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Article

Enhancing In Vitro Multiplication and Acclimatization of Blackberry (Rubus L.) Through Sterilization Optimizing and Growth Regulator Use

by
Natalya Malakhova
1,
Botakoz Tezekbayeva
1,
Vladimir Kiyan
2 and
Yuliya Yefremova
3,*
1
M.A. Aitkhozhin Institute of Molecular Biology and Biochemistry, Almaty 050012, Kazakhstan
2
Laboratory of Biodiversity and Genetic Resources, National Center for Biotechnology, Astana 010000, Kazakhstan
3
Educational Institution, Kazakh-German University in Almaty, Almaty 050010, Kazakhstan
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(12), 1422; https://doi.org/10.3390/horticulturae11121422
Submission received: 15 October 2025 / Revised: 21 November 2025 / Accepted: 23 November 2025 / Published: 25 November 2025
(This article belongs to the Section Propagation and Seeds)
Browse Figures
Versions Notes

Abstract

Blackberry (Rubus L.) cultivation in Kazakhstan is constrained by the limited availability of certified planting material and the absence of standardized micropropagation protocols adapted to local conditions. This study aimed to optimize the key stages of in vitro culture for the cultivars ‘Natchez’, ‘Black Magic’, ‘Osage’, and ‘Heaven Can Wait’, including explant sterilization, culture initiation, shoot multiplication, and acclimatization. A sequential sterilization scheme using 70% ethanol followed by 1% sodium hypochlorite ensured high explant survival. Shoot initiation was most efficient on MS medium supplemented with 0.1 mg/L BAP, whereas multiplication was enhanced by 0.5 mg/L BAP and 0.1 mg/L GA3. In the subsequent rooting stage, microcuttings formed stable root systems under ex vitro conditions in agroboxes, confirming that the optimized protocol ensured not only high survival during initiation but also a successful transition to the rooting phase, which is essential for further acclimatization. During ex vitro acclimatization, the application of humic acid, nanosilicon, or succinic acid improved survival under agrobox microventilation. The developed approach provides a reliable framework for producing healthy, adapted plants of the evaluated cultivars and contributes to establishing domestic propagation systems for reducing reliance on imported planting material.

1. Introduction

In recent years, the horticulture sector in Kazakhstan has expanded rapidly, accompanied by growing consumer demand for nutritious fruit and berry products [1]. At the same time, interest has increased in crops whose fruits are rich in biologically active and health-promoting compounds, reflecting broader trends toward functional nutrition and diversification of horticultural production [2,3,4,5,6]. Given the environmental challenges typical of many regions of the country, ensuring access to foods containing essential vitamins, minerals, and other bioactive substances remains a priority for improving dietary quality and public health [7,8,9,10,11,12]. Therefore, a key objective of modern horticulture is to broaden the assortment of berry crops suitable for cultivation across diverse climatic zones, thereby meeting the demand for high-quality, affordable, and health-enhancing products [13,14,15].
One promising direction for increasing berry production in Kazakhstan is the introduction and expansion of crops that are well established internationally but remain underrepresented locally. Blackberry (Rubus L.) is widely cultivated in many countries owing to its excellent organoleptic characteristics, rich biochemical profile, and high productivity potential; however, its cultivation in Kazakhstan remains limited, primarily due to the lack of domestically produced certified planting material and nursery infrastructure [16,17,18,19]. Blackberry fruits possess high nutritional value, containing elevated levels of vitamins, polyphenols, organic acids, and minerals that contribute to their antioxidant and health-promoting properties [20,21,22]. In addition to fresh consumption, blackberries are widely used in food processing, making the crop economically attractive. These characteristics emphasize the need to expand commercial blackberry production in Kazakhstan and highlight the importance of developing efficient propagation systems. Moreover, blackberry could serve as a valuable addition to the berry crops already grown in the country [23]. The species also demonstrates adaptive potential, supported by the presence of wild Rubus forms in several regions of Kazakhstan, which further justifies its suitability for local cultivation [24,25]. Blackberry fruits are visually attractive, possess excellent taste and aroma, and can be consumed fresh or processed into various food products [26,27,28,29]. At present, blackberries are cultivated industrially in Serbia, Hungary, Poland, Germany, the United Kingdom, France, the United States, Canada, and Russia [30]. However, the demand for these nutritionally rich berries significantly exceeds domestic production—by approximately five to six times [31,32,33].
In Kazakhstan, commercial blackberry cultivation remains limited, with no large-scale plantations established to date. The total cultivated area does not exceed several dozen hectares, and current yields are insufficient to satisfy domestic demand, making the market heavily dependent on imports. A similar situation exists with planting material: the scarcity of domestically produced blackberry stock remains one of the major barriers to commercial production. This is largely due to underdeveloped local propagation infrastructure and the absence of high-quality nursery plants, forcing growers to rely on imported material. Such reliance increases the risk of introducing quarantine pests and pathogens and often results in poor field performance due to limited adaptation to local soil and climatic conditions. Traditional propagation methods, such as layering and cuttings, do not address these challenges because of their inherently low multiplication rates, the need for extensive mother plantations, high labor intensity, and the potential transmission of diseases [34,35,36]. These constraints underscore the need for modern biotechnological approaches, particularly in vitro micropropagation, which enables the production of genetically uniform, pathogen-free, and regionally adapted planting material [36,37,38].
In developed countries, the establishment of productive blackberry plantations relies on high-quality, virus-free plant material derived from elite cultivars. Such material is typically produced via modern biotechnological methods—primarily in vitro microclonal propagation [34,38,39]. This technique provides numerous advantages, including genetic uniformity, improved plant performance, elimination of viruses and other pathogens through meristem culture, rapid production of large quantities of planting material, and year-round operation under controlled conditions, which facilitates scheduled plant release and international exchange of pathogen-free stock [35,40,41,42,43,44,45]. Although several protocols exist for blackberry in vitro culture [46,47,48,49,50], numerous studies emphasize that species- and cultivar-specific responses require optimization of media composition, growth regulator combinations, and acclimatization conditions at each stage of micropropagation [35,40,51,52].
Recent attention has also focused on the use of biostimulants during rooting and acclimatization, namely, humic acid, nanosilicon, and succinic acid because they act through different but complementary physiological mechanisms. Humic acid promotes root initiation, modulates auxin signalling, and contributes to reactive oxygen species (ROS) homeostasis [53]. Nanosilicon improves morphogenic responses, enhancing shoot and root formation as well as acclimatization efficiency [54,55], whereas succinic acid stimulates mitochondrial activity and energy metabolism, supporting overall plant vigor [20,21,22]. Together, these agents provide multifaceted stimulation during rooting and acclimatization that conventional synthetic auxins cannot fully ensure.
To evaluate the phytosanitary status of blackberry (Rubus L.) samples, a molecular screening was performed for viruses most commonly detected in Rubus species. The diagnostic panel included Raspberry bushy dwarf virus (RBDV), Raspberry leaf blotch virus (RLBV), and Raspberry ringspot virus (RpRSV). RBDV is the most widespread virus among Rubus crops and has previously been identified in Kazakhstan on raspberry (Rubus idaeus L.) [56]. RLBV and RpRSV have been identified mainly in raspberry [57], but their inclusion in the diagnostic panel is justified by the close phylogenetic relationship between raspberry and blackberry, which implies potential cross-susceptibility. Additionally, the frequent use of imported planting material increases the risk of latent or undetected infections. Given the ongoing expansion of commercial blackberry cultivation in Kazakhstan, preventive molecular screening is essential to ensure phytosanitary safety. Therefore, assessing the presence of these viruses in blackberry plants provides a comprehensive evaluation of their health status and minimizes the risk of pathogen introduction into production systems.
To address the limited availability of certified blackberry planting material in Kazakhstan, reliable propagation systems are required to support the establishment of commercial plantations. Conventional vegetative propagation techniques, such as layering and cuttings, have low multiplication rates and provide insufficient phytosanitary control, making them unsuitable for large-scale production.
Biotechnological approaches based on in vitro culture are widely recognized as an effective way to obtain genetically uniform and pathogen-free plants; however, existing protocols for Rubus spp. often exhibit strong cultivar-specific responses and therefore require further optimization.
Refining micropropagation conditions for locally relevant cultivars is thus essential for establishing a stable system for producing high-quality planting material.
The aim of this study was to optimize the conditions for in vitro culture initiation, microclonal propagation, rooting, and acclimatization of blackberry cultivars ‘Natchez’, ‘Black Magic’, ‘Osage’, and ‘Heaven Can Wait’ using agroboxes with adjustable microventilation.

