An Alternative Strategy for Clonal Micropropagation of the Garden Strawberry

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RESEARCH ARTICLE

An Alternative Strategy for Clonal Micropropagation of the Garden Strawberry

Sergey Kornatskiy1 , * Open Modal iD Ulyana Titarenko1 Zhanna Serebryakova1 Valeriy Burlutskiy1
Authors Info & Affiliations
The Open Biotechnology Journal • 16 Mar 2026 • RESEARCH ARTICLE • DOI: 10.2174/0118740707455704260310055813
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Abstract

Introduction/Objective

The aim of the study was to develop a technological approach for the production, storage, and preparation of strawberry microplants, facilitating their simultaneous acclimatization before planting in a greenhouse when favorable temperature and lighting conditions are established.

Methods

The initial material for the experiment was obtained via clonal micropropagation. Preparation, accumulation, and preservation of strawberry plants in vitro for delayed acclimatization were performed by periodically repeating the procedure of removing roots and leaves from previously rooted microplants and transferring them in the form of vegetating buds onto a fresh nutrient medium for further rehabilitation. The mass acclimatization of microplants was carried out under substrate-free flow-through hydroponic conditions, following which they were transplanted into a greenhouse in early spring.

Results

The survival rate of microplants during the periodically repeated procedures of removing roots and leaves from previously obtained microplants reached 100% for all studied cultivars. Active leaf and root recovery began on days 3–7 and 10–14, respectively. A 1-month acclimatization period on flow-through hydroponics allowed for a 2–2.5-fold increase in microplant height, a 2-fold increase in root length, and an almost 3-fold increase in weight.

Discussion

The repetitive removal of microplant roots and leaves, with their further transplantation on fresh nutrient medium as vegetating buds for subsequent rehabilitation during periods of unfavorable environmental conditions, does not reduce the viability of microplants, allowing the preservation of planting material without loss until acclimatization is advisable. Acclimatization using flow-through hydroponics enables the elimination of fungal pathogenesis that occurs during the use of solid substrates. This protocol also increases the predictability of acclimatization outcomes and ensures successful transplantation into a greenhouse.

Conclusion

The developed alternative strategy of strawberry clonal micropropagation allows prediction of the output volume of acclimatized microplants throughout a calendar year with a high degree of accuracy. The estimated quantity of such material can be compactly stored in a lighted tissue culture room, significantly saving time and resources. Microplants are well preserved until a favorable moment for acclimatization in a viable state as vegetating buds. After the rehabilitation cycles are complete, the microplants can be simultaneously transferred for acclimatization on flow-through hydroponics.

Keywords: Strawberry, Clonal micropropagation, Growth regulator, Vegetating bud, Flow-through hydroponics.

1. INTRODUCTION

Garden strawberry is a widespread berry crop that is successfully used as a model plant for scientific research. This crop is currently well-studied, but the development of new cultivation technologies using high-quality planting material remains a topical issue. Above all, strawberries are highly regarded for their excellent fruit taste, early fruiting, and ripening [1]. In temperate climate zones, commercial strawberry plantations are mainly found in open fields. However, the protected cropping area has been constantly expanding recently. This has become a common occurrence in the subtropical zone due to the use of imported planting material. It has been made possible through the development of new seedling production technologies, which allow for a commercial harvest in the year of planting.

The clonal micropropagation method can be successfully applied in this case, since it enables achieving a distinctively high reproduction rate and promotes the introduction of new, promising, and highly productive commercial cultivars to the industry [2-5]. However, despite significant methodical improvements and research experience, especially regarding obtaining virus-free initial planting material, not all the potential of this method has been implemented to date. For quite a long time, scientific research aimed at improving the efficiency of strawberry clonal micropropagation has been focused on optimizing nutrient medium composition and culture conditions [6-11]. The issues of initial explant selection and their surface sterilization methods have been thoroughly studied [12-16]. Numerous studies devoted to the production of constantly propagating microplants have been conducted [17-23], along with studies aimed at the investigation of cultivar specificity during microcutting rhizogenesis [24, 25] and the selection of growth regulator types and effective concentrations [26-34]. Various techniques were developed to reduce production costs of clonal micropropagation, including a system for growing strawberries in bioreactors [35]. In addition, the issue of ensuring and controlling the genetic stability of the obtained material has been raised repeatedly, since it is essential both under laboratory conditions [36-39] and after transplantation into the field [40-44]. All these studies are undoubtedly important for the evolution and improvement of this method; however, their application has proven insufficient for the development of a stable and reproducible plant production technology. It is well known that in vitro plant cultivation is carried out within culture vessels with humidity levels close to 100%. Moreover, the relatively low illumination level (4–7 klx) in tissue culture rooms poorly promotes microplants’ transition to autotrophic nutrition. This mainly explains the systemic problems that arise during microplant acclimatization, which is often accompanied by significant material loss.

