Application of Arbuscular Mycorrhizal Fungi for Improved Growth and Acclimatization of Micropropagated Fegra Fig (Ficus palmata Forssk.) Plantlets

Abstract

Fegra fig ( Ficus palmata ) is an important fruit-yielding crop and potential rootstock for grafting Ficus carica . The acclimatization phase is a pivotal step during the micropropagation of plants. During this study, the mycorrhization of micropropagated fegra fig plants with two arbuscular mycorrhizal fungi (AMF; Gigaspora margarita and Gigaspora albida ) to enhance their growth and survival during the acclimatization stage was investigated. The AMF were mixed in equal proportions and the acclimatizing fegra fig plantlets were treated for 8 weeks. The leaf pigments, i.e., chlorophyll a [2.56 mg·g ⁻¹ fresh weight (FW)], chlorophyll b (1.08 mg·g ⁻¹ FW), total chlorophyll (3.67 mg·g ⁻¹ FW), and carotenoid (1.34 mg·g ⁻¹ FW), of AMF-treated plants were higher than those of non-AMF plants. The number of stomata per unit was higher in the AMF-treated plants (16.00), the density of stomata per unit area (88.40 mm ² ) of AMF-treated plants was similar to that of non-AMF treated plants, and the number of epidermal cells (79.00) was higher in the AMF-treated plants. The AMF-treated plants were taller and had more leaves, a greater leaf area, and higher shoot FW and dry weight. The AMF-treated plants also had the greatest total root length values, greatest surface areas of roots, and greatest total root volume and diameter compared to those of non-AMF plants. Additionally, the AMF-treated plants had a 100% survival rate, whereas a survival rate of 95% was recorded for non-AMF plants. These findings emphasize the importance of biological acclimatization of micropropagated fegra fig plants with AMF.

Full text

HORTSCIENCE 59(11):1674–1681. 2024. https://doi.org/10.21273/HORTSCI18211-24
Application of Arbuscular Mycorrhizal
Fungi for Improved Growth and
Acclimatization of Micropropagated
Fegra Fig (Ficus palmata Forssk.)
Plantlets
Y. H. Dewir, A. A. Al-Aizari, R. S. Al-Obeed, T. S. Alshahrani,
M. M. Habib, J. A. Malik, and A. A. Al-Qarawi
Plant Production Department, College of Food and Agriculture Sciences,
King Saud University, Riyadh 11451, Saudi Arabia
M. S. Alwahibi
Department of Botany and Microbiology, College of Science, King Saud
University, Riyadh 11495, Saudi Arabia
Keywords. acclimatization, Gigaspora, hardening, mycorrhization, rooting
Abstract. Fegra fig(Ficus palmata) is an important fruit-yielding crop and potential root-
stock for grafting Ficus carica. The acclimatization phase is a pivotal step during the mi-
cropropagation of plants. During this study, the mycorrhization of micropropagated fegra
fig plants with two arbuscular mycorrhizal fungi (AMF; Gigaspora margarita and Giga-
spora albida) to enhance their growth and survival during the acclimatization stage was in-
vestigated. The AMF were mixed in equal proportions and the acclimatizing fegra fig
plantlets were treated for 8 weeks. The leaf pigments, i.e., chlorophyll a [2.56 mg·g
21
fresh
weight (FW)], chlorophyll b (1.08 mg·g
21
FW), total chlorophyll (3.67 mg·g
21
FW), and
carotenoid (1.34 mg·g
21
FW), of AMF-treated plants were higher than those of non-AMF
plants. The number of stomata per unit was higher in the AMF-treated plants (16.00), the
density of stomata per unit area (88.40 mm
2
)ofAMF-treatedplantswassimilartothatof
non-AMF treated plants, and the number of epidermal cells (79.00) was higher in the
AMF-treated plants. The AMF-treated plants were taller and had more leaves, a greater
leaf area, and higher shoot FW and dry weight. The AMF-treated plants also had the
greatest total root length values, greatest surface areas of roots, and greatest total root vol-
ume and diameter compared to those of non-AMF plants. Additionally, the AMF-treated
plants had a 100% survival rate, whereas a survival rate of 95% was recorded for non-
AMF plants. These findings emphasize the importance of biological acclimatization of mi-
cropropagated fegra fig plants with AMF.