2. Materials and Methods

2.1. Optimization of Sterilization Method for Plant Explants

2.1.1. Plant Material and Explant Source

The study was conducted using four blackberry (Rubus L.) cultivars: ‘Natchez’, ‘Black Magic’, ‘Osage’, and ‘Heaven Can Wait’. Actively growing shoots were collected from healthy donor plants cultivated at the nursery of LLP “Semirechye” (Almaty Region, Kazakhstan). Nodal segments with axillary buds (1.0–1.5 cm in length) were used as explants.

2.1.2. Surface Sterilization Protocols

Three sterilization protocols were tested, combining different concentrations and exposure times of 0.05% Tween-20 (v/v) (Helicon, Moscow, Russia), 1% sodium hypochlorite (Erkan Chemical, Almaty, Kazakhstan), and their combinations with a pre-treatment in 70% ethanol. After sterilization, explants were rinsed three times with sterile distilled water. The efficiency of each protocol was assessed by recording the percentage of aseptic, surviving, and contaminated explants after 14 days. Each treatment was applied to 150 explants (three biological replicates of 50 explants each). After sterilization, explants were transferred to initiation medium, and observations were made after 14–21 days. The percentage of viable sterile explants (i.e., free from contamination and surviving) was calculated for each treatment. For statistical evaluation, a chi-square test of independence (χ2) was used to compare proportions of sterile and viable explants across treatments.
Initially, one-year-old shoots were excised, leaves were removed, and segments containing a single bud were carefully cut (Figure 1A,B). At the first stage, explants in all variants were immersed in a soap solution and agitated on a laboratory shaker for 30 min (Figure 1C). This was followed by rinsing under running water for a similar duration.

2.2. Optimization of the Composition of the Nutrient Medium for Blackberry Culture Initiation

Culture Media and Growth Regulators

For culture initiation and multiplication, Murashige and Skoog (MS) basal medium (Phyto Tech Labs, Lenexa, KA, USA) was supplemented with various concentrations and combinations of 6-benzylaminopurine (BAP) (Phyto Tech Labs, Lenexa, KA, USA), kinetin (Kin) (Phyto Tech Labs, Lenexa, KA, USA), and indole-3-butyric acid (IBA) (Phyto Tech Labs, Lenexa, KA, USA). Sucrose (Titan biotech Ltd., Rajasthan, India) (30 g L−1) served as the carbon source, and agar (Phyto Tech Labs, Lenexa, KA, USA) (7 g L−1) was used as the gelling agent. The pH of the medium was adjusted to 5.8 before autoclaving at 121 °C for 20 min.
Three different hormonal compositions were tested:
-
Medium 1: MS + 0.1 mg/L BAP;
-
Medium 2: MS + 0.5 mg/L BAP + 0.1 mg/L α-naphthaleneacetic acid (NAA) (Phyto Tech Labs, Lenexa, KA, USA);
-
Medium 3: MS + 1 mg/L BAP + 0.5 mg/L Kin.
Explants were cultured under controlled conditions (25 ± °C, 16 h photoperiod, light intensity of 3000–3500 lx). The effectiveness of each medium was assessed based on explant survival rate and initial shoot formation after 3–4 weeks of cultivation.
To assess differences in survival rates between media treatments, a chi-square test (χ2) was applied. Each treatment consisted of 50 explants per cultivar and was conducted in three biological replicates (n = 150 per cultivar × medium combination). Pairwise comparisons were performed between media Treatments (Treatment 1 vs. Treatment 2 and Treatment 1 vs. Treatment 3). Differences were considered statistically significant at p < 0.05. The analysis was based on the absolute number of surviving and non-surviving explants in each replicate.
Survival-rate data are presented as mean ± standard error (SE), calculated from three biological replicates (n = 150), whereas all morphometric traits (shoot height, number of shoots, number of roots, root length, etc.) are presented as mean ± standard deviation (SD) from three independent replicates.
Standard errors (SE) were calculated using the binomial distribution formula, assuming each explant to be an independent observation:
SE = √[p(1 − p)/n]
where p is the proportion of surviving explants and n = 150.
To evaluate the effect of hormonal composition on blackberry microshoot growth during the multiplication stage, experiments were conducted using different concentrations of BAP and GA3. Each treatment included 30 explants (microshoots), divided into three biological replicates of 10 explants each, and cultured in test tubes containing nutrient medium. Shoot height and number of shoots were recorded after 30 days of cultivation. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey’s Honest Significant Difference (HSD) test for post hoc multiple comparisons. Differences were considered statistically significant at p < 0.05.

