SJA_42_4_1527-1537

Introduction

Orchids are ecologically well adapted to various habitats on the earth (Ramya et al., 2020). Orchidaceae is one of the largest and most diverse families of ﬂowering plants with more than 28,000 species classified around the different climatic zones of the world (Christenhusz et al., 2016). Although orchids are absent in polar and desert regions, they are abundant in the wet tropical regions, which divide them into two major categories, i.e., terrestrial and epiphytic orchids (Ogliore, 2024; Pérez-Escobar et al., 2024). Many of the orchid species are only locally distributed, whereas many wild species have become endangered (Ogliore, 2024). Associated with a large number of Orchidaceae species, orchids have extraordinary ﬂoral diversity representing a highly advanced and terminal ﬂoral evolution (Pérez-Escobar et al., 2024). Orchids are highly valued because of their long ﬂoral lifespan and exquisite ﬂowers of high diversity in floral size, variable colors, pleasant fragrance, aroma, ﬂoral form, and texture (Ramya et al., 2020). The unique interaction between pollinators and orchid ﬂower contribute to the richness of orchid species (Kirillova et al., 2023).

Thailand dominates the global orchid industry around the globe. In 2023, the top exporters of orchids were Thailand ($69.8M), Netherlands ($59.5M), Chinese Taipei ($35.3M), Vietnam ($7.46M), and Malaysia ($6.16M). The top importers of Orchids were Japan ($56.3M), United States ($24.7M), Vietnam ($14.4M), Italy ($13M), and Germany ($13M) (OEC, 2025). Between 2022 and 2023, the exports of Orchids grew the fastest in Netherlands ($3.19M), Slovenia ($2.62M), China ($606k), Chinese Taipei ($580k), and Denmark ($102k). Germany ($2.62M), Belgium ($1.4M), Zimbabwe ($1.26M), Macau ($382k), and Bulgaria ($322k) are the fastest-growing importers of orchids, as this data is published by OEC (2025).

Orchids are also used to treat medical complications, such as in the West Indies; residual water of boiled Bletia pupurea is used to treat food poisoning from fish and seafood (Kumaraswamy et al., 2022). Orchids are also part of the food in various regions of the world, such as in Malaysia; the leaves of Dendrobium salaccenseare cooked as a seasoning with rice (Tiwari et al., 2024). In certain parts of the Asian tropics, the tubers of some species of Gasrtrodia are eaten like potatoes Vanila planifolia Andr is an orchid, commercially cultivated for pods (beans) from which the popular flavoring substance called vanillin is extracted. Vanillin is mainly used in flavoring ice cream, soft drinks, condiments and oleoresins (Tiwari et al., 2024).

In vitro plant growth and development are mainly regulated by auxins and cytokinins (Kimoto, 2003; Hou et al., 2025). The interaction between two plant growth regulators is important to control the plant developmental processes. The meristems’ development is an important step to establish the whole plant body (Mohammadi et al., 2019). For instance, shoot meristems produce aboveground parts and root meristems produce underground plant parts (Zhang et al., 2023). Recent studies provide a better understanding of the molecular mechanisms of auxin and cytokinin interaction in the regulation of plant growth and meristem development, as described (Zhang et al., 2020). Similarly, auxin supplementation of the growth medium shows a synergistic effect for improving growth and root proliferation in Dendrobium orchids (Tini et al., 2025). Similarly, exogenous kinetin and auxin have a synergistic effect on root formation, while BAP and auxin have a synergistic effect on shoot formation (Manokari et al., 2021). Moreover, plant hormones such as auxin and cytokinin are critical for in vitro plant regeneration, with cytokinin playing an instrumental role in shoot organogenesis (Lee et al., 2022). The cytokinin signaling pathway represents a potential target for manipulating de novo shoot organogenesis and in vitro plant regeneration (Pokimica et al., 2024).

Plant tissue culture and micropropagation techniques play an important role in conservation programs and management of botanical collections (Oseni et al., 2018). Micro-propagation is a mass vegetative plant propagation system on an artificial nutrient medium under a controlled sterile environment to ensure pathogen-free, true-to-type, and rapid production (Rafique et al., 2012, 2013; Somaya et al., 2025). The production of these commodities through seeds further leads to variations and undesirable plant traits. Tissue culture has revolutionized the orchid industry due to the rapid and efficient multiplication rate under in vitro growth conditions. It is a significantly important alternative to conventional vegetative propagation of orchids through division (Lal et al., 2023). Vegetative propagation technique in orchids through division contains different hurdles, such as a high rate of mortality, a low rate of adventitious root formation, and high plant infectivity with nematodes and fungi. The plants produced through tissue culture provide much export potential as they are shipped internationally with limited quarantine restrictions (Rafique et al., 2013).

