Biological Acclimatization of Micropropagated Al-Taif Rose (Rosa damascena f. trigintipetala (Dieck) R. Keller) Plants Using Arbuscular Mycorrhizal Fungi Rhizophagus fasciculatus

Abstract

Tissue culture is used to multiply Al-Taif rose (Rosa damascena f. trigintipetala (Dieck) R. Keller) plants in order to meet the demands of the fragrance, cosmetic, and floriculture industries. The use of arbuscular mycorrhizal fungus (AMF) could potentially improve plant growth and acclimatization performance to ex vitro conditions. Thus, in the current study, we investigated how AMF Rhizophagus fasciculatus influences the growth, establishment, and physiological performance of micropropagated Al-Taif rose plants during the acclimatization stage. The growth and physiological parameters of the AMF-treated plants were evaluated after a 12 week growth period in the growth chambers. The plants treated with AMF exhibited greater height (25.53 cm) and biomass growth values for both shoot fresh weight (0.93 g/plant) and dry weight (0.030 g/plant), more leaves (11.3/plant), more leaf area (66.15 cm²), longer main roots (15.05 cm/plant), total root length (172.16 cm/plant), total root area (64.36 cm²/plant), and biomass from both fresh weight (383 mg/plant) and dry weight (80.00 mg/plant) of the plants. The plants treated with AMF also exhibited increased rates of net CO₂ assimilation, stomatal conductance, and transpiration compared to the control plants. The proline content in the leaves and roots was significantly lower in the AMF-treated plants than untreated plants. The Fv/Fm ratio, which serves as an indicator of the intrinsic or maximal efficacy of Photosystem II (PSII) demonstrated a notable decline in the untreated Al-Taif rose plants. These results elucidate the advantageous impact of AMF colonization on micropropagated Al-Taif rose plants, thereby enhancing their resilience against adverse ex vitro conditions.

1. Introduction

Plant tissue culture is one of the most important applications of modern biotechnology in horticulture, especially for plant propagation and multiplication. In comparatively short amounts of time, high-quality, disease-free, and uniform planting material could be quickly propagated by in vitro plant propagation [1,2]. Large-scale propagation could be accomplished by applying bioreactor technologies and employing liquid cultures, which offer various unique advantages not attainable through conventional propagation [3]. Liquid medium and bioreactor cultures are advantageous for micropropagation because they can be used to produce and scale up large numbers of plantlets; cultures are always in contact with the medium, which facilitates easy nutrient uptake and increases growth rate; forced air supply in bioreactor cultures promotes growth and metabolism of cultured cells and organs; and handling the cultures, including inoculation and harvesting, is simple and saves time and labor [3,4]. However, a necessary step for the commercial application of in vitro technology is the successful acclimatization of micropropagated plants and their subsequent transfer to the field [5]. Micropropagated plants require acclimatization in order for the plantlets to successfully establish and survive after being transplanted to ex vitro environments, where they are subjected to changes in temperature, light intensity, and water stress conditions. In order to gradually acclimate transplants from culture to ambient relative humidity and light levels, the ex vitro acclimatization environment is typically adjusted [6]. One method for successfully acclimating plants is the use of a bioinoculant, such as arbuscular mycorrhizal fungus (AMF), during the ex vitro transplantation of micropropagated plants. The fungus will be able to absorb carbon compounds from the photosynthetic host plants when AMF is applied as a bioinoculant, and the host plants will gain from the fungal mycelium’s ability to transport water and mineral nutrients [7]. Several studies have shown that the colonization of AMF during the transplantation of micropropagated plantlets from axenic to ex vitro conditions led to improved rooting and survival of micropropagated plants, a reduction in transplantation shock, a shortened weaning phase, an increase in vegetative growth, modified root morphology, and a decrease in transplantation mortality, injury, and disease occurrence [8]. Successful application of mycorrhization in horticultural plants during acclimatization has been demonstrated in Citrus tangerine [9], Cocos nucifera [10], Colocasia esculenta [11], Etlingera elatior [12], Gloriosa superba [13], Hylocereus polyrhizus [14], Malus prunifolia [15], Musa accuminata [16], Philodendron bipinnatifidum [17], Physalis peruviana [18], Rubus fruitcosus [19], and Satureja khuzistanica [20].