The genus Ficus is classified within the
Moraceae family, encompassing a diverse as-
semblage of approximately 800 species and
2000 variations of woody trees, shrubs, and
vines (Aati et al. 2022). The fegra fig(Ficus
palmata) represents a fruit-bearing species
within the Ficus genus, exhibiting a wide geo-
graphical distribution across Nepal, Somalia,
Egypt, India, Iran, Sudan, Ethiopia, and Saudi
Arabia (Khan et al. 2011). The fruit of the fe-
gra fig serves as a nutritious food source and
finds application in various industrial products
(Guasmi et al. 2006). Additionally, this tree
is a source of fuel wood and has been
traditionally used for the efficacious treatment
of numerous ailments (Sabeen and Ahmad
2009). Phytochemical investigations of the
aerial portions of the fegra fig have unveiled a
range of pharmacological properties (Aati et al.
2022; Alouffiet al. 2023; Al-Qahtani et al.
2023; Sharma et al. 2022). Furthermore, it has
been documented as a promising rootstock for
the grafting of Ficus carica (Al-Aizari et al.
2024; Hosomi 2015). The fig fruits exhibit an
independence from pollination for complete
maturation and development; consequently, the
likelihood of producing viable seeds remains
exceedingly low, thereby constraining propa-
gation through seed methods. An alternative
propagation method involves the use of cut-
tings; however, this approach is frequently
hindered by suboptimal rooting efficiency,
resulting in a survival rate that ranges from
merely 20% to 30% (Kumar et al. 1998). Fur-
thermore, cuttings display heightened sensi-
tivity to environmental fluctuations, such as
significant alterations in temperature and mois-
ture levels, which further diminishes their
overall survival rate (Dolgun and Tekintas
2008). Therefore, conventional propagation of
fig is not commercially viable and rather prop-
agated by tissue culture techniques.
Ex vitro hardening is a critical step in mi-
cropropagation. In vitro plants exhibit a di-
minished capacity for photosynthesis as a
result of their reliance on heterotrophic nutri-
tion. Consequently, micropropagated plantlets
encounter numerous environmental stresses
during the processes of hardening and acclima-
tization to ex vitro conditions, which adversely
affect their growth and survival rates. It has
been documented that a variety of strategies, in-
cluding the ventilation of culture vessels, the re-
duction of sucrose supplementation, and the
enhancement of light intensity, have been im-
plemented to optimize the physiological perfor-
mance of these plants. The application of
arbuscular mycorrhizal fungi (AMF) has dem-
onstrated efficacy in facilitating the biological
acclimatization of micropropagated plantlets.
AMF establish a symbiotic association with
plants that guarantees the availability of water
and nutrients, fosters robust vegetative growth,
and diminishes mortality rates during this criti-
cal developmental phase (Declerck et al. 2002;
Hazarika and Bora 2006; Kapoor et al. 2008;
Smith and Read 2008). Furthermore, AMF
confer protection to host plants against para-
sites, pathogenic fungi, and nematodes by trig-
gering the production of defensive compounds
and expanding the root exploration area. AMF
enhance the translocation of water from the
soil to the plant while also improving the
physical and chemical properties of the soil
through the incorporation of organic matter
and the formation of aggregates via the adhe-
sion of soil particles (Smith and Smith 2011a,
2011b). Additionally, the duration of the accli-
matization period for micropropagated plant-
lets may be effectively shortened through the
application of AMF (Salamanca et al. 1992).
The acclimatization phase can influence the
photosynthetic efficiency of micropropagated
plants, compromise their defenses against
pathogens, and impede the proper develop-
ment of root system (Kapoor et al. 2008).
It has been established that numerous fruit
crops form a symbiotic relationship with my-
corrhizal fungi, significantly relying on this
symbiosis for optimal growth and enhanced
performance in agricultural settings (Smith
and Smith 2011b). AMF are obligate bio-
trophs, and the symbiotic interactions primarily
revolve around nutrient exchange, whereby the
plant supplies carbon derived from photosyn-
thetic products, while the AMF facilitate the
transfer of nutrients from the soil to the plants
(Smith and Smith 2011a). Generally, AMF rap-
idly extend their hyphae through the soil over
considerable distances, efficiently absorbing nu-
trients for plant uptake. These characteristics are
particularly advantageous for plants during the
critical acclimatization phase. Due to these ben-
efits, mycorrhization and AMF inoculation are
extensively advocated for the successful ex vitro
establishment of horticultural plants (Azc
on-
Aguilar and Barea 1997; Kapoor et al. 2008).
