Application of Encapsulation Technology: In Vitro Screening of Two Ficus carica L. Genotypes under Different NaCl Concentrations

Abstract

Salinity stress represents an increasing issue for agriculture and has a great negative impact on plant growth and crop production. The selection of genotypes able to tolerate salt stress could be a suitable solution to overcome the problem. In this context, in vitro cultures can represent a tool for identifying the NaCl tolerant genotypes and quickly producing large populations of them. The possibility of exerting selection for tolerance to NaCl by using encapsulation technology was investigated in two genotypes of fig: ‘Houmairi’ and ‘Palazzo’. The effects of five concentrations of NaCl (0, 50, 100, 150 and 200 mM) added to the artificial endosperm were tested on the conversion of synthetic seeds and on the growth of derived shoots/plantlets. Moreover, proline (Pro) and malondialdehyde (MDA), the enzymatic activities of catalase (CAT), guaiacol peroxidase (POD), and EL (Electrolytic Leakage), as well as the chlorophyll content, flavanols, anthocyanins, and Nitrogen Balance Index (NBI) were determined on shoots/plantlet. The obtained results clearly showed that ‘Houmairi’ and ‘Palazzo’ could tolerate salt stress, although a strong difference was found depending on each specific physiological pathway. Indeed, ‘Houmairi’ was revealed to be more tolerant than ‘Palazzo’, with different response mechanisms to salt stress. The use of encapsulated vitro-derived explants proved to be a useful method to validate the selection of genotypes tolerant to salinity stress. Further investigation in the field must validate and confirm the legitimacy of the approach.

Full text

Citation: Granata, I.; Regni, L.;
Micheli, M.; Silvestri, C.; Germanà,
M.A. Application of Encapsulation
Technology: In Vitro Screening of
Two Ficus carica L. Genotypes under
Different NaCl Concentrations.
Horticulturae 2023,9, 1344. https://
doi.org/10.3390/horticulturae9121344
Academic Editor: Zhenchang Liang
Received: 6 October 2023
Revised: 5 December 2023
Accepted: 14 December 2023
Published: 16 December 2023
Copyright: © 2023 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/).
horticulturae
Article
Application of Encapsulation Technology: In Vitro Screening
of Two Ficus carica L. Genotypes under Different
NaCl Concentrations
Irene Granata 1, * , Luca Regni 2, Maurizio Micheli 2, * , Cristian Silvestri 3and Maria Antonietta Germanà1
1Department of Agricultural, Food and Forestry Sciences (SAAF), University of Palermo, Viale delle Scienze,
Ed. 4, 90128 Palermo, Italy; mariaantonietta.germana@unipa.it
2Department of Agricultural, Food and Environmental Sciences, University of Perugia, Borgo XX Giugno 74,
06121 Perugia, Italy; luca.regni@unipg.it
3Department of Agriculture and Forest Sciences, University of Tuscia, Via San Camillo de Lellis snc,
01100 Viterbo, Italy; silvestri.c@unitus.it
*Correspondence: irene.granata@unipa.it (I.G.); maurizio.micheli@unipg.it (M.M.)
Abstract:
Salinity stress represents an increasing issue for agriculture and has a great negative impact
on plant growth and crop production. The selection of genotypes able to tolerate salt stress could
be a suitable solution to overcome the problem. In this context,
in vitro
cultures can represent a
tool for identifying the NaCl tolerant genotypes and quickly producing large populations of them.
The possibility of exerting selection for tolerance to NaCl by using encapsulation technology was
investigated in two genotypes of fig: ‘Houmairi’ and ‘Palazzo’. The effects of five concentrations of
NaCl (0, 50, 100, 150 and 200 mM) added to the artificial endosperm were tested on the conversion
of synthetic seeds and on the growth of derived shoots/plantlets. Moreover, proline (Pro) and
malondialdehyde (MDA), the enzymatic activities of catalase (CAT), guaiacol peroxidase (POD), and
EL (Electrolytic Leakage), as well as the chlorophyll content, flavanols, anthocyanins, and Nitrogen
Balance Index (NBI) were determined on shoots/plantlet. The obtained results clearly showed that
‘Houmairi’ and ‘Palazzo’ could tolerate salt stress, although a strong difference was found depending
on each specific physiological pathway. Indeed, ‘Houmairi’ was revealed to be more tolerant than
‘Palazzo’, with different response mechanisms to salt stress. The use of encapsulated vitro-derived
explants proved to be a useful method to validate the selection of genotypes tolerant to salinity stress.
Further investigation in the field must validate and confirm the legitimacy of the approach.
Keywords:
salinity; abiotic stress; synthetic seed; micropropagation; fig tree; genotype selection;
climate change
1. Introduction
Salinity represents one of the most harmful and critical factors among the abiotic
stresses, limiting plants growth and crop production of numerous species of agricultural
interest [
1
]. Recent studies [
2
], based on available data from the Harmonized World Soil
Database, estimate that over one billion hectares of arable lands worldwide are affected
by salinity and sodicity. This occurs mainly in arid and semi-arid regions where irrigation
is essential to ensure satisfactory production levels, both in terms of quality and quantity,
although it is carried out with poor quality or brackish irrigation water, worsening the
phenomenon. Soil salinization, caused by both natural and anthropogenic activities, is
estimated to increase with time at a rate of 10% [
2
,
3
]. The excess of soluble salts—most
notably NaCl—in water and soil causes complex and detrimental effects on biochemical
pathways of plants resulting in morphological and physiological alterations [
4
,
5
]. Salin-
ity stress generally affects plants development in two possible ways [
6
]. The first one is
described as ion accumulation (when the uptake of Na
+
and Cl
−
are excessively) in plant
Horticulturae 2023,9, 1344. https://doi.org/10.3390/horticulturae9121344 https://www.mdpi.com/journal/horticulturae

