Innovative Protocols for Blackberry Propagation: In Vitro Cultivation in Temporary Immersion Systems with Ex Vitro Acclimatization

Abstract

Optimized in vitro cultivation offers a sustainable solution to enhance blackberry (Rubus spp.) production while reducing pathogen contamination during propagation. This study developed and validated protocols for in vitro cultivation and ex vitro acclimatization of the Tupy, Brazos, and Kiowa cultivars at the Instituto Tecnológico Superior de Los Reyes, Michoacán. A 20 min treatment with 2% sodium hypochlorite (NaOCl) reduced contamination by below 10% and achieved explant survival rates exceeding 95%. Temporary Immersion Systems (TIS) with four to six immersion cycles of 5 min each maximized survival (above 95%) while minimizing necrosis and hyperhydricity and increasing fresh mass. Shoot development was significantly enhanced with 2 mg L⁻¹ 6-benzylaminopurine, and 1 mg L⁻¹ indole-3-butyric acid promoted optimal root formation. Acclimatization success rates exceeded 90% in covered trays compared to significant losses in uncovered trays during early stages. These protocols enabled robust plant development and yields exceeding 10 t ha ⁻¹ during vegetative and reproductive stages, providing a scalable framework for sustainable blackberry production and broader applications in crop propagation.

1. Introduction

The blackberry (Rubus spp.), a member of the Rosaceae family, is an economically significant fruit crop cultivated worldwide for its high nutritional value and health-promoting properties. Known for their rich content of bioactive compounds such as polyphenols, stilbenoids, lignans, and triterpenoids, blackberries are highly sought after in global markets due to their antioxidant properties and ability to mitigate oxidative stress and inflammation in animal cells [1]. The rising consumer demand for functional foods has further propelled blackberry production, with Mexico leading as the largest global producer, contributing 23.3% of the world’s total output [2,3]. This underscores the critical role of blackberries not only in agricultural economies, but also in meeting the growing demand for nutrient-rich fruits.

Despite their economic importance, blackberry cultivation faces several challenges that limit productivity and scalability. Conventional propagation methods, including root cuttings and vegetative propagation, are prone to phytosanitary issues such as infections by soil-borne pathogens like Fusarium oxysporum f. sp. mori and species of Lasioliplodia, which cause significant crop losses and hinder disease-free plant production [4,5]. Furthermore, these traditional methods often result in low propagation efficiency, poor genetic uniformity, and limited adaptability to diverse environmental conditions [6]. These limitations underscore the urgent need for innovative propagation strategies that can ensure high-quality, disease-free planting material while addressing economic and agronomic constraints.

The history of the in vitro propagation of horticulturally important species, including blackberries, dates back to the 1970s, with pioneering studies laying the foundation for modern micropropagation techniques. Early work by Schelkunova and Popov [7] marked the first report published by Rubus in vitro propagation. Subsequent studies expanded on this work, with notable contributions from Broome and Zimmerman [8], who demonstrated the feasibility of blackberry propagation in vitro. Similarly, Skirvin et al. [9] reported successful protocols for propagating thornless trailing blackberries, emphasizing the potential of tissue culture for commercial applications. Other foundational studies include Babić and Nešković [10], who propagated three blackberry cultivars from small apical buds, and Fernandez and Clark [11], who optimized protocols for the erect thornless “Navaho” blackberry cultivar. These early efforts underscored the potential of tissue culture for commercial applications, paving the way for further advancements.

In recent decades, biotechnological innovations have refined micropropagation techniques, particularly through the use of Temporary Immersion Systems (TIS). TIS have gained significant attention for their ability to optimize the nutrient uptake, reduce hyperhydricity, and promote robust plant growth during in vitro cultivation [12,13,14]. Research on TIS for blackberry micropropagation has primarily focused on immersion cycles, media composition, and rooting procedures [15,16]. However, despite these advancements, the transition from the laboratory to the field remains a critical challenge, with acclimatization success rates often limiting the scalability of in vitro protocols [17].

Bibliometric analyses conducted by Regni et al. [18] using Scopus and Web of Science databases reveal that research interest in blackberry micropropagation has grown steadily since the late 1990s, driven by increasing consumer demand and advanced production technologies. A total of 78 scientific documents were published between 1998 and 2024, with key contributions from Brazil, Romania, Canada, and Serbia. The most frequently studied stages include stabilization, multiplication, and rooting, with the Murashige and Skoog (MS) medium being the most widely used basal formulation. Emerging trends highlight increasing focus on ex vitro adaptation, light quality optimization, and phenolic compound production from in vitro tissues—areas that align closely with current industry needs for scalable, cost-effective, and genetically stable propagation systems.

One underexplored area in blackberry micropropagation is the role of hormesis—an adaptive response to controlled stress in plants. Investigating this phenomenon could provide new insights into optimizing cultivation protocols and improving plant resilience [19]. Hormetic responses to mild oxidative or osmotic stress may enhance shoot proliferation, root development, and acclimatization success, especially when applied within dynamic environments like TIS [20].

This study aims to investigate the role of hormesis in blackberry (Rubus spp.) micropropagation within TIS to optimize in vitro cultivation protocols. Specifically, the research will assess the effects of controlled stress exposure—such as sub-lethal NaOCl treatments, regulated immersion cycles, and antioxidant supplementation—on plant growth, physiological responses, and overall propagation efficiency. By integrating advanced biotechnological tools with tailored acclimatization strategies, this study seeks to develop scalable and sustainable protocols for blackberry production. These innovations hold significant potential for addressing the challenges faced by conventional propagation methods and supporting the global demand for high-value blackberry crops.

2. Materials and Methods

This study was conducted at the Biotechnology Laboratory of the Technological Institute of Los Reyes, located in Michoacán, as part of the National Technological Institute of Mexico. The field experiment was carried out at La Tulera farm, situated at coordinates 19°32′02.13″ N, 102°29′51.01″ E, at an elevation of approximately 1600 m above sea level. The blackberry cultivars Tupy, Brazos, and Kiowa were selected for this study due to their agronomic performance and adaptability.

Tupy: Developed by Alverides Santos at EMBRAPA (Brazilian Agricultural Research Corporation), this cultivar is widely cultivated for its high yield potential, large fruit size, and excellent post-harvest quality [21]. Its adaptability to tropical and subtropical climates has made it a popular choice for commercial growers in Latin America. Kiowa: Bred by the University of Arkansas in 1983, Kiowa is known for its exceptionally large fruit size, extended shelf life, and late-season harvest. It is particularly valued in fresh fruit markets due to its superior fruit quality and flavor profile [22]. Brazos: Originating from Texas A&M University, Brazos is a thorny, early-maturing cultivar characterized by its vigorous growth, high yield, and adaptability to warm climates. Although primarily grown for processing, its robust performance under diverse conditions makes it a reliable choice for both commercial and small-scale cultivation [23].

These cultivars share several common characteristics that contribute to their commercial viability. Notably, they possess thorny stems, which can impact management practices and harvesting efficiency. Additionally, all three cultivars demonstrate high survival rates under field conditions, exceeding 97%, highlighting their resilience and adaptability to diverse cultivation environments. While each cultivar presents distinct advantages, their shared traits make them valuable cultivars for commercial blackberry production.

2.1. Initiation and Stabilization of In Vitro Cultures

2.1.1. Selection of Plant Material and Media Preparation

The plant material comprised three blackberry cultivars cultivated under greenhouse conditions. The selected plants measured between 40 and 50 cm in height, possessed 10 to 15 functional leaves, and had a fresh weight ranging from 10 to 12 g. Stems were sectioned into fragments of approximately 1 cm in length, each containing at least one bud, following the methodology described by Badr-Elden et al. [24].

