased the number of embryogenic callus for Ferula jaeschkeana (Sharma and Arun 2020) and Malaysian wild
rice at 3 g L-1 (Paramasivam
and Harikrishna 2020). However, increasing the
induction yield by combining PGRs for sorghum has not been reported, specifically somatic embryogenesis of Indonesian Numbu cv.
Another problem for the
micropropagation of sorghum is an ability of embryogenic callus to regenerate
into whole plants due to browning caused by phenolic compound. Phenolic
compound is produced as a result of oxidation stress that interfere enzyme activities, poison to the plant tissues and inhibit
cellular growth (Feng et al. 2007). It also causes a low number or unsuccessful callus regeneration into
shoot and root. Several cytokinins have been used to regenerate sorghum callus into plantlet, such as kinetin (Assem et al. 2014), BAP (Dreger et al. 2019), Zeatin (Chou et al. 2020), and TDZ (Karumba 2021). Furthermore, when the cytokinin concentration is higher than auxin, it triggers cell division as well as tissues or organ development in sorghum (Liu et al. 2015; Flinn et al. 2020). Previous study showed that supplementing an MS medium with 1 mg L-1 BAP and 1 g L-1
IAA can regenerate 70% callus of sorghum
variety, namely Tx430 (Kanani and Sayadat 2020). Karumba (2021) also reported a high
regeneration rate when 4 mg L-1 BAP was added to the medium. However, the toxic effect of browning or phenolic compound production on explants during regeneration cannot be avoided. The addition of L-proline to the culture medium reduces the production of the toxic pigment and also helps to obtain a more friable embryogenic callus. Proline acts as an antioxidant to retard the browning of explants caused by
phenolic oxidation (Blistrubienė et al. 2020).
This study aims to improve propagation of sorghum Numbu cv. using
the explant seed and meristematic leaf whorl. The 2,4-D was combined with proline to induce embryogenic callus. The combination of NAA and BAP was further used for
regeneration, while proline was added as a phenolic
retardant to accelerate
the process. The
result showed that the combination of PGRs is an
important treatment, which helps induce embryogenic callus development and its regeneration into plantlets.
Materials and Methods
Plant material and sterilization
Sorghum Numbu
cv seeds were provided by the Indonesian Cereals Research
Institute (Balitserealia) and two types of explants were used for the callus
induction, namely the seed and meristematic leaf whorl of a two-month old plant. The seeds were sterilized by washing twice with 90% alcohol for
10 min followed by immersion in commercial bleach containing 0.5% natrium hypochlorite for 30 min. The seeds then were rinsed
three times with sterile distilled water and then
imbibed by soaking in distilled water for 24 h. The leaf whorl was sterilized by spraying the outer layer with 70% alcohol followed by burning for 3 sec. The middle meristematic of
leaf whrol was isolated using a scalpel and
sliced from the apical meristem into a 3 mm thick disk.
Callus induction
Callus
induction for the seed explant was initiated by culturing the
imbibed sterile seed on a solid MS medium that was supplemented with 30 g L-1 sucrose and 0.5 mg L-1
kinetin. Combination of various concentrations of 2,4-D (2, 3, 4 and 5 mg L-1) and proline
(3000, 4000 and 5000 mg L-1) were added thereafter into the
medium. Meanwhile, the meristematic leaf whorl was cultured on an MS medium
supplemented with 30 g L-1
sucrose, 500 mg L-1
casein hydrolysate and various concentrations of 2,4-D (2, 3, 4 and 5 mg L-1). The medium's pH was then adjusted to 6.2 and 11 g L-1
agar was added as the gelling
agent. The cultures were incubated in a dark
condition for callus induction and proliferation
at 24˚C ± 2˚C for two weeks. Subsequently,
the induced callus was subcultured with the same medium and
the percentage of callus formation and induction time were recorded.
