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 mm2) 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.
The genus Ficus is classified within the Moraceae family, encompassing a diverse assemblage 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 geographical distribution across Nepal, Somalia, Egypt, India, Iran, Sudan, Ethiopia, and Saudi Arabia (Khan et al. 2011). The fruit of the fegra 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; Alouffi et 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 propagation through seed methods. An alternative propagation method involves the use of cuttings; 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). Furthermore, cuttings display heightened sensitivity to environmental fluctuations, such as significant alterations in temperature and moisture levels, which further diminishes their overall survival rate (Dolgun and Tekintas 2008). Therefore, conventional propagation of fig is not commercially viable and rather propagated by tissue culture techniques.
Ex vitro hardening is a critical step in micropropagation. In vitro plants exhibit a diminished capacity for photosynthesis as a result of their reliance on heterotrophic nutrition. Consequently, micropropagated plantlets encounter numerous environmental stresses during the processes of hardening and acclimatization to ex vitro conditions, which adversely affect their growth and survival rates. It has been documented that a variety of strategies, including the ventilation of culture vessels, the reduction of sucrose supplementation, and the enhancement of light intensity, have been implemented to optimize the physiological performance of these plants. The application of arbuscular mycorrhizal fungi (AMF) has demonstrated 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 critical 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 parasites, pathogenic fungi, and nematodes by triggering 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 adhesion of soil particles (Smith and Smith 2011a, 2011b). Additionally, the duration of the acclimatization period for micropropagated plantlets 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 development of root system (Kapoor et al. 2008). It has been established that numerous fruit crops form a symbiotic relationship with mycorrhizal fungi, significantly relying on this symbiosis for optimal growth and enhanced performance in agricultural settings (Smith and Smith 2011b). AMF are obligate biotrophs, and the symbiotic interactions primarily revolve around nutrient exchange, whereby the plant supplies carbon derived from photosynthetic products, while the AMF facilitate the transfer of nutrients from the soil to the plants (Smith and Smith 2011a). Generally, AMF rapidly extend their hyphae through the soil over considerable distances, efficiently absorbing nutrients for plant uptake. These characteristics are particularly advantageous for plants during the critical acclimatization phase. Due to these benefits, mycorrhization and AMF inoculation are extensively advocated for the successful ex vitro establishment of horticultural plants (Azcón-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 as walnut (Mortier et al. 2020), strawberry (Fragaria ×ananassa) (Taylor and Harrier, 2001), pomegranate (Singh et al. 2012), prunus (Monticelli et al. 2000), apple (Cavallazzi et al. 2007), blackberry (Rubus fruticosus ‘P45’) (Dewir et al. 2023a), and red dragon fruit (Hylocereus polyrhizus) (Dewir et al. 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 orchards conditions were colonized by indigenous 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. Additionally, Caruso et al. (2021) reported that Ficus carica was positively responsive to the mycorrhizal inoculation but with cultivar-dependent 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 University. The experiments were implemented using 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 autoclaving (at 121 °C and 1.2 kg·cm−2 pressure for 15 min). The cultures were incubated at 25 ± 2 °C under a 16-h photoperiod provided by cool-white fluorescent lights at 35 μmol·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 optimal auxin concentration for their rooting (Al-Aizari et al. 2024). The cultures were incubated at 25 ± 2 °C for 4 weeks in the dark, followed by 3 weeks of incubation under light (16-h photoperiod provided by cool-white fluorescent lights at 35 μmol·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 material in this study (Fig. 1B).
Preparation of AMF inoculum.
The AMF used in this investigation were isolated from soil associated with the roots of Cynodon dactylon growing in the King Saud University Botanical Garden located in Riyadh, Saudi Arabia (lat. 24°44′31.79″N, long. 46°51′25.19″E). The gathered samples 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 isolated AMF spores were classified based on morphological characteristics (i.e., shape, surface ornamentation, color, internal contents, and wall architecture) (Schenck and Perez-Collins 1990; Walker 1997) and compared with the morphological classifications of species documented by the International Culture Collection of Vesicular-Arbuscular Mycorrhizal Fungi (INVAM 2023) and supplementary scholarly references (Redecker et al. 2013; Schüßler et al. 2001; Schüβler and Walker 2010). Subsequently, the identified spores (Gigaspora albida and G. margarita) 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 determine the occurrence and abundance of spores in accordance with established methodologies 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 and amended with 5% (w/w) AMF inoculum soil. Two treatments (with or without AMF inoculation) were applied. The applied inoculum comprising AMF species Gigaspora margarita and Gigaspora albida (Fig. 2) with a density of 33.4 spores/g dry soil was acquired 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% relative humidity, and 100 µmol·m−2·s−1 PPFD (16-h:8-h photoperiod under white fluorescent 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, chlorophyll content, stomatal density, mycorrhizal 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 represented by a pot containing one micropropagated plantlet.
Measurements of chlorophyll and carotenoid contents.
Fresh leaves (0.1 g) of acclimatized plants were placed in a test tube containing 10 mL of 80% acetone and kept in the refrigerator at 4 °C for 48 h in the dark. After checking the turbidity of the extract, the absorbances of chlorophyll a, chlorophyll b, and carotenoids were measured at wavelengths of 663.2, 646.8, and 470.0 nm, respectively, using a spectrophotometer (T60 ultraviolet/Visible Spectrophotometer; PG Instruments Ltd., Lutterworth, UK). The quantities of chlorophyll 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.