All MS media were prepared using a full-strength formulation (4.2 g L⁻¹ MS salts) as per the original recipe by Murashige and Skoog (1962) [25], except during acclimatization, where half-strength MS medium (2.1 g L⁻¹ MS salts) was used to reduce salt stress. The medium was supplemented with 30 g L⁻¹ sucrose and solidified using 5 g L⁻¹ Gelrite^® (Sigma-Aldrich, St. Louis, MO, USA). The pH was adjusted to 5.8 prior to autoclaving at 121 °C and 118 kPa pressure for 20 min. The MS basal salt mixture was sourced from PhytoTechnology Laboratories^® (Cat. No. M524), and plant growth regulators included 6-Benzylaminopurine (BAP, Sigma-Aldrich, St. Louis, MO, USA; Cat. No. B3408) and Indole-3-Butyric Acid (IBA, Sigma-Aldrich, St. Louis, MO, USA; Cat. No. I5386).

2.1.2. Disinfection of Plant Material

The disinfection protocol involved sequential treatments to eliminate contaminants. Primary stem segments (approximately 3 cm in length) were initially washed with distilled water and liquid soap to remove dust and impurities, followed by a rinse with bottled water. Subsequently, the segments were immersed in a 3 g L⁻¹ (v/v) solution of Captan^® fungicide (Bayer, Leverkusen, Germany) for 20 min, as described by Barrios et al. [26] and Kefayeti et al. [27]. After treatment, the segments were rinsed with distilled water for 2 min to remove fungicide residues, followed by immersion in a 70% ethanol (v/v) solution for 5 min. The final sterilization step involved treatment with 2% sodium hypochlorite (NaOCl) (Cloralex^® 6%, S.A. de C.V., Ciudad de México, México) (v/v) for varying durations (0, 5, 10, 15, and 20 min). After NaOCl treatment, the explants were rinsed three times with sterile distilled water to remove any residual NaOCl.

For each disinfection treatment, five explants were placed in Gerber glass jars containing semi-solid MS culture medium [25], consisting of 2.1 g L⁻¹ MS salts, 30 g L⁻¹ sucrose, and 5 g L⁻¹ Gelrite^® (Sigma-Aldrich, St. Louis, MO, USA).

Each jar contained five explants, and there were four jars per replication, with five replications per treatment. This results in a total of 100 explants per treatment (5 explants × 4 jars × 5 replications = 100). The explants were incubated in an in vitro culture chamber at 25 ± 2 °C, under a 16 h photoperiod provided by LED lamps (wavelengths of 460 and 560 nm), with a photosynthetic photon flux (PPF) of 45 ± 5 µmol m⁻² s⁻¹ [28].

2.1.3. Evaluation of Contamination, Survival, and Regeneration

Contamination was evaluated 5 days post-establishment, following AbdAlla and Mostafa [29]. The percentage of contaminated jars was calculated based on the total number of initial jars. Survival was assessed 7 days after implantation by determining the percentage of green explants from the initial total, as described by Badr-Elden et al. [24]. Regeneration was evaluated 45 days post-establishment, based on the percentage of sprouted buds from the initial total of explants treated with 2% NaOCl [30].

2.2. In Vitro Multiplication Stage

2.2.1. Multiplication of Plant Material

The multiplication stage was conducted under controlled conditions to optimize shoot proliferation and ensure uniform development. After 45 days, shoots approximately 6 cm in length were excised from axillary buds under aseptic conditions in a laminar flow hood and transferred to 500 mL polyethylene jars [31]. Each jar contained 60 mL of full-strength MS medium, prepared using MS Basal Salt Mixture (PhytoTechnology Laboratories^®, Shawnee Mission, KS, USA; Cat. No. M524) at a concentration of 4.2 g L⁻¹, supplemented with 30 g L⁻¹ sucrose and solidified with 5 g L⁻¹ Gelrite^® (Sigma-Aldrich, St. Louis, MO, USA). The medium was supplemented with appropriate growth regulators as required for the experiment. The pH was adjusted to 5.8 before autoclaving at 121 °C for 20 min. To prevent excessive competition for nutrients and space, each jar was inoculated with five explants, following established protocols for optimized blackberry micropropagation. The cultures were maintained under controlled environmental conditions at 25 ± 2 °C, with a 16 h photoperiod and a PPF of 45 ± 5 µmol m⁻² s⁻¹, ensuring efficient shoot multiplication while minimizing the risks of hyperhydricity and necrosis. Measurement protocol: shoots per bud were counted at 20 days, while buds per shoot were evaluated at 40 days to account for developmental progression.

2.2.2. Establishment of TIS

The TIS was established using a twin-flask system with a total capacity of 2 L. Each bioreactor was filled with 900 mL of liquid full-strength MS medium, prepared using MS Basal Salt Mixture (PhytoTechnology Laboratories^®, Shawnee Mission, KS, USA; Cat. No. M524) at a concentration of 4.2 g L⁻¹ and supplemented with 30 g L⁻¹ sucrose to support optimal in vitro propagation conditions [32]. To ensure uniform propagation, 10 explants per cultivar were introduced into each bioreactor, with 15 replicates per treatment, leading to a total of 150 explants per experimental condition. To prevent microbial contamination, 2 mg L⁻¹ of Plant Preservation Mixture (PPM) (Plant Cell Technology, Washington, D.C., USA) was added to the culture medium at the time of establishment. The immersion cycles were regulated at frequencies of 2, 4, and 6 per day, with immersion durations of 5, 10, and 15 min. A control group with no immersion (0 min) was also included, maintained on semi-solid MS medium without immersion, but supplemented with the same concentration of PPM (2 mg L⁻¹) to ensure comparable conditions. During both shoot multiplication and rooting stages, the explants were not physically anchored; they were freely suspended in the liquid medium, relying on turgor pressure and gravitropism to maintain an upright position during immersion cycles. The cultures were maintained under controlled environmental conditions, including a temperature of 25 ± 2 °C, a 16 h photoperiod, and a PPF of 45 ± 5 µmol m⁻² s⁻¹, ensuring optimal conditions for shoot proliferation and root induction. After 40 days, several physiological and morphological parameters were evaluated, including survival rates, necrotic explants, hyperhydric leaves, and total plant mass per jar, to determine the efficiency of the TIS in enhancing blackberry (Rubus spp.) micropropagation.

2.2.3. Effect of 6-Benzylaminopurine (6-BAP) on TIS

To assess the effect of 6-BAP on shoot proliferation in the TIS, a liquid full-strength MS medium was utilized. The medium was prepared using MS Basal Salt Mixture (Phyto Technology Laboratories^®, Cat. No. M524) at a concentration of 4.2 g L⁻¹ and supplemented with 30 g L⁻¹ sucrose. Each bioreactor contained a total volume of 900 mL of this medium, which was further supplemented with 0 mg L⁻¹ (control), 1 mg L⁻¹, 2 mg L⁻¹, and 3 mg L⁻¹ of 6-BAP (Sigma-Aldrich, Cat. No. B3408), following the methodology described by Mirzabe et al. [15] and da Silva and Biasi [16]. After 40 days, explants from each bioreactor were evaluated based on proliferation rate, survival rates, necrotic explants, hyperhydric leaves, and total plant mass per jar to determine the optimal 6-BAP concentration for blackberry (Rubus spp.) micropropagation under TIS conditions. Number of shoots per bud is defined as count of new shoots emerging from a single original axillary bud explant. Number of buds per shoot is defined as count of newly formed auxiliary buds observed along each elongated shoot.