Plantlet regeneration and phenolic
control
The embryogenic
callus regeneration was carried out using an MS solid medium containing 30 g L-1
sucrose, 100 mg L-1
glutamine, 500 mg L-1
casein hydrolysate and combination of PGRs. The NAA concentrations at 0.1 and 0.3 mg L-1 were combined with BAP at 2 and 3 mg L-1 and applied to induce callus regeneration into plantlet. The callus was
cultured in the combination medium and
placed in a dark room at 24±1oC
for 2 weeks. It was then transferred into a
regeneration room where it was exposed to 16 h light at an intensity of 1500–1600 lux as well as 8 h in a dark
condition. Proline concentrations at 150,
300, 450 and 560 mg L-1
were added to the combination as the phenolic retardation agent. The
percentage of greenspot callus, planlet and
browning callus were then recorded once
a week.
Microscopy observation and
histological analysis
The
development of callus was monitored using LEICA
EZ4 W stereomicroscope. The callus
was placed on a sterile petri dish, sealed and then viewed under the
microscope. The observation was performed throughout somatic
embryogenesis stages at globular,
scutellar and coleoptillar phases.
The
histological analysis was carried out to evaluate the
development stage of somatic embryogenesis. The embryogenic
calli were fixed for 24 h using FAA solution containing formaldehyde, acetic acid, and
100 mg L-1
ethanol in a ratio of 17:1:2. The fixed calli were washed with aquades, dehydrated with ethanol, and then saturated with the toluene-paraffin mixture and pure
paraffin. They were sliced into 7–8 μm sections using a microtome and
then molded with paraffin. The
sections were glued to the preparate
glass slides using a toluene-paraffin
solvent (Sass 1951). After removing paraffin, the preparate was stained with hematoxylin and eosin. Subsequently, they were observed under a light
microscope (Olympus CX31 Japan) and then
photographed.
Acclimatization
The healthy planlets completed
with root and
shoot were washed with sterile water and then soaked in fungicide (Dhytane) for 5 min. After
drained, the planlets were adapted
to grow in the bottles containing sterilized sand, which
was watered with liquid nutrition (Growmore) and incubated in a growth chamber for 2-3 weeks. The adapted plants were then transferred to a polybag containing a mixture of sand and soil in a ratio of 1:1 and grown in green house. They were watered twice a week and fertilized once a month until harvesting.
Experimental design and statistical analysis
Complete
randomized design (CRD) was applied to determine the importance of different
culture media combinations. Each treatments had three replications. For each
replication, around 10–11 seeds were
plated on callus induction media and 7 clumps or embryoids were plated on
regeneration media. All data were collected and presented as means ± standard error from three
replicates. Statistical significance was then calculated
using ANOVA and Tukey’s test. A p-value
of 0.05 (p<0,05) was considered for determining
the significance.
Result
Induction of embryogenic callus by combining PGRs
Seeds
and leaf whorl explants were used to examine the combined effects of 2,4-D and proline on embryogenic callus induction. The formation of embryogenic callus was observed after incubated for two weeks on the combination medium ranging from 10–68% (Table 1). The increase of both 2,4-D and proline led to an increase in the percentage of embryogenic callus. Combining 3 mg L-1 of 2,4-D and 5000 mg L-1 of
proline (D2P3) had the highest percentage followed by 3 mg L-1 2,4-D and
4000 mg L-1 proline (D2P2). Although increasing PGRs induced callus formation, the highest
induction was observed after combining 3 mg L-1 2,4-D with 5000 mg L-1
proline. The D2 level was assumed to be the optimal level for 2,4-D because a
lower concentration decreased the callus formation, while higher levels did not
accelerate the process. Increasing proline concentration
induced embryogenic callus and P3 level showed the highest induction.