Horticulturae 2023,9, 1344 2 of 15
cells, which causes an ion imbalance. The other one is the osmotic stress, which ensues
by reducing the water potential, limiting the water and other soluble salts uptake by the
roots due to osmotic pressure [
6
]. Toxic ions are accumulated in plants cells, exceeding
the threshold level and causing ion toxicity [
7
,
8
]. Thus, as a result of water deficits, this
leads to stomatal closure, decreasing CO
2
availability, and decreasing photosynthesis rate;
an oxidative stress increasing the likelihood of reactive oxygen species (ROS) formation
in plants also occurs. Salinity stress leads to several consequences regarding morpholog-
ical traits [
9
], such as growth reduction—including the number and size of leaves and
roots—due to the trigger of both chlorophyll content decrease and cell membrane damage
caused by ion imbalance and cations outflow [
10
,
11
]. A low photosynthetic rate and high
levels of osmotic stress leads to an excess of reactive oxygen species (ROS), causing cyto-
toxic effects and promoting specific defense pathways. The major strategies that plants
use to overcome these stresses include the control of water loss through stomata closure,
metabolic adjustment, toxic ion homeostasis, and osmotic adjustment [
7
] through the ac-
tivation of enzymatic and non-enzymatic activities such as superoxide dismutase (SOD),
catalase (CAT), peroxidase (POD), malondialdehyde (MDA), carotenoids, flavonoids, and
the osmolyte proline, all to reduce oxidative stress and prevent further damage [12–14].
Most fruit tree species are sensitive to salinity, with only a few being considered to be
moderately tolerant [
4
,
12
]. Even so, conventional methods for the selection and propagation
of salinity-tolerant genotypes are both costly and time-consuming. To overcome this
constraint,
in vitro
culture techniques represent useful tools that allow the selection and
rapid validation of suitable genotypes under controlled conditions [
8
]. Therefore, the
in vitro
culture approach is helpful in investigating the impact of abiotic stress, especially
salinity, in numerous Mediterranean species, owing to the significant body of literature
focusing on the 0 to 400 mM range [
15
–
17
]. Several types of plant material such as cell
suspension, meristem tip culture, or axillary buds can be employed for this purpose.
Among the latest advancements in tissue culture applications, encapsulation is a recent
technology that can be very useful in combining the benefits of
in vitro
culture—such
as a high proliferation rate and mass production of true-to-type plants—with the easy
handling and conservation of genetic resources. Additionally, this technology is now being
considered as an efficient method for short-term and medium-term storage, as well as
for the simplified exchange of plant material between tissue culture laboratories and for
mass clonal propagation of important commercial plant species by producing synthetic
seeds [
18
,
19
]. The fig tree (Ficus carica L.), belonging to the Moraceae family, is one of the
most ancient and traditionally cultivated fruit trees in the Mediterranean region, grown
for fresh and dried fruits. In southern Italy, the fig tree is often cultivated together with
other species (i.e., olive, almond, pomegranate, apricot and grapevine). This plant can
be considered moderately tolerant to salt and drought stress, even though common salt
stress conditions reduce the number and length of newly formed shoots [
20
] and negatively
affect the plant growth and fruit yield, especially in plants derived from the cuttings
propagation method as they are characterized by a shallow and fibrous root system [
21
].
Therefore, the selection, conservation, and propagation of a wide range of salt tolerance
genotypes is essential for the availability of new varieties and for their use in breeding
programs, considering that the phenomenon of salinity is expected to increase with climate
change over the next few years. In this context, few study have been conducted to assess
genetic diversity among the Italian fig germplasm [
22
,
23
]. This study aims to improve
the understanding of the physiological and morphological effects of salt stress at five
different NaCl concentrations (0, 50, 100 and 200 mM) in two traditional fig genotypes (cvs.
Houmairi and Palazzo) typical of the Mediterranean area, and for the first time, report on
the application of encapsulation technology for synthetic seed production.

Horticulturae 2023,9, 1344 3 of 15
2. Materials and Methods
2.1. Plant Material
The experimental material used were derived from
in vitro
proliferated shoots of
two Ficus carica L. genotypes, cvs. Houmairi and Palazzo. Subcultures were performed
every 45 days in 500 mL glass jars containing 100 mL of a hormone free, full-strength MS
medium [
9
]. In each jar, 9 binodal shoots were cultured, and the vessels were placed into a
growth chamber at 23
±
2
◦
C, under a photon flux density of 40
µ
mol
−2
s
−1
with a 16/8 h
light/dark photoperiod. From the proliferated shoots derived from a 45-days-old single
subculture, microcuttings (uninodal segments 3–4 mm long with lateral bud) were excised
and leaflets were removed and selected as the explant source.
2.2. Encapsulation Procedure and Culture Conditions
The microcuttings described above were initially dipped into a (2.5% w/v) sodium
alginate (Sigma Aldrich, St. Louis, MI, USA, medium viscosity) solution for a few minutes
for the coating phase, then soaked in a (1.1% w/v) calcium chloride (CaCl
2·
2H
2
O) complex-
ing solution for 35 min. Finally, the encapsulated microcuttings were rinsed with a sterile
washing solution (artificial endosperm solution) for 15 min.
All three solutions were enriched with the components of artificial endosperm com-
posed by MS basal salts, including vitamins [
9
] at half strength, 50 g L
−1
of sucrose, and
supplemented with five different sodium chloride (NaCl) (Sigma Aldrich, St. Louis, MI,
USA) concentrations (0, 50, 100, 150 and 200 mM). The pH was corrected to 5.8 with HCl
and KOH 1 M solutions before autoclaving sterilization.
The hardened capsules were sown in 100
×
15 mm Petri dishes (five capsules in each
plate) containing 10 mL of sterile culture media composed by a full strength MS medium,
enriched with 30 g L
−1
of sucrose and 7 g L
−1
of agar (Plant Agar, Duchefa, BH Haarlem,
Netherlands). The pH was adjusted to 5.8 before sterilization (autoclave, 20 min, 121 ◦C).
The obtained Petri dishes were placed in a growth chamber at 23
±
2
◦
C, under a
photon flux density of 40 µmol−2s−1with a 16/8 h light/dark photoperiod.
All the plant material handlings were carried out in sterile conditions using a horizon-
tal laminar flow cabinet.
2.3. Data Collection
The
in vitro
culture of both fig genotype capsules was carried out using five repli-
cates (single Petri dish) per treatment, in addition to a ‘control group’ consisting of non-
encapsulated propagules. In each replicate, five synseeds were sown, while NaCl concen-
trations consisting of 0, 50, 100, 150 and 200 mM were applied in the five treatments.
Data were collected at the end of 45 days of experimental observation, and the fol-
lowing parameters were evaluated: (1) Viability (% of explants with a green appearance),
(2) Regrowth (% of explants that produced shoots at least 4 mm long), (3) Conversion (% of
explants that produced both shoots and roots), (4) shoots and roots number per explant,
(5) shoot and root length regenerated from single explants (cm).
2.3.1. Measurement of Photosynthetic Pigments and Biochemical Traits
To evaluate the physiological response of the encapsulated explants to NaCl salinity,
the determination of chlorophyll content, flavanols, anthocyanins, and Nitrogen Balance
Index (NBI) have been carried out by using Dualex
®
Scientific Polyphenols and a Chloro-
phyll Meter (FORCE-A, Orsay, France). NBI is given as a ratio between the amounts of
chlorophyll and flavonoids, the latter expressed as the ratio of chlorophyll and flavanols
according to the protocols reported by Silvestri et al. (2017) and Bashir et al. (2021) [
24
,
25
].
The level of lipid peroxidation has been expressed as malondialdehyde (MDA) content,
and was determined as TBA (2-thiobarbituric acid) reactive metabolites according to Astolfi
et al. [
26
]. Briefly, fresh tissues (0.2 g) were homogenized in 10 mL of 0.25% TBA made
in 10% TCA (trichloroacetic acid). The extracts were heated at 95
◦
C for 30 min and then
quickly cooled on ice. After centrifugation at 10,000
×
gfor 10 min, the absorbance of the