2.3. Rooting Stage

The rooting stage aimed to evaluate the efficiency of root induction under different conditions, focusing on the effect of IBA on root formation, root length, and overall rooting success. At 40 days, plants from TIS were transferred to 500 mL polypropylene containers containing semi-solid full-strength MS medium. The medium was prepared using MS Basal Salt Mixture (PhytoTechnology Laboratories^®, Shawnee Mission, KS, USA; Cat. No. M524) at a concentration of 4.2 g L⁻¹, supplemented with 30 g L⁻¹ sucrose, and solidified with 5 g L⁻¹ Gelrite^® (Sigma-Aldrich, St. Louis, MO, USA). The plants were maintained for 20 days at 25 ± 2 °C under the same light conditions as previously described. The effect of IBA on rooting was evaluated using two methods: (i) rooting on solid medium, using full-strength MS medium supplemented with 0, 0.5, 1, and 1.5 mg L⁻¹ IBA, following the methodology of Dönmez et al. [33], and (ii) rooting in TIS, where explants were immersed in liquid full-strength MS medium with identical IBA concentrations. Each treatment contained 10 plants per replicate, with three replicates per treatment. Rooting percentage, root number per plant, and root system length (defined as the longest root) were measured after 20 days.

2.4. Acclimatization and Field Performance

The acclimatization and field performance stage aimed to evaluate the ex vitro survival rate, plant growth, physiological adaptations, and overall yield to assess the long-term viability of the optimized propagation protocols.

2.4.1. Ex Vitro Acclimatization

The ex vitro acclimatization phase was conducted over 70 days, divided into three consecutive sub-stages to facilitate a controlled transition from in vitro to ambient greenhouse conditions. This progressive approach minimized physiological stress, promoted photomorphogenic adaptation, and enhanced survival rates.

Plantlets from both rooting systems were transferred to plastic trays (40 cm × 60 cm) or polypropylene cups (360 cm³) containing a sterile 1:1 peat-perlite substrate. Transparent plastic/nylon covers maintained high humidity initially.

A micro-sprinkler system (AquaSmart 2002; 2 L m⁻² min⁻¹ flow rate) irrigated plants twice daily (09:00 and 14:00 h) for 10 min/cycle. Half-strength MS salts (2.1 g L⁻¹) were added to irrigation water to mitigate osmotic stress.

Environmental parameters, particularly light intensity and relative humidity, were closely regulated throughout the acclimatization process. Light was modulated using neutral-density shade fabrics provided by Ludvig Svensson (Sweden). Two shading levels were employed: the LS-77 shade cloth with 25% light transmission (200 g m⁻²) and a custom-designed fabric with 75% light transmission (250 g m⁻²). The spectral transmission properties of both fabrics were validated within the photosynthetically active radiation (PAR) range (400–700 nm) using a calibrated spectroradiometer (Apogee SR-999, USA; wavelength range 350–800 nm; five scans per sample at 1 nm resolution).

To monitor irradiance conditions, a quantum sensor (LI-COR LI-190R, USA) was employed to collect weekly light intensity measurements at three canopy-level points per replicate. The sensor, featuring ±5% measurement accuracy, was factory-calibrated annually and field-zeroed daily to ensure data reliability. These procedures ensured that light conditions remained consistent and reproducible across all experimental units.

The acclimatization process was carried out in a greenhouse constructed with 6-mm-thick UV-stabilized polycarbonate panels (Palram SolarSoft™, Israel), offering a certified PAR transmission of 52 ± 2%. The structure was oriented along an east–west axis to optimize natural light exposure. External shading from nearby structures was minimal, allowing stable and uniform irradiance throughout the photoperiod.

The specific conditions and rationale for each sub-stage were as follows:

Stage 1 (Days 1–10): During the initial stage, plantlets were maintained under the LS-77 shade cloth (25% transmission) at a relative humidity of 90 ± 3% and a temperature of 26.5 °C. Light intensity was maintained at 250 ± 30 µmol m⁻² s⁻¹. These conditions were designed to minimize transplant shock and water loss while promoting initial recovery and tissue stabilization following transfer from in vitro culture.

Stage 2 (Days 11–40): At the start of this stage, covers were replaced with the custom 75% transmission fabric to gradually increase irradiance and stimulate photosynthetic activity. Environmental conditions were adjusted to a relative humidity of 70–80% and a constant temperature of 26.5 °C. Light intensity during this period averaged 800 ± 30 µmol m⁻² s⁻¹. This intermediate stage was critical for promoting functional leaf development, chloroplast differentiation, and stomatal regulation.

Stage 3 (Days 41–70): In the final stage, plantlets were exposed to diffuse natural sunlight filtered through the greenhouse’s UV-stabilized polycarbonate panels. Recorded light intensity averaged 100 ± 50 µmol m⁻² s⁻¹, with peak values observed between 12:00 and 14:00 h. The air temperature was maintained at 30.5 ± 1.5 °C. The relatively low light intensity during this phase was consistent with the ~52% PAR transmission of the polycarbonate panels and the inherently diffused lighting typical of greenhouse environments. This final stage facilitated the acclimation of plantlets to natural photoperiods and irradiance fluctuations, preparing them for subsequent field transplantation.

At the conclusion of the 70-day acclimatization period, plantlet viability was assessed based on morphological vigor, physiological condition, and survival rates. These outcomes validated the efficacy of the protocol in enhancing plantlet survival and readiness for field conditions.

2.4.2. Field Performance

Plants from the acclimatization process were transplanted into the field following the methodology described by Wu et al. [34]. The experimental design was a randomized complete block design, with each cultivar serving as a treatment and replicated four times. Plants measuring 30 cm in height were planted in furrows covered with nylon mesh. A planting spacing of 0.50 m between plants and 2 m between furrows was maintained, resulting in a planting density of 10,000 plants ha⁻¹ [35,36,37]. The soil characteristics are presented in

Table 1. Chemical characteristics of the soil used in this research.

Chemical Property	Value for a Vertisol
pH in H2O	6.12
Organic Matter (OM)	3.5%
Cation Exchange Capacity (CEC)	35.22 cmol kg−1
CEC at Base Saturation	43.18 cmol kg−1
Exchangeable Aluminum (Al)	0.8 cmol kg−1
Calcium (Ca)	32.43 cmol kg−1
Magnesium (Mg)	16.22 cmol kg−1
Potassium (K)	0.38 cmol kg−1
Sodium (Na)	2.85 cmol kg−1
Base Saturation	73.62%
Electrical Conductivity (EC)	1.86 dS m−1
Available Phosphorus (P)	15.22 mg kg−1
Total Nitrogen (N)	0.25%
C:N Ratio (Carbon:Nitrogen)	12:1
Soil Texture	60% clay, 30% silt, 20% sand

The soil analysis was conducted by the Mexican Soil Laboratory Network (MEXSOLAN), which is integrated into both the Global Soil Laboratory Network (GLOSOLAN) and the Latin American Soil Laboratory Network (LATSOLAN).

.

During this stage, plants of the three cultivars underwent cultural practices as outlined by Cárdenas et al. [38]. Agroproductive indicators were assessed, including survival rate, plant height, stem diameter, number of branches, fresh plant mass, and dry plant mass during the vegetative stage [39]. In the reproductive stage, the following parameters were evaluated: time to anthesis, number of flowers per plant, fertilization percentage, number of fruits per plant, fruit mass per plant, and yield [40].

2.5. Statistical Analysis

Data were analyzed using IBM SPSS version 22. Results are expressed as the mean ± standard error (SE). Parametric tests, including one-way and two-way ANOVA, were conducted to determine statistical significance (p ≤ 0.05). Tukey’s HSD test was used for pairwise comparisons when significant differences were detected. Statistically significant differences are indicated by different lowercase letters (a, b, c) in tables and figures. Percentage data were transformed using the formula y′ = 2 arcsine ((y/100)^1/2).

