Blackberry (Rubus sp., family: Rosaceae) is an important fruit-yielding plant cultivated worldwide. Blackberry fruits are rich in nutrients and bioactive substances, such as anthocyanins and other phenolic compounds (Kaume et al. 2011). The flavonols and ellagitannins contained in them are major compounds with antioxidant, antiobesity, antidiabetic, antimicrobial, and anti-inflammatory effects (Zia-Ul-Haq et al. 2014). Blackberry is one of the most popular blackberry species, and it is cultivated in various regions of the world. It is conventionally propagated by softwood cutting, suckers, and layering (Dziedzic and Jagla 2012). However, micropropagation techniques are adopted to achieve rapid and efficient propagation. The in vitro propagation of blackberry depends on several factors, including the physiological conditions of explants, the composition of the culture medium, and the plant growth regulators added to the medium (AbdAlla and Mostafa 2015; Hunkova et al. 2016; Hunkova et al. 2018). Critical steps in blackberry micropropagation are the acclimatization of plants on ex vitro transplantation and plant growth (Dewir et al. 2022).
The mycorrhization of in vitro-propagated plants using arbuscular mycorrhizal fungi (AMF) is beneficial for micropropagated plants, especially during acclimatization. AMF provide several benefits to the host plants by transferring nutrients efficiently from the soil to facilitate plant photosynthesis, growth, and development (Smith and Read 2008; Smith and Smith 2011a, 2011b). Additionally, AMF protect host plants from parasites, pathogenic fungi, and nematodes by stimulating them to produce defensive compounds and increasing the area of exploration of the roots, thus increasing the flow of water from the soil to the plant, as well as by enhancing the physical and chemical properties of the soil through the addition of organic matter and formation of aggregates through the adhesion of soil particles (Smith and Smith 2011a, 2011b). AMF have been successfully used to improve the acclimatization and growth of micropropagated fruit-bearing species such as walnut (Mortier et al. 2020), strawberry (Taylor and Harrier 2001), pomegranate (Singh et al. 2012), prunus (Monticelli et al. 2000), apple (Cavallazzi et al. 2007), and red dragon fruit (Dewir et al. 2023a).
The natural symbiotic relationship with AMF has been documented for Rubus alpinus Macfad, R. floribundus Kunth, R. bogtensis Kunth, and R. utricifolius Poir (Rincon et al. 2022). Additionally, Taylor and Harrier (2000) reported the beneficial effects of various AMF on the development and nutrition of micropropagated red raspberry plants (Rubus idaeus L. ‘Glen Prosen’). However, these types of studies have not been conducted for R. fruticosus. Therefore, the aim of the present investigation was to evaluate the effects of AMF on the vegetative growth, root growth, and development of micropropagated blackberry (R. fruticosus ‘P45’) plants during acclimatization.
Materials and Methods
Plant material.
This study was conducted at the plant tissue culture laboratory of the King Saud University College of Food and Agricultural Sciences (Saudi Arabia). Blackberry (R. fruticosus ‘P45’) plants were micropropagated based on the method described by Dewir et al. (2022). Blackberry microshoots (1.5–2.0 cm in length; 9 explants per culture vessel) were cultured in Magenta GA-7 culture vessels (77 × 77 × 97 mm; Sigma Chemical Co., St. Louis, MO, USA) containing 60 mL of Murashige and Skoog (MS) medium (Murashige and Skoog 1962) supplemented with indole-3-butyric acid (1 mg⋅L−1), naphthalene acetic acid (0.5 mg⋅L−1), and sucrose (30 g⋅L−1) for 8 weeks for root induction. The MS medium was gelled using 0.8% (weight/volume) agar, and its pH was adjusted to 5.8 before autoclaving at 121 °C and 1.2 kg⋅cm−2 for 15 min. The cultures were incubated under a mixture of blue and red light-emitting diodes (LEDs) with a 2:1 spectral ratio (Shenzhen Lumini Technology Co., Ltd, Shenzhen, China). The LED light was provided under a 16:8-h (light:dark) photoperiod with a light intensity of 50 μmol⋅m−2⋅s−1 photosynthetic photon flux density (PPFD). After 8 weeks, the plantlets were used as plant material for the experiments (Fig. 1).