Horticulturae 2023,9, 1344 4 of 15
supernatant was measured at 532 nm. Correction of non-specific turbidity was made by
subtracting the absorbance value taken at 600 nm. The level of lipid peroxidation was
expressed as mmol g−1fresh weight by using an extinction coefficient of 155 mM cm−1.
Freshly harvested leaf samples (100 mg of fresh weight) were collected, and pro-
line concentration was determined using a spectrophotometer according to Bates et al.
(1973) [
27
]. Briefly, for colorimetric determination, based on the proline’s reaction with
ninhydrin, a 1:1:1 solution of proline, ninhydrin acid, and glacial acetic acid was incu-
bated at 100
◦
C for 1 h. The reaction was arrested in an iced bath and the chromophore
was extracted with 4 mL toluene, and the absorbance at 520 nm was determined with a
spectrophotometer EVO 60 (Thermo Fischer Scientific Inc., Waltham, MA, USA).
For ROS-scavenging enzymes, shoot tissues (1 g) were powdered in a pre-chilled
mortar with liquid N2. A cold extraction buffer, containing 50 mM HEPES-KOH (pH 7.4),
5 mM MgCl
2
, 1 mM EDTA, 10% (v/v) glycerol, 0.1% (v/v) Triton X-100, 5 mM dithiothreitol
(DTT), 1 mM phenylmethylsulphonyl fluoride (PMSF), and 1% (w/v) polyvinylpyrrolidone
(PVP) was added in a ratio of 1:7 (w/v) (Silvestri et al., 2021) [28].
Peroxidase activity was measured spectrophotometrically at 470 nm using guaiacol
as the hydrogen donor [
29
]. Catalase activity (E.C. 1.11.1.6) was evaluated by measuring
the decrease in absorbance at 240 nm due to the decomposition of H
2
O
2
, as described by
Santangelo [
29
]. All reported enzyme activities were linear with time and proportional to
the amount of extract used. Protein content was estimated according to Bradford (1976) [
30
],
using BSA as the standard.
Furthermore, electrolyte leakage (EL) was used to assess cell membrane permeability
(Lutts et al., 1996) [
31
]. Leaves were placed in 20 mL of deionized water and incubated
overnight at 25
◦
C on a rotary shaker. The electrical conductivity of the solution (EL
0
)
was determined after 24 h. Samples were then autoclaved at 120
◦
C for 20 min and a last
conductivity reading (EL1) were performed. The electrolyte leakage was expressed (L0/L1)
as a percentage.
2.3.2. Statistical Analysis
The experiment was developed with a complete randomized design, with two factors
studied (genotype and treatment) and their interaction.
Statistical analysis was conducted with the IBM SPSS Statistics software ((IBM Inc.,
New York, NY, USA; 28.0.1.1 version) using analysis of variance (two-way ANOVA), and
the means were separated with Tukey’s test (p< 0.05). Data on percentages were arcsine-
transformed before performing statistical analysis [
32
]. The results are expressed as the
mean ±standard error (SE).
3. Results
3.1. Effect of Salt Concentrations on Growth Traits
At the end of the experiment, synthetic seeds of fig genotypes ‘Houmairi’ and ‘Palazzo’
showed significant differences, cv in particular. Houmairi proved to be more tolerant
than cv. Palazzo was in all the studied morphological descriptors as shown in Table 1.
Both genotypes achieved satisfactory percentages, especially for viability (97.6% and 84%)
and regrowth (90.4% and 65.6%); however, ‘Houmairi’ reached valuable performances,
especially for rooting and conversion rates (66.4%), compared to ‘Palazzo’ (40.0 and 37.6%).
The different salt concentrations, as expected, clearly affected all the examined parameters
(showed in Table 1), reducing the growth rates of both genotypes, especially in the 200 mM
treatments for rooting (28%) and conversion (26%), compared to the 80% of the control.