3. Results

3.1. Initiation and Stabilization of In Vitro Cultures

The initiation and stabilization of in vitro cultures were evaluated based on the contamination levels, survival rates, and regeneration efficiency following surface sterilization with 2% (v/v) NaOCl. Figure 1 illustrates the effects of the NaOCl exposure time on these parameters across the three blackberry cultivars (Tupy, Kiowa, and Brazos). A significant reduction in contamination was observed with increasing exposure time, with the lowest contamination levels (<10%) recorded at 20 min of immersion (Figure 1A,D,G). This decrease in contamination corresponded with an increase in survival (Figure 1B,E,H) and regeneration rates (Figure 1C,F,I), suggesting that 20 min of NaOCl exposure was the optimal sterilization duration. However, exposure beyond 20 min resulted in a decline in both survival and regeneration, indicating potential cytotoxic effects of prolonged NaOCl exposure.

The statistical analysis further supports these findings. Linear regression analysis demonstrated a strong correlation between the NaOCl exposure time and contamination levels, with an R² value exceeding 0.95, indicating a highly predictable relationship (Figure 2A). Additionally, the regression analysis between the contamination and survival rates revealed a significant negative impact of contamination on survival. A strong positive correlation (R = 0.97) was observed between survival and regeneration rates, further confirmed by an R² value greater than 0.95 (Figure 2B). These results reinforce the effectiveness of the sterilization protocol in optimizing the initial establishment of in vitro cultures, while highlighting the importance of balancing the disinfection efficiency with the explant viability.

3.2. In Vitro Multiplication Stage

The in vitro multiplication stage in TIS was evaluated based on contamination rates, survival percentages, morphological indicators, and the effect of 6-BAP on shoot proliferation. Throughout the multiplication phase, contamination remained below 10% across all three blackberry cultivars, with no significant differences observed between treatments (Figure 3A). The immersion cycles effectively minimized contamination levels, supporting aseptic growth conditions. However, the survival rates varied with the frequency of immersion. The lowest survival rates were recorded at two immersion cycles per day, whereas survival increased significantly with four immersion cycles and reached its peak at six cycles, with no statistical differences among the cultivars (Figure 3B).

The morphological responses were significantly influenced by the immersion frequency (

Table 2. Effect of immersion cycles in TIS on the morphological indicators of explants from three blackberry cultivars (all treatments used 2 mg L⁻¹ 6-BAP with 5 min immersion).

Cultivars	Immersion Cycles per Day	Number of Necrotic Explants	Number of Leaves with Hyperhydricity	Total Fresh Mass of Shoots (g/per Culture Vessel)	Total Dry Mass of Shoots (g/per Culture Vessel)
Tupy	0 (Control)	0.0 ± 0.0 c	0.0 ± 0.0 c	2.33 ± 1.20 c	0.065 ± 0.001 c
	2	5.5 ± 0.6 a	0.0 ± 0.0 c	6.22 ± 1.70 b	0.186 ± 0.003 b
	4	2.2 ± 0.5 b	1.0 ± 0.5 b	9.34 ± 0.60 a	0.280 ± 0.001 a
	6	3.1 ± 0.5 b	2.8 ± 0.8 a	9.40 ± 0.60 a	0.281 ± 0.002 a
Kiowa	0 (Control)	0.0 ± 0.0 c	0.0 ± 0.0 c	1.88 ± 1.22 c	0.042 ± 0.001 b
	2	6.0 ± 0.7 a	0.3 ± 0.5 c	7.22 ± 0.40 b	0.194 ± 0.001 b
	4	3.0 ± 0.5 b	1.3 ± 0.3 b	10.28 ± 0.70 a	0.282 ± 0.002 a
	6	4.0 ± 0.8 b	3.0 ± 1.0 a	10.35 ± 0.70 a	0.281 ± 0.001 a
Brazos	0 (Control)	0.2 ± 0.1 c	0.0 ± 0.0 c	2.07 ± 1.25 c	0.067 ± 0.001 b
	2	5.8 ± 0.8 a	1.3 ± 0.5 b	5.55 ± 0.72 b	0.184 ± 0.001 b
	4	2.8 ± 0.5 b	1.3 ± 0.5 b	8.97 ± 0.05 a	0.311 ± 0.002 a
	6	3.5 ± 0.7 b	3.8 ± 1.0 a	9.12 ± 0.05 a	0.312 ± 0.001 a

Data are presented as mean ± standard error (SE) (n = 50 per treatment). Different lowercase letters within a column indicate statistically significant differences (two-way ANOVA, Tukey’s test, p ≤ 0.05).

). The highest fresh and dry mass accumulation was observed at six immersion cycles per day, which also corresponded to the lowest percentage of necrotic explants and hyperhydric leaves. In contrast, the highest percentage of necrotic explants and hyperhydric leaves, along with the lowest fresh and dry mass values, were recorded at two immersion cycles per day.

The morphological responses of the blackberry explants were significantly affected by the frequency of the immersion cycles (Table 2). Specifically, six immersion cycles per day resulted in the highest fresh and dry mass accumulation across all cultivars, with a concomitant reduction in the percentage of necrotic explants and leaves exhibiting hyperhydricity. Conversely, two immersion cycles per day resulted in the lowest fresh and dry mass values, coupled with the highest percentage of necrotic explants and hyperhydric leaves. These findings suggest that an increased immersion frequency positively influences shoot growth while minimizing undesirable morphological abnormalities.

The morphological responses were significantly influenced by the immersion frequency (Table 2). The highest fresh and dry mass accumulation was observed at six immersion cycles, which also resulted in the lowest percentage of necrotic explants and hyperhydric leaves. Conversely, the most necrotic explants and hyperhydric leaves, along with the lowest fresh and dry mass values, were recorded at two immersion cycles per day.

Table 3. Effect of immersion duration on contamination, survival, fresh mass, and dry mass of explants from three blackberry cultivars (all treatments used 2 mg L⁻¹ 6-BAP with 4–6 immersion cycles per day).

Cultivars	Immersion Time (min)	Contamination (%)	Survival (%)	Total Fresh Mass of Shoots (g/ per Culture Vessel)	Total Dry Mass of Shoots (g/per Culture Vessel)
Tupy	0 (Control)	1.64 ± 0.00 a	96.80 ± 0.38 a	2.37 ± 0.03 c	0.069 ± 0.001 c
	5	1.88 ± 0.01 a	97.26 ± 0.42 a	9.82 ± 0.05 a	0.186 ± 0.002 a
	10	1.44 ± 0.00 a	97.88 ± 0.43 a	8.46 ± 0.06 b	0.080 ± 0.010 b
	15	1.32 ± 0.00 a	98.02 ± 0.52 a	8.40 ± 0.05 b	0.081 ± 0.020 b
Kiowa	0 (Control)	1.32 ± 0.01 a	97.83 ± 0.46 a	1.88 ± 0.02 c	0.042 ± 0.020 c
	5	1.39 ± 0.00 a	97.45 ± 0.45 a	9.94 ± 0.04 a	0.194 ± 0.002 a
	10	1.45 ± 0.00 a	98.03 ± 0.47 a	7.88 ± 0.07 b	0.082 ± 0.010 b
	15	1.36 ± 0.01 a	98.01 ± 0.48 a	7.35 ± 0.06 b	0.281 ± 0.010 b
Brazos	0 (Control)	1.58 ± 0.02 a	98.21 ± 0.42 a	2.07 ± 0.01 c	0.067 ± 0.010 c
	5	1.65 ± 0.03 a	98.03 ± 0.39 a	9.85 ± 0.04 b	0.184 ± 0.030 c
	10	1.73 ± 0.01 a	97.88 ± 0.38 a	8.97 ± 0.03 a	0.078 ± 0.020 b
	15	1.84 ± 0.02 a	97.92 ± 0.42 a	8.12 ± 0.04 a	0.082 ± 0.010 b

Data are presented as mean ± standard error (SE) (n = 50 per treatment). Different lowercase letters within a column indicate statistically significant differences (two-way ANOVA, Tukey’s test, p ≤ 0.05).

summarizes the effects of the immersion duration on the contamination, survival, fresh mass, and dry mass of explants. Across all treatments, contamination remained below 10%, and survival rates exceeded 95%, indicating that the immersion durations did not negatively impact explant viability. The highest fresh and dry mass values were recorded at 5 min of immersion, with a notable decline in both parameters observed at 10 and 15 min, particularly for fresh mass. For Tupy, Kiowa, and Brazos, the 5 min immersion duration consistently yielded the highest fresh mass and dry mass, significantly outperforming the longer immersion times (10 and 15 min). While the fresh mass showed a consistent decrease with longer immersion times, the dry mass values were more variable, with Kiowa showing a significant increase in dry mass at 15 min, compared to the 10 min duration. These findings suggest that a 5 min immersion time optimizes shoot growth in all cultivars, while extended immersion durations (10 and 15 min) do not further enhance, and may even hinder, shoot development.