Since microplants are cultivated in the clonal micropropagation laboratory continuously and throughout the year, new batches of rooted microplants are produced regularly. These plants require subsequent acclimatization, which involves a lot of manual labor. It should be noted that a postponement of this procedure for more than 1–1.5 months after microcuttings are transplanted for the rooting stage leads to progressive material loss inside the culture vessels. In a temperate climate zone, the expediency of strawberry microplant acclimatization during the period from September to February is extremely low. This is explained by the fact that transferring plants to a permanent location is considered impossible during this period due to low temperatures, even in a greenhouse if it is not designed for all-season use. In addition, the acclimatization method itself plays a crucial role in the procedure outcome. The complex of stress factors at this stage continues to be the main subject of research, as it appears to be key to finding an effective and universal solution.

Many published research papers provide examples of various techniques used to improve the efficiency of the strawberry microplant acclimatization process. However, these achievements were typically limited to a local level and could not be replicated on a larger scale [45-50]. It is well known that the effectiveness of acclimatization also depends on the physical parameters of microplants, as well as the substrate type used for transferring them to non-sterile conditions. Most often, peat-sand or peat-perlite mixes were used, which require sterilization to eliminate pathogenic microflora. This measure is essential for protecting plants during the initial period of acclimatization. Without this, the microplants’ roots become colonized by fungi and bacteria within 3–5 days, following which the prognosis for their survival becomes unfavorable. The high labor intensity of the described process, combined with its low efficiency, requires closer consideration of the well-established method of growing plants, known as hydroponics, in order to solve the problem of acclimatization [51-53].

Hydroponics clearly has similar principles to clonal micropropagation, as both methods require mineral salt solutions as the basis for plant cultivation, with some differences depending on the specific growing conditions. In terms of clonal micropropagation, agar-agar – the gel-forming component – is added to the nutrient solution, which significantly increases the technological efficiency of the process. There are also certain similarities in the root development parameters of plants obtained using these methods. It is well known that problems that occur during the acclimatization of in vitro plants arise due to the combination of abiotic and biotic factors. A particularly problematic factor is the change from liquid to solid substrate, for which the in vitro plant root system is not physiologically ready. Considering that, hydroponics appears to be much more preferable for microplant acclimatization, since the root environment does not change drastically as it does in traditional schemes. Basically, the roots of microplants are transferred from one solution to another, and the latter is additionally aerated, which has a beneficial effect on the plants’ condition. It is also extremely important that microplants have no contact with pathogens specific to soil substrates, which ensures a high plant survival rate [54].

The study aimed to develop a technological strategy for the preparation, accumulation, and preservation of strawberry plants in vitro, to perform a delayed acclimatization under hydroponic conditions, which should be carried out before planting in a greenhouse during favorable temperature and lighting conditions.

2. MATERIALS AND METHODS

The following strawberry cultivars of European selection were studied: Kimberly, Asia, and Florence. Aseptic cultures were maintained at a temperature of 23 ± 1 °C, a 16-hour photoperiod, and an illuminance of 5–6 klx at the plant culture level. Microplants were maintained on Murashige and Skoog nutrient medium (MS) [55]. Subculturing was performed at intervals of 3–5 weeks. Proliferation of plant tissue cultures was carried out all year round on a nutrient medium supplemented with 1.0 mg L−1 6-benzylaminopurine (BAP) in 200×21 mm test tubes. For the elongation stage, 100 ml flasks with 30 ml of a nutrient medium supplemented with 0.05 mg L−1 BAP were used. One month after transplantation to the elongation stage, microshoots with a base diameter of 2–5 mm and 3–5 leaves were selected and transferred from the flasks for rooting, which was carried out in 150×16 mm test tubes with a nutrient medium containing 1.0 mg L−1 indolebutyric acid (IBA).