Roses are a valuable commodity for the floriculture, food, pharmaceutical, cosmetic, and medical industries [21,22]. They are available in a variety of cultivars on the global market [23]. Only a small number of the more than 200 species in the Rosa genus are used to produce essential oils. Among these, the damask rose (Rosa damasceana Mill.), a hybrid of Rosa moschaata and Rosa gallica, is grown for commercial rose oil production. Its main industrial products are rose water, oil, concentrate, and pure oil [24]. Rose essential oil is useful in the fragrance and cosmetics sectors, but it also has various medical benefits, including antibacterial, cytotoxic, and antioxidant qualities [25]. Furthermore, damask roses are quite popular as cut flowers and potted ornamental plants. Stem cuttings are typically used for propagation. Nevertheless, a key barrier to large-scale proliferation is the stem cuttings’ poor rooting ability. Additionally, plants grown traditionally showed low oil content and production [25,26]. As a result, roses propagated in vitro have been essential in producing healthy plants and large-scale multiplication of cultivars with desired features [27]. Rosa damascena f. trigintipetala (Dieck) R. Keller, also known as the Al-Taif rose, is typically propagated by micropropagation. In our earlier research, we examined the effects of cytokinins, light and dark incubation, and the use of bioreactor cultures for the regeneration of shoots in Al-Taif roses using nodal explants [28]. We showed that benzyl aminopurine (BAP) at 0.5 mg/L was optimal for shoot regeneration in Al-Taif roses, and that gibberellic acid (GA₃) treatment encourages the appearance of axillary buds. Additionally, our earlier findings demonstrated that dark incubation was preferable to light for shoot proliferation and that continuous immersion bioreactor cultures increased the quantity, length, and fresh weight of the shoots. The low rooting percentage was a primary issue encountered in in vitro propagation of Al-Taif rose. Therefore, in an additional set of experiments, we examined the effects of various auxins, including 2,4-dichlorophenoxyacetic acid (2,4-D), indole acetic acid (IAA), indole butyric acid (IBA), and naphthaleneacetic acid (NAA), at concentrations of 0–0.4 mg/L; salt strength (full- and half-strength Murashige and Skoog (MS) medium); sucrose concentration (20–80 g/L); light spectra (a 2:1 or 1:2 blue/red spectral ratio, cool or warm white light at a 1:1 ratio, and fluorescent light); light intensity (photosynthetic photon flux density (PPFD) values of 25, 50, and 100 μmol·m⁻²·s⁻¹); and activated charcoal (i.e., 0 and 0.5 g/L). The best conditions for 100% induction of adventitious roots were found to be half-strength MS media supplemented with 0.2 mg/L NAA, 80 g/L sucrose, 0.5 g/L activated charcoal, and 50 μmol·m⁻²·s⁻¹ PPFD [29].

Numerous studies, as previously mentioned, have shown that AMF symbiosis has positive impacts on plant growth, water intake, nutrient uptake, and tolerance to environmental challenges, particularly during the acclimatization of micropropagated plants. Conversely, there are no reports on the consequences of mycorrhizal inoculation of Al-Taif rose plants grown in vitro. Rhizophagus fasciculatus (Thaxt.) C. Walker & Schüßler, earlier belonged to the genus Glomus [30], is among the prominent AMF species that form symbiotic associations with a wide range of terrestrial plants and, thereby, aid in acquisition of essential nutrients and water, as well as increase the resistance of plants to biotic and abiotic stressors [31,32,33]. The current study hypothesized that inoculation with AMF is essential for enhanced growth of micropropagated plants of Al-Taif rose during the acclimatization phase. Therefore, we aimed to ascertain how AMF R. fasciculatus affected the physiological performance and growth of micropropagated Al-Taif rose plants, particularly during acclimatization.