Statistical comparisons of the untreated controls (0 mg L⁻¹ 6-BAP) were performed to quantify baseline morphogenic variability among cultivars. These differences (e.g., Tupy’s higher shoot proliferation vs. Kiowa’s moderate vigor) provide a reference framework for evaluating PGR-induced responses and optimizing genotype-specific propagation proto-cols (

Table 4. Effect of 6-BAP on shoot multiplication of three blackberry cultivars in TIS (all treatments used 6 immersion cycles per day of 5 min duration).

Cultivars	Concentration BAP (mg L−1)	Shoots per Initial Bud	Shoot Length (cm)	Buds per New Shoot	Total Fresh Mass of the Shoots per Culture Vessel (g)
Tupy	0 (Control)	2.90 ± 0.90 d	3.0 ± 0.6 c	2.4 ± 0.8 c	9.34 ± 0.40 c
	1	23.30 ± 1.30 b	5.5 ± 1.5 a	5.2 ± 0.5 a	10.22 ± 0.80 b
	2	34.01 ± 1.20 a	5.4 ± 1.2 a	5.1 ± 0.7 a	11.15 ± 0.60 a
	3	9.53 ± 0.50 c	3.0 ± 0.8 b	3.0 ± 0.8 b	6.34 ± 1.70 c
	4	8.42 ± 0.24 d	2.3 ± 0.6 c	2.3 ± 0.7 c	4.42 ± 1.02 d
	5	6.87 ± 0.18 e	1.7 ± 0.4 d	1.9 ± 0.5 d	3.22 ± 0.87 e
Kiowa	0 (Control)	1.80 ± 0.60 d	3.5 ± 0.4 c	2.9 ± 0.5 c	10.28 ± 0.70 c
	1	20.70 ± 1.50 a	8.4 ± 2.5 a	6.3 ± 1.1 a	11.92 ± 0.90 b
	2	20.60 ± 1.26 a	5.4 ± 1.9 b	5.6 ± 0.5 a	12.22 ± 0.50 a
	3	7.00 ± 1.80 b	2.7 ± 0.5 c	4.4 ± 0.6 b	7.35 ± 0.90 d
	4	5.45 ± 1.20 c	2.6 ± 0.5 c	3.12 ± 0.4 b	5.54 ± 0.71 e
	5	5.57 ± 1.20 c	1.8 ± 0.3 d	2.88 ± 0.3 b	4.75 ± 0.40 f
Brazos	0 (Control)	2.30 ± 1.00 c	4.4 ± 0.9 b	3.2 ± 0.4 b	9.17 ± 0.04 c
	1	18.50 ± 0.80 b	6.9 ± 0.7 a	5.8 ± 0.6 a	10.38 ± 0.80 b
	2	20.00 ± 1.30 a	6.5 ± 0.5 a	5.3 ± 0.6 a	11.42 ± 0.41 a
	3	8.30 ± 0.70 d	3.6 ± 0.4 c	3.5 ± 0.5 b	5.22 ± 1.40 d
	4	6.22 ± 0.40 e	3.1 ± 0.2 d	3.5 ± 0.5 b	4.18 ± 1.11 e
	5	6.18 ± 0.40 e	2.7 ± 0.2 e	3.5 ± 0.5 b	3.82 ± 1.41 f

Shoots per initial bud quantifies multiplication efficiency; buds per shoot indicates subsequent branching potential. Means followed by different lowercase letters within columns differ significantly ((two-way ANOVA), Tukey, p ≤ 0.05). Each data point represents the mean for n = 50. Statistical comparisons among controls are presented to illustrate inherent genetic variability and do not reflect treatment effects.

). The 2 mg L⁻¹ 6-BAP treatment showed optimal proliferation, yielding 34.01 ± 1.20 shoots per initial bud (primary multiplication) while maintaining 5.1 ± 0.7 buds per new shoot (secondary branching capacity) in the Tupy cultivar. Across all cultivars, 2 mg L⁻¹ 6-BAP yielded the highest shoot number per bud, shoot length, number of buds per shoot, and total fresh mass across all cultivars. However, increasing the 6-BAP concentration beyond 2 mg L⁻¹ resulted in a decline in the shoot multiplication efficiency, indicating possible inhibitory effects at higher concentrations.

These results indicate that six immersion cycles per day and 5 min immersion durations provide optimal conditions for in vitro shoot multiplication. Additionally, 2 mg L⁻¹ of 6-BAP is the most effective concentration for promoting shoot proliferation in blackberry (Rubus spp.) TIS cultures.

3.3. Rooting Stage

The effect of IBA on root induction was evaluated in both TIS and rooting on solid medium methods. For TIS rooting, plants were maintained under the same immersion protocol as the multiplication stage: 6 cycles per day of 5 min immersion duration, with 1 mg L⁻¹ IBA added to the liquid MS medium. Figure 4 illustrates the response of the three blackberry cultivars to different IBA concentrations. All tested IBA concentrations significantly enhanced the root emission, root number per plant, and root system length compared to the control. The highest rooting efficiency was observed at 1 mg L⁻¹ IBA, which resulted in the greatest root formation across all cultivars in both TIS (Figure 4A–C) and rooting on a solid medium (Figure 4D–F). However, higher IBA concentrations led to a decline in root development, suggesting a possible inhibitory effect.

The rooting efficiency of the blackberry (Rubus spp.) plantlets was evaluated under two different in vitro conditions: TIS and rooting on solid medium methods. The results, summarized in

Table 5. Root emission parameters of in vitro-grown blackberry plants using TIS and solid medium methods.

Rooting Method	Cultivar	Root System Length (cm)	Number of Roots per Plant	Root Emission (%)
TIS *	Tupy	1.14 ± 0.08	3.97 ± 0.20	96.25 ± 0.81
	Kiowa	1.16 ± 0.09	3.98 ± 0.18	97.22 ± 0.78
	Brazos	1.18 ± 0.07	4.01 ± 0.21	96.28 ± 0.76
On solid Medium *	Tupy	1.17 ± 0.08	3.92 ± 0.17	96.12 ± 0.80
	Kiowa	1.14 ± 0.07	3.96 ± 0.18	96.18 ± 0.88
	Brazos	1.15 ± 0.09	3.95 ± 0.19	96.32 ± 0.87

One-way ANOVA followed by Tukey’s multiple comparison test (p ≤ 0.05). Data are presented as mean ± standard error, with n = 100 per treatment. * No statistically significant differences were observed between rooting methods.