2.1. Principles of Preparing Explants for Transplantation

Further work with plants was performed according to our methodology. Starting in September, microplants of the studied cultivars obtained after 1.5 months of microshoot rooting were prepared for further subcultivation in the following manner. For transplantation, a fundamentally new type of strawberry explant was used, which was named the “vegetating bud.” These explants were obtained by removing the existing roots and leaves from fully developed microplants with a base diameter of 4–5 mm. Surgical scissors 20 cm long were used to perform the removal. The base diameter was measured using a caliper with specially prepared jaws, so that the working part could be sterilized. After the described procedure, the vegetating buds were transferred onto a fresh MS nutrient medium supplemented with 0.1 mg L−1 IBA for the rehabilitation stage. This stage involved placing 6–7 explants into each 250 ml flask containing 70 ml of nutrient medium. The protocol was repeated periodically 1–3 times with an interval of 1.5–2 months, depending on the starting point of the procedure and the condition of the recovered plants.

2.2. The Use of Flow-through Hydroponics during Microplants’ Acclimatization

A hydroponic system consisting of plastic pallets measuring 0.6 × 0.4 × 0.04 m (with a volume of 9 L) was used to acclimatize the microplants. To maintain the plants above the solution, a thin non-woven polymer fabric with a density of 17 g/m2 was stretched over specially prepared frames. Translucent plastic film covers were placed on top of the frames, creating a high-humidity chamber in the inner volume (Fig. 1).

Before planting, the surface of the non-woven fabric was perforated with 3–4 mm diameter planting holes, which formed a 3.5 × 3.5 cm planting pattern. The total number of microplants in each pallet was 140 for all cultivars. The circulation of the solution was provided for 5 minutes every 3 hours around the clock. The nutrient solution was pumped into a 10-liter tank located at a height of 2 m. The solution then flowed by gravity through a tube system into hydroponic pallets, after which it was collected in a lower 20-liter tank. The hydroponic solution was prepared according to a recipe for growing aquatic vegetable crops [56]. Before transplantation, the microplants were removed from flasks, following which their roots were gently washed free of the agar nutrient medium. The roots of the microplants were then passed through the planting holes using thin tweezers, leaving the microcrowns above the surface of the non-woven fabric. After planting, the frames were placed on top of hydroponic pallets and covered with transparent film caps. The acclimatization was carried out for 1 month, after which the plants were transferred into a greenhouse in early spring and planted in a non-sterile soil substrate consisting of peat and perlite in a 2:1 ratio. The difference in experiments was assessed using Duncan’s multiple range test at a 5% significance level [57].

Fig. (1).

Construction of a flow-through hydroponic system: a - hydroponic tray, b - frame with non-woven fabric, c - translucent cover.

3. RESULTS AND DISCUSSION

3.1. Principles of Propagation of Plant Material for the Experiment that was Required

To follow the accepted research concepts, great attention was paid to obtaining a sufficient quantity of material for subcultivation. For this purpose, the number of proliferating units for each cultivar was maintained at the level of 120–140 tubes per month. Typically, 20 test tubes with an average multiplication coefficient of 6.0 at the end of each proliferation subcultivation were sufficient to form the total volume of material for the next subcultivation. The remaining material (approximately 100 tubes of each cultivar) was transplanted into 40–50 flasks for elongation. It is known that to stabilize shoot formation and ensure the production of well-developed microplants during the elongation stage, it is necessary to monitor the microplants’ illuminance and maintain it at a level of at least 6–7 klx.

The most important elongation outcome was the production of shoots (microcuttings) with a 2–5 mm base diameter and normally developed leaves 3–5 cm long. The number of such microcuttings amounted to 3–4 pieces per flask. This resulted in an opportunity to receive 120–200 microcuttings of each cultivar monthly, indicating the efficiency of rooting and the suitability of the material for subsequent operations. The principle for microcutting differentiation was based on previous research, where it was found to be counterproductive to use microplants that were less developed than the described ones. Rhizogenic activity was normally observed 5–8 days after transplantation onto a nutrient medium with 1.0 mg L−1 IBA in the case of microcuttings of all studied cultivars.