2.3. Rooting and Ex Vitro Acclimatization of Regenerated Plants in Experimental Agroboxes

For rooting and adaptation to natural light and temperature conditions, regenerated in vitro plantlets of the studied blackberry cultivars (‘Natchez’, ‘Black Magic’, ‘Osage’, and ‘Heaven Can Wait’) were transferred to experimental agroboxes.
Agroboxes (10 × 24 × 15 cm) (LLP ‘Hitex Fabrik’, Kazakhstan) are modular systems consisting of removable cassettes filled with a peat–perlite substrate (3:1) and placed inside transparent, sealed plastic containers. The cassette cells measure 2 × 2 cm, though this size may vary depending on the cultivated plant species. Each plastic insert includes drainage holes and side handles to facilitate easy removal of the plants. The lower part of the container is narrower than the middle and upper sections, which helps to keep the root system above the liquid level. The lid is made of transparent plastic and includes ventilation holes (0.3–0.5 cm in diameter), which are drilled as needed during the plant acclimatization phase. The agroboxes were specifically designed for this study using ordinary food-grade plastic containers as the base (Figure 2).
The rooting substrate was sterilized by autoclaving at 121 °C for 30 min. To ensure uniform experimental conditions and to eliminate the influence of uncontrolled microbial communities that may vary between substrate batches, the rooting substrate was sterilized prior to use. This approach was chosen to standardize the physical and chemical properties of the substrate and to minimize variability in early root development attributable to microbial contamination. Prior to planting, to ensure uniform baseline rooting support, all microcuttings were treated with a commercially available rooting powder (‘Kornevin’). This step was applied identically across all variants and was not considered an experimental factor, serving only to standardize initial rooting conditions at the moment of ex vitro transfer. Three different nutrient and rooting solutions were used to moisten the substrate and stimulate root formation: variant 1—0.01% humic acid, variant 2—0.01% nanosilicon (NanoSilicon, Moscow, Russian), variant 3—0.1% succinic acid (Bioabsolut, Golitsyno, Russian). The concentrations of biostimulants (0.01% humic acid, 0.01% nanosilicon, and 0.1% succinic acid) were selected based on published studies reporting positive physiological responses at comparable low-dose levels (≈50–150 ppm for humic substances and nanosilicon; ≈500–1000 ppm for succinic acid) in in vitro and seedling experiments [58,59,60,61,62]. These doses were used here as non-phytotoxic baseline values to evaluate their potential applicability. A full dose–gradient experiment was not conducted within this study and is identified as an important direction for future optimization.
In each treatment, 35 microcuttings were planted, with experiments conducted in triplicate. The agroboxes were placed in a controlled-environment chamber under the following conditions: photoperiod: 18 h of light; light intensity: 3000–5000 lux; relative humidity: 70%; temperature: +25 °C (day)/+22 °C (night). Root development was monitored weekly by inspecting the substrate for visible root emergence. Upon the appearance of the first roots, four small ventilation holes were made in the lids of the agroboxes to gradually expose the plantlets to ambient air. To promote stomatal adaptation, four additional holes were introduced every 2–3 days until full lid removal. Plants were watered with the respective stimulant solutions as the substrate began to dry. After eight weeks of acclimatization, the rooted plantlets were transferred to pots containing a soil mix composed of 70% peat and 30% perlite for further growth under greenhouse conditions.

2.4. Virus Detection by PCR

Leaf and young shoot samples of blackberry were used for total RNA extraction employing the RNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. RNA concentration and purity were determined spectrophotometrically using a Nano-500B spectrophotometer (Hangzhou Allsheng Instruments Co., Ltd., Hangzhou, China). Reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Vilnius, Lithuania) with oligo(dT) primers. Amplification of viral genome fragments was carried out by PCR using published primers: RBDV—according to Martin et al. [63]; RLBV—according to McGavin et al. [57]; RpRSV—according to Tang et al. [64].
Amplification was performed on a Gentier 96E PCR instrument (Tianlong Science & Technology, Xi’an, China) under the following cycling conditions: initial denaturation at 94 °C for 5 min, followed by 40 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s, with a final extension at 72 °C for 10 min. Fluorescence data were collected at the end of each cycle, and data analysis was conducted using the Real-time PCR System Version 1 software (Tianlong Science & Technology, Xi’an, China).

2.5. Experimental Design and Statistical Analysis

All experiments were arranged in a Completely Randomized Design (CRD) with three replicates per treatment. Data were analyzed using one-way ANOVA, and treatment means were compared using Tukey’s HSD test at a significance level of p < 0.05. Statistical analyses were performed using Microsoft Excel 2021 (Microsoft Corporation, Redmond, WA, USA). Survival-rate data are shown as mean ± SE, whereas morphometric traits are presented as mean ± SD.

3. Results

3.1. Optimization of the Method of Sterilization of Plant Explants

Three sterilization treatments were compared in this experiment (Treatment 1, Treatment 2, Treatment 3; Table 1). To ensure clarity and consistency, all results in this section are reported using these treatment numbers. Among the tested sterilization treatments, Treatment 2 (70% ethanol for 2 min + 1% NaOCl for 15 min) demonstrated the highest effectiveness, providing the lowest contamination rate and the highest proportion of healthy explants across all cultivars. Treatments using lower concentrations of sodium hypochlorite or shorter exposure times produced moderate contamination levels, whereas the least effective schemes were those lacking the ethanol pre-treatment, which consistently showed the highest contamination percentages. These results clearly demonstrate that contamination rate serves as a reliable indicator of explant health, and the optimized protocol greatly improved the establishment of clean in vitro cultures. The detailed data for all four cultivars are presented in Table 1.
The results of the experiment demonstrated significant differences in the effectiveness of the tested sterilization protocols. Treatment 1, (70% ethanol 1 min + 1% NaOCl 10 min) resulted in a moderate number of sterile explants (35%). Treatment 2 yielded the highest proportion of sterile explants among the tested protocols, with 85.0% sterile explants. This variant ensured both effective surface disinfection and minimal tissue damage, leading to high survival and sterility rates.
In contrast, Treatment 3, (70% ethanol 3 min + 1% NaOCl 20 min), resulted in the lowest survival rate—only 21.3% of the explants remained viable. This suggests that prolonged and intensive exposure to sterilizing agents caused damage to the plant tissues and inhibited subsequent in vitro development.
It is important to note that the efficiency of sterilization treatments differed slightly among cultivars. Although Treatment 2 consistently produced the highest proportion of sterile explants in all four cultivars, the absolute values varied: ‘Natchez’ and ‘Black Magic’ showed the highest survival after disinfection, whereas ‘Osage’ and ‘Heaven Can Wait’ exhibited a slightly higher sensitivity to prolonged exposure to ethanol and sodium hypochlorite. These cultivar-specific differences highlight the importance of adjusting sterilization parameters according to varietal responses.

