Optimization of in vitro multiplication of Dendrobium sp. through application of Gibberellin (GA₃)

 Corresponding author: Endang Nurcahyani Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Optimization of in vitro multiplication of Dendrobium sp. through application of Gibberellin (GA₃) Mita Ardelia, Endang Nurcahyani *, Yulianty and Mahfut Applied Biology Study Program, Faculty of Mathematics and Natural Sciences, University of Lampung, Bandar Lampung, Indonesia. World Journal of Advanced Research and Reviews, 2025, 27(01), 1056-1062 Publication history: Received on 29 May 2025; revised on 05 July 2025; accepted on 07 July 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.27.1.2515 Abstract Dendrobium sp. is a widely popular ornamental orchid valued for its aesthetic appeal and diverse varieties. In vitro multiplication is a method of propagating new plants by stimulating shoot growth, either directly or via callus induction. Gibberellic acid (GA₃), a type of plant growth regulator (PGR), is commonly used to enhance plant growth and development. This study aimed to evaluate the effect and optimal concentration of GA₃ on the in vitro growth of Dendrobium sp. plantlets. The experiment was arranged in a Completely Randomized Design (CRD) with five concentrations of GA₃: 0 mg/L, 1 mg/L, 2 mg/L, 3 mg/L, and 4 mg/L. Each treatment was replicated five times, with one plantlet per replicate. Data were analyzed using one-way ANOVA in SPSS, followed by Tukey’s post hoc test at a 5% significance level. The results indicated that GA₃ application significantly affected plantlet survival rate, height, number of leaves, and number of roots. However, no significant effect was observed on total carbohydrate content. Among the tested concentrations, 2 mg/L GA₃ was identified as the most effective in promoting the growth and development of Dendrobium sp. plantlets in vitro. Keywords: Dendrobium sp.; Gibberellic Acid; In vitro Culture; Orchid Propagation; Plant Growth Regulator 1. Introduction Dendrobium sp. is one of the most popular orchid genera due to its adaptability to lowland environments, minimal maintenance requirements, ease of flowering, diversity of floral forms, and the rapid blooming characteristics of certain hybrids [1]. Most Dendrobium species exhibit racemose-type inflorescences, in which the flowers bloom sequentially from the base to the apex along a single main axis [2, 3]. Furthermore, large compound flowers are particularly favored by consumers, as they enhance the ornamental appeal of the plant [3, 4]. Plant tissue culture is an in vitro propagation technique conducted aseptically on artificial media. This method involves culturing explants derived from various parts of the parent plant, such as shoots, roots, callus, seeds, embryos, pollen, seedlings, or even single cells [5]. The culture medium is typically supplemented with plant growth regulators (PGRs), including auxins, cytokinins, gibberellins, ethylene, abscisic acid, and other related hormones [6]. Multiplication refers to the phase of in vitro propagation aimed at generating new plantlets by stimulating shoot proliferation prior to acclimatization. This process is performed by transferring the developing plantlets to fresh culture vessels containing media enriched with PGRs that promote shoot formation and development [7]. One of the plant growth regulators (PGRs) commonly used to stimulate plant growth and development is gibberellic acid (GA₃) [8]. Gibberellin, specifically GA₃, plays a vital role in promoting growth by significantly enhancing cell World Journal of Advanced Research and Reviews, 2025, 27(01), 1056-1062 1057 elongation in the apical meristem. In addition to stimulating cell elongation, gibberellins also activate cell division and differentiation, break dormancy, and accelerate flowering [9]. Research on the in vitro multiplication of Dendrobium sp. plantlets supplemented with gibberellic acid (GA₃) remains limited. Therefore, this study aims to investigate the effect and optimal concentration of GA₃ on the in vitro growth of Dendrobium sp. plantlets. 2. Materials and Methods 2.1. Materials and Equipment The materials used in this study included Dendrobium sp. plantlets, Vacin and Went (VW) ready-to-use solution, 1 N potassium hydroxide (KOH), 1 N hydrochloric acid (HCl), 70% ethanol, agar, sucrose, activated charcoal, gibberellic acid (GA₃) at various concentrations, glucose solution, sulfuric acid (H₂SO₄), standard glucose solution, and phenol. The equipment used consisted of an autoclave, culture bottles, heat-resistant plastic, rubber stoppers, stirring rods, beakers, Bunsen burners, Petri dishes, measuring cylinders, Erlenmeyer flasks, hot plate, oven, laminar air flow (LAF) cabinet, magnetic stirrer, pH meter, forceps, scalpels, analytical balance, Whatman No. 1 filter paper, millimeter ruler, and UV-Vi’s spectrophotometer. 