The present study shows that continuous contact with auxin contributed to thickening of the shoot bases due to active root and callus formation, especially in microcuttings with a base diameter of more than 2 mm. Presumably, this improved the nutrition of the developing leaves and stems, which promoted plant growth. However, the maintenance of rooted microplants on a nutrient medium supplemented with auxin for more than 1–1.5 months negatively affected their subsequent viability. The reason for this was that a so-called dedifferentiation effect occurred in cells of the root epidermis, causing their subsequent destruction, which led to further microplant loss. A similar consequence of long-lasting contact of plant tissues with auxins is well-known, expected, and requires proper and timely management.

The acclimatization of such microplants, especially under insufficient illumination levels, can take 2–3 months and be accompanied by the development of competitive pathogenic microflora, leading to a decrease in the survival rate of rooted microplants to 20–30% or even less. At the same time, such plants require mandatory manual care, which is very complicated and time-consuming, even for small quantities. This care cannot be terminated until the plants are transferred to a greenhouse or planted in the open field, where they can develop relatively independently under appropriate conditions.

3.2. Features of Using Vegetating Buds for Transplantation

The discussed set of issues led to a revision of the principles of working with the plant material obtained during periods that are unfavorable for acclimatization. It was decided not to acclimatize each batch of microplants after rooting, but to periodically transplant them with an interval of 1.5–2 months onto a fresh nutrient medium in the form of vegetating buds. To obtain them, as indicated in the methods, we completely removed the already formed leaves and roots from the microplants (Fig. 2).

This approach allows working individually with specific plants, rather than with the general mass of material, the propagation outcome of which is difficult to predict. Transplantation onto a rehabilitation nutrient medium containing 0.1 mg L−1 IBA ensured the successful recovery of leaves and roots on all vegetating buds (Table 1, Fig. 3a).

Table 1.
Dynamics of leaves and roots recovery on vegetating buds after their transplantation on a nutrient medium for rehabilitation (MS with 0.1 mg L-1 IBA).
Strawberry Cultivar The Number of Days After Planting Before the Start of Vegetative Organs Recovery Morphometric Parameters of Microplants After Planting On Survivalrate, %)
Tenth Day Thirtieth Day Tenth Day Thirtieth Day
Leaves Roots Number of Leaves, pcs Number of Roots, pcs.
Kimberly 3-5 10-12 1,9 5,4ns 1,1 5,8b 100
Asia 3-6 10-12 1,6 5,2ns 0,9 5,4ab 100
Florence 5-7 12-14 1,1 4,9ns 0,4 4,5a 100

Note: *Means followed by different letters in the column differ from Duncan’s multiple range test (P<0.05).

Fig. (2).

Algorithm for carrying out the rehabilitation of the material: plants after 1.5 months of cultivation on a rehabilitation medium (a), the contents of 1 flask before preparing “vegetating buds” (b), “vegetating buds” prepared for planting (c), and planted (d) for the next rehabilitation (Kimberly cultivar).

Fig. (3).

Vegetating buds 10 days (a) and 30 days (b) after planting for rehabilitation (Florencecultivar).

Recovery (rehabilitation) took up to 30 days, which corresponds to the duration of subculture for propagation, and the survival rate of vegetating buds amounted to 100% for all cultivars. By the end of the observation, there were no significant differences recorded in the development parameters of microplants of the studied cultivars. The rehabilitation of the Florence cultivar proceeded slower, but it should be noted that its microcuttings were initially objectively less developed.

3.3. Acclimatization of Strawberry Microplants in Flow-through Hydroponic Conditions

Taking into account the seasonal increase of light flux in the temperate climate zone at the end of February, the acclimatization of microplants was planned for this period. It is well known that strawberry shoots can root easily and quickly form new crowns when in contact with moist soil or substrate ex vitro. Therefore, flow-through hydroponics was used in order to model similar conditions during microplants’ acclimatization. A non-woven fabric fixed in a frame above the nutrient solution was used as a wet supporting surface. An advantage of working with strawberry microplants is that the crown form of their shoots eliminates the need for vertical positioning. The leaf rosette serves as a natural support for microplants, simplifying the planting process and preventing excessive immersion in the nutrient solution. After planting, only the roots come into contact with the solution, while the leaves remain above the surface of the non-woven fabric in a high-humidity condition, which eliminates the need for additional moisturizing and simplifies care.