Observation of the induction time showed that neither the increase of 2,4-D nor
proline concentration resulted in a faster embryogenic callus development (Table
1). However, combining 3 mg L-1
2,4-D with 3000 or 5000 mg L-1 proline significantly produced the quickest embryogenic formation with
an induction time of approximately 3 weeks. These results indicate that the
combination of 3 mg L-1 2,4-D and 5000 mg L-1 proline is the effective
concentration for producing a large number of embryogenic callus in sorghum.
To compare embryogenic callus induction, the leaves whorl explant was
incubated in a MS medium, which was supplied with the same PGR combination. The addition of proline produced a
dark brown callus at the early stages of incubation (Supplement 1).
Therefore, the leaves whorl explant was incubated in a medium that was only
supplied with various concentrations of 2,4-D. Observation of the induction
time showed that increasing 2,4 D concentration resulted in a longer development
of embryogenic callus. As a consequence, the lowest 2,4-D with concentration of
2 mg L-1 produced the quickest incubation time of approximately 2–3 weeks (Table 2). Furthermore, the percentage of embryogenic callus formed was not
affected by 2,4-D concentrations in the leaf whorl explant.
Based on the induction time and percentage of the callus
induced, lowest concentration of 2 mg L-1
might the effective level for callus induction
using the leaf whorl explant.
Table 1: Effect of 2,4-D and proline concentrations on percentage of callus induction from seed explants. The
percentage values are presented as means ± SD for three independent
observations and the different lowercase denote significant differences
(Tukey’s test, p≤0.05)
|
2.4-D (mg L-1) |
Proline (mg
L-1) |
Callus
induction frequency (%) |
Callus
induction time (WAG) |
|
2 |
3000 (P1) |
10 ± 7d |
8.3 ± 0.47a |
|
4000 (P2) |
20 ± 7d |
8.0 ± 0.80ab |
|
|
5000 (P3) |
30 ± 7cd |
3.3 ± 0.47cd |
|
|
3 |
3000 (P1) |
50 ± 7c |
2.7 ± 0.47d |
|
4000 (P2) |
60 ± 10.8ab |
6.0 ± 0.80b |
|
|
5000 (P3) |
68 ± 4a |
3.0 ± 0.80d |
|
|
4 |
3000 (P1) |
45 ± 4cd |
4.3 ± 0.47c |
|
4000 (P2) |
60 ± 7ab |
6.0 ± 0.80b |
|
|
5000 (P3) |
60 ± 7bc |
6.0 ± 0.80bc |
|
|
5 |
3000 (P1) |
45 ± 10.8cd |
4.3 ± 1.20cd |
|
4000 (P2) |
50 ± 7c |
5.0 ± 0.80c |
|
|
5000 (P3) |
63 ± 7ab |
6.0 ± 0.47b |
Morphological of embryogenic
callus
To
observe the development of embryogenic callus, the morphological
character of sorghum during incubation on the medium was viewed using stereomicroscope. The features of embryogenic callus were observed as dry
friable, yellowish and a globular structure after two weeks on the appropriate
medium (Table 3). In the seed explant, all combinations of 2,4-D with proline induced
callus, as shown in Table 1. However, morphological observation showed that the
combination of PGRs at the lowest concentration produced a brown, wet, and not
friable callus. Yellow, dry and friable callus was
induced on the medium with increasing 2,4-D concentration in the addition of
proline. These results are consistent with the works of Ramulifho et al. (2019)
that the embryogenic callus was formed on the medium
with increasing 2,4-D concentration. However, combining the highest 2,4-D and
proline concentrations produced a white, wet, and not friable callus, which
indicates that it was not an embryogenic callus. Similar results were
also observed when the callus was induced from the leaf whorl explant (Table 3). The concentration of 2,4-D at 3 and 4 mg L-1 produced a yellow, dry and friable embryogenic callus, but at the lower and higher concentration, a wet and not
friable callus was obtained.