Horticulturae 2023,9, 1344 5 of 15
Table 1.
Effect of five different mM concentrations of NaCl added to the artificial endosperm of
synthetic seeds of Ficus carica L. cvs. Houmairi and Palazzo on viability, regrowth, rooting, and
conversion, observed after 45 days.
Genotype Viability (%) Regrowth (%) Rooting (%) Conversion (%)
Houmairi NaCl concentrations 97.6 ±0.17 a 90.4 ±0.34 a 66.4 ±0.41 a 66.4 ±0.41 a
Palazzo NaCl concentrations 84 ±0.29 b 65.6 ±0.34 b 40 ±0.28 b 37.6 ±0.31 b
Treatment
0 (Control) Genotypes average 96.0 ±0.19 88.0 ±0.28 80.0 ±0.3 a 80.0 ±0.3 a
50 Genotypes average 96.0 ±0.20 84.0 ±0.36 62.0 ±0.46 ab 60.0 ±0.47 ab
100 Genotypes average 92.0 ±0.27 82.0 ±0.39 54.0 ±0.34 ab 54.0 ±0.34 ab
150 Genotypes average 88.0 ±0.32 66.0 ±0.36 42.0 ±0.17 bc 40.0 ±0.19 bc
200 Genotypes average 82.0 ±0.27 70.0 ±0.45 28.0 ±0.3 c 26.0 ±0.34 c
Genotype ×NaCl
concentrations NaCl (mM)
Houmairi 0 (Control) 100 ±0.00 100 ±0.00 92 ±0.25 92 ±0.25
50 100 ±0.00 100 ±0.00 84 ±0.31 84 ±0.31
100 100 ±0.00 100 ±0.00 76 ±0.28 76 ±0.28
150 96 ±0.25 72 ±0.37 52 ±0.19 52 ±0.19
200 92 ±0.25 80 ±0.37 28 ±0.30 28 ±0.30
Palazzo 0 (Control) 92 ±0.25 76 ±0.10 68 ±0.19 68 ±0.19
50 92 ±0.25 68 ±0.34 40 ±0.37 36 ±0.37
100 84 ±0.31 64 ±0.37 32 ±0.12 32 ±0.12
150 80 ±0.37 60 ±0.40 32 ±0.12 28 ±0.12
200 72 ±0.12 60 ±0.48 28 ±0.33 24 ±0.40
Significance of ANOVA
Genotype *** *** *** ***
NaCl concentrations ** * *** ***
Genotype ×NaCl concentrations ns ns ns ns
Mean values shown in columns according to Tukey’s test at p
≤
0.05. Data are presented as mean
±
SD. Mean
values followed by the same letter are not significantly different at p
≤
0.05. ns, ***, **, *: non-significant or
significant at p≤0.001, p≤0.01, p≤0.05, respectively.
Similarly, significant differences between the two fig varieties were observed for the
other morpho-physiological traits investigated, as presented in Table 2.
Significant differences were found in the number of shoots produced during the
experiment between the two studied cultivars. In cv. Houmairi in particular, every average
value significantly differed from the control (0 mM), which had the higher value (1.40).
Meanwhile, the mean number of shoots identified in cv. Palazzo was not significantly
different from the control. Moreover, these observations were comparable to the lowest
mean exhibited by ‘Houmairi’ (under a concentration of 200 mM).
For the shoot length parameter, cv. Houmairi showed an average length of 2.40 cm in
the control; the treatment with 50 mM yielded the highest length value (3.14 cm), while
subsequent treatments demonstrated decreasing values until the lowest was reached at
200 mM concentration (0.34 cm). On the contrary, cv. Palazzo showed no significant
differences among treatments, as all mean values were similar to Houmairi’s shorter length.
Differences in root number and length between treatments were also observed
(Figures 1and 2).
The cv. Houmairi started to show a significant decrease in the number of newly formed
roots and root length at the concentration of 150 mM (1.12 and 1.17 cm) in comparison to
the control (2.20 and 4.30 cm). In contrast, cv. Palazzo exhibited a significant reduction,
since the lower salt concentration (50 mM) (0.60) and all the remainder salt concentrations
were equivalent to the least mean values recorded for ‘Houmairi’ at 200 mM (0.32 and
0.57 cm), as presented in Table 2.

Horticulturae 2023,9, 1344 6 of 15
Table 2.
Effect of five different concentrations of NaCl added to the artificial endosperm synthetic
seeds of Ficus carica L. cvs. Houmairi and Palazzo on proliferation and rooting performance, observed
after 45 days.
Genotype Number of
Shoots
Shoots Length
(cm)
Number of
Roots
Roots Length
(cm)
Houmairi NaCl concentrations 0.82 ±0.50 a 1.60 ±1.39 a 0.59 ±0.85 b 3.06 ±2.19 a
Palazzo NaCl concentrations 0.28 ±0.16 b 0.23 ±0.19 b 0.66 ±0.64 a 0.94 ±1.35 b
Treatment
0 (Control) Genotypes average 0.92 ±0.59 a 1.29 ±1.30 ab 1.92 ±0.58 a 3.78 ±1.13 a
50 Genotypes average 0.70 ±0.41 ab 1.81 ±1.77 a 1.42 ±1.01 ab 2.82 ±2.64 ab
100 Genotypes average 0.46 ±0.40 bc 0.64 ±0.62 bc 1.24 ±0.92 b 2.29 ±2.46 b
150 Genotypes average 0.40 ±0.36 bc 0.58 ±0.94 bc 0.72 ±0.49 c 0.70 ±0.56 c
200 Genotypes average 0.26 ±0.13 c 0.24 ±0.17 c 0.32 ±0.23 c 0.40 ±0.43 c
Genotype ×NaCl
concentrations NaCl (mM)
Houmairi 0 (Control) 1.40 ±0.40 a 2.40 ±0.92 ab 2.20 ±0.42 a 4.30 ±0.81 a
50 1.10 ±0.30 ab 3.14 ±1.61 a 2.24 ±0.52 a 4.96 ±1.88 a
100 0.72 ±0.41 bc 1.14 ±0.50 bc 2.08 ±0.36 a 4.29 ±1.90 a
150 0.56 ±0.48 bc 1.03 ±1.23 bc 1.12 ±0.30 bc 1.17 ±0.35 bc
200 0.36 ±0.09 c 0.34 ±0.18 c 0.32 ±0.18 c 0.57 ±0.52 c
Palazzo 0 (Control) 0.44 ±0.22 c 0.21 ±0.11 c 1.64 ±0.62 ab 3.28 ±1.25 ab
50 0.36 ±0.09 c 0.49 ±0.26 c 0.60 ±0.60 c 0.67 ±0.82 c
100 0.21 ±0.14 c 0.15 ±0.11 c 0.40 ±0.14 c 0.28 ±0.13 c
150 0.24 ±0.09 c 0.14 ±0.05 c 0.32 ±0.23 c 0.23 ±0.17 c
200 0.16 ±0.09 c 0.14 ±0.10 c 0.32 ±0.30 c 0.22 ±0.26 c
Significance of ANOVA
Genotype *** *** *** ***
NaCl concentrations *** *** *** ***
Genotype ×NaCl concentrations ** *** *** ***
Mean values shown in columns according to Tukey’s test at p
≤
0.05. Data are presented as mean
±
SD. Mean
values followed by the same letter are not significantly different at p
≤
0.05. ***, **: significant at p
≤
0.001,
p≤0.01, respectively.
Horticulturae 2023, 9, x FOR PEER REVIEW 7 of 17
Figure 1. (a) ‘Houmairi’ control; (b) ‘Palazzo control; (c) ‘Houmairi’ 50 mM; (d) ‘Palazzo’ 50 mM, at
the end of the experiment.
Figure 1. (a) ‘Houmairi’ control; (b) ‘Palazzo control; (c) ‘Houmairi’ 50 mM; (d) ‘Palazzo’ 50 mM, at
the end of the experiment.