Observations did not reveal significant differences in plants’ growth parameters during acclimatization of microplants of different cultivars in flow-through hydroponic conditions. The microplants of the Kimberly and Asia cultivars developed slightly more dynamically than the Florence cultivar, but the latter were initially weaker, with lower height and weight indices. In general, according to the studied parameters, the Kimberly cultivar stood out, with the increase in plant height and root length significantly exceeding the measurements of other cultivars. At the end of the acclimatization period, the Asia microplants had the highest weight, although the increase in this parameter relative to the starting value turned out to be significantly lower than in other cultivars (Table 2, Fig. 4).

Fig. (4).

General appearance of microplants at the end of the acclimatization period (a) and the root system of acclimatized plants (b) (Florence cultivar).

Table 2.
Development of microplants of different strawberry cultivars in the acclimatization process to non-sterile conditions using flow-through hydroponics.
Morphometric Parameters of Microplants Moment of Measurement Strawberry Cultivar
Kimberly Asia Florence
Plant height, cm When planting in hydroponics 4.0 4.2 3.8
after 1 month of acclimatization 9.9 8.5 7.9
Parameter increment, % - 147b 102a 107a
root length, cm When planting in hydroponics 2.8 2.9 3.4
After 1 Month of acclimatization 6.5 5.9 6.8
Parameter increment, % - 132b 103a 100a
plant weight, g When planting in hydroponics 0.8 1.1 0.7
after 1 monthofacclimatization 2.1 2.5 1.9
Parameter increment, % - 162b 127a 171b
survivalrate, % - 100 100 100

Note: *Means followed by different letters in the column differ from Duncan’s multiple range test (P<0.05).

Based on the results obtained, it should be noted that the most significant outcome of the experiment is a high survival rate (reaching 100%) of microplants during the acclimatization process, which is impossible to achieve in the case of large-scale acclimatization according to the traditional scheme of using solid substrates. Figure 3b clearly shows that roots formed during the acclimatization process are well developed, characterized by a lighter tone and greater length, which appear to be the basis for the high survival rate.

CONCLUSION

The developed alternative strategy for strawberry clonal micropropagation allows for predicting the production outcome of acclimatized microplants throughout the calendar year with a high degree of accuracy. The estimated quantity of such material can be compactly stored in a lighted tissue culture room, significantly saving time and resources. This can be achieved because microplants are well-preserved in a viable state as vegetating buds until the moment favorable for acclimatization. After synchronizing the rehabilitation cycles, microplants can be simultaneously transferred for acclimatization onto flow-through hydroponics.

An obvious reduction in the process complexity during microplants’ acclimatization to non-sterile conditions is achieved by avoiding the use of any solid substrate. This eliminates the need for its preliminary energy-intensive preparation through heat sterilization to remove pathogenic microflora. The transplantation of microplants was also greatly simplified by reducing the involvement of highly qualified personnel. The high microplants’ viability during the acclimatization period is ensured by the immersion of plant roots in water containing the necessary mineral salts, which periodically circulates in a closed hydroponic system.

AUTHORS’ CONTRIBUTIONS

The authors confirm contribution to the paper as follows: S.K.: Study conception and design; U.T., Z.S.: Data collection; V.B.: Analysis and interpretation of results; S.K.: Draft manuscript. All authors reviewed the results and approved the final version of the manuscript.

LIST OF ABBREVIATIONS

MS = Murashige and Skoog medium
IBA = Indolebutyric Acid
BAP = 6-benzylaminopurine

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

Not applicable.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

The data and supportive information are available within the article.

FUNDING

This publication was prepared with the support of the Ministry of Science and Higher Education of the Russian Federation (project number FSSF-2023-0014).

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

Declared none.