Table 2:
Effect of 2,4-D concentration on percentage and callus induction time from leaf
whrol explants. The values are presented as means ±
SD for three independent observations and the different lowercase denote
significant differences (Tukey’s test, p≤0.05).
|
2,4-D
(mg L-1) |
Callus induction frequency (%) |
Callus induction
time (WAP) |
|
2 (D1) |
37±4.7bc |
2.3±0.47b |
|
3 (D2) |
47±4.7a |
3.3±0.94b |
|
4 (D3) |
40±4.7ab |
4.3±0.47ab |
|
5 (D4) |
33±0.0b |
5.7±0.47a |
Table 3: Effect of 2,4-D and proline concentration on characteristic
of embryogenic callus generated from seed and leaf whorl
explants
|
Explants |
2,4-D (mg L-1) |
Proline (mg L-1) |
Callus
color |
Surface
structure |
|
Seed |
2 (D1) |
3000 (P1) |
Browning |
Wet, no
friable |
|
4000 (P2) |
Browning |
Wet, no
friable |
||
|
5000 (P3) |
White |
Dry,
friable |
||
|
3 (D2) |
3000 (P1) |
White |
Dry,
friable |
|
|
4000 (P2) |
White |
Dry,
friable |
||
|
5000 (P3) |
Yellowish |
Dry,
friable |
||
|
4 (D3) |
3000 (P1) |
Yellowish |
Dry,
friable |
|
|
4000 (P2) |
Yellowish |
Dry,
friable |
||
|
5000 (P3) |
Yellowish |
Dry,
friable |
||
|
5 (D4) |
3000 (P1) |
Yellowish |
Dry,
friable |
|
|
4000 (P2) |
White |
Wet, no
friable |
||
|
5000 (P3) |
White |
Wet, no
friable |
||
|
Leaf whrol |
2 (D1) |
|
White |
Wet, no
friable |
|
3 (D2) |
Yellowish |
Dry,
friable |
||
|
4 (D3) |
Yellowish |
Dry,
friable |
||
|
5 (D4) |
White |
Wet, no
friable |
To observe embryogenic callus development, the multi-step process starting from pre-embryo
mass (PEM), globular, scutellar and coleoptillar stages
were viewed under a stereomicroscope and then photographed. The seed explants
were incubated for 7 days in a combination medium that required germination
before the embryogenic callus development started (Fig. 1a). Furthermore, the globular
stage was developed from the germinated seed coleoptile after 30 days
incubation in the combination medium (Fig. 1e). It continued to the scutellar and coleoptillar phases after 45- and 60-days incubation,
respectively (Fig. 1f and g). Interestingly, the embryogenic callus produced from seed
explants regenerated into a green whole plant (Fig. 1h).
The
leaf whorl explants have similar the SE development
stages, which consist of the PEM, globular, secutelar
and colleoptillar phases. They showed swelling and developed into a pre-embryo mass (PEM) after 7-
and 10-days incubation in the medium (Fig. 1b–d). Subsequently, PEM further developed
into the globular stage on the 16th day (Fig. 1e). The next stage was the scutellar stage
characterized by a heart shape structure, which was observed on the 41st day,
and then followed by the choleoptillar stage, which
produced a young greening shoot (Fig. 1f–g). The multi-step development of embryogenic callus from the explant leaf whorl was also observable in the four stages,
but the explant was unable to regenerate into a whole plant due to browning
(Fig. 1i).
%20301-307_files/image002.jpg)
Fig. 1: Somatic embryogenesis (SE)
phases of sorghum: (a, b) seeds and leaf whorl explants; (c, d) pre-embryo mass (PEM) from seeds and leaf whorl explants; (e)
globular; (f) secutelar; (g) coleoptillar; (h, i) shoot regeneration from seed explant
and shoot browning from leaf whorl explant.