2. Materials and Methods

2.1. Study Location and Plant Materials

This work was carried out at King Saud University’s College of Food and Agricultural Sciences’ plant tissue culture facility. Al-Taif rose axillary shoots were regenerated in vitro using Murashige and Skoog’s medium (MS) [34], supplemented with 30 g/L sucrose and 0.5 mg/L 6-benzyl amino purine, and incubated for 6 weeks in a dark environment [28]. The individual clumps of axillary shoots, measuring 3.5–4.0 cm, were separated and cultivated in a half strength MS medium (Figure 1a), which was supplemented with 80 g/L sucrose, 0.5 g/L activated charcoal, and 0.2 mg/L naphthaleneacetic acid (NAA) to aid in rooting [29]. The medium was solidified using agar-agar (Duchefa, Haarlem, The Netherlands), and its pH was adjusted to 5.8 before it was autoclaved for 15 min at 121 °C and 118 kPa pressure. The cultures were kept at 25 ± 2 °C with a 16:8 h (light:dark) photoperiod and a photosynthetic photon flux density (PPFD) of 50 μmol·m⁻²·s⁻¹ supplied by a cool white fluorescent tube. The plantlets were taken out of the gelled media after six weeks (Figure 1b,c), washed with tap water, and then put into plastic pots (Figure 1d) that were 21.5 cm long, 4 cm tall, and 1.7 cm wide. The pots were then filled with sterile sand and soil mixture (1:1).

2.2. Preparation of AMF Inoculum and Arbuscular Mycorrhizal Fungi (AMF) Treatment

The inoculum used in this experiment contained the spores of single AMF species R. fasciculatus acquired from the rhizosphere soil of Aeluropus lagopoides in the saline Sabkha habitat of Aushazia, AlQasim region (26°03′58″ N 44°07′55.6″ E). The spores were extracted from the collected samples as per the methods of Gerdman and Nicolson [35] and a trap culture was set using sterilized sand:soil mixture (1:1; w/w) as the substrate and Zea mays as a host plant. The trap culture was harvested in the sixth month of cultivation and spores were extracted from the substrate soil [35]. The extracted spores were subjected to identification on the basis of morphology (i.e., shape, surface ornamentation, color, contents, and wall structure) [36,37] and compared to the morphological descriptions of species presented in the International Culture Collection of Vesicular-Arbuscular Mycorrhizal Fungi (INVAM) and other literature [30,36,38,39,40], which resulted in the predominant occurrence of R. fasciculatus in the soil. Later, R. fasciculatus spores were further mass multiplied as single species inoculum with maize as a host plant. After five months, the maize vegetative part was discarded and the soil substrate containing spores (25 spores/g dry soil), infected roots, and hyphae of R. fasciculatus was used as inoculum. There were two treatment options: mycorrhizal inoculation with or without R. fasciculatus. Al-Taif rose plantlets were given an inoculum consisting of 35 spores per gram of dry soil in the Rangeland Laboratory of the Plant Production Department at King Saud University in Saudi Arabia. The autoclaved AMF inoculum was administered to the non-AMF plantlets at the same dosage. The potted plants were grown in a growth room with the following conditions: 25 ± 2 °C, 50–60% RH, and 100 µmol m⁻² s⁻¹ PPFD (16:8 h photoperiod under white fluorescent lamps). The plants received regular irrigation with phosphorus-free Hoagland nourishment solution. The vegetative growth of the acclimated plantlets was evaluated twelve weeks following their relocation into the growth chamber. There were thirty replications for each treatment, and each replicate was represented by a container holding a single plantlet.

2.3. Mycorrhizal Evaluations Following Host Plant Bio-Inoculation

Al-Taif rose plant roots were collected, separated, and thoroughly cleansed using distilled water. Following a 30 min treatment with 10% potassium hydroxide (KOH) at 80 °C, they were rinsed again, exposed to 3% hydrogen peroxide (H₂O₂) for 3 min, and then acidified for 10 min with 1% HCl. They were then stained with trypan blue for 20 min at 80 °C [41]. After drying the root segments, they were put on glass slides using lactoglycerol solution. Several characteristics of the segments were examined at 400× magnification under an Olympus-BX53 optical microscope (Olympus global, Tokyo, Japan)). To evaluate intraradical colonization, at least fifty root segments from each Al-Taif rose sample were examined.

2.4. Vegetative Growth Analysis of AMF-Treated and Control Plants

Following a twelve week period of cultivation, the fresh and dry weights of the shoots (g), plant height (cm), number of leaves, and leaf area (cm²) per plantlet were used to measure the growth responses. A portable area meter (CI-202; CID, Inc., Vancouver, WA, USA) was used to calculate the leaf area. Ten Al-Taif rose plants were chosen at random, and measurements were made of each plant in triplicate. Toluidine red was used to dye the roots for eight hours, and then a flatbed scanner (Cannon unit 101; Cannon, Green Island, NY, USA) was used to scan them. WinRHIZO (version 5.0; Regent Instruments, Quebec, QC, Canada) was used to analyze the acquired pictures. Root system parameters were measured, including root surface area, root diameter, total root length, fresh and dried weight (g), and root volume.