Although some previous studies have focused on micropropagation of orchids, there is still a need for an improved in vitro regeneration method. Orchid propagation, considering the effect of plant growth regulators, can help with large-scale multiplication, which can reduce the import of this plant. Therefore, the present research was carried out to develop an efficient protocol for mass multiplication of orchids by adjusting the auxin and cytokinin ratio in growth media.

Materials and Methods

Growth conditions and PGR treatments:

The experiments on standardization of protocols were conducted in the Plant Tissue Culture Cell at the Institute of Horticultural Sciences, University of Agriculture, Faisalabad. In vitro leaf disc cultures were used as an explant for the experiment. Uniform-sized leaf segments were cultured in test tubes containing 10 ml growth medium of pH 5.8. Sucrose 3% was added as a carbohydrate source, along with 0.8% agar was as a gelling agent for callus induction. The details on regeneration protocols for orchids were presented (Rafique et al., 2012, 2013). MS-Medium was supplemented with different auxins and cytokinins in variable concentrations, i.e. IBA+BAP and NAA+BAP (0.0+0.0, 0.5+2.0, 1.0+2.0, 1.5+2.0, 2.0+2.0, 2.0+0.5, 2.0+1.0 and 2.0+1.5 mg/L). The cultures were sub-cultured after four weeks on the same nutrient medium, and after 2 weeks, protocorm-like bodies (PBLs) were sub-cultured for organogenesis, in a growth room with temperature adjusted at 25 ± 2 ºC, 2000 lux light intensity, and a 16-hour photoperiod was maintained during the experiment.

Acclimatization of plants

Plantlets with four expanded leaves and roots were hardened on 1/2 strength basal MS medium for 4-6 weeks in glass jars and were transferred to small plastic pots containing 1:1 (w/w) mixture of peat moss and compost (autoclaved at 121°C for 30 min). Transplanted plants in pots were covered with polyethylene bags to maintain high humidity and partially cut after 2 weeks and fully opened after another 2 weeks for better acclimatization and transferred to the greenhouse. Potted plants were kept at 25 ± 1°C temperature and artificial light in a growth room 4–6 weeks. During this period, plantlets were moistened using a diluted 1/2 MS macro nutrient solution for better growth.

Growth parameters

Orchid cultures were observed daily for the whole experimental period days and changes in growth and development were observed. Data for traits: callus induction probability on visual observation, number of days to induce callus formation, Callus growth (mg), days to callus induction, fresh weight of callus (mg) after 20, 40, and 60 days, number of days for embryogenesis and embryogenesis percentage. Similarly, the number of emerged shoots, shoot length (cm) and number of roots were also observed.

Statistical analysis

All experiments were conducted in controlled conditions and laid out in a Completely Randomized Design (CRD) with four replications and six tubes per replication. Statistical analysis of experiments was performed using analysis of variance (ANOVA), and differences among treatment means were compared using the Least Significant Difference (LSD) test at the 5 % probability (P) level, with Statistix 8.1 software.

Results and Discussions

Effect of Media modification (MS-Medium + (NAA+BAP mg/L)) for better in vitro response of Dendrobium orchid

Callus induction from leaf disc culture

The interactive radar graph in Figure 1 indicates the callus fresh weight growth after 20, 40, and 60 days on MS-supplemented medium with different NAA and BAP concentrations, along with control, i.e., no auxin or cytokinins added. The results after twenty (20) days of culturing showed significantly higher callus weight (228 mg) in MS-medium modified with Naphthalene acetic acid and 6-Benzyleaminopurine (NAA+BAP) @1.5+2.0 mg/L compared to control (107.67 mg), i.e., unmodified MS-Medium. After 60 days of inoculation, higher calli weight was observed in MS medium modified with 1.5 + 2.0 mg/L (401.00 mg) and 2.0 + 2.0 mg/L (413.67 mg) compared with

the control (246.17 mg) (Table 1).

Days to callus initiation and callus growth rate

The results of our experiment also indicate that Naphthalene acetic acid and 6-Benzyleaminopurine (NAA+BAP) in modified MS-Medium at 2.0 + 2.0 mg/L showed early callus induction in (10.83 days), 1.5+ 2.0 mg/L (12.50 days) followed by (NAA+BAP), 2.0 + 1.5 mg/L (17.5 days) and 2.0 + 0.5 (16.66 days) mg/L compared with unmodified MS-medium (23.33 days). Callus induction was significantly earlier in MS-Medium 2.0 + 2.0 mg/L (Figure 3). Similarly, the growth rate of callus per day was higher for the 1st growth interval, i.e., initial 20 days of culture, than the growth rate for the 2nd growth interval (40 days). Rapid cell multiplication and callus growth rate per day have been recorded for the 3rd growth interval (60 days) as indicated in Figure 2. Moreover, callus induction probability was higher at 2.0 + 2.0 mg/L and 1.5+2.0 mg/L NAA+BAP treatments in comparison with other treatments and control.