(%20301-307_files/image004.png){kind=small}
= 1 mm)
The histological analysis confirmed the presence of a large parenchymal
cell (PC) without a visible nucleus, which was surrounded by small meristematic
cells during the initial stage of embryogenic callus development (Fig. 2a). Subsequently, the cell formed
the globular callus that was characterized by a nodular shape (Fig. 2b) and then further developed into
a hearth-shape or embryo structure due to protuberances of mitosis (Fig. 2c). Occurred embryo structure division led to the formation of cotyledon
as well as initiation of vascular bundle, which in turn formed the shoot, young
leaf, shoot apical meristem, bud primordial, and vascular tissue
(Fig. 2d–e). This histological analysis
supported SE callus development stage in sorghum.
Callus regeneration into plantlet
A preliminary
study showed that callus was
regenerated in an MS medium
containing BAP and NAA. Regeneration was successfully performed from callus
obtained from the seed explant, while leaf whorl did not regenerate into a plant, but only produced a green
spot. To find the most appropriate combination
of NAA and BAP concentrations for regeneration of seed
callus, the PGRs concentration were varied.
The combination of 0.1 mg L-1 NAA and 3 mg L-1 BAP (N1B2) had the
highest green spot formation and regeneration percentage with 78% of the callus being converted into plantlet (Fig. 3a). The addition of proline to the
regeneration medium containing N1B2 combination improved the regeneration rate of seed
explant (Fig. 3b). The addition of proline reduced the browning and increased plantlet regeneration.
Callus obtained from sorghum can have a characteristic brown color due to the presence of phenolic compound. However, proline can significantly reduce phenol levels and it also has antioxidant activity in
stevia-plant callus (Blistrubienė et al. 2020). Adding approximately 560 mg L-1
proline into the N1B2 medium doubled the plantlet regeneration rate compared to the medium
without the amino acid.
%20301-307_files/image006.jpg)
Fig.
2: Histological analysis of SE
development phases in sorghum; (a)PEM; (b) globular; (c) scutellar with heart-like shape; (d) cotyledonary; (e) regenerated shoot.
MC, meristematic cell; PC, parenchym
cell; GC, globular cell; VB, vascular
bundle; YL, young leaf; BP, bud
primordial; SAM, shoot apical
meristem; VT, vascular
tissue ( %20301-307_files/image008.png){kind=small}
200 μm)
%20301-307_files/image010.jpg)
Fig. 3: Number of plantlets in the combining NAA and BAP medium (A) and
acceleration of callus regeneration by proline (B). Number plantlet is
calculated as percentage (%) of plantlets number per callus. Acceleration of
callus regeneration are expressed as relative values of the number of
regenerated plantlets in combining medium N1B2 without proline
Acclimatization
A number
of regenerated plantlets were successfully acclimatized to ex vitro conditions (Supplement 2). The plantlets regenerated from embryogenic callus were acclimated in
a growth chamber for 21 days and then exposed to natural conditions in a
greenhouse. The plant showed a remarkable growth performance during three
months of observation. Morphologically, there were no differences between the
growth, size, and leaf of the adapted in
vitro plant and the plant obtained from the natural seed. The adapted plant
had a height of approximately 2–3 meters and also produced normal seed that was
harvested a month later.
Discussion
Sorghum Numbu
cv is a superior hybrid sorghum in Indonesia and its tissue culture method needs to be improved
because of prerequisites for genetic enhancement, transgenic
production, and clonal propagation (Aregawi et
al. 2021). It has been
reported that cereals including
sorghum are recalcitrant to in vitro propagation. This study used two types of explant, namely seed
and leaf whorl to induce somatic embryogenesis callus and regeneration in sorghum. The results
showed that the callus induced from the seed explant in a
medium containing combination of 2,4-D and proline was friable,
yellowish, and had a globular structure after
two weeks (Table 1, 3). The embryogenic callus further developed into
greening callus and finally into a plant after sub-cultured in a regeneration
medium. In other hand, the addition of proline to the
explant leaf whorl produced a dark brown callus, but was produced in an MS
medium supplied with only 2,4-D. The results indicate that the seed explant quickly developed
embryogenic callus, which has many
advantages including its high availability and
easy to prepare.