3.2. Optimization of Nutrient Medium Composition for Blackberry Introduction

It was found that the earliest axillary bud regeneration occurred on days 9–10 across all varieties when cultured on MS nutrient medium supplemented with 0.1 mg/L BAP. In contrast, in the second variant, which contained 0.5 mg/L BAP and 0.1 mg/L NAA, initial bud regeneration was observed only on days 16–18. In the third variant of the nutrient medium, which included both BAP and kinetin, callus formation occurred in the majority of blackberry explants. The data presented in Table 1 demonstrate that the type and concentration of growth regulators have a significant effect on the survival rate of blackberry explants. Survival rates of blackberry explants varied significantly depending on the culture medium used. All cultivars demonstrated high survival on Medium 1 (84–90%), moderate survival on Medium 2 (40–62%), and low survival on Medium 3 (12–24%).
Pairwise comparisons using the chi-square test confirmed that the differences between Medium 1 and Medium 3 were statistically significant for all cultivars (p < 0.0001). Significant differences were also observed between Medium 1 and Medium 2 (p < 0.005), particularly for ‘Black Magic’ and ‘Heaven Can Wait’.
In Medium 2, the cultivar ‘Natchez’ had a significantly higher survival rate compared to ‘Heaven Can Wait’ (p = 0.01), while differences among other cultivars within the same medium were either statistically non-significant or borderline significant.
The marked decrease in explant survival when transitioning from Medium 1 to T Medium 2 and 3 highlights the critical importance of nutrient medium composition for successful in vitro culture of blackberry. The consistent reduction in survival across all tested cultivars suggests a general sensitivity to media composition changes and confirms the robustness of the observed effect. Statistical analysis demonstrated that Medium 1 resulted in the highest survival rates observed in this experiment. To clarify the scope of the study, no callus formation or spontaneous organogenesis was observed under the selected media compositions and cultivation conditions. Browning symptoms were also absent in all treatments. Contamination rates were evaluated only during the initial sterilization trials and were not quantified for the multiplication or acclimatization stages.

3.3. Optimization of Nutrient Medium Composition for Shoot Multiplication

The analysis showed that a nutrient medium supplemented with 0.5 mg/L BAP and 0.1 mg/L GA3 was the most favourable medium composition under our experimental conditions for promoting shoot elongation and increasing the multiplication rate across all studied blackberry varieties (Figure 3, Table 2).
Each treatment included 30 explants (microshoots), divided into three replicates, and cultured in test tubes containing nutrient medium.
The four nutrient-medium variants tested at the multiplication stage differed in the concentration of BAP and GA3, resulting in distinct morphogenic responses across all cultivars (Table 2). The highest shoot height was consistently obtained on Variant 1 (0.5 mg/L BAP + 0.1 mg/L GA3), where explants of all four cultivars (‘Natchez’, ‘Black Magic’, ‘Osage’, and ‘Heaven Can Wait’) produced elongated shoots ranging from 5.27 to 5.43 cm, with no statistically significant differences among cultivars (p < 0.05). Variant 2 (0.5 mg/L BAP + 0.3 mg/L GA3) resulted in moderate shoot height, with values between 3.36 and 4.07 cm, showing a noticeable decline compared to Variant 1. Variant 3 (0.5 mg/L BAP + 0.5 mg/L GA3) produced shoots of similar height to Variant 2 (3.62–3.87 cm), indicating that higher GA3 concentrations did not promote further elongation. Variant 4 (1.0 mg/L BAP + 0.5 mg/L GA3) showed the lowest shoot height, declining to 3.45–3.92 cm, particularly in the cultivar ‘Black Magic’, which demonstrated the shortest shoots under this condition.
When considering the number of shoots per explant, the trends were similar:
Variant 1 produced the highest shoot numbers (5.2–5.8 shoots per explant), representing the most favorable morphogenic response across cultivars. Variants 2 and 3 showed intermediate shoot numbers (3.1–4.9), with statistically significant reductions relative to Variant 1. Variant 4 resulted in the lowest shoot numbers (2.1–2.9), demonstrating that increasing BAP concentration to 1.0 mg/L suppressed both shoot multiplication and elongation.
Overall, Variant 1 (0.5 mg/L BAP + 0.1 mg/L GA3) provided the most effective balance of shoot height and shoot proliferation, and thus represents the most efficient medium formulation for the multiplication stage of the four studied cultivars.
In fact, plants grown under the highest hormone concentrations exhibited noticeably reduced vigor, characterized by shorter and thinner shoots, compact internodes, and poorly developed leaves. These morphological changes rendered the shoots unsuitable for further subculturing and propagation (Figure 4B). In contrast, plants cultured with the lowest hormone treatment (0.5 mg/L BAP and 0.1 mg/L GA3) showed vigorous growth and normal morphology, indicating optimal conditions for further multiplication (Figure 4A).

3.4. Virus Diagnostics of Donor and Micropropagated Plants

To confirm the phytosanitary status of the source material used for in vitro culture establishment, all donor and micropropagated blackberry plants were screened for the presence of RBDV, RLBV and RpRSV (Figure 5). PCR analysis was performed using specific primers (RBDV, RLBV, RpRSV). No viruses were detected in either the source plants or the micropropagated plants. No amplification of specific genome fragments of RBDV, RLBV, or RpRSV was observed in any of the tested blackberry samples. Control reactions with positive samples (RBDV-infected raspberry) produced the expected fragments, confirming the correctness of the amplification conditions.
Thus, the molecular screening confirmed the absence of the specified viruses in the studied blackberry cultivars, indicating their virological cleanliness within the sensitivity limits of the applied method. Although the primary objective of this study was to optimize the stages of in vitro micropropagation, the production of pathogen-free planting material represents an integral quality criterion of any propagation technology. Therefore, molecular virus screening was performed to confirm the sanitary status of the donor plants used in culture initiation. This step ensures that all subsequent results—explant establishment, multiplication, and acclimatization—are based exclusively on virus-free source material and that the developed protocol is suitable for generating healthy, contamination-free plants for practical application.