The AMF have been successfully used to
improve the acclimatization and growth of
micropropagated fruit-bearing species such
Received for publication 13 Sep 2024. Accepted
for publication 27 Sep 2024.
Published online 25 Oct 2024.
We acknowledge the researchers supporting pro-
ject number (RSP-2024R375), King Saud Uni-
versity, Riyadh, Saudi Arabia.
Y.H.D. is the corresponding author. E-mail:
ydewir@ksu.edu.sa.
This is an open access article distributed under
the CC BY-NC license (https://creativecommons.
org/licenses/by-nc/4.0/).
1674 HORTSCIENCE VOL. 59(11) NOVEMBER 2024

as walnut (Mortier et al. 2020), strawberry
(Fragaria ×ananassa) (Taylor and Harrier,
2001), pomegranate (Singh et al. 2012), pru-
nus (Monticelli et al. 2000), apple (Cavallazzi
et al. 2007), blackberry (Rubus fruticosus
‘P45’) (Dewir et al. 2023a), and red dragon
fruit (Hylocereus polyrhizus)(Dewiretal.
2023b). Among fruit crops, fig has been less
studied to determine the effects of AMF;
however, Tabassum et al. (2016) found that
the root system of fig trees grown under or-
chards conditions were colonized by indige-
nous AMF. Furthermore, Comlekcioglu et al.
(2008) observed a positive effect on the root
system growth of the fig cultivar Alkuden in
response to different Glomus species. Addi-
tionally, Caruso et al. (2021) reported that
Ficus carica was positively responsive to the
mycorrhizal inoculation but with cultivar-de-
pendent patterns. However, AMF studies of
fegra fig have not been conducted. Therefore,
the present study aimed to apply AMF for the
biological acclimatization of micropropagated
fegra fig.
Materials and Methods
Study location and experimental design.
This study was conducted in the plant tissue
culture laboratory at the College of Food and
Agriculture Sciences of King Saud Univer-
sity. The experiments were implemented us-
ing a completely randomized design.
Plant material. In vitro axillary shoots of
F. palmata were multiplied using Murashige
and Skoog’s medium (Murashige and Skoog
1962) supplemented with 30 g/L sucrose and
2 mg/L 6-benzylaminopurine (BAP). The pH
of the medium was adjusted to 5.8 before
autoclavin g (at 121 C and 1.2 kg·cm
2
pres-
sure for 15 min). The cultures were incubated
at 25 ± 2 C under a 16-h photoperiod pro-
vided by cool-white fluorescent lights at
35 mmol·m
2
·s
1
photosynthetic photon flux
density (PPFD) and relative humidity of 50%
to 60%. Multiple axillary shoots (Fig. 1A)
were individually separated and cultured in
Murashige and Skoog’s medium containing
3% sucrose and 1.5 mg/L activated charcoal
and 1 mg/L indole-3-acetic acid as the opti-
mal auxin concentration for their rooting
(Al-Aizari et al. 2024). The cultures were
incubated at 25 ± 2 Cfor4weeksinthe
dark, followed by 3 weeks of incubation un-
der light (16-h photoperiod provided by cool-
white fluorescent lights at 35 mmol·m
2
·s
1
PPFD). After 7 weeks, the plantlets were
gently removed from the gelled medium,
cleaned using tap water, and used as plant ma-
terial in this study (Fig. 1B).