Horticulturae 2023,9, 1344 7 of 15
Horticulturae 2023, 9, x FOR PEER REVIEW 7 of 17
Figure 1. (a) ‘Houmairi’ control; (b) ‘Palazzo control; (c) ‘Houmairi’ 50 mM; (d) ‘Palazzo’ 50 mM, at
the end of the experiment.
Figure 2.
(
a
) ‘Houmairi’ 100 mM; (
b
) ‘Palazzo’ 100 mM; (
c
) ‘Houmairi’ 150 mM; (
d
) ‘Palazzo’
150 mM; (e) ‘Houmairi’ 200 mM; (f) ‘Palazzo’ 200 mM at the end of the experiment.
3.2. Effect of Alginate Coating Treatment on Growth Activity
The synthetic seeds exposed at 0 mM of NaCl (control) were also compared to the
propagules lacking the encapsulation matrix; at the end of the experiments, growth perfor-
mance was evaluated. In Table 3, percentages of viability, regrowth, rooting, and conversion
are detailed.
No statistical differences have been observed in the viability and regrowth rates
between the varieties ‘Houmairi’ and ‘Palazzo’. On the other hand, there were differences
in rooting and conversion rates between the cultivars. Indeed, ‘Houmairi’ yielded more
rooted plantlets compared with the cv. Palazzo (84 and 58%). Additionally, providing the
coating treatment resulted in the highest mean values for both genotypes when compared
to non-coated propagules. The number of roots and their length prove to be closely related
to genotype, as illustrated in Table 3. ‘Houmairi’ showed higher average values (2.36 and
4.33 cm) according to the other morphological traits previously described.
Likewise, the number and length of shoots were positively influenced by the alginate
coating treatment, especially in ‘Houmairi’ genotype (0.94 and 1.46 cm) as shown in Table 4.

Horticulturae 2023,9, 1344 8 of 15
Table 3.
Effect of coating treatment on Ficus carica L. cvs. Houmairi and Palazzo microcuttings on
viability, regrowth, rooting and conversion, and rooting performance observed after 45 days.
Genotype Viability
(%)
Regrowth
(%) Rooting (%) Conversion
(%)
Number of
Roots
Roots Length
(cm)
Houmairi
Treatments average
100 ±0.00 94 ±0.13 84 ±0.21 a 84 ±0.21 a 2.36 ±0.69 a 4.33 ±1.48 a
Palazzo
Treatments average
96 ±0.20 82 ±0.15 58 ±0.20 b 58 ±0.20 b 1.26 ±0.67 b 2.32 ±1.42 b
Treatment
No coating Genotypes average 100 ±0.00 88 ±0.30 62 ±0.4 b 62 ±0.4 b 1.70 ±1.1 2.90 ±2.2
Coating Genotypes average 96 ±0.20 88 ±0.30 80 ±0.3 a 80 ±0.3 a 1.90 ±0.6 3.80 ±1.1
Genotype ×Treatment
Houmairi No coating 100 ±0.00 88 ±0.18 76 ±0.26 76 ±0.26 2.52 ±0.91 4.36 ±2.06
Coating 100 ±0.00 100 ±0.00 92 ±0.11 92 ±0.11 2.21 ±0.42 4.30 ±0.81
Palazzo No coating 100 ±0.00 88 ±0.18 48 ±0.18 48 ±0.18 0.88 ±0.52 1.37 ±0.85
Coating 92 ±0.11 76 ±0.09 68 ±0.18 68 ±0.18 1.64 ±0.62 3.27 ±1.25
Significance of ANOVA
Genotype ns ns ** ** * *
Treatment ns ns * * ns ns
Genotype ×Treatment ns ns ns ns ns ns
Mean values shown in columns according to Tukey’s test at p
≤
0.05. Data are presented as mean
±
SD. Mean
values followed by the same letter are not significantly different, at p
≤
0.05. ns, **, *: non-significant or significant
at p≤0.01, p≤0.05, respectively.
Table 4.
Effect of coating treatment on Ficus carica L. cvs. Houmairi and Palazzo microcuttings on
proliferation activities observed after 45 days.
Genotype Treatment Number of Shoots Shoots Length (cm)
Houmairi Treatments average 0.94 ±0.61 a 1.46 ±1.23 a
Palazzo Treatments average 0.44 ±0.16 b 0.20 ±0.08 b
Treatment
No coating Genotypes average 0.5 ±0.3 b 0.4 ±0.50 b
Coating Genotypes average 0.9 ±0.6 a 1.3 ±1.03 a
Genotype ×Treatment
Houmairi No coating 0.48 ±0.39 b 0.56 ±0.70 b
Coating 1.40 ±0.40 a 2.37 ±0.92 a
Palazzo No coating 0.44 ±0.09 b 0.19 ±0.05 b
Coating 0.44 ±0.22 b 0.21 ±0.11 b
Significance of ANOVA
Genotype *** ***
Treatment *** ***
Genotype ×Treatment ** ***
Mean values shown in columns according to Tukey’s test at p
≤
0.05. Data are presented as mean
±
SD. Mean
values followed by the same letter are not significantly different at p
≤
0.05. ***, **: significant at p
≤
0.001,
p≤0.01, respectively.
3.3. Variability on Photosynthetic Pigments and Biochemical Traits
The results of the analysis of variance suggest that the increasing salt concentrations
had a significant effect on all the parameters studied (Table 5). Additionally, regarding
the chlorophyll content, no significative differences have been found between the cv.
Houmairi and Palazzo, but an increase in salt concentration at 200 mM induce an increase
in chlorophyll content (30.11 and 30.97 µg/cm2).
Flavanols and anthocyanins, two important traits related to the secondary metabolism
of the shoots, showed clear differences due to the cultivars, but also due to the salt con-
centration used. Furthermore, a significant interaction between factors (cv
×
treatments)
were observed. In control conditions (0 mM NaCl), flavanols contents were not different
between the cv. Houmairi and Palazzo (0.42 and 0.39 µg/cm2, respectively).
As the concentration of NaCl increased, the cultivars showed different behaviors:
in cv. Houmairi, in fact, no significant difference was observed between the control and
increasing concentrations of NaCl, while in the cv. Palazzo at the NaCl concentration of
100 mM, a significant increase in this parameter was observed compared to the control