REFERENCES

1
López-Aranda JM, Soria C, Santos BM, Miranda L, Domínguez P, Medina-Mínguez JJ. Strawberry production in mild climates of the world: A review of current cultivar useIntl. Int J Fruit Sci 2011; 11(3): 232-44.
2
Ather-uz-Zaman Z, Al-Khayri JM. Genetic improvement of strawberry (Fragaria ananassa Duchesne). In: Al-Khayri J, Jain S, Johnson D, Eds. Advances in Plant Breeding Strategies: Fruits 2018; 217-75.
3
Liu Z, Liang T, Kang C. Molecular bases of strawberry fruit quality traits: Advances, challenges, and opportunities. Plant Physiol 2023; 193(2): 900-14.
4
Mukherjee E, Gantait S. Strawberry biotechnology: A review on progress over past 10 years. Sci Hortic (Amsterdam) 2024; 338: 113618.
5
Banerjee D, Singh V, Thakur R. Micro propagation on strawberry: A review. E3S Web Conf 2023; 453: 01019.
6
Dhukate MR, Kher MM, Vadawale AV, Giri P. Protocol for micropropagation of strawberry (Fragaria × ananassa) cv. ‘Sweet Charlie’ and ‘Winter Dawn’. Environ Exp Biol 2021; 19(1): 1-6.
7
Sabbadini S, Marcellini M, Mezzetti B, Capocasa F. Establishing micropropagation protocols for new strawberry ( Fragaria × ananassa ) breeding lines. Acta Hortic 2021; (1309): 573-8.
8
Ramlal A, Quan PW, Rajendran A, Subramaniam S. Impact of biophysicochemical factors on micropropagation, haploidy and doubled haploidy in strawberry (Fragaria sp.): A critical revisit. Curr Plant Biol 2025; 44: 100555.
9
Li Y, Huang P, Qiu X, et al. Integration of Green and Far-Red Light with Red-Blue Light Enhances Shoot Multiplication in Micropropagated Strawberry. Horticulturae 2025; 11(6): 701.
10
Kadhimi AA, Alhasnawi AN, Mohamad A, Yusoff WMW, Binti CR, Zain CM. Tissue culture and some of the factors affecting them and the micropropagation of strawberry. Life Sci J 2014; 11(8): 484-93. [http://www.lifesciencesite.com].
11
Park SW, Kwack Y, Chun . Growth of runner plants grown in a plant factory as affected by light intensity and container volume. Weonye Gwahag Gisulji 2017; 35(4): 439-45.
12
Munir M, Iqbal S, Baloch JUD, Khakwani AA. In vitro explant sterilization and bud initiation studies of four strawberry cultivars. J Appl Hortic 2015; 17(3): 192-8.
13
Palei S, Das AK, Dash DK, Rout GR. Effect of surface sterilant for reducing microbial contamination of field grown strawberry explants intended for in vitro culture. Int J Chem Stud 2017; 5(4): 1476-9.
14
Gonzales Arteaga JJ, Rodríguez Layza J, Romero Rivas LC, Párraga Quintanilla A. Establecimiento in vitro de Fragariax ananassa var. Aroma a partir de yemas de corona. Manglar (Tumbes) 2022; 19(4): 301-8.
15
Kumar M, Rajbhar YP, Kumar V, et al. Studies on the effect of different surface sterilization agents under in-vitro culture of Strawberry (Fragaria × ananassaDuch.) variety “Chandler”. J Pharmacogn Phytochem 2020; 9(6): 1833-5.
16
Tung HT, Thuong TT, Cuong DM, et al. Silver nanoparticles improved explant disinfection, in vitro growth, runner formation and limited ethylene accumulation during micropropagation of strawberry (Fragaria ananassa ). Plant Cell Tissue Organ Cult 2021; 145(2): 393-403.
17
Aguirre-Noyola JL, Ramírez-Mosqueda MA, Cadena-Zamudio JD, Caamal-Velázquez JH, Cruz-Gutiérrez EJ, Armenta-Medina A. Green-Synthesized Silver Nanoparticles (AgNPs) enhance in vitro multiplication and rooting of strawberries (Fragaria ananassa Duchesne). BioTech 2025; 14(2): 45.
18
Kryukov LA, Vodolazhsky DI, Kamenetsky-Goldstein R. Micropropagation of Grapevine and Strawberry from South Russia: Rapid Production and genetic uniformity. Agronomy 2022; 12(2): 308.
19
Karmaker S, Hossain MM, Hoque MA, Kaium MA, Al Amin M, Islam MS. In vitro propagation of three strawberry cultivars through runner tips culture. Am J Plant Sci 2023; 14(11): 1296-304.
20