Preparation of AMF inoculum. The AMF
used in this investigation were isolated
from soil associated with the roots of Cyn-
odon dactylon growing in the King Saud
University Botanical Garden located in
Riyadh, Saudi Arabia (lat. 2444031.7900N,
long. 4651025.1900E). The gathered sam-
ples were mixed with sterile sand to create
a trap culture with maize (Zea mays)as
the host mycotrophic plant. Following a
duration of 6 months, spores were isolated
from the substrate of the trap culture using
the wet sieving and decanting technique
(Gerdemann and Nicolson 1963). The iso-
lated AMF spores were classified based on
morphological characteristics (i.e., shape,
surface ornamentation, color, internal con-
tents, and wall architecture) (Schenck and
Perez-Collins 1990; Walker 1997) and com-
pared with the morphological classifications
of species documented by the International
Culture Collection of Vesicular-Arbuscular
Mycorrhizal Fungi (INVAM 2023) and sup-
plementary scholarly references (Redecker
et al. 2013; Sch€
ußler et al. 2001; Sch€
ubler
and Walker 2010). Subsequently, the identi-
fied spores (Gigaspora albida and G. marga-
rita) were used to establish single spore
cultures with maize serving as the host plant
in sterile sand. After a period of 6 months,
the cultures were dried and examined to de-
termine the occurrence and abundance of
spores in accordance with established meth-
odologies and subsequently used as inoculum.
In vitro rooting, AMF inoculation, and
acclimatization of micropropagated fegra fig
plantlets. The plantlets were transplanted into
conical plastic pots (upper diameter, 4 cm;
bottom diameter, 1.7 cm; length, 21.5 cm)
filled with sterilized sand:soil (1:1) mixture
andamendedwith5%(w/w)AMFinoculum
soil. Two treatments (with or without AMF in-
oculation) were applied. The applied inoculum
comprising AMF species Gigaspora marga-
rita and Gigaspora albida (Fig. 2) with a
density of 33.4 spores/g dry soil was ac-
quired from the Rangeland Laboratory of
the Plant Production Department of King
Saud University. The non-AMF plantlets
received the same dosage of autoclaved
AMF inoculum. Thereafter, the potted plants
were grown at 25 ± 2 C, 50% to 60% rela-
tive humidity, and 100 mmol·m
2
·s
1
PPFD
(16-h:8-h photoperiod under white fluores-
cent lamps) in a growth chamber with the
pots covered with transparent polyethylene
for the first 4 weeks. The plantlets were
regularly irrigated with Hoagland nutrient
solution without phosphorus. The plantlet
growth, root growth characteristics, chloro-
phyll content, stomatal density, mycorrhi-
zal condition/status, and survival rate were
evaluated 8 weeks after being transferred
to the growth chamber. Each treatment had
20 replicates, and each replicate was repre-
sented by a pot containing one micropro-
pagated plantlet.
Measurements of chlorophyll and caroten-
oid contents. Freshleaves(0.1g)ofaccli-
matized plants were placed in a test tube
containing 10 mL of 80% acetone and kept in
the refrigerator at 4 Cfor48hinthedark.Af-
ter checking the turbidity of the extract, the ab-
sorbances of chlorophyll a, chlorophyll b, and
carotenoids were measured at wavelengths of
663.2, 646.8, and 470.0 nm, respectively, us-
ing a spectrophotometer (T60 ultraviolet/Visi-
ble Spectrophotometer; PG Instruments Ltd.,
Lutterworth, UK). The quantities of chloro-
phyll a, chlorophyll b, total chlorophyll, and
carotenoids in leaves were calculated using the
Arnon (1949) method. All measurements were
performed in triplicate.
Microscopic observation of stomata. Mi-
croscopic examination of stomatal structures.
Leaf cuticle strips were prepared in accor-
dance with the methodology of Cotton
(1974). The desiccated leaves underwent a
soaking process for 24 h, after which the thin,
transparent epidermal layer was carefully ex-
cised using pointed forceps and subsequently
positioned on a glass slide. This layer was
then subjected to staining with a mixture
comprising 0.1 g of triaryl methane dye and
2 mL of glacial acetic acid in 100 mL of dis-
tilled water (yielding a light-green dye) for a
brief duration before being covered with a
cover slip. The glass slides were examined to
ascertain types of stomata, dimensions of sto-
mata (quantified with an ocular ruler), and
the density of stomata (number of stomata
per unit area) utilizing an optical microscope
outfitted with a SwiftCam 20 Megapixel
Fig. 1. Axillary shoot multiplication of fegra fig on Murashige and Skoog’s (MS) medium supple-
mented with 30 g/L sucrose, 2 mg/L blood agar (BAP) (A), and rooting onto MS medium contain-
ing 1 mg/L indole-3-acetic acid (IAA) (B).