Horticulturae 2023,9, 1344 9 of 15
(0.54 vs. 0.39
µ
g/cm
2
). At higher NaCl concentrations (150 and 200 mM), the average
flavanol content was lower than the treatment with 100 mM NaCl.
However, regarding the anthocyanin content, different behavior of the two cultivars
was observed when increasing the salt treatments (as also confirmed by the significance of
the interaction between the two factors). In cv. Houmairi, in fact, at the salt concentration
of 100 mM, a significant increase in anthocyanin content was observed. In cv. Palazzo, on
the other hand, a significant decrease in anthocyanin content was observed in the presence
of different salt concentrations.
As the NBI parameter is an index that takes into account the relationship between
the primary and secondary metabolism, it depends on the content of chlorophylls, antho-
cyanins, and flavanols. In this case, however, apart from a statistical difference between the
cultivars, this parameter showed no difference among the treatments.
Table 5.
Estimates of physiological parameters detected through the Dualex
®
tool on fig genotypes
‘Houmairi’ and ‘Palazzo’ exposed to five different salt (NaCl) concentrations.
Genotype Chlorophyll
(µg/cm2)
Flavanols
(µg/cm2)
Anthocyanin
(µg/cm2)NBI (Chl./Flav.)
Houmairi NaCl
concentrations 28.65 ±3.83 0.42 ±0.06 b 0.07 ±0.05 70.27 ±15.76 a
Palazzo NaCl
concentrations 27.60 ±4.90 0.46 ±0.09 a 0.07 ±0.03 62.56 ±14.48 b
Treatment
0 (Control)
Genotypes average
25.63 ±4.57 b 0.41 ±0.05 c 0.08 ±0.04 ab 63.57 ±12.67
50
Genotypes average
29.00 ±4.00 ab 0.44 ±0.06 ab 0.06 ±0.03 bc 66.90 ±15.60
100
Genotypes average
28.50 ±3.68 ab 0.48 ±0.11 a 0.05 ±0.03 c 62.68 ±17.25
150
Genotypes average
27.63 ±4.87 ab 0.44 ±0.07 ab 0.10 ±0.03 a 64.75 ±17.29
200
Genotypes average
30.11 ±3.88 a 0.41 ±0.06 c 0.07 ±0.04 abc 74.17 ±13.03
Genotype ×NaCl
concentrations NaCl (mM)
Houmairi 0 (Control) 26.05 ±6.95 0.42 ±0.05 b 0.07 ±0.04 bc 62.63 ±22.69
50 28.69 ±5.83 0.45 ±0.06 ab 0.06 ±0.04 bc 65.81 ±20.78
100 28.89 ±4.07 0.42 ±0.06 b 0.04 ±0.02 c 70.82 ±17.86
150 30.38 ±3.67 0.42 ±0.09 b 0.13 ±0.02 a 74.93 ±15.25
200 29.25 ±8.59 0.39 ±0.06 b 0.07 ±0.05 bc 77.14 ±26.75
Palazzo 0 (Control) 25.22 ±5.35 0.39 ±0.06 b 0.10 ±0.04 ab 64.52 ±5.86
50 29.31 ±3.96 0.44 ±0.06 b 0.06 ±0.02 bc 68.00 ±9.24
100 28.12 ±4.57 0.54 ±0.12 a 0.07 ±0.03 bc 54.54 ±11.25
150 24.89 ±4.53 0.46 ±0.05 ab 0.07 ±0.02 bc 54.56 ±8.41
200 30.97 ±3.81 0.44 ±0.05 b 0.07 ±0.02 bc 71.19 ±5.71
Significance of ANOVA
Genotype ns ** ns **
Treatment * ** ** ns
Genotype ×NaCl concentrations ns ** ** ns
Mean values shown in columns according to Tukey’s test at p
≤
0.05. Data are presented as mean
±
SD. Mean
values followed by the same letter are not significantly different at p
≤
0.05. ns, **, *: non-significant or significant
at p≤0.01, p≤0.05, respectively.
Many biochemical signals are generally produced in plant tissues due saline stress,
and Table 6shows the data of the major involved traits.
The electrolyte leakage (EL), an early indicator of cell membrane damage, showed
different trends depending on the cultivar: in the case of cv. Houmairi, in fact, no significant
differences were observed among treatments, unlike in the case of cv. Palazzo, where when
salt concentration increased, particularly at NaCl concentrations of 100, 150, and 200 mM,
a significant increase in this trait was observed, confirming higher cellular damage in
‘Palazzo’ than the ‘Houmairi’.