3.5. Adaptation of Regenerated Blackberry Plants Under In Vitro—Ex Vitro Conditions in Experimental Agroboxes

The overall survival rate of regenerated blackberry plants transferred to the soil substrate was 86.4% across all studied varieties. Rooting efficiency of the Rubus blackberry varieties (‘Natchez’, ‘Osage’, ‘Black Magic’, and ‘Heaven Can Wait’) under ex vitro conditions was assessed eight weeks after planting in agroboxes. Each agrobox contained 35 microcuttings, with three replicates per treatment, ensuring consistent experimental conditions while allowing future work to refine the dose–response relationships. The assessment was based on the following morphometric parameters: shoot length (cm), number of internodes, number of roots, and average root length (cm) (Figure 6).
Analysis of the data (Table 3) showed that plant development was more intensive when substrates were supplemented with different biostimulant agents (1—0.01% humic acid; 2—0.01% nanosilicon; 3—0.1% succinic acid) compared to the control (soil with water only).
Shoot length (cm): Across all cultivars, the addition of biostimulants increased shoot elongation compared with the control. Nanosilicon produced the most pronounced effect, resulting in the longest shoots in ‘Natchez’, ‘Osage’, ‘Black Magic’, and ‘Heaven Can Wait’. Humic acid had a moderate positive influence, whereas succinic acid produced only a slight improvement.
Number of internodes: The pattern matched shoot length: nanosilicon consistently produced the highest number of internodes across all cultivars. Humic acid resulted in intermediate values, and succinic acid demonstrated the weakest effect.
Number of roots: Nanosilicon again demonstrated the strongest stimulatory effect, significantly increasing root number in all four cultivars compared with control and other treatments. Humic acid produced high root numbers particularly in ‘Natchez’ and ‘Heaven Can Wait’. Succinic acid resulted in lower values, indicating suboptimal performance at the tested concentration.
Average root length (cm): Humic acid led to the longest individual roots in several cultivars, demonstrating its effectiveness for root elongation. Nanosilicon produced moderately long roots, while succinic acid again showed the weakest response.
Survival rate (%): Survival was highest in the nanosilicon treatment: ‘Natchez’—86.6% (91/105 plants), ‘Osage’—87.6% (92 plants), ‘Heaven Can Wait’—95.2% (100 plants), ‘Black Magic’—90.4% (95 plants).
In the substrate supplemented with a 0.01% nanosilicon solution, the survival rate was 86.6% for the cultivar ‘Natchez’ (91 out of 105 plants), 87.6% for ‘Osage (92 plants), 95.2% for ‘Heaven Can Wait’ (100 plants), and 90.4% for ‘Black Magic’ (95 plants). In the control variant, the survival rate was 50.0% for ‘Natchez’ (52 out of 105 plants), 52.0% for ‘Osage’ (54 out of 105 plants), 59.0% for ‘Heaven Can Wait’ (62 out of 105 plants), and 54.2% for ‘Black Magic’ (57 out of 105 plants).
The biostimulants, including humic acid, nanosilicon, and succinic acid, showed varying effects on the morphometric parameters of blackberry microshoots. Notably, nanosilicon significantly enhanced within the scope of this experiment the number of roots and internodes in all tested varieties, while humic acid resulted in the highest average root length and root number for several cultivars. Succinic acid, in contrast, had a generally less favorable effect on the development of blackberry plants. Although humic acid, nanosilicon, and succinic acid produced distinguishable effects on shoot and root development, it is important to note that the concentrations used in this study represent baseline, literature-supported levels rather than fully optimized doses. Therefore, the differences observed among treatments may, in part, reflect suboptimal concentrations, particularly in the case of succinic acid.