HORTSCIENCE VOL. 59(11) NOVEMBER 2024 1675

camera (DeltaPix, Smørum, Denmark). Micro-
scopic images of the leaf surfaces were captured
at a magnification of 40×. In the microscopic
field of view, the type of stomata, stomatal den-
sity, measurements of aperture length and width,
the quantity of epidermal cells, and the stomatal
index were systematically determined. A total of
30 measurements were conducted for the evalua-
tion of stomatal characteristics derived from ran-
domly selected plants with different leaves.
Symbiotic development and AMF spore
count. To quantify the percentage of root coloni-
zation, fresh fine roots were carefully selected,
stained, and studied according to the methods of
Phillips and Hayman (1970), with some modifi-
cations (Al-Qarawi et al. 2012). Spores were iso-
lated according to the methods of Gerdemann
and Nicolson (1963). The total spore population
of each treatment was calculated based on 100 g
of dry soil (Sch€
ubler and Walker 2010).
Measurement of the root growth parame-
ters. The roots of fegra fig plantlets (with or
without mycorrhizal inoculation) were ex-
tracted from their pots and thoroughly
washed with tap water to facilitate the estab-
lishment of three root replicates for three
individual plants from each treatment group.
Prior to scanning, the roots were subjected to
staining with toluidine red for approximately
8h.Aflatbed scanner (Cannon unit 101,
Green Island, NY, USA) was employed for
the scanning process, and the resulting im-
ages were analyzed using WinRHIZO soft-
ware (V5.0; Regent Instruments, Quebec,
QC, Canada). Various root system character-
istics were quantified, including the number
of roots per plantlet, the number of root tips
per plantlet, the length of the primary root per
plantlet, total root length, root fresh weight,
Fig. 2. Photomicrographs showing arbuscular mycorrhizal fungi spores collected from the trap culture: crushed spore spores of Gigaspora albida (Aand B)
and crushed spores of Gigaspora margarita (Cand D).
Fig. 3. Photomicrographs indicating the root colonization structures and colonization status of the roots of fegra fig plants after 8 weeks of acclimatization
(A–D). Ar 5arbuscules; ArT 5arbuscular trunk; EH 5external hyphae; ES 5extraradical intact spores; IS 5intraradical spores.
1676 HORTSCIENCE VOL. 59(11) NOVEMBER 2024

root dry weight, root diameter, root volume,
and root surface area.
Experimental design and data analysis.
The experiments were conducted using a
completely randomized design. The data were
analyzed using an analysis of variance,
Tukey’s multiple range test, and Student’s
unpaired ttest. The mean values were com-
pared using SAS (version 9.4; SAS Institute,
Inc., Cary, NC, USA) (P#0.05–0.001).
Results and Discussion
Mycorrhizal colonization of acclimatized
fegra fig plantlets. After 8 weeks of acclimati-
zation, the roots of treated fegra figplants
with G. margarita and G. albida were har-
vested and examined to determine colonization
with the host plants. The microscopic observa-
tion of the mycorrhizal status of fegra figplants
indicated the presence of predicted AMF struc-
tures (mycelium, arbuscules, and spores) in
roots (Fig. 3). The analysis of mycorrhizal col-
onization showed the following colonization
percentages: mycelium, 37.77%; vesicles, 0%;
and arbuscules, 17.77%. The total spore count
was also recorded as 181/100 g soil (Fig. 4).
Similarly, Etilingera elatior micropropagated
plants showed good colonization of AMF
fungi Gigospora albida and Claroideogloums
etunicatum after treatment with these fungi
(Melo et al. 1999). Microporpagated Musa
spp. ‘Grand Naine’plants also showed good
colonization of AMF fungi Gigaspora marga-
rita and Gigospora albida (Rui et al. 2021).