2.2. Experimental Design The experiment was arranged in a completely randomized design (CRD) with a single factor: GA₃ concentration. Five levels of GA₃ were tested: 0 mg/L, 1 mg/L, 2 mg/L, 3 mg/L, and 4 mg/L. Each treatment was replicated five times. Observations were conducted over a period of four weeks after planting to evaluate the most effective GA₃ concentration for in vitro cultivation of Dendrobium sp. The data collected comprised both qualitative and quantitative variables. Qualitative data were presented descriptively and supported by photographic documentation. Quantitative data for each parameter were analyzed using the Statistical Package for the Social Sciences (SPSS) with analysis of variance (ANOVA). When significant differences were found, Tukey’s post hoc test was applied at a 5% significance level. 2.3. Preparation and Dilution of GA₃ Gibberellic acid (GA₃) stock solutions were prepared by dissolving GA₃ powder in 1 liter of distilled water for each concentration level: 0 mg/L, 1 mg/L, 2 mg/L, 3 mg/L, and 4 mg/L. The 0 mg/L solution, used as the control, consisted of distilled water without GA₃. The 1 mg/L solution was prepared by dissolving 1 mg of GA₃ powder in 1 liter of distilled water; similarly, 2 mg, 3 mg, and 4 mg of GA₃ were dissolved in 1 liter of distilled water to obtain the 2 mg/L, 3 mg/L, and 4 mg/L solutions, respectively. Once fully dissolved and homogeneous, the solutions were filtered using Whatman No. 1 filter paper into sterilized culture bottles. Subsequently, 1 mL of each GA₃ solution was added to the Vacin and Went (VW) medium prepared for in vitro culture. 2.4. Sterilization of Dendrobium sp. Dendrobium sp. plantlets were individually transferred into an Erlenmeyer flask using sterile forceps and initially rinsed with sterile distilled water for 5 minutes. They were then surface-sterilized using 10 mL of Bayclin solution (a commercial bleach containing 5.25% sodium hypochlorite, NaOCl) for 3 minutes. After sterilization, the plantlets were rinsed twice with 100 mL of sterile distilled water to remove any residual disinfectant. 2.5. Multiplication of Dendrobium sp. Plantlets The washed plantlets were placed in Petri dishes lined with millimeter grid paper to measure their initial height. Each culture bottle received a single explant, which was sealed near a flame from a Bunsen burner and covered with plastic wrap to maintain sterility. Planting was carried out in a laminar air flow (LAF) cabinet at room temperature (24°C– 26°C). The culture bottles containing the explants were then incubated on shelves in a growth room under optimal lighting conditions (1,000 lux, equivalent to a 40-watt fluorescent lamp) and maintained at a constant temperature of 24°C–26°C [10]. World Journal of Advanced Research and Reviews, 2025, 27(01), 1056-1062 1058 2.6. Percentage of Live Plantlets and Plantlet Visualization Observations were carried out four weeks after planting to determine the effective concentration of GA₃ for the in vitro multiplication of Dendrobium sp. The following parameters were assessed [11]: Percentage of live plantlets = Number of surviving plantlets Total number of plantlets x 100% 2.7. Plantlet Height The height of the plantlets was measured from the base of the stem to the tip of the longest leaf. Measurements were taken at both the beginning and the end of the observation period [12]. 2.8. Number of Leaves The number of leaves was determined by manually counting all leaves present on each plantlet, including both newly emerged and fully developed leaves [12]. 2.9. Number of Roots The number of roots was determined by observing the root development of each plantlet. All roots emerging from the base of the plantlet were counted manually, including those that were newly formed or still short in length [12]. 2.10. Total carbohydrate analysis Total carbohydrate content was analyzed using the phenol-sulfuric acid method. Leaves from Dendrobium sp. plantlets were collected and weighed to 0.1 grams. The samples were ground using a mortar and pestle, then mixed with 10 mL of distilled water. The mixture was filtered through Whatman No. 1 filter paper, and the resulting filtrate was transferred into a test tube. Subsequently, 1 mL of the filtrate was mixed with 1 mL of concentrated sulfuric acid (H₂SO₄) and 2 mL of phenol solution. The reaction mixture was transferred to a cuvette and its absorbance was measured at 490 nm using a UV-Vis spectrophotometer. Total soluble carbohydrate content was quantified using a standard glucose calibration curve prepared from known glucose concentrations and measured at the same wavelength [13]. 