, indicate that the application of 1 mg L⁻¹ IBA significantly enhanced root development across all tested cultivars. No statistically significant differences were observed between TIS and rooting agar-solidified medium methods in terms of the root emission percentage, number of roots per plant, and root system length, demonstrating the comparable effectiveness of both approaches. Across all cultivars, root emission exceeded 96%, with Kiowa exhibiting the highest percentage (97.22% in TIS and 96.18% in the rooting on a solid medium system). The number of roots per plant remained consistent between methods, with values ranging from 3.92 to 4.01. Similarly, the root system length showed minimal variation, with Brazos displaying the longest average root length (1.18 cm in TIS and 1.15 cm in the rooting using the solid medium method).

These findings confirm that 1 mg L⁻¹ IBA is the optimal concentration for inducing robust root formation in blackberry micropropagation. Furthermore, the comparable performance of TIS and rooting on solid medium methods underscores the potential of TIS as a scalable alternative, offering improved automation and resource efficiency without compromising rooting success.

3.4. Acclimatization and Field Performance

3.4.1. Effect of Acclimatization Conditions on Survival Rates

The acclimatization phase was critical in determining the ex vitro survival and adaptation of in vitro-propagated blackberry plants. Figure 5 illustrates the survival rates of the Tupy, Kiowa, and Brazos cultivars under different acclimatization conditions. Plants derived from both TIS and rooting on solid medium methods, when placed in trays with covers, exhibited survival rates exceeding 95%. In contrast, those acclimatized without covers displayed significantly lower survival percentages across all cultivars, highlighting the importance of controlled humidity in the early stages of ex vitro adaptation.

Figure 5 further details survival percentages across the three acclimatization stages (first, second, and third). At each stage, plants placed in trays with covers exhibited significantly higher survival rates compared to those grown without covers. The absence of covers resulted in a progressive decline in survival rates, indicating the necessity of high-humidity conditions during the initial acclimatization phase.

A comparative analysis between the two rooting methods revealed that TIS-derived plants showed comparable survival rates to solid-medium plants in covered trays (both ~95%), indicating that both methods can achieve excellent results under optimal humidity conditions. However, TIS plants exhibited marginally better resilience in uncovered trays during early acclimatization (Figure 5A–F), suggesting potential advantages in suboptimal conditions. These results emphasize that proper humidity control remains critical for successful acclimatization, regardless of the rooting method.

3.4.2. Optimization of In Vitro Propagation Protocol

The different stages of the optimized in vitro propagation protocol for the three blackberry cultivars are illustrated in Figure 6. The establishment of shoots from stem segments (D) under in vitro conditions demonstrated efficient regeneration, while the use of TIS with six immersion cycles per day (E–G) significantly enhanced shoot development, minimizing necrosis and promoting robust growth.

The rooting stage (I–L) was successfully optimized using 1 mg·L⁻¹ IBA in both TIS (K,L) and rooting on solid medium methods (I,J). Strong root emission and well-developed root systems were observed in both cases, confirming that TIS and rooting on solid medium methods are equally effective when an optimal IBA concentration is applied.

During acclimatization (M,N), plants successfully transitioned to ex vitro conditions. Those placed in trays with covers during the first acclimatization stage (M) exhibited higher survival, while plants in the third stage (N) demonstrated full adaptation to ambient conditions, confirming the effectiveness of the acclimatization process.

3.4.3. Vegetative Growth Under Field Conditions

During the vegetative phase, plants from the three blackberry cultivars exhibited significant differences in morphological indicators, as detailed in

Table 6. Morphological indicators of three blackberry cultivars during the vegetative phase under field conditions.

Indicator	Cultivar
	Tupy	Kiowa	Brazos
Survival (%)	98.42 ± 0.82 a	97.52 ± 0.83 a	97.55 ± 0.81 a
Plant height (cm)	1.48 ± 0.58 a	1.32 ± 0.58 b	1.26 ± 0.58 b
Stem diameter (mm)	2.89 ± 0.48 a	2.53 ± 0.47 b	2.58 ± 0.46 b
Fresh plant mass (kg)	2.96 ± 0.67 a	2.22 ± 0.68 b	2.15 ± 0.67 b
Dry plant mass (kg)	0.089 ± 0.006 a	0.064 ± 0.005 b	0.058 ± 0.007 b

Data are presented as mean ± standard error (SE), where SE values represent variability among replicates (n = 100 per treatment). Means with different letters indicate significance (two-way ANOVA, Tukey, p ≤ 0.05).

. Survival rates exceeded 97% across all cultivars, with no significant differences observed, confirming high resilience and successful establishment under field conditions.

The Tupy cultivar demonstrated a superior performance across all growth parameters, exhibiting the tallest plants (1.48 cm) and the largest stem diameter (2.89 cm). Additionally, it showed the highest fresh plant mass (2.96 kg) and dry plant mass (0.089 kg), significantly outperforming Kiowa and Brazos. In contrast, Kiowa and Brazos cultivars displayed lower growth indicators, with Kiowa presenting the shortest plants, smallest stem diameter, and reduced biomass accumulation. These findings suggest that Tupy may be the most suitable cultivar for environments requiring rapid vegetative growth, whereas Kiowa and Brazos may require additional agronomic interventions to optimize their performance.

3.4.4. Reproductive Growth and Yield Performance

Significant differences in the reproductive growth and yield were observed across the three blackberry cultivars (

Table 7. Reproductive performance of three blackberry cultivars under field conditions.

Indicator	Cultivar
	Tupy	Kiowa	Brazos
Anthesis stage (days)	187 ± 1.25 c	192 ± 1.27 b	202 ± 1.26 a
Number of flowers per plant	292 ± 4.22 a	262 ± 4.20 b	268 ± 4.21 b
Flower fertilization (%)	89.93 ± 1.12 a	86.52 ± 1.11 b	87.57 ± 1.12 b
Number of fruits per plant	268 ± 3.87 a	227 ± 3.88 b	232 ± 3.88 b
Fruit mass per plant (g)	3.96 ± 0.13 b	4.32 ± 0.14 a	3.82 ± 0.14 b
Yield (t ha−1)	12.88 ± 0.34 a	10.38 ± 0.33 b	10.65 ± 0.34 b

Data are presented as mean ± standard error (SE), where SE values represent variability among replicates (n = 100 per treatment). Means with different letters indicate significance (one-way ANOVA, Tukey, p ≤ 0.05).

). Tupy exhibited the earliest anthesis (187 days), compared to Kiowa (192 days) and Brazos (202 days), suggesting an extended fruiting period and higher production potential. Tupy also produced the highest number of flowers per plant (292), significantly exceeding Kiowa (262) and Brazos (268). This advantage was reflected in higher flower fertilization rates (Tupy: 89.93%, Kiowa: 86.52%, Brazos: 87.57%), which led to an increased number of fruits per plant.

Although Tupy exhibited the highest fruit production per plant (268 fruits), its average fruit mass (3.96 g) was lower than that of Kiowa (4.32 g), suggesting that its higher yield was driven by the fruit quantity rather than the individual fruit size. However, the total yield per hectare was significantly higher for Tupy (12.88 t ha −¹) compared to Kiowa (10.38 t ha −¹) and Brazos (10.65 t ha −¹), confirming its higher productivity potential.

These findings highlight significant genotypic differences in the vegetative and reproductive performance among the three blackberry cultivars. Tupy emerged as the most productive genotype, benefiting from earlier flowering, greater flower production, higher fertilization rates, and a superior total yield. Its productivity was largely driven by a high fruit count rather than the individual fruit size, making it an ideal choice for commercial-scale production focused on maximizing the output per hectare. Kiowa, despite exhibiting a lower number of fruits per plant, produced the largest average fruit mass (4.32 g), making it potentially more suitable for fresh fruit markets where a larger fruit size is preferred. Brazos displayed intermediate reproductive indicators, with a slightly lower yield and fruit production than Tupy, but a greater fruit mass than Tupy, positioning it as a balanced option between the yield potential and fruit quality.