Effects of AMF on vegetative growth char-
acteristics, stomata density, and leaf pigments
of acclimatized fegra fig plantlets. The AMF
significantly (P#0.05) affected various veg-
etative growth parameters of fegra figafter
8 weeks of acclimatization (Table 1). The
AMF-treated plants had a significantly greater
plant height (21.70 cm), number of leaves
(21.30 per plant), leaf area (190.81 cm
2
),
shoot FW (4.12 g) and dry weight (0.65 g), as
well as total FW per plant (8.28 g/plantlet)
and total dry weight per plant (1.05 g/plantlet)
compared with those of non-AMF treated
(control) plants (Table 1). Figure 5 shows the
promoting effect of vegetative growth of
AMF plants compared with that of non-AMF
plants. The leaf pigment, i.e. chlorophyll a
(2.56 mg·g
1
FW), chlorophyll b (1.08 mg·g
1
FW), total chlorophyll (3.67 mg·g
1
FW), and
carotenoid (1.34 mg·g
1
FW), levels in AMF-
treated plants were higher than those in non-
AMF plants (Table 3). The number of stomata
per unit of the AMF-treated plants was higher
(16.00), the density of stomata per unit area
(88.40 mm
2
) of AMF-treated plants was sim-
ilar to that of non-AMF-treated plants, and
the number of epidermal cells (79.00) of the
AMF-treated plants was higher. However,
the aperture length of the stomatal appara-
tus of the non-AMF plants was higher than
that of AMF-treated plants (Table 2, Fig. 6).
The AMF-treated fegra fig plants had a higher
stomatal density with more stomata per unit
area. Inoculation with G. margarita and G.
albida increased the number of stomata per
unit area in AMF-treated Philodendron bipin-
natifidum plants, whereas the aperture length
and aperture width of the stomatal apparatus of
both AMF and non-AMF-treated plants were
nearly similar (Dewir et al. 2023c). Stomatal
density is an important characteristic that
enables CO
2
assimilation, stomatal conduc-
tance, and transpiration in plants (Salmon et al.
2019). The high stomatal frequency of AMF-
treated fegra fig plants was accompanied by a
higher chlorophyll content, higher leaf area,
and a greater number of leaves compared with
those of non-AMF plants. Previous studies
suggested a positive correlation between the
chlorophyll content and net photosynthetic rate
(Bhusal et al. 2018; Massonnet et al. 2007).
The symbiotic relationship between AMF and
host plants is recognized because of its capac-
ity to modify stomatal behavior (Aug
eetal.
2016). This modification in stomatal behavior
encompasses enhanced stomatal conductance
and gas exchange, both of which are funda-
mentally governed by the dynamics of the sto-
matal pore and the density of stomata. The
ameliorated stomatal behavior observed in
AMF-inoculated plants may be attributed to
the augmented uptake of phosphorus (Koide
1985; Nagarajah and Ratnasuriya 1978). Fur-
thermore, the proliferation of roots facilitated
by extraradical hyphae provides plants with su-
perior access to water, which may elucidate the
enhanced stomatal activity observed in AMF-
inoculated plants (Duan et al. 1996). Overall,
the AMF–symbiosis-induced mechanisms drive
changes in stomata (Aug
e 2001; Smith and
Read 2008). Our results showed that inocula-
tion with mycorrhizal species increased the leaf
pigments and improved the vegetative growth
as compared with those of non-AMF fegra fig
plantlets. This increment in the chlorophyll con-
tent is associated with a high photosynthetic ca-
pacity resulting in vigorous vegetative growth
during acclimatization (Fig. 6). Additionally,
the increased carotenoid content in AMF plant-
lets implies their greater ability to survive
stressful conditions during acclimatization
compared with that of non-AMF plantlets.
Carotenoids act as accessory light-harvesting
pigments and play a role in scavenging
singlet oxygen and other toxic oxygen spe-
cies formed within the chloroplast (Young
1991). Previous reports have suggested that
inoculation of micropropagated plants with
AMF during the acclimatization phase sig-
nificantly enhanced leaf pigments. For ex-
ample, AMF-inoculated Vitis vinifera plants
demonstrated increased chlorophyll and ca-
rotenoid contents in the leaves, and all the
AMF treatments either singly or in combination
Fig. 4. Arbuscular mycorrhizal fungi (AMF) root colonization (Mycelium, and arbuscules) and spore
count of acclimatized fegra fig plants.
Table 1. Vegetative growth characteristics and leaf pigments of fegra fig in response to arbuscular
mycorrhizal fungi after 8 weeks of acclimatization.