4. Discussion

In vitro propagation has become the primary approach for blackberry multiplication [65,66]. Traditionally, blackberries were propagated vegetatively, mainly through layering and cuttings [38,67], but these techniques are constrained by the need for large plantation areas, significant labor, and intensive weed control [36]. In contrast, in vitro propagation allows for large-scale production of genetically uniform, disease-free plants, which is essential for commercial cultivation and germplasm preservation [36,37,68].
Micropropagation protocols have been developed for numerous blackberry cultivars; however, the wide genetic diversity within the genus Rubus, which includes more than 740 described species worldwide [67], results in varied responses to tissue culture [65].
Consequently, fine-tuning the culture medium, nutrient composition, and combinations of plant growth regulators (PGRs) remains critical for optimizing results [36]. Auxins and cytokinins, in particular, have a decisive impact on shoot proliferation, root formation, and overall plant development [37].
The earliest micropropagation attempts were carried out by Vujović [69] and Rani [70]. Their initial work with the ‘Bedford Giant’ blackberry and the ‘Tayberry’ hybrid faced limitations due to a low multiplication index, which reduced the effectiveness of the method. Despite these early difficulties, protocols for blackberries and hybrid berries have been significantly improved. Initially, research focused on rapid plant multiplication, pathogen elimination, and enhancement of vegetatively propagated varieties [54]. Today, micropropagation is the method of choice for large-scale, rapid production, playing a key role in commercial blackberry cultivation [66].
Sterilization of plant material is a critical step in establishing successful in vitro cultures, as contamination can significantly reduce the efficiency of micropropagation protocols. Numerous studies have explored different approaches to surface sterilization. For example, some researchers recommend pre-treatment under running tap water for 15–30 min, followed by washing on a magnetic stirrer with a detergent solution, and final disinfection with 0.1% sodium hypochlorite for 15 min [51,71]. Others suggest using 70% ethanol for 2 min as an effective surface sterilizing agent [52]. These approaches are in line with our results. However, these methods differ in their efficacy depending on the Rubus plant species and the sensitivity of explant tissues.
Our study confirmed that the efficiency of blackberry micropropagation depends on a careful balance between sterilization conditions, hormonal composition of the nutrient medium, and ex vitro rooting treatments. The tested sterilization protocols clearly demonstrated the trade-off between microbial elimination and tissue viability. Excessive exposure to disinfectants reduced explant survival, while insufficient treatment failed to suppress contamination. The optimal balance was achieved using 70% ethanol (2 min) followed by 1% sodium hypochlorite (15 min), which ensured 85% sterile, viable explants. This agrees with previous findings indicating that moderate ethanol exposure combined with hypochlorite is optimal for Rubus tissues, balancing sterility with tissue viability [51,52,71].
This protocol provided optimal disinfection while maintaining tissue viability, indicating a favorable balance between antimicrobial action and plant tissue tolerance. In contrast, Treatment 1 (70% ethanol for 1 min and 1% sodium hypochlorite for 10 min) resulted in only 35.0% sterile explants, suggesting that the shorter exposure time was insufficient for complete microbial elimination. In contrast, prolonged exposure (Treatment 3) sharply reduced explant survival, consistent with reports that excessive contact with hypochlorite can cause oxidative damage and impair organogenesis in Rubus spp. [70]. Thus, the results of our study align with previously reported data, confirming that sterilization protocols must be carefully optimized for each species and explant type. Protocols that strike a balance between sterility and tissue preservation are essential for the effective establishment of in vitro cultures. This result highlights the necessity of tailoring disinfection procedures to the sensitivity of blackberry tissues, a finding consistent with previous reports but specific to the cultivars studied.
Another critical factor for successful establishment of in vitro cultures is the optimization of the nutrient medium composition during the initiation stage. The hormonal composition of the culture medium had a decisive impact on shoot initiation and multiplication. Low BAP concentration (0.1 mg/L) was optimal for culture initiation, supporting early axillary bud regeneration and survival rates above 84–90% across all cultivars. This result is fully consistent with observations reported for other Rubus species, where low cytokinin levels promote bud activation without inducing callus formation [36,47,65]. In contrast, the combination of BAP and kinetin promoted excessive callus formation, a response that has also been previously observed when kinetin disrupts shoot organogenesis in Rubus spp. [46].
The results are consistent with previously reported studies on various berry crops and blackberry cultivars, which emphasize the importance of cytokinin type and concentration in achieving high regeneration and survival rates during the culture initiation phase [47]. The successful response observed in our study may be attributed to the specific role of BAP in stimulating cell division and shoot initiation without excessive callus formation, which can hinder further morphogenesis. This reinforces the importance of tailoring hormonal balances in the nutrient medium to the specific physiological requirements of each cultivar.
At the multiplication stage, medium containing 0.5 mg/L BAP and 0.1 mg/L GA3 produced the highest number of shoots (5.3–5.8 per explant) with adequate shoot elongation (5.27–5.43 cm). Similar cytokinin–gibberellin combinations have been reported to enhance shoot elongation and prevent vitrification in blackberry and raspberry microshoots [36,66]. Importantly, morphological observations confirmed that shoots were robust, with well-developed leaves and uniform coloration, indicating high physiological quality. Excessive GA3 concentrations caused weaker shoots, a pattern also noted by Dewir et al. and Hunková et al., who showed that high cytokinin or GA3 doses negatively affect leaf development and internode elongation [40,52]. These findings suggest that the hormonal response of blackberry cultivars must be evaluated not only in terms of multiplication index but also with respect to plant morphology and vigor.
The acclimatization results demonstrated pronounced cultivar-specific responses to the tested biostimulants. Nanosilicon improved shoot vigor and root initiation, whereas humic acid enhanced root elongation, collectively contributing to higher survival during ex vitro transfer. These findings emphasize that acclimatization is strongly influenced by both pre-acclimation physiological status and substrate composition, supporting similar observations reported for Rubus species in ex vitro adaptation studies.
Improved acclimatization outcomes observed in this study were also influenced by the optimized sterilization protocol and the hormonal composition used during earlier stages of micropropagation. Effective surface disinfection reduces initial microbial load, preventing latent contamination that often impairs root development during transfer to ex vitro conditions. Likewise, the balanced cytokinin–gibberellin regime applied during multiplication produced physiologically stronger shoots with well-developed leaves and internodes, which are known to exhibit higher survival under acclimatization stress.
Virus diagnostics revealed no presence of RBDV, RLBV, or RpRSV in the four blackberry cultivars examined in this study (‘Natchez’, ‘Black Magic’, ‘Osage’, and ‘Heaven Can Wait’). This aligns with earlier reports indicating lower virus occurrence in blackberry compared to raspberry [57,58], and supports the known effectiveness of micropropagation in maintaining virus-free status in Rubus crops [66].
From a practical standpoint, the virological cleanliness of the material confirms the effectiveness of biotechnological methods (in vitro microclonal propagation and ex vitro adaptation) for obtaining healthy planting material specifically for the cultivars included in this work.
From a scientific perspective, the findings highlight the need for continued monitoring of Rubus spp. viruses, including BLMaV, BCRV and BYVaV, with the aim of creating a national database on viral infections in Kazakhstan’s berry crops. These data provide a foundation for developing a certification system for virus-free blackberry planting material. However, the present results should not be generalized beyond the four cultivars studied here, as broader surveillance and testing across additional genotypes will be required to draw population-level or species-wide conclusions.
The results obtained demonstrate that substrate composition and the type of growth stimulant used have a significant impact on the efficiency of ex vitro rooting and the morphometric characteristics of regenerated blackberry plants.
The rooting and acclimatization stage demonstrated that biostimulants strongly influence morphometric parameters. Nanosilicon significantly promoted shoot development and root number. These effects are consistent with recent findings showing that nanosilicon enhances antioxidant defense systems, stabilizes cellular structures, and supports morphogenesis under ex vitro stress [58,59]. The use of nanosilicon at a concentration of 0.01% provided comparatively stronger stimulation for promoting shoot elongation and increasing the number of internodes in all studied varieties. Among the treatments, the substrate with nanosilicon solution demonstrated the best performance in terms of shoot length and number of internodes for the ‘Natchez’ and ‘Osage’ varieties (8.47 cm and 8.10 cm; 7.70 and 7.57 internodes, respectively). For the ‘Heaven Can Wait’ and ‘Black Magic’ varieties, shoot lengths were 7.10 cm and 7.15 cm, with 7.30 and 7.21 internodes, respectively. Additionally, the nanosilicon treatment resulted in high numbers of roots (≥13.70 per plant) across all varieties. However, root lengths on this substrate were relatively modest, ranging from 3.80 to 4.07 cm. These findings are consistent with published evidence showing that nanosilicon improves acclimatization performance by enhancing antioxidant defense systems, mitigating oxidative stress, and contributing to the stabilization of cellular structures, thereby supporting overall plant recovery and establishment under ex vitro conditions [60,61,62].
Although the nanosilicon treatment produced shorter roots, the total number of roots per plant was notably high, suggesting improved root initiation capacity. Conversely, humic acid treatment was more effective for stimulating root elongation, highlighting the complementary benefits of different biostimulants at the rooting stage. For the ‘Natchez’, ‘Osage’, ‘Heaven Can Wait’, and ‘Black Magic’ varieties, root lengths reached 6.23 cm, 6.50 cm, 6.43 cm, and 6.35 cm, respectively. This matches experimental evidence that humic substances activate H+-ATPase, modify root architecture, and exhibit auxin-like activity [53].
The application of succinic acid at 0.1% concentration did not yield favorable results in terms of either shoot or root development. Shoot lengths ranged from 3.61 to 4.60 cm, and the number of internodes varied from 3.30 to 3.66 in the ‘Natchez’ and ‘Black Magic’ varieties, and from 3.41 to 3.45 in the ‘Osage’ and ‘Heaven Can Wait’ varieties. Similar inhibitory effects at higher concentrations were reported in cereals and other species when succinate disturbed metabolic regulation [58,59]. Based on the overall set of morphometric parameters, the most efficient rooting and acclimatization were observed on a peat–perlite (3:1) substrate supplemented with 0.01% nanosilicon, making it the most suitable formulation for the ex vitro adaptation of regenerated blackberry plants. Although the biostimulant concentrations used here were guided by previously published low-dose ranges, the absence of a full dose–response gradient represents a methodological limitation. Future studies should incorporate multi-level concentration testing to determine cultivar-specific optima and to validate whether the observed effects are proportional to dose [58,59,60,61,62].
It is important to note that the present study used sterilized substrate to ensure full experimental control; however, several authors have reported that non-sterile substrates may enhance rooting or acclimatization by preserving beneficial microbial communities. Considering this, future experiments should include a comparative evaluation of sterile versus non-sterile substrates to assess potential microbiome-mediated effects on blackberry rooting and early ex vitro development.
Overall, our findings emphasize that the optimization of blackberry micropropagation protocols requires a multidimensional evaluation that integrates quantitative indices (survival rate, multiplication index, rooting percentage) with qualitative morphological traits (shoot robustness, leaf development, root system architecture). This integrative approach ensures that regenerated plants are not only numerous but also physiologically vigorous, which is essential for successful ex vitro adaptation and future field performance.