Yeasmin S, Banu TA, Goswami B, et al. In vitro regeneration of strawberry plant from leaf explants via callus induction. Plant Tissue Cult Biotechnol 2022; 32(1): 67-75.
21
Valliath AS, Mondal R. Micropropagation of strawberry crop (Fragaria ananassa): A review. Bhartiya Krishi Anusandhan Patrika 2023; 38(Of): 41-4.
22
Bettoni JC, Wang MR, Wang QC. In vitro regeneration, micropropagation and germplasm conservation of horticultural plants. Horticulturae 2024; 10(1): 45.
23
Ashrafuzzaman M, Faisal SM, Yadav D, Khanam D, Raihan F. Micropropagation of strawberry (Fragaria ananassa) through runner culture. Bangladesh J Agric Res 2013; 38(3): 467-72.
24
Chaouch R, Soufi S, Kthiri Z, Bettaieb T. Auxin-like potential of essential oils in strawberry micropropagation: A sustainable approach to root induction and photosynthetic performance. S Afr J Bot 2025; 177: 352-62.
25
Sirijan M, Pipattanawong N, Chaiprasart P. Effect of 1-naphthalene acetic acid and 6-benzyladenine on micropropagation of strawberry cultivar ‘Praratchatan No. 80’. Agr Nat Resour 2019; 53: 355-63.
26
Gonzales-Arteaga JJ, Rodríguez-Layza J, Romero-Rivas LC, Párraga-Quintanilla A, Olivera-Soto JA. Role of BA and IAA on regeneration and shoot proliferation in vitro of three strawberry (Fragaria ananassa Duch.) varieties. Agroindustrial Sci 2023; 13(2): 93-102.
27
Kotsupiy O, Karpova E, Trofimova E, Novikova T, Ambros E. Transformation of strawberry plants’ phenolic profile after treatment with a mechanocomposite based on silicon chelates in the course of development under in vitro, ex vitro, and in vivo conditions. Horticulturae 2023; 9(2): 157.
28
Saridaş MA, Baktemur G, Tas¸kın H, Kargi SP. Effect of plant hormones on micropropagation potential of superior strawberry genotypes and their parents via shoot-tip culture. Acta Sci Pol Hortorum Cultus 2021; 20(3): 63-75.
29
Cadena-Zamudio JD, Cruz-Cruz CA, Ramírez-Mosqueda MA, Cruz-Gutiérrez EJ, Hernández-Domínguez E. Effect of meta-Topolin on the in vitro propagation of strawberry (Fragaria AnanassaDuch). Agro Product 2024; 17(11): 237-43.
30
Diengngan S, Murthy BNS. Influence of plant growth promoting substances in micropropagation of strawberry cv. Festival. Bioscan 2014; 9(4): 1491-4. [https://thebioscan.com/index.php/pub/article/view/871].
31
Dutta C. Effect of different plant growth regulators on in-vitro regeneration in varieties of strawberry. International J Environ Climate Change 2022; 12(11): 1178-87.
32
El-Sayed SF, El-Sawy AM, Taha SS. Effect of Benzylaminopurine concentration and number of subcultures on behavior of some strawberry cultivars in vitro. Egypt J Plant Breed 2017; 21(1): 1-12.
33
Hussein MA, Mohamed FE, Abdoun MI. Multiplication and regeneration potential in strawberry genotypes using different in vitro culture methods and growth regulators. Hort Sci J Suez Canal University 2018; 7(2): 47-54.
34
Wang F, Li Y, Pang Y, Hu J, Kang X, Qian C. Thidiazuron enhances strawberry shoot multiplication by regulating hormone signal transduction pathways. Int J Mol Sci 2025; 26(9): 4060.
35
Badjakov I, Georgiev V, Georgieva M, et al. Bioreactor technology for in vitro berry plant cultivation. In: Ramawat KG, Ekiert HM, Goyal S, Eds. Plant Cell and Tissue Differentiation and Secondary Metabolites: Fundamentals and Applications 2021; 383-431.
36
Naing AH, Kim SH, Chung MY, Park SK, Kim CK. In vitro propagation method for production of morphologically and genetically stable plants of different strawberry cultivars. Plant Methods 2019; 15(1): 36.
37
Thakur A, Nath AK, Sharma V. Genetic Homogeneity analysis in tissue culture raisedFragaria ananassaDuch. Revealed through PCR based molecular markers. Erwerbs-Obstbau 2024; 66: 441-51.
38