Horticulturae 2023,9, 1344 10 of 15
Table 6.
Electrolyte leakage (EL), total protein (PROT), malondialdehyde (MDA), guaiacol peroxidase
(POD), catalase (CAT), and proline contents detected in fig genotypes shoots ‘Houmairi’ and ‘Palazzo’
exposed to five different salt (NaCl) concentrations.
Genotype EL
(%)
PROT
(mg g−1FW)
MDA
(µg g−1FW)
POD
(∆470 min−1-mg−1
prot)
CAT
(∆240 min−1-mg−1
prot)
Proline
(µg g−1FW)
Houmairi NaCl
concentrations 18.41 ±3.16 b 5.74 ±0.91 4.36 ±0.85 b 7.25 ±3.21 2.12 ±0.23 3.57 ±0.79
Palazzo NaCl
concentrations 33.25 ±5.41 a 5.67 ±1.52 4.60 ±1.21 a 8.08 ±4.05 2.25 ±0.23 4.01 ±1.78
Treatment
0 (Control) Genotypes
average 24.26 ±5.82 4.98 ±1.06 d 4.24 ±0.90 c 4.21 ±0.35 b 2.07 ±0.23 2.59 ±0.72 b
50 Genotypes
average 23.74 ±6.21 4.86 ±0.26 d 3.67 ±0.06
d5.03 ±0.63 b 2.22 ±0.23 3.27 ±0.84 b
100 Genotypes
average 28.52 ±10.31 6.89 ±1.55 a 3.77 ±0.69
d6.45 ±2.15 b 2.31 ±0.20 3.18 ±0.54 b
150 Genotypes
average 25.62 ±11.72 6.56 ±0.84 b 5.72 ±1.10 a 10.81 ±3.40 a 2.11 ±0.26 4.73 ±1.17 a
200 Genotypes
average 27.01 ±10.28 5.22 ±0.39 c 5.01 ±0.26 b 11.83 ±1.40 a 2.23 ±0.25 5.18 ±1.47 a
Genotype ×NaCl
concentrations NaCl (mM)
Houmairi 0 (Control) 20.07 ±3.14 bcd 5 .15 ±0.12 b 5.05 ±0.10 b 4.14 ±0.24 d 2.16 ±0.30 2.87 ±0.96 c
50 19.19 ±3.85 bcd 5.06 ±0.21 b 3.63 ±0.05
d4.68 ±0.44 cd 2.07 ±0.14 3.15 ±0.30 bc
100 19.37 ±0.63 bcd 5.49 ±0. 16 c 3.14 ±0.08 e 7.5 ±2.56 bc 2.21 ±0.23 3.26 ±0.82 c
150 15.49 ±3.03 d 7.32 ±0.12 a 4.72 ±0.05 c 7.98 ±1.93 b 1.93 ±0.05 3.84 ±0.74 bc
200 17.92 ±4.01 cd 4.88 ±0.12 c 5.24 ±0.04 a 11.94 ±1.85 a 2.22 ±0.34 3.93 ±0.83 bc
Palazzo 0 (Control) 28.45 ±4.71 ab 4.01 ±0.04 e 3.42 ±0.06 e 4.28 ±0.49 c 1.98 ±0.13 2.30 ±0.35 c
50 28.28 ±4.42 abc 4.66 ±0.07 d 3.70 ±0.05
d5.38 ±0.65 c 2.36 ±0.23 2.60 ±0.58 c
100 37.68 ±3.74 a 8.30 ±0.12 a 4.40 ±0.10 c 5.38 ±1.27 c 2.40 ±0.12 3.11 ±0.17 c
150 35.75 ±5.11 a 5.80 ±0.11 b 6.72 ±0.08 a 13.64 ±1.05 a 2.30 ±0.25 5.63 ±0.68 ab
200 36.10 ±0.60 a 5.57 ±0.12 c 4.77 ±0.12 b 11.72 ±1.20 b 2.23 ±0.21 6.42 ±0.16 a
Significance of ANOVA
Genotype ** ns ** ns ns ns
Treatment ns ** ** ** ns **
Genotype ×NaCl concentrations * ** ** ** ns **
Mean values shown in columns according to Tukey’s test at p
≤
0.05. Data are presented as mean
±
SD. Mean
values followed by the same letter are not significantly different at p
≤
0.05. ns, **, *: non-significant or significant
at p≤0.01, p≤0.05, respectively.
The total protein content trend—a crucial estimate that provides information on the
amount of stress-related proteins—followed a similar pattern for both genotypes, but
‘Palazzo’ showed a spike in the 100 mM salt treatment, while ‘Houmairi’ showed an
increase in 150 mM treatment.
Also, the content of malondialdehyde could be useful to evaluate the oxidative stress
as a result of lipid peroxidation. In this case, ‘Houmairi’ seems to have not been affected by
salt stress because at 50–100 mM of NaCl, it showed lower mean values than the control,
and only in the higher salt treatments (200 mM) was an important increase of MDA content
observed compared to the control (11.94 and 4.14 respectively). At the same time, cv.
Palazzo showed a significative increase of MDA content, also at a concentration of 150 mM.
A central role for ROS detoxifying signaling pathways, especially for H
2
O
2
, is the
upregulation of the two groups of enzymes; i.e., catalases and peroxidases. In our findings,
both POD and CAT activities in both cv showed no differences in control condition, but
when subjected to salt stress, their responses were different. Regarding the POD activity,
both varieties showed a significant increase in the activity of this enzyme as salt concen-
tration increased, particularly at the 150 and 200 mM concentrations. On the other hand,
neither cultivar showed any differences regarding CAT activities, not among them nor or
as a function of imposed salt stress.
One of the adaptive responses to prevent salt toxicity is the osmotic adjustment
through the synthesis and accumulation of low-molecular-weight metabolites known as
osmolytes, especially free proline (PRO), a proteinogenic amino acid that plays a crucial role.
In our experiments, the cultivar Houmairi did not show significant differences, maintaining
a constant threshold in all treatments observed. The cv. Palazzo, contrarily, showed a

Horticulturae 2023,9, 1344 11 of 15
significant increase in proline contents when increasing the concentration of NaCl, in
particular at higher concentrations of 150 and 200 mM.
4. Discussion
The obtained findings clearly show that fig genotypes ‘Houmairi’ and ‘Palazzo’ are
affected differently by salinity stress. Both genotypes showed a medium-high level of
viability even under the most severe concentrations; however, ‘Houmairi’ proved to be more
tolerant than ‘Palazzo’ to salt stress in almost all the morphological indicators evaluated.
The number of shoots and their length were negatively affected by the increasing salt
concentrations, according to other similar studies [
9
,
33
]. However, the number of roots
and their length showed a different trend; therefore, cv. Houmairi starts to be affected
by salt stress from the third level equal to 150 mM. This contrasts with the findings of
Vijayan et al. (2003) [
34
], but is in agreement with another study [
35
] that suggests a
different Na ion accumulation in shoots and roots. Our findings are also confirmed by
the assessment of polyphenolic compounds conducted by the Dualex; i.e., the chlorophyll
content of ‘Houmairi’ shoots is still the same in all the applied treatments while ‘Palazzo’
shoots indicate a significantly lower value under 150 mM of salt concentration. It is fully
demonstrated that a high concentration of NaCl affects the photosynthesis rate [
36
]. Many
authors agree with our results [
9
,
37
,
38
]; Ekinci et al. (2012) [
39
] suggest that an increase in
the chlorophyll content under salt stress conditions could be due to the increased number
of chloroplasts and to the accumulation of NaCl in the chloroplast.
The nitrogen balance index (NBI), which is expressed as the ratio of chlorophyll to
epidermal flavonoids belonging to epidermal polyphenolics (Chl/Flav), represents a sen-
sitive indicator of plant nitrogen N status [
40
], because when plants are subjected to a
low availability of nitrogen, the content of (Chl) decreases while epidermal polypheno-
lics (Phen) increase; and so, it represents an useful method for estimating stress-related
metabolism parameters. In this study, both flavanols content and chlorophyll content were
not significantly different in ‘Houmairi’ in all the treatments; cv. Palazzo, on the contrary,
shows a different trend; flavanols content indeed had a growing value compared to both
the control and cv. Houmairi. This evidence is almost the same for the electrolyte leakage.
When cells are critically damaged and lose the integrity of the membrane, electrolytes—
K ions in particular—leak out of the cell, and it is possible to measure the electrolytic con-
ductivity of the water in order to assess the relative of dead cells in response to stresses [
41
].
Salt stress induces an increase in the concentration of Na
+
inside the cells, and this re-
sults in an excess of Na
+
in the cytosol. At the same time, it induces a K
+
efflux, thus
allowing the drastic decrease of the cytosolic K
+
/Na
+
ratio [
42
]. The EL content of the
‘Houmairi’ genotype was not significant for all treatments, while ‘Palazzo’ shows greater
values than ‘Houmairi’ and the control. Electrolyte leakage may be induced by a wide
range of factors [
43
] in many stress-tolerant species in order to avoid stress-induced loss
of K
+
. Higher levels of K
+
are maintained in stress conditions [
44
]; this can be achieved
through increased K
+
selectivity of total plasmatic membrane conductance [
43
]. Demidchik
(2014) [
43
] suggests that the decrease in cytosolic K
+
could play the role of a ‘switch’ that
reduces the energy consumption for anabolic reactions and stimulates ‘energy-releasing’
for catabolic processes. So, this mechanism stops plant growth and ‘redirects’ the energy
flow to adaptation and reparation needs. This represents an interesting hypothesis and
could be a critical step in plant cell adaptation to any stress factor, as plants which are
subjected to stress stop growing and use the released energy to fight stress-induced in-
juries [
43
]. Different studies [
45
,
46
] reported that salinity-tolerant plants limit the excess
of soluble salts in vacuoles, or accumulate essential ions in different tissues. The study of
Yassin et al., 2019) [
47
] suggests that the low uptake of Na
+
and high uptake of K
+
denote
salinity tolerance in higher plants. Probably, ‘Houmairi’ and ‘Palazzo’ could have different
mechanisms to avoid salt stress.