Embryogenesis in callus cultures

Modified MS-medium supplemented with NAA+BAP (1.5 + 2.0 mg/L) showed100% embryogenesis, which was found to be the best treatment, followed by 1.0 + 2.0 mg/L with 95% embryogenesis (Figure 3). Lower response of embryogenesis was recorded at simple MS-medium as greater number of days, i.e., 22.5, were taken for embryo formation compared with modified

Organogenesis from PLBs

The organogenesis showed interesting results at varying NAA and BAP concentrations (Figure 4). Significantly higher shoot length (cm) was observed in MS-medium modified with 1.5 + 2.0 mg/L. (4.43 cm) compared with simple MS-medium (2.83 cm) followed by 1.0 + 2.0 mg/L (3.70 cm), 2.0 + 1.5 mg/L (3.66 cm) and 0.5 + 2.0 mg/L (3.60cm). Number of shoots significantly increased compared to control, and a greater number of shoots (16.16) were recorded in MS-medium modified with 2.0+2.0 mg/L NAA+BAP followed by 1.0 + 2.0 mg/L (15.13), 1.5+2.0 mg/L (14.33), 2.0+0.5 mg/L (6.58), and 0.5 +2.0 mg/L (7.08) compared to simple MS-medium. More roots were observed in MS-medium with 1.5 +2.0 mg/L, i.e., 2.2 per plantlet, followed by 2.0+1.0 mg/L, i.e., 2.1 per plantlet, compared with unmodified MS-medium, i.e., 0.35 per plantlet.

Effect of media modification (MS-Medium + IBA+BAP (mg/L)) for better vitro response in dendrobium orchid

Callus induction from leaf disc culture

The interactive radar graph indicates varying callus fresh weight gain after 20, 40, and 60 days on MS-supplemented medium with different NAA and BAP concentrations in comparison with the control (Figure 5). The results after twenty days of inoculation showed (<0.05) significantly higher callus weight in MS-medium modified with 2.0+ 2.0 mg/L IBA+BAP (224 mg), followed by 1.5 +2.0 mg/L IBA+BAP (220 mg). After forty days of inoculation, fresh callus formation was significantly high in MS-medium modified with 1.5 + 2.0 mg/L IBA+BAP (326 mg) and 2.0 + 2.0 mg/L IBA+BAP (316 mg).

After 60 days, significantly high callus fresh weight was recorded in MS-medium modified with 1.5 +2.0 mg/L IBA+BAP (396 mg) and 2.0+2.0 mg/L IBA+BAP (399 mg) compared with the control.

Number of days to callus initiation and callus growth rate:

The results of our experiment indicate that Indole acetic acid and 6-Benzyleaminopurine (IAA+BAP) in modified MS-Medium with 1.5+ 2.0 mg/L IAA+BAP showed early callus induction i.e., 10.8 days, for 2.0 + 2.0 mg/L 12.5 days, followed by IAA+BAP @ 2.0 + 0.5 mg/L 15 days, and for 2.0 + 1.0 mg/L 15.8 days compared with unmodified MS-medium, i.e., 23 days. Callus induction is significantly earlier in MS-Medium supplemented with 1.5 + 2.0 mg/L IAA+BAP (Figure 6). Rapid cell multiplication and callus growth rate per day have been recorded for the 3rd growth interval (60 days) as indicated in Figure 6 and Table 2. Furthermore, callus induction probability is higher for 2.0 + 2.0 mg/L and 1.5+2.0 mg/L IAA+BAP treatments in comparison with other treatments and the control.

Embryogenesis in callus cultures

Modified MS-medium with IAA+BAP (1.5 + 2.0 mg/L and 2.0 + 2.0 mg/L) with 100% embryogenesis was found to be the best followed by 1.0 + 2.0 mg/L with 95% embryogenesis, compared with various levels of Media modification in addition to control (Figure 7). Lower response of embryogenesis was recorded at unmodified MS-medium as a greater number of days, i.e., 22.5 days, were taken for embryo formation compared with MS-medium modified with 2.0 + 1.0 mg/L, i.e., 13.3 days, 2.0 + 1.0 mg/L, i.e., 19.1 days, 2.0 + 0.5 mg/L, i.e., 19.2 days and 1.5+2.0 mg/L, i.e.,

19.2 days as indicated in Figure 7. 2.0 mg/L IAA+BAP (17.66). The maximum number of roots was produced in MS-medium modified with IBA+BAP at 1.5 +2.0 mg/L, i.e., 2.3 per plantlet, compared to the control, as shown in Figure 8.