5. Conclusions

This study optimized the key stages of Rubus L. micropropagation under the conditions of Kazakhstan, including explant sterilization, culture initiation, shoot multiplication, and acclimatization using agroboxes with adjustable microventilation. The results confirmed that the developed protocol ensures the production of healthy, physiologically stable, and well-adapted planting material of the cultivars ‘Natchez’, ‘Black Magic’, ‘Osage’, and ‘Heaven Can Wait’. The practical significance of this work lies in establishing a foundation for large-scale production of virus-free blackberry plants, reducing reliance on imported planting material, minimizing the risk of pathogen introduction, and supporting the expansion of domestic berry cultivation. Future research should focus on a more detailed physiological assessment during acclimatization and on testing a broader set of cultivars to further refine and validate the technology.
For the blackberry cultivars examined in this study (‘Natchez’, ‘Black Magic’, ‘Osage’, and ‘Heaven Can Wait’), the following practical recommendations can be made. Effective surface sterilization is achieved using 70% ethanol followed by 1% sodium hypochlorite. Reliable culture initiation occurs on MS medium supplemented with 0.1 mg/L BAP. During the multiplication stage, optimal morphogenic responses were obtained on medium containing 0.5 mg/L BAP and 0.1 mg/L GA3. During ex vitro rooting and acclimatization, the use of humic acid, nanosilicon, or succinic acid enhanced survival and early root development under agrobox conditions. These recommendations apply specifically to the cultivars included in this study and should not be generalized beyond the tested genotypes.

Author Contributions

Conceptualization, N.M. and Y.Y.; methodology, N.M., B.T. and Y.Y.; software, B.T.; validation, N.M., Y.Y. and B.T.; formal analysis, N.M., B.T., V.K. and Y.Y.; investigation, N.M., B.T. and Y.Y.; resources, B.T.; data curation, N.M. and B.T.; writing—original draft preparation, Y.Y. and V.K.; writing—review and editing, Y.Y. and V.K.; visualization, N.M. and B.T.; supervision, N.M.; project administration, N.M.; funding acquisition, N.M. All authors have read and agreed to the published version of the manuscript.

Funding

The research was carried out with financial support from the Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan within the framework of the scientific and technical program BR21881942 “Development of biotechnological approaches to control phytopathogens with the aim of increasing the productivity of agricultural crops.”

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

All authors are very thankful to Alexandra Khanseitova for technical support.

Conflicts of Interest

The authors declare that they have no conflicts of interest to disclose.