Horticulturae 2023,9, 1344 12 of 15
Generally, salinity stress causes a significant increase in protein content in plant tissues,
and their increased production is particularly important to stabilize cell membranes for cell
survival, especially under salt stress conditions.
Salt tolerant cultivars have higher protein concentration due to their higher osmotic
regulation mechanism efficiency, which in turn causes a decrease of sodium toxicity in the
cytoplasm compared to susceptible ones. The result is the prevention of proteins reduction
under salt stress [
48
]. In the present study, both cultivars—Houmairi and Palazzo—showed
a significant amount of total protein content.
Lipid peroxidation (MDA) is an oxidative salt-stress related indicator; indeed, higher
concentrations of MDA are related to cell membrane damages. In salt-tolerant plants, there
is lower MDA expression, as well as lower ROS production—especially H
2
O
2
—due to
the presence of an efficient protection mechanism and a high scavenging capacity [
6
]. In
our study, we found that ‘Palazzo’ had a significant increase of MDA, especially in the
range 100–200 mM, but the highest value was registered at a 150 mM level. This finding,
according to [
9
], is probably due to the fact that when NaCl concentration was equal to
200 mM, some stress-related genes were induced in leaves and the MDA accumulation was
lower. On the other hand, ‘Houmairi’ showed an opposite trend, since in all the treatments,
with the exception of 150–200 mM, the content of MDA was lower than the control. A
recent study [
36
] suggests that a sustained decrease of MDA expression might be caused
by the activation of PSII (photosystem II) core proteins (which are responsible for water
splitting, oxygen evolution, and plastoquinone reduction of the enzyme RuBisCo) [49].
Plants control the concentrations of ROS under no stressful conditions using an array
of constitutive enzymatic and non-enzymatic antioxidants such as catalase (CAT) and
guaicol peroxidase (POD). Generally high antioxidant activities are interpreted as symp-
toms of oxidative stress (the plant upregulates the antioxidant enzymes as a result of the
increased production of ROS), but the higher antioxidant activity could also be interpreted
as a tolerance mechanism to oxidative stress, since these plants avoid oxidative stress by
maintaining higher antioxidant activity [
6
]. In our study, we found a higher increase of
POD with the 50 mM treatment, associated with a non-significant accumulation of CAT for
both genotypes ‘Houmairi’ and ‘Palazzo’. This result is in accordance with [6,50].
Salinity occurs with excess ions in the root zone that causes a detrimental effect on
water uptake, resulting in osmotic stress and high concentrations of toxic ions inside
the cells. Plants have developed complex strategies to optimize adaptive responses to
reduce salt toxicity. One of these mechanisms is osmotic adjustment through the synthesis
and accumulation of low-molecular-weight metabolites known as osmolytes, in order to
maintain metabolic activities [
51
] such as soluble sugars, proteins, and GSH. The large
availability of these compounds in the cells could be helpful in the selection for salt
tolerance genotypes [
52
]. Proline is a multifunctional amino acid that accumulates in
plant cells subjected to several kinds of stress [
51
]. It may adjust ion balance, scavenge
free radicals, and can act as a non-enzymatic antioxidant [
53
]. Many studies [
49
,
53
,
54
]
reported that proline content increases with increasing salt stress levels. In the present
study, ‘Houmairi’ showed a slow increase, but resulted in substantial differences among
all salt treatments, while ‘Palazzo’ had an increase from the first salinity concentration
(50 mM), and in particular, shoots exposed to 150 and 200 mM had higher values.
5. Conclusions
Salinity is a major constraint that negatively affects soil fertility and crop production.
The aim of this study was to assess the tolerance of fig nodal explants to salt stress when
encapsulated in a nutrient matrix containing varying concentrations of NaCl. The study
evaluated both vegetative and rhizogenic activity of the synthetic seeds, as well as chemi-
cal factors to better understand the effects on the two varieties studied (‘Houmairi’ and
‘Palazzo’). The results indicate that both genotypes can tolerate salt stress, but through
different physiological pathways. ‘Houmairi’ was found to be more tolerant than ‘Palazzo’,
and both varieties showed variability, possibly due to genetic differences. Synthetic seed

Horticulturae 2023,9, 1344 13 of 15
technology and tissue cultures proved to be a useful method for validating the selection of
valuable genotypes.
Author Contributions:
M.A.G., C.S. and I.G. are responsible for the conceptualization and experi-
mental design; M.M. and C.S. for methodology, I.G., C.S. and L.R. conducted the experiment and
data analysis, I.G, C.S. and M.M. draft the manuscript, M.M and L.R acquire publication costs. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to no public repository for raw data is
provided.
Conflicts of Interest: The authors declare no conflict of interest.
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