In vitro plantlets of Rubus fruticosus ‘P45’.
Citation: HortScience 58, 8; 10.21273/HORTSCI17211-23
Transplantation of plantlets to potting medium, AMF inoculation, and plant growth conditions.
After 8 weeks of in vitro rooting, the plantlets were gently removed from the gelled medium and cleaned using tap water. Two treatments, with and without AMF inoculation, were applied. The plantlets were transplanted into conical plastic pots (upper diameter, 4 cm; bottom diameter, 1.7; length, 21.5 cm) filled with a sterilized sand:soil (1:1) mixture and amended with 5% (weight/weight) AMF inoculum soil. The applied inoculum, which consisted of equal proportions of G. margarita and G. albida (Fig. 2) with a density of 33.4 spores/g dry soil, was acquired from the Rangeland Laboratory at the Plant Production Department of King Saud University (Saudi Arabia). The untreated plantlets were inoculated with the same dosage of the autoclaved AMF inoculum. Then, the potted plants were grown at 25 °C ± 2 °C, 50% to 60% relative humidity, and 100 μmol⋅m−2⋅s−1 PPFD (16:8-h photoperiod under white fluorescent lamps) in a growth chamber; for the first 2 weeks, the pots were covered with transparent polyethylene. The plantlets were regularly irrigated with Hoagland nutrient solution without phosphorus. After 8 weeks, the plantlet growth, mycorrhizal condition/status, and survival were evaluated. A total of 30 replicates were conducted for each treatment (with and without mycorrhizal inoculation), with each replicate being represented by a pot containing one micropropagated plantlet. To obtain a general microbial population free of AMF propagules, non-AMF plants received the same amount of autoclaved AMF inoculum and filtrate. The total spore population in each treatment was obtained from calculations performed using 100 g of dry soil (Schussler and Walker 2010).
Arbuscular mycorrhizal fungi (AMF) spores used for the inoculation of micropropagated blackberry plants. (A) Crushed spores of G. albida. (B) Crushed spores of G. margarita.
Citation: HortScience 58, 8; 10.21273/HORTSCI17211-23
Mycorrhizal assessments.
The roots of blackberry plants were collected, separated, and cleaned with distilled water. Then, they were treated with 10% potassium hydroxide (KOH) for 30 min at 80 °C, rinsed once more, and exposed to 3% hydrogen peroxide (H2O2) for 3 min before being acidified with 1% HCl for 10 min. Then, trypan blue was used to stain them for 20 min at 80 °C (Phillips and Hayman 1970). Subsequently, the dyed root segments were placed in a lactoglycerol solution, and then on glass slides. An optical microscope was used to examine various features of the segments (at 400× magnification). At least 50 root segments from each blackberry sample were analyzed to assess intraradical colonization. The presence of mycelium, vesicles, and arbuscules was detected. The ratio and level of intraradical mycorrhizal colonization (development of mycelium, vesicles, and arbuscules) within the roots were calculated (Al-Qarawi et al. 2012; Trouvelot et al. 1986).
Measurement of leaf gas exchange.
Net CO2 assimilation, stomatal conductance, and transpiration were measured in both untreated and AMF-treated blackberry plantlets after 8 weeks of acclimatization, as described by Dewir et al. (2023b). An LI-6400 portable photosynthesis system (LI-COR Inc., Lincoln, NE, USA) outfitted with a typical 2- × 3-cm leaf cuvette and a LI-COR LI-6400–02B light source was used to collect the data. The leaf temperature was 23 °C, and photosynthetic parameters were assessed in inflow air with a CO2 concentration of 350 μmol and relative humidity of 60%. Ten randomly selected plants from each treatment were used for the measurements, which were performed in triplicate. Dry weight was recorded after the plants were oven-dried for 2 d at 70 °C.
Measurement of vegetative parameters.