2.5. Analysis of the Gas Exchange Characteristics of Leaves

As per the protocol of Dewir et al. [19], measurements of net CO₂ assimilation, stomatal conductance, and transpiration were made at twelve weeks of acclimatization for both non-AMF and AMF-treated Al-Taif rose plants. The data was collected using a Li-Cor LI-6400 portable photosynthesis system (Li-Cor, Inc., Lincoln, NE, USA) equipped with a standard 2 × 3 cm leaf cuvette and a Li-Cor LI-6400-02B light source. Inflow air with a 350 µmol CO₂ concentration and 60% relative humidity was used to measure the photosynthetic parameters when the leaf temperature was 23 °C. The measurements were obtained in triplicate using ten randomly selected plants from each treatment.

2.6. Estimation of Chlorophyll Fluorescence

Using a portable chlorophyll fluorescence instrument, the Hansatech Handy PEA (Hansatech Instruments, Pentney, UK), chlorophyll fluorescence characteristics were measured on the abaxial surface of Al-Taif rose leaves. Using light with an intensity of less than 0.1 µmol m⁻² s⁻¹, the initial fluorescence (F₀) was measured for 30 min in leaves that had been acclimated to darkness. The maximum fluorescence (Fm) was then measured on the same leaves after applying a saturating pulse of light (>3500 µmol m⁻² s⁻¹) for one second. Using the methodology outlined by Dewir et al. [19], the maximal variable fluorescence (Fv = Fm − F₀) and the photochemical efficiency of photosystem II (Fv/Fm) were calculated for the dark-adapted leaves. A standard leaf chamber was utilized in conjunction with a random selection of three plants, specifically the fully expanded young leaves. Each treatment consisted of three separate replications, each involving one leaf.

2.7. Microscopic Observation of Stomata

Following the procedure outlined by Cotton et al. [42], strips from the cuticle of the leaves of non-AMF and AMF-treated Al-Taif rose plantlets were prepared. After soaking the leaves for twenty-four hours in distilled water, the translucent, thin layer of the leaf’s epidermal layer’s surface cells was carefully removed using pointed forceps and put on a glass slide. After that, they were stained for a few seconds using a mixture of 0.1 g triaryl methane dye and 2 mL glacial acetic acid in 100 mL distilled water (a light-green dye), and then a cover glass was placed over the epidermal strips. The glass slides were inspected under an optical microscope equipped with a SwiftCam 20 Megapixel camera for microscopes (Del-taPix, Smørum, Denmark) in order to determine the types of stomata, their sizes (measured with an ocular ruler), and their stomatal density (number of stomata per unit area). The leaf surfaces’ microscopic pictures were obtained at a magnification of 40×. The type of stomata, stomatal density, and aperture length and width were determined in the microscopic view field. A total of 30 measurements were conducted for the analysis of stomatal characteristics from randomly selected plants with different leaves (10 × 3 = 30).

2.8. Estimation of Proline Content

The measurement of free proline content followed Abraham et al. [43] guidelines. After weighing and homogenizing 0.5 g of Al-Taif rose leaves and roots in 3 mL of 3% 5-sulfosalyicilic acid and liquid nitrogen with a prechilled mortar and pestle, the samples were centrifuged for 10 min at 4000 rpm. After that, 2 mL of the supernatant was taken and incubated for 1 h at 100 °C using a ninhydrin reagent (0.125 g of ninhydrin, 3 mL of glacial acetic acid, and 2 mL of 6 M H₃PO₄) in order to estimate the proline concentration. After 15 min, the sample was submerged in an ice-cold bath to cease the reaction. For the extraction process, 4 mL of toluene was used, and the absorbance at 520 nm was measured. Finally, proline concentration was estimated using a standard curve.

2.9. Experimental Design and Data Analysis

With 30 replicates for each treatment, the experiments were set up in a completely randomized design. Each replicate was represented by a pot that contained one plantlet. The treatment effects were statistically evaluated using ANOVA and the unpaired t-test in SAS software (version 9.4; SAS Institute, Inc., Cary, NC, USA).