Suvalaxmi P, Mandakini K, Gyana RR, et al. Optimized in vitro regeneration and genetic fidelity assessment of strawberry (Fragaria × ananassaDuch. cv. Chandler). Plant Sci Today 2025; 12(3): 1-10.
39
Pang WQ, Lai CS, Mad’ Atari MF, et al. Effect of graphene oxide nanoparticles on in vitro growth of Fragaria ananassa (Cameron Highlands white Strawberry) and evaluation of genetic stability using DAMD and ISSR markers. Plant Physiol Biochem 2023; 204: 108104.
40
Yaman M, Yilmaz KU. The effects of different chemicals on runner yield and quality of ‘Kabarla’ strawberry young plants grown in Cappadocia region. Erwerbs-Obstbau 2022; 64(1): 85-90.
41
Capocasa F, Balducci F, Marcellini M, Bernardini D, Navacchi O, Mezzetti B. Comparing nursery behavior, field plant yield and fruit quality of in vitro and in vivo propagated strawberry mother plants. Plant Cell Tissue Organ Cult 2019; 136(1): 65-74.
42
Thakur M, Shylla B. Influence of different growing media on plant growth and fruit yield of strawberry (Fragaria × Ananassa Duch.) Cv. chandler grown under protected conditions. Int J Curr Microbiol Appl Sci 2018; 7(4): 2724-30.
43
Karmaker S, Hossain MM, Hoque MA, Kaium MA, Ali TB, Ferdous M. In vitro propagation of three strawberry varieties and field evaluation. Am J Plant Sci 2023; 14(11): 1214-22.
44
Burak Ceran M, Yildiz E, Yaman M. Determination of the yield and quality characteristics of some strawberry varieties growed in the open field of Yahyali (Kayseri) ecological conditions. Curr Trends Nat Sci 2023; 12(23): 36-41.
45
Dev R, Singh SK, Dayal V, Kumar K, Singh T. Standardization of in vitro hardening strategies for tissue cultured wine grape (Vitis vinifera L) Genotypes. Int J Curr Microbiol Appl Sci 2019; 8(2): 2108-17.
46
Dwiyani R, Fitriani Y, Ana FGS, Bimantara PO. A Study of acclimatization media on strawberry (Fragaria x ananassaDuch.) plantlets produced from meristem culture. Nutrition Public Health 2024; 5(2): 85-91.
47
Ambros EV, Toluzakova SY, Shrainer LS, Trofimova EG, Novikova TI. An innovative approach to ex vitro rooting and acclimatization of Fragaria × ananassa Duch. microshoots using а biogenic silica- and green-tea-catechin-based mechanocomposite. In Vitro Cell Dev Biol Plant 2018; 54(4): 436-43.
48
Bag N, Palni LMS, Nandi SK. An efficient method for acclimatization: In vitro hardening of tissue culture-raised tea plants (Camellia sinensis (L.) O. Kuntze). Current Science 2019; 117(2): 288-93.
49
Sharma N, Nishant Kumar , James J, Sonika Kalia , Joshi S. Strategies for successful acclimatization and hardening of in vitro regenerated plants: Challenges and innovations in micropropagation techniques. Plant Sci Today 2023; 10(sp2): 90-7.
50
Neri JC, Meléndez-Mori JB, Tejada-Alvarado JJ, et al. An optimized protocol for micropropagation and acclimatization of strawberry (Fragaria × ananassa Duch.) variety ‘Aroma’. Agronomy 2022; 12(4): 968.
51
Asao T. Hydroponics: A Standard Methodology for Plant Biological Researches 2012; 244.
52
Batukaev AA, Kornatskiy SA, Gaplaev MS. Block-container system for growing strawberry planting material in greenhouses. IOP Conf Ser Earth Environ Sci 2021; 624(1): 012116.
53
Treder W, Tryngiel-Gać A, Klamkowski K. Development of greenhouse soilless system for production of strawberry potted plantlets. Hortic Sci (Prague) 2015; 42(1): 29-36.
54
Kornatskiy S, Kuzmina M, Serebryakova Z, Kudrina A. The effectiveness of flow through substrateless hydroponics in the adaptation of strawberry microplants. Bio Web Conf 2024; 121: 01016.
55
Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plant 1962; 15(3): 473-97.
56
Bentley M. Commercial hydroponics 1959; 750.
57
Steel RGD, Torrie JH. Principles and Procedures of Statistics: A Biometrical Approach 2nd. 1980; 245.