Organogenesis from PLBs

Organogenesis of the Dendrobium orchids indicates very fascinating results on varying IAA and BAP concentrations (Figure 8). Significantly more shoot length was observed in MS-medium modified with 1.5 + 2.0 mg/L IAA+BAP, i.e., 4.30 cm compared with simple MS-medium, i.e., 2.83 cm, followed by 1.0 + 2.0 mg/L, i.e., 3.86 cm, 2.0 + 2.0 mg/L, i.e., 3.62 cm, and 0.5 + 2.0 mg/L, i.e., 3.60cm. Results show that the maximum number of shoots was observed at 2.0 + 4.3

Discussion

Dendrobium orchid is used for direct and indirect somatic embryogenesis for mass multiplication of orchids (Deng et al., 2024). Cymbidium forrestii sps. have a significant response against 6-Benzyleadenine for cytoplasmic zone induction from apical meristem in direct and early leaf formation (Mosoh et al., 2024). Direct shoot induction is possible from leaf tips, callus driven from the shoot tip in orchids in MS-medium supplemented with NAA, BA, and IBA and this method is used for multiple shoot induction in present research. In orchids, direct shoot induction is the most significant method of plant regeneration. In direct regeneration, the shoots that first appeared are small, green protuberances at the leaf bases and develop into shoots without any callus or PLB formation, as shown in Figure 9. These PLBs are normally confused by callus formation, as described by Ahlawat et al. (2022). The PBLs are, in fact, in vitro seeds that can be further used for multiplication and mass regeneration of plants. The PLBs started rapid growth in 3–4 weeks after being placed on culture media, as shown in Figure 9.

During the present research, the least days, i.e., 10.8, were observed in MS-Medium modified with 2.0+2.0 (mg/L) of NAA+BAP and IBA+BAP for PLBs and calli formation. These results are consistent with Salem et al. (2022), who showed a similar response of growth regulators during in vitro propagation. The observations in the present research showed that the callus mass (413.67g) for 60 days after inoculation was increased two-fold higher 20 days after inoculation in MS-Medium modified with NAA+BAP with 2.0+2.0 mg/L. Embryos or PBLs in general, germinate freely comparatively better than the mature botanical nutrient flow, which is obstructed by the seed coat and seed dormancy (Lee et al., 2023). Moreover, the nutritional requirements of embryos during the initial stages of development are quite simple, facilitating their germination (Jolman et al., 2022). The present research showed excellent embryo formation and germination in MS-medium modified with NAA+BAP (21.6) and IBA+BAP (23.3) at the level of 2.0+1.5. The tendency of germination, development and protocorm proliferation using for growth regulators has been reported in some other orchid species, including Cymbidium aloifolium, C. giganteum C. pendulum, Dendrobium aphyllum and Epidendrum ibaguense as discussed by Guo et al. (2024) and Balilashaki et al. (2023). Previous studies show

fewer weak roots from multiplied adventitious shoots in MS-Medium modified with IAA, as described by Hossain et al. (2012) and Mastuti et al. (2017). The same rooting behavior was observed in orchids during the present research, as a few roots were developed in MS-medium modified with NAA+BAP, while the rooting response was slightly better in IBA+BAP (2.25) compared with NAA+BAP (2.22).

Conclusions and Recommendations

The present study standardizes an efficient in vitro protocol from leaf disc cultures through promoting PLB formation in supplemented MS-medium that would help the private sector in minimizing orchid imports. It is concluded that MS-media modification with BAP + IBA is better in terms of plantlet development and growth in the in vitro environment compared with different levels of NAA+BAP. MS-medium supplemented with 1.5+2.0 mg/L NAA+BAP or IAA+BAP performed well. Rooting was successfully induced on MS-medium supplemented with 1.5+2.0 mg/L of NAA+BAP or IAA+BAP. The optimized protocol will enable local production of orchid plant material for growers and other stakeholders at a lower cost and with less threat of invasive diseases, and would contribute towards a sustainable floriculture industry.

Acknowledgements

The authors highly acknowledge the infrastructural, financial and technical support of Plant Tissue Culture Cell (PTCC), IHS, University Agriculture, Faisalabad, Pakistan.

Novelty Statement

The study provides a simple but novel and efficient in

vitro propagation protocol for orchids that can effectively be used at a commercial scale.

Author Contribution

Rizwan Rafique: The research work was conducted.

Bilquees Fatima: Supervised

Muhammad Usman, Monis Hussain Shah, Tanzila Rafique and Muhammad Rafique Sajjad: Have contributed to analyzing results, drafting, and reviewing the paper.

Generative AI and AI-assisted technology statement

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

Declaration of competing interest

There exists no conflict of interest, and all co-authors agree with this submission. Authors adhere to and follow the publishing policy of the Journal.

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