Growth parameters Non-AFM AFM-treated Pvalues
Plant height (cm) 17.3 b 21.7 a 0.044*
Number of leaves/plants 9.0 b 21.3 a 0.001*
Leaf area/plant (cm
2
) 139.23 b 190.81 a 0.000*
Fresh weight/plant (g) 2.68 b 4.12 a 0.006*
Shoot 2.68 b 4.12 a 0.006*
Root 2.55 b 4.15 a 0.004*
Total 5.23 b 8.28 a 0.003*
Dry weight/plant (g) 0.46 a 0.65 a 0.068
NS
Shoot 0.46 a 0.65 a 0.068
NS
Root 0.25 b 0.40 a 0.003*
Total 0.71 b 1.05 a 0.012*
Chlorophyll a (mg·g
1
FW) 1.88 b 2.56 a 0.004*
Chlorophyll b (mg·g
1
FW) 0.73 b 1.08 a 0.003*
Total chlorophyll (mg·g
1
FW) 2.63 b 3.67 a 0.001*
Carotenoids (mg·g
1
FW) 0.78 1.34 0.000*
Values followed by the same letter in each row are not significantly different at P#0.05 according
to Student’s unpaired ttest. NS and * 5nonsignificant and significant at P#0.05, respectively.
AMF 5arbuscular mycorrhizal fungi.
HORTSCIENCE VOL. 59(11) NOVEMBER 2024 1677

were significantly superior to no inoculation
(Krishna et al. 2005). A high chlorophyll con-
tent was also reported for AMF-treated plantlets
compared with that of Coffea arabica (Fonseca
et al. 2020), Musa spp. ‘Grand Naine’(Rui
et al. 2021), and red dragon fruit (Hylocer-
eus polyrhizus) plantlets without AMF
(Dewir et al. 2023b). Our results showed that
AMF have beneficial effects on the micropro-
pagated fegra fig plants and help them to im-
prove their physiological adjustments during
acclimatization.
Effects of AMF on root growth character-
istics of acclimatized fegra fig plantlets. Root
growth characteristics of AMF-treated plants
were compared with those of non-AMF
treated plants (Table 3, Fig. 5). The number of
roots per plantlet (16.3), length of the main root
(21.7 cm), total root length/plantlet (393.0 cm),
number of root tips/plantlet (1032.7), average
root diameter/plantlet (1.45 mm), total root
surface area (160.4 cm
2
), total root volume/
plantlet (5.2 cm
3
), root FW (4.15 g), and
root dry weight (0.40 g) were all signifi-
cantly higher when compared with those of
non-AMF plants, except the number of root
tips/plantlet (Table 3). Figure 6 shows the
promoting effect of root growth in AMF-
treated plants compared with that of non-
AMF plants. In the present study, AMF-
treated plants had a 100% survival rate,
whereas non-AMF plants had a 95% survival
rate following their acclimatization. Previ-
ous studies highlighted that micropropagated
Ficus carica had a survival rate ranging from
80% to 100% during acclimatization (80%:
Abdolinejad et al. 2020; Shatnawi et al. 2019;
Shekafandeh and Shahcheraghi 2017; 90.25%:
Sen and Patel 2018; 98%: Al-Zahrani et al.
2018; 100%: Ling et al. 2022; Prabhuling and
Huchesh 2018; Rasheed and Toma 2023). The
AMF-treated fegra fig plants exhibited signifi-
cantly higher fresh biomass and dry biomass
values of shoots. The root growth parameters
of AMF-treated fegra figplantshadsignifi-
cantly higher values (i.e., number of roots per
plantlet, root length, total root length per plant-
let, surface area of roots, root volume, root di-
ameter, FW of roots, and dry weight of roots)
compared with those of non-AMF-treated
plantlets. It can be concluded that these mycor-
rhiza species (Gigospora albida and G. mar-
ginata) significantly enhanced fegra figplant
growth during the acclimatization stage. Inocu-
lation with AMF is extensively acknowledged
for its profound influence on the growth of
plants and particularly for increasing both root
and shoot biomass (Begum et al. 2019). This
enhancement in growth can be explained by
the capacity of AMF-inoculated plants t o i m-
prove the absorption of nutrients and water
(Rouphael et al. 2015). Moreover, it is be-
lieved that AMF colonization may also cause
alterations in root morphology by penetrating
the cells and extending its hyphal network be-
yond the availability of vital nutrients, thus fa-
cilitating the plants’ability to accumulate a
comparatively greater biomass (Bowles et al.