Overall, these results suggest that Tupy is the most suitable cultivar for high-yield cultivation, whereas Kiowa may be better suited for markets that prioritize a larger fruit size. These insights are essential for selecting optimal blackberry cultivars for different agricultural and commercial production systems.

3.4.5. Final Field Establishment and Morphological Development

The successful transition of in vitro-propagated blackberry (Rubus spp.) plants to field conditions was assessed based on their morphological development and adaptation. Figure 7 illustrates the final stage of acclimatization and establishment of the Tupy, Kiowa, and Brazos cultivars under ex vitro and field conditions. At the end of acclimatization, all three cultivars exhibited morphological integrity, a fully functional foliar system, and a well-developed root system (Figure 7, panels A–C). The absence of necrosis, leaf deformation, or growth abnormalities indicates that the acclimatization process was effective, allowing plants to successfully transition from controlled greenhouse conditions to natural environments.

Once transplanted into the field, the plants maintained uniform growth and development, with no visible impairments in their vegetative structure (Figure 7, panels B–D–F). These findings confirm the robustness and adaptability of the in vitro propagation protocol, reinforcing its effectiveness in generating high-quality planting material suitable for large-scale agricultural production.

These results emphasize the success of the acclimatization process and the field viability of the propagated blackberry plants, demonstrating that in vitro propagation techniques can effectively produce plants with strong adaptability and high survival rates in commercial cultivation systems.

4. Discussion

This study refines blackberry (Rubus spp.) micropropagation by optimizing sterilization protocols, TIS conditions, plant growth regulator (PGR) regimes, and acclimatization strategies. These refinements address persistent challenges in blackberry tissue culture and align with global research trends identified through a comprehensive bibliometric analysis [18]. A total of 78 scientific publications (1998–2024) highlight increasing interest since the late 2000s, driven by rising demand for high-quality planting material and advancements in propagation technologies [41,42,43,44].

Surface sterilization remains one of the most critical and challenging steps in in vitro plant propagation. In our study, contamination levels were successfully reduced to below 10% using a combination of 2% NaOCl and 2 mg L⁻¹ PPM, confirming the effectiveness of this protocol for commercial applications. This outcome was primarily attributed to the disinfection treatment rather than the TIS itself. While TIS enhances nutrient uptake and shoot proliferation [45], it does not inherently control microbial contamination, which must be addressed through complementary antimicrobial measures such as PPM supplementation.

The statistical analysis further supports these findings. Linear regression revealed a strong predictive relationship between NaOCl exposure time and contamination levels (R² > 0.95), indicating a consistent and reproducible response. Additionally, survival and regeneration rates improved with increasing exposure time up to 20 min. Beyond this point, however, both parameters declined, suggesting that prolonged exposure induces cytotoxic effects. These results highlight the importance of balancing effective disinfection with explant viability, as excessive exposure can damage tissue and reduce regeneration potential.

Interestingly, the three blackberry genotypes—Tupy, Kiowa, and Brazos—showed distinct responses to NaOCl treatment: Tupy exhibited a decline in survival and regeneration at exposure times beyond 20 min, suggesting a relatively narrow tolerance range. Brazos maintained high viability—over 85%—even after 25–30 min of exposure, indicating greater resistance to the disinfectant. Kiowa showed an intermediate pattern, consistent with its previously reported adaptability in in vitro conditions.

These differences suggest that each genotype responds uniquely to surface sterilization, likely due to variations in their physiological or structural characteristics. While the exact mechanisms behind these differences were not investigated in this study, the results highlight the importance of adjusting sterilization protocols according to genotype-specific sensitivity to ensure optimal explant survival and regeneration.

Our findings support the integration of broad-spectrum biocides into sterilization protocols to improve sterility without resorting to complex nanomaterials. Unlike Spinoso-Castillo et al. [46], who used silver nanoparticles in TIS media for microbial suppression, our approach with PPM achieved comparable sterility levels while remaining compatible with routine propagation workflows—making it more suitable for commercial application.

The use of NaOCl in the sterilization process aims to eliminate all microorganisms that may affect the tissue and to ensure viability and regenerative capacity [47]. However, it is important to establish the concentration, application duration, temperature, and type of disinfectant agent in any in vitro protocol. In the research, only the time was considered, with the goal of minimizing contamination and maximizing survival and regeneration at 20 min.

These differential sensitivities underscore the importance of incorporating genotype-specific sterilization thresholds into protocol design and highlight how genetic makeup interacts with disinfection methods to shape outcomes [46]. The observed responses fit a hormetic model, where moderate NaOCl exposure stimulates effective disinfection without inducing oxidative damage, whereas excessive exposure surpasses physiological limits.

The interaction between genotype and sterilization treatments plays a critical role in determining success in micropropagation, with disinfectants like HgCl₂, NaOCl, and H₂O₂ inducing oxidative stress that affects explant viability [47]. Disinfectants generate ROS (H₂O₂, O₂⁻), which in sensitive genotypes exceed tolerance thresholds, damaging membranes and DNA [48,49].

This relationship can be observed in Nymphaea colorata when HgCl₂ (0.1%, 15 min) was used, resulting in only 10% contamination and 90% survival [50]. These concentrations maximize shoot multiplication within two weeks, and during rooting, produced the best response in terms of roots/shoot, ensuring a robust root system. The use of gamma irradiation at 40 Gy reduced contamination and significantly improved rooting and vegetative growth in blackberry seedlings (Rubus fruticosus) [51]. These studies highlight the hormetic response of the plant material.

To further improve outcomes—particularly for recalcitrant cultivars—future protocols may benefit from combining NaOCl with antioxidant supplements or exploring alternative sterilants like ozone [52], supporting the ongoing shift toward tailored, genotype-informed disinfection strategies in modern micropropagation design.

TIS significantly improved shoot proliferation compared to static liquid or semi-solid media. However, hyperhydricity—a known risk in liquid culture—was genotype-dependent. Tupy developed hyperhydric symptoms when exposed to more than six immersions daily, whereas Brazos maintained normal morphogenesis under identical conditions. These findings highlight the importance of tailoring immersion schedules to cultivar-specific needs. This variation may be linked to differences in aquaporin expression, as suggested by Muñoz-Concha et al. [43], who associated PIP2;1 overexpression with water uptake regulation in Rubus. This indicates that some genotypes are more prone to fluid imbalance under dynamic liquid environments, particularly for species with high sensitivity to excess water absorption [52,53,54,55].

Improper aeration and immersion timing can exacerbate hyperhydricity. Our data show that four to six immersions per day (5–10 min each) balanced growth and hyperhydricity reduction, corroborating previous findings [54,55]. Vessel design also played a role: mesh-supported explants improved gas exchange and reduced hyperhydricity by up to 40%, as noted by Vujović et al. [56].

Despite these benefits, root morphology differed between the TIS and solid medium methods. Roots derived from TIS were thinner, likely due to suppressed lignin biosynthesis in low-mechanical-stress environments [57]. This phenomenon has been increasingly recognized in recent literature [18], reinforcing the need for mechanical conditioning techniques such as air-pruning or auxin pulse treatments before acclimatization.

Therefore, future work should focus on fine-tuning immersion frequencies, vessel design, and antioxidant supplementation [58,59,60] to improve physiological resilience and shoot quality under TIS conditions. Exploring alternative carbohydrate sources or mechanical hardening techniques could also enhance root and shoot development before acclimatization.