After 8 weeks of cultivation, growth responses were measured in terms of fresh and dry weights of shoots (g), plant height (cm), number of leaves, and leaf area (cm2) per plantlet. The leaf area was calculated using a portable area meter (CI-202; CID, Inc., Vancouver, WA, USA). Measurements were conducted in triplicate for 10 randomly selected blackberry plantlets. The roots of both untreated and AMF-treated plantlets were removed from the pots and washed with tap water to establish three root replicates for three plants from each treatment. The roots were dyed with toluidine red for ∼8 h before they were scanned using a flatbed scanner (Cannon unit 101; Cannon, Green Island, NY, USA). The images obtained were analyzed using WinRHIZO (version 5.0; Regent Instruments, Quebec, QC, Canada). The characteristics of the root system, including root fresh and dry weights, total root length, root diameter, root volume, and root surface area, were measured.
Experimental design and data analysis.
The experiments were conducted using a completely randomized design with 30 replicates per treatment. Each replicate was represented by a pot containing one plantlet. The treatment effects were assessed statistically using an analysis of variance and unpaired t test.
Results
The blackberry plants that were transplanted to potting medium and treated with AMF grew well during the 8-week experimental period (Fig. 3). Microscopic examination of the roots of the acclimatized AMF-treated plants showed the presence of mycelium, arbuscules, and spores (Fig. 4). The analysis of mycorrhizal association revealed proportions of mycelia, vesicles, and arbuscules of 68.88%, 15.00%, and 24.44%, respectively. The spore count was 201 per 100 g of soil (Fig. 5).
(A) Vegetative growth and (B) root growth of untreated and arbuscular mycorrhizal fungi (AMF)-treated R. fruticosus plantlets after 8 weeks and (C) micropropagated plantlets after 12 weeks of acclimatization.
Citation: HortScience 58, 8; 10.21273/HORTSCI17211-23
(A–D) Photomicrographs of stained roots of R. fruticosus showing spores, mycelia, and arbuscules. Ar = arbuscules; ArT = arbuscular trunk; ES = extraradical intact spores; IS = intraradical intact spores.
Citation: HortScience 58, 8; 10.21273/HORTSCI17211-23
Spore and mycelial count in R. fruticosus roots colonized by arbuscular mycorrhizal fungi (AMF).
Citation: HortScience 58, 8; 10.21273/HORTSCI17211-23
The growth parameters of both AMF-treated and control plants were measured 8 weeks after transplantation. The treated plants exhibited higher growth values compared with the control plants (Figs. 5 and 6), and the shoot fresh (2.76 g/plantlet) and dry (0.77 g/plantlet) weights as well as the total fresh weight per plant (3.97 g/plantlet) were higher compared with the non-AMF-treated (control) plants. Plant height (15.33 cm), number of leaves (14.33 per plantlet), and leaf area (143.66 cm2/plantlet) of ASMF-treated plants were significantly higher (Fig. 6).
Vegetative growth characteristics of R. fruticosus plants in response to arbuscular mycorrhizal fungi (AMF) after 8 weeks of acclimatization. (A) Number of leaves per plantlet. (B) Leaf area per plantlet. (C) Plant height. (D) Shoot fresh weight per plantlet. (E) Shoot dry weight per plantlet. (F) Total fresh weight per plantlet.
Citation: HortScience 58, 8; 10.21273/HORTSCI17211-23
Data regarding growth parameters associated with root biomass are presented in Table 1. The length of the main root, total root length, root surface area, average root diameter, root volume, and number of root tips of AMF-treated plants were 21.33 cm/plantlet, 752.64 cm/plantlet, 733.03 cm2/plantlet, 3.17 mm/plantlet, 57.35 cm3/plantlet, 420.33/plantlet, respectively. Similarly, root fresh (1.21 g/plantlet) and dry (0.295 g/plantlet) weights of treated plants were also higher. These results clearly demonstrate that both shoot, leaf, and root growth parameters increased with the AMF treatment.
Root growth characteristics of R. fruticosus plants in response to arbuscular mycorrhizal fungi (AMF) treatment after 8 weeks of acclimatization.
The net CO2 assimilation, stomatal conductance, and transpiration rates of the AMF-treated and control plants were measured to assess their physiological conditions, and the data are reported in Fig. 7. The net CO2 assimilation recorded 9.10 µmol CO2 m−2⋅s−1 and 5.9 µmol CO2 m−2⋅s−1 for AMF-treated and control plants, respectively. For the AMF-treated and control plants, the stomatal conductance values were 0.06 mol H2O mg−2⋅s−1 and 0.04 mol H2O mg−2⋅s−1, respectively. The transpiration rate of the treated plants was also higher (1.25 mol H2O m−2⋅s−1) compared with that of control plants (Fig. 7).