PGRs
are important substances for callus
induction, callus proliferation, and plant regeneration. The PGRs can also be combined with
amino acids to increase the
number of callus induction and regeneration.
However, the concentration is very important for
controlling the development of cereal somatic embryogenesis (Schnablová et
al. 2006). The higher 2,4-D concentration increased
the callus formation, and the concentration of 3 mg L-1 2,4-D to be
most effective concentration for the callus induction in both seed and leaf
whorl explant. The addition of proline improved callus induction and the
highest concentration at of 5000 mg L-1 proline produced 68% induced
callus. The embryogenic callus produced in a medium containing 2,4-D and
proline showed the characteristics crumble, yellowish, and not watery on callus
surface (Table 3). Proline is an essential amino acid for supporting plant growth and
acts as antioxidant to retard browning of explant caused by phenolic oxidation (Khokhar et al.
2021). Therefore, the combination of 2,4-D and
proline is a determinant to produce a high number of embryogenic callus in
sorghum.
The efficiency of plant regeneration was improved by adding PGRs, such
as NAA, BAP, and proline as the amino acid supplement. The combination of NAA
and BAP had a significant effect on the callus regeneration. The optimal
concentration was 0.1 mg L-1 NAA and 3 mg L-1 BAP, which was able to regenerate 78% callus into sorghum plantlet (Fig. 3a). The addition of 560 mg L-1 proline doubled the regeneration
rate compared to the medium without proline (Fig. 3b). This result indicates that proline
improve regeneration rate of callus, as reported in Brassica
napus (Ahmadi and Mehran 2015) and in sugarcane (Kaur and Manish 2016). Amino acid proline has been
postulated has a role to suppress enzymatic browning and improve plant
regeneration.
Microscopic and histological observations confirmed that the embryogenic
callus was proliferated using the combination of PGRs. Furthermore, combining
2,4-D with proline to proliferate and develop callus into the SE
phases, namely globular, scutellar, colleoptillar, and shoot formation was observed (Fig. 1). The histological observation
showed that there were significant changes in the cell structure, component,
and shape at each phase (Fig. 2). The pre-embryo mass
was characterized by large parenchymal cells and
dense protoplasm, which then develop
into a globular callus. The scutellar
stage was characterized by cotyledon structure development as well as the formation of a
heart-shaped structure by the embryo due to mitosis protuberances. Successful divisions of the embryo poles
started the development of embryos and to be mature cotyledons. The cells
differentiation also led to
vascular bundles formation in the colleoptillar stage. All the SE development phases were confirmed by morphological and
histological observation. The regenerated shoots that were completed with the
root system were adapted to greenhouse condition
and grew normally in the soil medium. These results indicate that the
embryogenic callus induction and subsequent regeneration in sorghum of Numbu cv were successfully improved by combining PGRs with
proline.
Conclusion
Embryogenic
callus and plantlet regeneration were successfully developed using seed explant. The combination of
3 mg L-1 of 2,4-D
and 5000 mg L-1 of proline
produced a 68% embryogenic
callus for 3 weeks. The combination of 0.1 mg L-1
NAA and 3 mg L-1 BAP regenerated the highest number of
planlet, namely 78%, while the addition of 560 mg L-1 proline doubled
the regeneration rate in sorghum.
Acknowledgment
This work was
supported by the Indonesian Ministry of Education and Culture under grant No.
972/UN25.3.1/LT/2020 and (SPPK)
116/E4.1/AK.04.PT/2021-World Class Research for BS.
Author Contributions
WNN and BS planned the experiments, WNN, DPR and BS interpreted
the results, DPR histological observation, WNN statistically analyzed the data
and made illustration, WNN and BS writing original draft and editing, BS
funding acquisition.
Conflict of Interest
All
authors declare no conflict of interest
Data Availability
Data
presented in this study will be available on a fair request to the
corresponding author.
Ethics Approval
Not applicable in this paper
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