3. Results and discussion 3.1. Percentage of Live Plantlets and Plantlet Visualization The survival of Dendrobium sp. plantlets was monitored weekly from the first to the fourth week after planting. Plantlets exhibiting green to yellowish coloration were classified as alive, whereas those showing brown discoloration were considered dead [14]. Table 1 Survival Percentage of Dendrobium sp. Plantlets at Various GA₃ Concentrations Treatment Percentage of Live Plantlets at Week (%) GA3 concentrations I II III IV 0 mg/L 100 100 100 100 1 mg/L 100 100 100 100 2 mg/L 100 100 100 100 3 mg/L 100 100 100 100 4 mg/L 100 100 100 80 Note 100 indicates 100% percentage of live Dendrobium sp. planlets from the first to fourth week. Based on Table 1. it was observed that from the first to the third week, all treatments—including the control (0 mg/L) and GA₃ concentrations ranging from 1 to 4 mg/L—exhibited a 100% survival rate of plantlets. This suggests that during the early stages of the culture period, Dendrobium sp. plantlets were able to tolerate the applied GA₃ concentrations effectively. World Journal of Advanced Research and Reviews, 2025, 27(01), 1056-1062 1059 Differences in survival began to emerge in the fourth week, particularly at the 4 mg/L GA₃ concentration, where the survival rate declined to 80%. In contrast, all other treatments maintained a 100% survival rate. The decline at 4 mg/L is likely due to the toxic effects of excessive GA₃, which may disrupt metabolic processes or induce physiological stress in the plantlets [15]. This trend is illustrated in Figure 1. Figure 1 Visualization of Dendrobium sp. plantlets in the fourth week on Vacin and Went (VW) medium with varying concentrations of GA₃. A = 0 mg/L (control), B = 1 mg/L, C = 2 mg/L, D = 3 mg/L, E = 4 mg/L 3.2. Plantlet Height Plantlet height was measured in the fourth week after the application of various GA₃ concentrations to the Vacin and Went (VW) medium. The effect of different GA₃ concentrations on plantlet height is presented in Table 2. Table 2 Average Height of Dendrobium sp. Plantlets in the Fourth Week After Application of Various GA₃ Concentrations Treatment Average height of planlets y± SE Fourth week 0 mg/L 4.56 ± 0.31ab 1 mg/L 5.58 ± 0.35b 2 mg/L 5.68 ± 0.31b 3 mg/L 4.86 ± 0.48b 4 mg/L 3.28 ± 0.32a Notes y= Average height of planlets; SE= Standard Error; Numbers followed by different letters indicate significant differences between treatments. Based on Table 2. the application of various concentrations of GA₃ to the VW medium had a significant effect on the average height of Dendrobium sp. plantlets after four weeks of treatment. The concentration of 2 mg/L GA₃ resulted in the highest average plantlet height, reaching 5.68 cm, indicating it was the most effective in promoting growth. This increase in height is likely attributed to the application of GA₃ at low to moderate concentrations, which can optimally stimulate growth in Dendrobium sp. plantlets. This finding is consistent with previous research, which reported that low concentrations of GA₃ promote both cell division and cell elongation [16]. In general, phytohormones at low concentrations tend to stimulate plant growth, while excessively high concentrations may inhibit plant height due to potential physiological stress or hormonal imbalance [15]. World Journal of Advanced Research and Reviews, 2025, 27(01), 1056-1062 1060 3.3. Number of Leaves The number of leaves is a critical parameter influencing various aspects of plant physiology and growth. Leaf number was recorded in the fourth week following the application of GA₃ to Dendrobium sp. plantlets. The results are presented in Table 3. Table 3 Average number of Dendrobium sp. leaves in the fourth week after GA3 addition Treatment Average number of leaves y± SE Fourth week 0 mg/L 3.8 ± 0.38a 1 mg/L 5.0 ± 0.45ab 2 mg/L 7.4 ± 0.83b 3 mg/L 5.6 ± 0.51ab 4 mg/L 3.8 ± 0.38a Notes y = Average number of leaves; SE= Standard Error; Numbers followed by different letters indicate significant differences between treatments. Based on Table 3. the application of various concentrations of GA₃ had a significant effect on the average number of leaves produced by Dendrobium sp. plantlets. The 2 mg/L concentration was found to be the most effective in promoting leaf formation. The increase in leaf number following GA₃ application aligns with the findings of [16], which reported that GA₃ at optimal