3. Results

3.1. Mycorrhizal Colonization

The AMF R. faciculaus-treated Al-Taif rose micropropagated plants displayed development vesicles, intraradical hype, intraradical spore, coiled hyphae, and arbuscular with the roots (Figure 2a–c). The mycorrhizal colonization analysis revealed 66.7% mycelium, 4.3% vesicles, and 66.7% arbuscules. It was found that 102/100 g of spores were present in the soil (Figure 3).

3.2. Growth Parameters

Twelve weeks following transplantation, the growth parameters of the AMF-treated and control plants were measured. When compared to control plants, the AMF-treated plants showed higher shoot growth values (

Table 1. Vegetative growth characteristics in non-AMF and AMF-treated Al-Taif rose plantlets after 12 weeks acclimatization.

Growth Parameters	Non-AMF	AMF-Treated
Plant height (cm)	8.51 ± 0.356	23.53 ± 0.697 *
Shoot fresh weight/plant (g)	0.240 ± 0.016	0.938 ± 0.036 *
Shoot dry weight/plant (g)	0.089 ± 0.005	0.302 ± 0.009 *
Number of leaves/plants	6.4 ± 0.452	11.3 ± 0.731 *
Leaf area/plant (cm2)	12.24 ± 0.938	66.15 ± 3.014 *

* = significant at p ≤ 0.05 according to Student’s unpaired t-test.

and Figure 4a). The AMF-treated plants had a height of 23.53 cm, which was 2.8 times greater than the height of the untreated plants. AMF-treated plants exhibited significantly greater shoot fresh weight (0.938 g/plant) and shoot dry weight (0.302 g/plant) compared to control plants (Table 1). With AMF-treated plants, there were 11.3 leaves per plant, but untreated plants had 6.4 leaves per plant. Compared to control plants (12.24 cm²), the leaf areas of AMF-treated plants were much greater (66.15 cm²). All the shoot and leaf parameters were statistically significant at p ≤ 0.05 (Table 1). It is noted that both non-AMF and AMF-treated Al-Taif rose plantlets showed high levels of survival 98% and 100%, respectively, following ex vitro acclimatization.

Figure 5 displays the results on growth parameters related to the root system. When comparing AMF-treated plants to control plants, the root growth was more robust (Figure 4b). With the AMF-treated plants, the length of the main root, total root length, number of root tips, total root area, total root volume, root fresh weight, and dry weight were, in order, 15.05 cm/plant, 172.16 cm/plant, 278/plant, 64.36 cm²/plant, 1.66 cm³/plant, 382 mg/plant, and 80.00 mg/plant (Figure 5). At p ≤ 0.05 level, each of these root growth values was statistically significant. Conversely, when compared to the control (1.04 mm/plantlet), the root diameter was at its maximum in non-AMF plants (1.16 mm/plantlet) but this parameter was not statistically significant. These findings unequivocally show that AMF-treated plants exhibited considerably higher vegetative development, as measured by shoot, leaf, and root growth indices, in comparison to the untreated plants.

3.3. Stomatal Frequency, Stomatal Conductance, Leaf Gas Exchange, and Transpiration Rate

Figure 6 and Figure 7 show the results of the assessment of the physiological status of both AMF-treated and untreated plants with respect to transpiration rate, stomatal frequency, stomatal conductance, net carbon dioxide assimilation, and the maximum potential quantum efficiency of photosystem II (Fv/Fm). As shown in Figure 6a–c, the stomatal density of AMF plants was 7.50 mm², while that of control plants was 12.33 mm². Plants treated with AMF had a greater stomatal aperture height of 15.33 µm compared to control plants’ height of 10.00 µm. Nonetheless, both the control (7.33 µm) and AMF-treated (8.00 µm) plants had equal stomatal aperture widths. The recorded net CO₂ assimilation values were 3.21 µmol CO₂ m⁻²·s⁻¹ and 1.43 CO₂ m⁻²·s⁻¹ for AMF treated and control plants, respectively (Figure 7a). The stomatal conductance in AMF and control plants was 0.021 mol H₂O mm⁻²·s⁻¹ and 0.013 mol H₂O m⁻²·s⁻¹, respectively (Figure 7b). The transpiration rate of the AMF-treated plants was also higher (1.40 mol H₂O m⁻²·s⁻¹) compared with that of the control plants (0.82 mol H₂O m⁻²·s⁻¹) (Figure 7c). When comparing the AMF-treated plants to the untreated plants, the Fv/Fm values were significantly higher (0.77) (Figure 7d). Furthermore, we calculated the proline content in the roots and leaves of the plants treated with AMF and the control plants. When compared to the proline concentration in the AMF-treated leaves and roots, the untreated leaves and roots had proline contents of 3.63 µg/g leaf fresh weight and 3.83 µg/g root fresh weight, respectively. These values were statistically significant (Figure 7e,f).