Microscopic examination of stomatal structures. Leaf cuticle strips were prepared in accordance 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 excised 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 distilled 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 stomata (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 camera (DeltaPix, Smørum, Denmark). Microscopic images of the leaf surfaces were captured at a magnification of 40×. In the microscopic field of view, the type of stomata, stomatal density, 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 evaluation of stomatal characteristics derived from randomly selected plants with different leaves.
Symbiotic development and AMF spore count.
To quantify the percentage of root colonization, fresh fine roots were carefully selected, stained, and studied according to the methods of Phillips and Hayman (1970), with some modifications (Al-Qarawi et al. 2012). Spores were isolated 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 (Schüβler and Walker 2010).
Measurement of the root growth parameters.
The roots of fegra fig plantlets (with or without mycorrhizal inoculation) were extracted from their pots and thoroughly washed with tap water to facilitate the establishment 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 8 h. A flatbed scanner (Cannon unit 101, Green Island, NY, USA) was employed for the scanning process, and the resulting images were analyzed using WinRHIZO software (V5.0; Regent Instruments, Quebec, QC, Canada). Various root system characteristics 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, 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 t test. The mean values were compared 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 acclimatization, the roots of treated fegra fig plants with G. margarita and G. albida were harvested and examined to determine colonization with the host plants. The microscopic observation of the mycorrhizal status of fegra fig plants indicated the presence of predicted AMF structures (mycelium, arbuscules, and spores) in roots (Fig. 3). The analysis of mycorrhizal colonization 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 margarita and Gigospora albida (Rui et al. 2021).
Effects of AMF on vegetative growth characteristics, stomata density, and leaf pigments of acclimatized fegra fig plantlets.
The AMF significantly (P ≤ 0.05) affected various vegetative growth parameters of fegra fig after 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 cm2), 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 mm2) of AMF-treated plants was similar 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 apparatus 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 bipinnatifidum 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 CO2 assimilation, stomatal conductance, 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 capacity to modify stomatal behavior (Augé et al. 2016). This modification in stomatal behavior encompasses enhanced stomatal conductance and gas exchange, both of which are fundamentally governed by the dynamics of the stomatal 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). Furthermore, the proliferation of roots facilitated by extraradical hyphae provides plants with superior 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 (Augé 2001; Smith and Read 2008). Our results showed that inoculation 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 content is associated with a high photosynthetic capacity resulting in vigorous vegetative growth during acclimatization (Fig. 6). Additionally, the increased carotenoid content in AMF plantlets 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 species formed within the chloroplast (Young 1991). Previous reports have suggested that inoculation of micropropagated plants with AMF during the acclimatization phase significantly enhanced leaf pigments. For example, AMF-inoculated Vitis vinifera plants demonstrated increased chlorophyll and carotenoid contents in the leaves, and all the AMF treatments either singly or in combination were significantly superior to no inoculation (Krishna et al. 2005). A high chlorophyll content 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 (Hylocereus polyrhizus) plantlets without AMF (Dewir et al. 2023b). Our results showed that AMF have beneficial effects on the micropropagated fegra fig plants and help them to improve their physiological adjustments during acclimatization.
Vegetative growth characteristics and leaf pigments of fegra fig in response to arbuscular mycorrhizal fungi after 8 weeks of acclimatization.
Stomata density of fegra fig in response to arbuscular mycorrhizal fungi after 8 weeks of acclimatization.
Root growth characteristics of non-AMF and AMF-treated fegra fig plants after 8 weeks of acclimatization.
Effects of AMF on root growth characteristics 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 cm2), total root volume/plantlet (5.2 cm3), root FW (4.15 g), and root dry weight (0.40 g) were all significantly 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. Previous 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 significantly higher fresh biomass and dry biomass values of shoots. The root growth parameters of AMF-treated fegra fig plants had significantly higher values (i.e., number of roots per plantlet, root length, total root length per plantlet, surface area of roots, root volume, root diameter, FW of roots, and dry weight of roots) compared with those of non-AMF-treated plantlets. It can be concluded that these mycorrhiza species (Gigospora albida and G. marginata) significantly enhanced fegra fig plant growth during the acclimatization stage. Inoculation 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 to improve the absorption of nutrients and water (Rouphael et al. 2015). Moreover, it is believed that AMF colonization may also cause alterations in root morphology by penetrating the cells and extending its hyphal network beyond the availability of vital nutrients, thus facilitating the plants’ ability to accumulate a comparatively greater biomass (Bowles et al. 2016). The AMF have successfully improved vegetative growth and survival of several micropropagated plant species under ex vitro conditions. However, the degree of fungus–plant compatibility is genetically controlled by the symbionts (Silveira et al. 1992). Comlekcioglu et al. (2008) reported that mycorrhizal inoculation increased the shoot, root dry weight, zinc, and phosphorus uptake of micropropagated plantlets of fig (Ficus carica). Caruso et al. (2021) confirmed that Ficus carica was positively responsive to the mycorrhizal inoculation but with cultivar-dependent patterns, and all root growth parameters of the cultivar Natalese treated with AMF were increased compared 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 micropropagated 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, it was reported that AMF inoculation significantly increased plant height, root and shoot weights, and leaf area at the end of the hardening phase (Quatrini et al. 2003). Banana plantlets inoculated with AMF had a greater height, leaf area, and FW of shoots and roots, as well as higher rates of photosynthesis and transpiration compared to those of controls (Melo et al. 1999). Similarly, Mortier et al. (2020) showed that early inoculation with AMF improved the survival and seedling performance of transplanted 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, inoculations 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 micropropagated fegra fig plantlets with G. margarita and G. albida mycorrhizal species significantly increased the contents of leaf pigments, 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 during acclimatization of micropropagated fruit trees.
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