Leaf gas exchange in R. fruticosus in response to arbuscular mycorrhizal fungi (AMF) after 8 weeks of acclimatization. (A) Net CO2 assimilation. (B) Stomatal conductance. (C) Transpiration rate.
Citation: HortScience 58, 8; 10.21273/HORTSCI17211-23
Discussion
AMF are a natural microbial component of most soils. They colonize the roots of plants and form a mutual symbiosis with most plant species, including micropropagated plants. It has been demonstrated that mycorrhizae improve nutrient intake (particularly phosphorus uptake), increase the volume of the rhizosphere, and reduce both biotic and abiotic stresses. As a result, AMF can work in three ways: as biofertilizers, as biocontrol agents, and as bioregulators (Smith and Smith, 2011a). By using bioinoculants ,such as AMF, for optimal hardening, the success rate of plantlets grown in tissue culture can be significantly increased. Several fruit crops establish a symbiotic association with mycorrhizal fungi and greatly depend on this symbiosis for normal development and enhanced field performance (Smith and Smith 2011b).
In the present study, G. margarita and G. albida were used as AMF, and the impact of their inoculation on the development and physiological characteristics of in vitro grown blackberry plants was evaluated. The results revealed that AMF influenced the growth of plantlets by increasing the fresh biomass and dry biomass of shoots. The overall total fresh weight of plantlets, plant height, number of leaves, and leaf area per plantlet also increased (Fig. 6). The root growth parameters of AMF-treated blackberry plants (i.e., root length, total root length per plantlet, surface area of roots, root volume, number root tips per plantlet, and fresh and dry weights of roots) also showed a general increasing trend (Table 1). It has been previously reported that inoculation with G. margarita and G. albida significantly improved growth and root development in micropropagated plantlets of red dragon fruit (Hylocereus polyrhizus) (Dewir et al. 2023a) and lacy tree philodendron (Philodendron bipinnatifidum) (Dewir et al. 2023b). Supplementation of micropropagated apple plants with Glomus etunicatum, Scutellospora pellucida, Acaulospora scrobiculata, and Scutellospora heterograma led to increased plant height, shoot biomass, and root biomass (Cavallazzi et al. 2007). Jaizma-Vega et al. (1991) reported increases in plant height, leaf area, and shoot dry matter of banana plants inoculated with A. scrobiculata, Glomus clarum, and G. etunicatum.
Positive effects of AMF on plant growth and performance have also been reported for many other fruit-yielding species such as plum (Prunus cerasifera Ehrh clone MrS 2/5) (Fortuna et al. 1992), in which mycorrhizal inoculation stimulated apical growth during the first month after transplanting, whereas noninoculated plants did not actively grow, and the apices remained undeveloped. They showed that mycorrhizal symbiosis not only affects the nutritional status but also may influence the hormonal balance. Regarding Citrus limon, it was reported that AMF inocula significantly increased plant height, root and shoot weights, and leaf area at the end of the weaning phase (Quatrini et al. 2003). Similarly, Mortier et al. (2020) showed that early inoculation with AMF improved survival and seedling performance of transplanted walnut trees. Improved physiological parameters, such as water relations, stomatal conductance, photosynthesis, and respiration, were recorded for Musa spp. ‘Pacovan’ (Yano-Melo et al. 1999), Punica granatum (Singh et al. 2012), Malus prunifolia (Costa et al. 2021), and Philodendron bipinnatifidum (Dewir et al. 2023b). In this study, plants inoculated with G. margarita and G. albida also demonstrated better CO2 assimilation, stomatal conductance, and transpiration rates, thereby corroborating the results of previous reports.
In conclusion, this study demonstrated that the inoculation of isolated AMF increased the growth of blackberry plants and influenced their physiological traits. These results indicate the potential usefulness of mycorrhization in micropropagated blackberry plants. In fact, this process is a viable method for preparing micropropagated blackberry plantlets for ex vitro settings and may accelerate their growth for eventual field transplantation. Further research is required to enable the necessary up-scaling of this method for commercial applications.
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