4. Discussion

Micropropagated plants present challenges with regard to plantlet growth, development, and survival throughout the acclimatization phase. Micropropagated plantlets suffer stress upon their transfer to ex vitro conditions, resulting in low survival rate. Soil infections often affect plants with stunted development, which prevents them from recovery [44,45,46]. Most floriculture and horticulture plant species are now propagated in vitro; unfortunately, 10–40% of these plants die when transplanted ex vitro, causing significant losses for the industry [47]. Mycorrhizal fungi are known to have the ability to increase agricultural productivity as biofertilizers and bioprotectors. This interaction may allow the plants to absorb more of the available nutrients in the soil, which would be advantageous for their growth. Mycorrhizal symbioses are therefore a desirable system in horticulture and floriculture [47]. AMF reduces disease invasion, increases drought stress tolerance, improves host plant uptake of minerals and nutrients, and promotes photosynthesis [48]. AMF beneficially affects host plant development, polyamine synthesis, osmotic adjustment, water transport, and drought tolerance [49]. Consequently, one of the biological techniques for acclimatization and establishment of micropropagated plants has been the use of AMF. We used AMF Rhizophagus faciculaus in the present study to transplant micropropagated Al-Taif rose plants, and we investigated the effects of AMF on the plants’ growth, development, and physiological traits following a 12 week transplanting period.

After 12 weeks of cultivation, the roots of AMF treated plants were harvested and examined for colonization with Al-Taif rose plants. The microscopic observation showed the development of vesicles, intraradical hype, intraradical spore, coiled hyphae, and arbuscular with the roots. In the mycorrhizal colonization analysis, 66.7% mycelium, 4.3% vesicles, and 66.7% arbuscules were found. These results are in concurrence with Etilingera elatior in vitro regenerated plants which showed very good colonization of AMF Gigospora albida and Claroideogloums etunicatum after treatment with these fungi [12]. Whereas, micropropagated plants of Philodendron bipinnatifidum demonstrated 66.66% mycelium, 11.11% vesicles, and 51.11% arbuscules with treatment of AMF Gigospora albida and G. marginata [17]. Numerous studies have shown that fertilizing AMF with in vitro regenerated plants following ex vitro transplantation promotes the plants’ development and growth as well as their ability to produce more biomass. In the current study, Rhizophagus faciculaus-treated Al-Taif rose micropropagated plants showed improved growth in terms of plant height, number, and area of leaves, as well as considerably greater fresh and dry weight of the shoots. In line with the present findings, studies on gerbera and heliconea [50], flame lilies [13], bananas [16], peonies [51], tree philodendron [17], and blackberries [19] have shown increases in plant height, leaf number and area, and shoot biomass.