2016). The AMF have successfully improved
vegetative growth and survival of several mi-
cropropagated plant species under ex vitro con-
ditions. However, the degree of fungus–plant
compatibility is genetically controlled by the
symbionts (Silveira et al. 1992). Comlekcioglu
et al. (2008) reported that mycorrhizal in oc u-
lation increased the shoot, root dry weight,
zinc, and phosphorus uptake of micropropa-
gated plantlets of fig(Ficus carica). Caruso
et al. (2021) confirmed that Ficus carica was
positively responsive to the mycorrhizal inocu-
lation but with cultivar-dependent patterns, and
all root growth parameters of the cultivar Nat-
alese treated with AMF were increased com-
pared with those not treated with non-AMF. It
has been previously reported that inoculation
with G. margarita and G. albida significantly
improved growth and root development of mi-
cropropagated fruit species such as blackberry
(Rubus fruticosus ‘P45’) (Dewir et al. 2023a)
and red dragon fruit (Hylocereus polyrhizus)
(Dewir et al. 2023b). The positive effects of
AMF on plant growth and performance have
also been reported for many other fruit-yielding
species such as apple and plum (Fortuna
et al. 1996). Regarding Citrus limon,itwasre-
ported that AMF inoculation significantly in-
creased plant height, root and shoot weights,
and leaf area at the end of the hardening phase
(Quatrini et al. 2003). Banana plantlets inocu-
lated with AMF had a greater height, leaf area,
and FW of shoots and roots, as well as higher
rates of photosynthesis and transpiration compared
Fig. 5. Leaf stomata density of non-arbuscular mycorrhizal fungi (AMF) (A) and AMF-treated (B) fegra fig plants after 8 weeks of acclimatization.
Table 2. Stomata density of fegra fig in response to arbuscular mycorrhizal fungi after 8 weeks of
acclimatization.
Growth parameters Non-AFM AFM-treated Pvalues
Stomata density (mm
2
) 66.3 b 88.4 a 0.000*
Number of epidermal cells 58 b 79 a <0.0001*
Aperture height (mm) 11 a 8.5 b 0.024*
Aperture width (mm) 8.6 a 7.0 a 0.063
NS
Values followed by the same letter in the same row are not significantly different at P#0.05 accord-
ing to Student’s unpaired ttest. NS and * 5nonsignificant and significant at P#0.05, respectively.
AMF 5arbuscular mycorrhizal fungi.
Table 3. Root growth characteristics of non-AMF and AMF-treated fegra fig plants after 8 weeks of
acclimatization.
Root parameters Non-AFM AFM-treated Pvalues
Number of roots/plantlets 12.0 b 16.3 a 0.031*
Number of root tips/plantlet 900.0 a 1032.7 a 0.062
NS
Length of the main root/plantlet (cm) 17.3 b 21.7 a 0.017*
Total root length (cm) 335.1 b 393 a 0.002*
Total of root surface area/plantlet (cm
2
) 123.2 b 160.4 a 0.007*
Total root volume/plantlet 3.4 b 5.2 a 0.037*
Root diameter/plantlet (mm) 1.0 b 1.45 a 0.012*
Values followed by the same letter in the same row are not significantly different at P#0.05 accord-
ing to Student’s unpaired ttest. NS and * 5nonsignificant and significant at P#0.05, respectively.
AMF 5arbuscular mycorrhizal fungi.
1678 HORTSCIENCE VOL. 59(11) NOVEMBER 2024

to those of controls (Melo et al. 1999). Sim-
ilarly, Mortier et al. (2020) showed that
early inoculation with AMF improved the
survival and seedling performance of trans-
planted walnut trees. It is well known that
natural growth and development depend on
the formation of arbuscular fungi in many
woody plants and trees. Therefore, inocula-
tions with AMF may help to overcome
problems associated with micropropagation
of woody and fruit trees, as reported by
Taylor and Harrier (2003).
In conclusion, inoculation of micropropa-
gated fegra fig plantlets with G. margarita
and G. albida mycorrhizal species signifi-
cantly increased the contents of leaf pig-
ments, stomatal density, and vegetative and
root growth characteristics. Additionally, all
AMF-treated plantlets (100%) survived
acclimatization to ex vitro conditions, whereas
non-AMF plants had a 95% survival rate.
Therefore, mycorrhizal inoculation can be
further used as a biotechnological tool to
improve growth and reduce mortality dur-
ing acclimatization of micropropagated fruit
trees.
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