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Figure 1. Preparation and sterilization of blackberry explants: (A) cut blackberry shoots; (B) preparation of explants for sterilization; (C) sterilization stage.
Figure 1. Preparation and sterilization of blackberry explants: (A) cut blackberry shoots; (B) preparation of explants for sterilization; (C) sterilization stage.
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Figure 2. Agrobox: (A)—Components of the agrobox: container, cell tray, and individual cells; (B)—Agrobox in assembled form.
Figure 2. Agrobox: (A)—Components of the agrobox: container, cell tray, and individual cells; (B)—Agrobox in assembled form.
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Figure 3. In vitro shoots of blackberry (Rubus L.) generated under the optimized multiplication conditions. The image represents a typical example shown for cultivar ‘Natchez’; other cultivars (‘Black Magic’, ‘Osage’, and ‘Heaven Can Wait’) exhibited comparable morphology under identical conditions. (A) microplant ready for cutting; (B) cultivation of microcuttings on the 3rd day after planting; (C) microplants after 25–30 days of growth.
Figure 3. In vitro shoots of blackberry (Rubus L.) generated under the optimized multiplication conditions. The image represents a typical example shown for cultivar ‘Natchez’; other cultivars (‘Black Magic’, ‘Osage’, and ‘Heaven Can Wait’) exhibited comparable morphology under identical conditions. (A) microplant ready for cutting; (B) cultivation of microcuttings on the 3rd day after planting; (C) microplants after 25–30 days of growth.
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Figure 4. Morphology of Rubus ‘Natchez’ microshoots after 30 days of cultivation under different hormonal treatments: (A) Plants cultured on medium with BAP 0.5 mg/L + GA3 0.1 mg/L, showing vigorous growth, well-formed shoots, and healthy leaf morphology. (B) Plants grown on medium with BAP 1.0 mg/L + GA3 0.5 mg/L, exhibiting reduced vigor, shortened internodes, and signs of morphological abnormalities.
Figure 4. Morphology of Rubus ‘Natchez’ microshoots after 30 days of cultivation under different hormonal treatments: (A) Plants cultured on medium with BAP 0.5 mg/L + GA3 0.1 mg/L, showing vigorous growth, well-formed shoots, and healthy leaf morphology. (B) Plants grown on medium with BAP 1.0 mg/L + GA3 0.5 mg/L, exhibiting reduced vigor, shortened internodes, and signs of morphological abnormalities.
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Figure 5. Real-time PCR amplification curves for detection of raspberry viruses in blackberry (Rubus L.) donor plants: RBDV, RLBV and RpRSV. Curves: Red curves—positive controls (virus-infected raspberry), showing characteristic exponential amplification. Blue and green curves—blackberry in vitro samples (Samples 1–4). Yellow line—baseline; orange line—threshold level. All blackberry samples demonstrated no detectable amplification within the quantification cycle range, whereas the positive control produced a clear exponential curve, confirming the specificity and correct performance of the assay.
Figure 5. Real-time PCR amplification curves for detection of raspberry viruses in blackberry (Rubus L.) donor plants: RBDV, RLBV and RpRSV. Curves: Red curves—positive controls (virus-infected raspberry), showing characteristic exponential amplification. Blue and green curves—blackberry in vitro samples (Samples 1–4). Yellow line—baseline; orange line—threshold level. All blackberry samples demonstrated no detectable amplification within the quantification cycle range, whereas the positive control produced a clear exponential curve, confirming the specificity and correct performance of the assay.
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Figure 6. Effect of various biostimulants on the morphometric parameters of regenerated ‘Heaven Can Wait ‘blackberry plants in agroboxes: (A) soil with water (control); (B) soil with 0.01% humic acid; (C) soil with 0.01% nanosilicon; (D) soil with 0.1% succinic acid.
Figure 6. Effect of various biostimulants on the morphometric parameters of regenerated ‘Heaven Can Wait ‘blackberry plants in agroboxes: (A) soil with water (control); (B) soil with 0.01% humic acid; (C) soil with 0.01% nanosilicon; (D) soil with 0.1% succinic acid.
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Table 1. Survival rate of blackberry explants 14–21 days after placement on various nutrient media.
Table 1. Survival rate of blackberry explants 14–21 days after placement on various nutrient media.
CultivarMedium 1, Pcs
Total/Survived (Percentage) 		Medium 2, Pcs
Total/Survived (Percentage)		Medium 3, Pcs
Total/Survived (Percentage)
‘Natchez’50/45 (90% ± 2.5%) a50/31 (62% ± 4.0%) b50/11 (22% ± 3.3%) c
‘Black Magic’50/45 (90% ± 2.5%) a50/23 (46% ± 4.1%) c50/12 (24% ± 3.4%) c
‘Osage’50/43 (86% ± 2.9%) a50/27 (54% ± 4.1%) b,c50/6 (12% ± 2.5%) d
‘Heaven Can Wait’50/42 (84% ± 3.0%) a50/20 (40% ± 4.0%) c50/8 (16% ± 2.9%) c,d
Note. Differences between values marked with different letters (a, b, c, d) are statistically significant. Each value is based on three biological replicates of 50 explants (n = 150). Values are presented as mean ± standard error (SE), calculated based on three replicates of 50 explants each (n = 150) using the binomial distribution formula.
Table 2. Influence of hormonal composition of the nutrient medium on the development of blackberry microshoots at the multiplication stage (after 30 days of cultivation).
Table 2. Influence of hormonal composition of the nutrient medium on the development of blackberry microshoots at the multiplication stage (after 30 days of cultivation).
Content and Concentration of Hormones, mg/LCultivarShoot Height, cmNumber of Shoots, (Per Explant).
BAP 0.5‘Natchez’5.43 ± 0.26 a5.8 ± 0.6 a
‘Black Magic’5.30 ± 0.06 a5.3 ± 0.2 a
GA3 0.1‘Osage’5.27 ± 0.30 a5.7 ± 0.5 a
Heaven Can Wait’5.32 ± 0.08 a5.5 ± 0.2 a
BAP 0.5‘Natchez’5.36 ± 0.58 a5.2 ± 0.4 a
‘Black Magic’4.07 ± 0.12 b4.8 ± 0.2 ab
GA3 0.3‘Osage’3.87 ± 0.03 b4.9 ± 0.2 ab
‘Heaven Can Wait’3.36 ± 0.09 c4.3 ± 0.4 c
BAP 0.5‘Natchez’3.80 ± 0.53 b3.9 ± 0.5 bc
‘Black Magic’3.47 ± 0.15 c3.4 ± 0.2 c
GA3 0.5‘Osage’3.87 ± 0.03 b3.1 ± 0.4 c
‘Heaven Can Wait’3.63 ± 0.14 bc3.7 ± 0.5 c
BAP 1.0‘Natchez’3.92 ± 0.04 b2.9 ± 0.5 d
‘Black Magic’3.45 ± 0.15 c2.5 ± 0.2 d
GA3 0.5‘Osage’3.62 ± 0.03 bc2.1 ± 0.4 d
‘Heaven Can Wait’3.76 ± 0.12 b2.6 ± 0.2 d
The data represent the mean ± standard deviation of three independent replicates. Values within the same column with different letters (a, b, c, d) are significantly different according to ANOVA followed by Tukey’s test (p < 0.05).
Table 3. Morphometric parameters of regenerated blackberry plants during ex vitro rooting depending on the substrate and growth stimulant used.
Table 3. Morphometric parameters of regenerated blackberry plants during ex vitro rooting depending on the substrate and growth stimulant used.
SubstrateCultivarShoot Length, cmNumber of Internodes, PcsNumber of Roots, PcsAverage Root Length, cm
Control, H2ONatchez2.37 ± 0.09 f6.60 ± 0.29 b6.57 ± 0.64 e4.60 ± 0.58 b
Osage2.30 ± 0.06 f6.23 ± 0.07 b6.27 ± 0.38 e5.50 ± 0.45 b
Heaven Can Wait2.03 ± 0.12 f6.27 ± 0.22 b5.53 ± 0.29 f5.27 ± 0.30 b
Black Magic2.21 ± 0.06 f6.45 ± 0.65 b6.35 ± 0.41 e5.36 ± 0.29 b
Humic acid
(0.01%)		Natchez5.43 ± 0.26 c6.73 ± 0.34 b8.30 ± 0.84 d6.23 ± 0.43 a
Osage3.87 ± 0.03 e5.43 ± 0.03 c12.03 ± 0.13 c6.50 ± 0.31 a
Heaven Can Wait5.30 ± 0.06 c6.40 ± 0.10 b11.77 ± 1.4 c6.43 ± 0.19 a
Black Magic4.65 ± 0.15 d6.25 ± 0.18 b9.64 ± 0.34 d6.35 ± 0.23 a
Nanosilicon
(0.01%)		Natchez8.47 ± 0.15 a7.70 ± 0.26 a15.67 ± 0.58 b3.83 ± 0.12 c
Osage8.10 ± 0.15 a7.57 ± 0.22 a13.70 ± 0.44 c3.80 ± 0.53 c
Heaven Can Wait7.10 ± 0.15 b7.30 ± 0.29 a17.67 ± 1.17 f4.07 ± 0.12 c
Black Magic7.15 ± 0.15 b7.21 ± 0.23 a14.71 ± 0.21 c3.93 ± 0.14 c
Succinic acid
(0.1%)		Natchez4.60 ± 0.21 d3.66 ± 0.45 e7.33 ± 0.05 e3.40 ± 0.08 d
Osage4.02 ± 0.16 d3.41 ± 0.61 e7.20 ± 0.12 e2.23 ± 0.41 e
Heaven Can Wait4.20 ± 0.18 d3.45 ± 0.36 e7.35 ± 0.24 e3.31 ± 0.23 d
Black Magic3.61 ± 0.09 e3.30 ± 0.34 e7.52 ± 0.11 e3.45 ± 0.24 d
The data are presented as mean ± standard deviation from three independent replicates. Values within the same column followed by different letters (a, b, c, d, e, f) are significantly different according to ANOVA followed by Tukey’s test (p < 0.05).
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Malakhova, N.; Tezekbayeva, B.; Kiyan, V.; Yefremova, Y. Enhancing In Vitro Multiplication and Acclimatization of Blackberry (Rubus L.) Through Sterilization Optimizing and Growth Regulator Use. Horticulturae 2025, 11, 1422. https://doi.org/10.3390/horticulturae11121422

AMA Style

Malakhova N, Tezekbayeva B, Kiyan V, Yefremova Y. Enhancing In Vitro Multiplication and Acclimatization of Blackberry (Rubus L.) Through Sterilization Optimizing and Growth Regulator Use. Horticulturae. 2025; 11(12):1422. https://doi.org/10.3390/horticulturae11121422

Chicago/Turabian Style

Malakhova, Natalya, Botakoz Tezekbayeva, Vladimir Kiyan, and Yuliya Yefremova. 2025. "Enhancing In Vitro Multiplication and Acclimatization of Blackberry (Rubus L.) Through Sterilization Optimizing and Growth Regulator Use" Horticulturae 11, no. 12: 1422. https://doi.org/10.3390/horticulturae11121422

APA Style

Malakhova, N., Tezekbayeva, B., Kiyan, V., & Yefremova, Y. (2025). Enhancing In Vitro Multiplication and Acclimatization of Blackberry (Rubus L.) Through Sterilization Optimizing and Growth Regulator Use. Horticulturae, 11(12), 1422. https://doi.org/10.3390/horticulturae11121422

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