Fortifying micropropagated plants with AMF enhances the rooting intensity and surface area of existing roots, which helps to develop a superior and stronger root system, according to Kapoor et al. [8] and Puthur et al. [52]. When AMF colonizes plant roots, it can alter the morphology of a root system in a structural, distinct, quantitative, and temporal way [53]. The adventitious roots of AMF colonization are highly branching, have larger diameters, and shorter specific root lengths, according to Berta et al. [54]. Rhizophagus faciculaus treatment resulted in a greater total length, number of roots, total root area, total root volume, fresh weight, and dry weight for the AMF treated Al-Taif rose micropropagated plants in this study. In a similar vein, treatment with AMF Glomus mosseae and Acaulospora laevis resulted in noticeably longer and stronger roots for flame lily micropropagated plants [13]. Wu et al. [9] have demonstrated the beneficial impact of AMF Glomus mossease on the growth and accumulation of dry biomass in Citrus tangerine in a different study. It has been found that in vitro regenerated plants have increased transpiration rate, stomatal conductance, and decreased net-CO₂ assimilation. Nevertheless, after a few weeks of transplantation, plants receiving AMF treatments have demonstrated the restoration of these physiological processes [55]. When compared to untreated plants, Al-Taif rose plants treated with AMF showed significantly reduced stomatal density (7.5 mm²), higher stomatal conductance (0.021 mol H₂O m⁻²·s⁻¹), and higher transpiration rate (1.40 mol H₂O m⁻²·s⁻¹) (Figure 6 and Figure 7). These findings show that the plants treated with AMF experienced physiological changes, including a decrease in the number of stomata per unit area and appropriate stomatal conductance and respiration. In line with the findings above, apple in vitro regenerated plants treated with Gigaspora albida, Acaulospora morrowiae, Acaulospora colombiana, and Claroideoglomus etunicatum demonstrated improved stomatal apparatus functioning in comparison to untreated plants [15].

The ratio of variable to maximum fluorescence, or Fv/Fm, is used to compute chlorophyll fluorescence, a metric of photosynthetic efficiency. When micropropagated plants are acclimating, the PSII’s principal photochemistry, which is highly susceptible to a range of environmental stressors, is measured by the Fv/Fm ratio. For instance, stress from light or salt may damage the PSII reaction center and interfere with electron transport in the plants’ photosynthetic machinery. A decrease in this parameter indicates down regulation of photosynthesis or photoinhibition [56]. Previous studies reported that mycorrhizal symbiosis could reduce the harmful effects of salinity on the PSII response center [57]. Compared to non-mycorrhizal plants, the mycorrhizal plants had a substantially higher Fv/Fm ratio [57,58]. Additionally, compared to uninoculated plants, the mycorrhiza-inoculated plants exhibited greater non-photochemical quenching [57] Processes that shield the leaf from light-induced damage may lead to an increase in non-photochemical quenching [59]. According to the current findings, Al-Taif rose plants treated with AMF exhibited greater Fv/Fm values, indicating improved plant acclimation following transplantation. Several studies have demonstrated that plants treated with AMF have greater net CO₂ assimilatio and higher rates of photosynthesis [8]. When compared to untreated plants, the AMF treated Al-Taif rose micropropagated plants in this study showed better net CO₂ assimilation 3.21 µmol CO₂ m⁻²·s⁻¹ and a higher chlorophyll florescence ratio of 0.77. Additionally, Gomez-Falcon et al. [10] have demonstrated that Cocus nucifera plantlets treated with Rhizoglomus intraradices and Acaulospora colombiana exhibited greater levels of CO₂ assimilation and optimal chlorophyll fluorescence. Numerous studies have indicated that adding AMF to micropropagated plants can help them resist a variety of biotic and abiotic challenges [60,61]. One of a plant’s primary responses to stress is the buildup of osmotic adjustment compounds like proline. Proline buildup was found to be less in plants infected with AM fungus than in control plants, such as roses [62,63]. Plants treated with AMF therefore accumulate less proline than control plants. The proline content of the AMF-treated and untreated plants was estimated in this study. The results showed that the proline content of the AMF-treated plants was significantly lower in the leaves (2.44 µg/g leaf fresh weight compared to 3.63 µg/g leaf fresh weight) and roots (2.03 µg/g root fresh weight tissue compared to 3.83 µg/g root fresh weight). These findings imply that AMF colonization increased the host plant’s tolerance to the ex vitro adverse environments.

5. Conclusions

AMF R. faciculaus affected growth, biomass accumulation in the shoots and roots, and physiological responses of micropropagated Al-Taif rose plants during the acclimatization stage. When compared to untreated plants, plants treated with AMF established well and witnessed vigorous growth. When AMF had been administered into the plants, the following parameters showed optimal growth: root length, total root volume, number of rootlets, fresh and dry biomass of roots, fresh and dry biomass of shoots, leaf number, and leaf area. The AMF-treated plants also showed increased transpiration rate and stomatal conductance, along with a reduced number of stomata. Likewise, AMF-treated plants exhibited low proline content and considerably increased net CO₂ assimilation and Fv/Fm values. These findings demonstrate the usefulness of AMF inoculation with in vitro regenerated plants during acclimatization.