concentrations enhances vegetative growth by stimulating leaf production. Gibberellic acid plays a vital role in promoting cell division and elongation, particularly in meristematic tissues, thereby accelerating the initiation of leaf primordia that develop into mature leaves. Additionally, GA₃ can stimulate shoot meristem activity, supporting the development of new vegetative organs, including leaves [16]. However, the use of GA₃ at excessively high concentrations may not only be ineffective in increasing leaf number but may also inhibit overall plantlet growth. This inhibition is attributed to phytotoxic effects, where hormonal imbalance leads to physiological stress, such as oxidative damage, disruption of photosynthetic processes, and suppression of cell division in meristematic regions. As a result, the formation of new leaves slows down, and the quality of the leaves declines, often appearing abnormally elongated, thin, or pale. Prolonged exposure to such conditions can significantly hinder plantlet development, including leaf production [16]. 3.4. Number of Roots The number of roots was recorded in the fourth week following the application of various GA₃ concentrations to Dendrobium sp. plantlets. The results are presented in Table 4. Table 4 Average Number of Roots in Dendrobium sp. Plantlets Treated with Various GA₃ Concentrations Treatment Average number of roots y± SE Fourth week 0 mg/L 4.40±0,51ab 1 mg/L 5.00±0,31ab 2 mg/L 6.00±054b 3 mg/L 4.60±0,67ab 4 mg/L 3.40±0,51a Notes y = Average number of roots; SE = Standard Error; Numbers followed by different letters indicate significant differences between treatments. World Journal of Advanced Research and Reviews, 2025, 27(01), 1056-1062 1061 Based on Figure 4. the application of various GA₃ concentrations had a significant effect on the average number of roots produced by Dendrobium sp. plantlets. The 2 mg/L GA₃ treatment resulted in the highest root number, indicating it was the most effective concentration for promoting root development. This increase can be attributed to the role of GA₃ in enhancing meristematic activity in root tissues and accelerating cell division. GA₃ is also known to stimulate the activity of enzymes and proteins associated with root initiation. However, excessive concentrations of GA₃ may induce phytotoxic effects, such as reduced physiological responses or hormonal imbalances particularly with hormones like auxins that are also involved in root formation ultimately inhibiting root development [17]. 3.5. Total Carbohydrate Analysis Total carbohydrate content serves as an indicator of the effectiveness of GA₃ treatment on plant metabolic activity. The total soluble carbohydrate content of Dendrobium sp. plantlets following the application of GA₃ to the Vacin and Went (VW) medium is presented in Figure 2. Figure 2 Average Total Carbohydrate Content in Dendrobium sp. Plantlets Treated with Various GA₃ Concentrations Based on Figure 2. the application of various GA₃ concentrations had no significant effect on the average total carbohydrate content of Dendrobium sp. plantlets. GA₃ is primarily effective in stimulating cell division in meristematic tissues and promoting stem elongation. However, it does not significantly influence leaf development after the budding stage, which may explain its limited impact on carbohydrate accumulation. Although GA₃ can enhance stem diameter through cell enlargement, this morphological change does not directly correlate with an increase in total carbohydrate content. Since leaves are the main organs responsible for photosynthesis, the limited number of leaves restricts the plant's ability to produce and accumulate carbohydrates efficiently [9]. 4. Conclusion Based on the results and discussion of this study, it can be concluded that GA₃ has a significant effect on the survival rate, root number, leaf number, and height of Dendrobium sp. plantlets cultured in vitro. The most optimal GA₃ concentration for promoting the growth of Dendrobium sp. plantlets was 2 mg/L. Compliance with ethical standards Acknowledgments The authors would like to express their sincere gratitude to the Botany Laboratory and the Plant Tissue Culture Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, University of Lampung, Bandar Lampung, Indonesia, for providing the facilities that enabled this research to be conducted. World Journal of Advanced Research and Reviews, 2025, 27(01), 1056-1062 1062 Disclosure of conflict of interest All authors have no conflicts of interest. References [1] Budiasih A, Amalia L, Parlinah L, Nurhayatini R, Nabila R. 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