Our findings underscore the critical role of genotype-specific optimization of PGRs, particularly cytokinins such as BAP, in refining blackberry micropropagation protocols. As highlighted by the bibliometric analysis conducted by Regni et al. [18], BAP remains the most frequently used cytokinin across blackberry tissue culture studies, appearing prominently in multiplication stages. However, its application in rooting phases is rare, with only one study—Titenkov et al. [60]—reporting combined use of BAP and IBA for root induction.

Despite its widespread use, BAP effectiveness varies significantly across genotypes, underscoring the need for tailored PGR strategies. In our experiments, Tupy responded optimally at 2 mg L⁻¹ BAP, yielding 34.01 ± 1.20 shoots per initial bud during primary multiplication, while maintaining 5.1 ± 0.7 buds per new shoot, indicating strong secondary branching potential. This aligns with earlier reports showing that BAP concentrations between 1–5 mg L⁻¹ promote shoot formation in Rubus species [61], although higher doses often result in metabolic inhibition or morphological abnormalities [51,62].

Importantly, we observed a clear hormetic response pattern with increasing BAP concentrations. While moderate levels stimulated shoot proliferation, higher concentrations (e.g., 3–5 mg L⁻¹) led to inhibitory effects, including reduced shoot number, shorter shoot length, and fewer auxiliary buds. These findings support the concept of hormesis —a dose-dependent biological response where low concentrations stimulate growth, but excessive levels become inhibitory [63]. Similar trends have been reported in other species, where excess cytokinins disrupted cellular metabolism and induced physiological stress [18,63,64].

In contrast, Brazos exhibited differential sensitivity, suggesting potential benefits from lower BAP concentrations or alternative cytokinins like TDZ, which can induce callusing and facilitate indirect regeneration [65]. Specifically, 0.5 mg L⁻¹ TDZ triggered callus formation in Brazos, possibly through activation of WUSCHEL-like regulators [66]. However, TDZ use must be carefully managed due to its association with increased somaclonal variation, a concern raised in 23% of recent studies [18].

This variability in PGR response among cultivars represents a major challenge in standardizing micropropagation protocols [18]. Therefore, future research should explore alternative cytokinins—such as zeatin—either alone or in combination with BAP, to evaluate whether synergistic effects could enhance shoot proliferation rates and overall plant quality [40,67].

Additionally, integrating dose-response matrices and gene expression profiling will help define cultivar-specific hormonal thresholds, enabling more precise and effective micropropagation strategies [44,57]. Such approaches may include assessing auxin-cytokinin interactions at the molecular level, particularly genes involved in meristem regulation and stress adaptation [43].

These tools are essential for transitioning from generic protocols to precision-based approaches that account for genetic differences and environmental responsiveness [18]. By adopting such strategies, researchers can better align micropropagation practices with emerging global research priorities focused on hormone dynamics, genotype × environment interactions, and ex vitro adaptation [68,69].

Root development is a critical determinant of successful acclimatization and ex vitro survival. This study confirms that 1 mg L⁻¹ IBA was the most effective concentration for root induction across all tested genotypes—Tupy, Brazos, and Kiowa—achieving rooting percentages above 85% with well-developed root systems. These findings corroborate earlier reports indicating that optimal auxin concentrations enhance root initiation, while excessive levels disrupt the endogenous hormone balance and inhibit elongation [70,71].

Notably, root morphology differed between culture systems: plants rooted in TIS developed thinner root structures compared to those grown on solid media, likely due to reduced mechanical resistance and suppressed lignin biosynthesis in liquid environments [72]. Despite this, TIS-derived plantlets showed comparable acclimatization success when transferred under controlled humidity conditions (70–80% relative humidity), suggesting that structural differences may not necessarily compromise field performance if supported by appropriate substrates during acclimatization.

However, certain limitations persist in TIS-based rooting. Root systems developed under continuous immersion often exhibited lower lignification and branching density, which could affect anchorage and water uptake efficiency under field conditions. To address this, future studies should explore alternative auxins such as naphthaleneacetic acid (NAA) or sequential auxin applications (e.g., IBA followed by NAA) to improve root robustness and enhance transition success to ex vitro and field environments [63].

Acclimatization remains one of the most critical phases in micropropagation, ensuring the transition from controlled in vitro environments to fluctuating external conditions [66,67]. In this study, plants acclimatized in covered trays-maintained survival rates exceeding 95%, whereas uncovered trays led to rapid dehydration and mortality, particularly in sensitive genotypes like Brazos. These findings emphasize the importance of humidity control during early acclimatization stages, as previously noted by Trofim et al., [73].

Acclimatization success exceeded 90% survival under controlled humidity (70–80% RH), consistent with prior studies emphasizing gradual adaptation to minimize water loss and stomatal stress [68,69]. However, significant genotype-specific differences emerged: ‘Tupy’ exhibited faster adaptation and higher survival rates, possibly due to thicker cuticles or superior stomatal regulation [31]; ‘Brazos’ required extended adaptation periods, highlighting its sensitivity to transpiration stress; and ‘Kiowa’ demonstrated intermediate tolerance, consistent with its known field resilience and historical use in rooting studies.

These results support earlier findings showing that acclimatization success depends on root system development, water regulation capacity, and the gradual adaptation of photosynthetic mechanisms [68,69]. Importantly, covered trays significantly improved survival (~95%), reinforcing the role of controlled humidity in reducing physiological stress during early adaptation.

To enhance ex vitro adaptation in diverse cultivars, future research should investigate strategies such as CO₂ enrichment, partial defoliation, and substrate modifications—approaches shown to reduce evaporative demand and promote root-to-shoot balance [70]. Such interventions could further refine acclimatization practices and ensure robust plant establishment in diverse growing environments.

Field evaluations confirmed that all three cultivars successfully transitioned to commercial production, achieving high yields in their first production cycle. Notably, ‘Tupy’ excelled in fruit mass, number per plant, and total yield, validating earlier reports of its adaptability and productivity in blackberry cultivation [71]. Rooting on solid medium appeared to confer slight advantages in root structure, potentially contributing to its superior field performance.

Despite these promising results, long-term monitoring remains essential. Future research should assess: multi-season yield stability and productivity, disease resistance and environmental stress tolerance, and molecular fidelity and physiological stability of micropropagated plants compared to conventional counterparts.

This will ensure that the benefits observed during in vitro and early acclimatization translate into sustainable, scalable field performance—a key objective for advancing blackberry propagation protocols.

Furthermore, our findings align with recent bibliometric trends identifying ex vitro adaptation and substrate optimization as emerging research priorities [18]. The integration of morpho-physiological assessments—including leaf gas exchange, root hydraulic conductivity, and antioxidant profiling—could further refine acclimatization strategies and support precision-based micropropagation tailored to individual cultivars.

5. Conclusions

This study investigated micropropagation protocols for the blackberry (Rubus spp.) cultivars Tupy, Brazos, and Kiowa using temporary immersion systems (TIS) with ex vitro acclimatization. The lowest contamination, with the highest survival and regeneration of explants, was obtained with 20 min in 2% NaOCl. In TIS, the use of four and six immersion cycles, with a duration of 5 min, ensured the highest survival rates, with fewer necrotic explants, fewer hyperhydric leaves, and a greater fresh mass. The concentration of 2 mg L⁻¹ of 6-BAP induced the greatest morphological development of the shoots. The use of 1 mg L⁻¹ promotes greater root system development in rooting on solid medium and TIS. During acclimatization, trays with plastic covers promoted higher survival rates: above 90% for the three cultivars. The greatest losses, regardless of the rooting method, occurred in the first stage of acclimatization, where trays and pots without covers stood out. The optimal conditions obtained during in vitro cultivation and acclimatization ensured plants with morphological development unaffected during the vegetative and reproductive stages, with yields above 10 t ha⁻¹. The results of the research generate a micropropagation protocol that demonstrates high efficiency in the in vitro stages, ex vitro acclimatization, and field conditions.