Atractylodes macrocephala Koidz.(A. macrocephala), is a wild perennial medicinal plant found in the cool climate regions of Southeast Asia, including Korea, China and Japan. It has been cultivated for over 700 years for its medicinal properties (Zhu et al., 2018). Known by various names “Baekchul in Korea, “Bai-zhu” in Chinese, and “Baikujustu” in Japanese, A. macrocephala belongs to the Atractylodes genus and Asteraceae family. In China, it is classified as a homologous resource for medicine and food, and has been widely applied within food-medicine continuum (Wu et al., 2020; Liu et al., 2022; Luo et al., 2025a). A. macrocephala is incorporated into a variety of products as a functional food ingredient. Currently, several health care products on the market contain A. macrocephala rhizome (AMR), tangerine peel, and villous amomum fruit. The plant contains a variety of bioactive components, including hydrophobic lactones, volatile oils, and hydrophilic carbohydrates that supports its application in functional foods (Luo et al., 2025a). To date, over 180 compounds have been isolated from A. macrocephala, spinning diverse classes such as sesquiterpenoids, triterpenoids, polyacetylenes, coumarins, phenylpropanoids, flavonoids, flavonoid glycosides, benzoquinones, polysaccharides, among other compounds. These compounds are responsible for A. macrocephala’s wide range of pharmacological activities, including anti-tumor, immunomodulatory, anti-inflammatory, anti-oxidant, and neuroprotective effects, as well as support for gastrointestinal functions. As such, A. macrocephala is widely recognized for its significant medicinal, nutritional, economic, and ecological values (Zhu et al., 2018; Luo et al., 2025a). The plant has traditionally been used to manage several conditions including diabetes mellitus, digestive system diseases that include spleen disorder, gastrointestinal function, food intake reduction, abdominal distention, bloating, diarrhoea, boosting immune system and repairing gastrointestinal mucosal damage. The plant has also been reported to support the treatment and management of bone hyperplasia, headache, and obesity (Liu et al., 2022; Yan and Li, 2023; Singh et al., 2024; Yang et al., 2025). Several reports have indicated the use of A. macrocephala in managing cancer, edema, dizziness, treating tumor, and relieving inflammation (Jain et al., 2015; Luo et al., 2025b). Initially harvested from the wild, the plant has long been incorporated into traditional Chinese medicine, and its domestication has been practiced for many years. To preserve its genetic stability, A. macrocephala was traditionally propagated from seeds and cultivated in the open field (Chen et al., 2019). The seeds were collected from mature A. macrocephala plants consisting multiple capitula, forming a clear distinguished “banquet” (a cluster of flower heads) arranged in a regular pattern. These plants, typically harvested from the naturally growing ecosystem, have traditionally provided the seeds for propagation (Ye et al., 2022).

However, conventional propagation of A. macrocephala through seeds faces various challenges including, low seed germination rates, delayed multiplication, limited seed sets even with manual pollination, environmental stress during seed germination due to climatic variability, and a short seed viability period (Yang et al., 2023), (Mao et al., 2009), (Lee et al., 2025). Given these challenges, In vitro propagation emerges as an urgent and viable alternative for the effective cultivation of A. macrocephala. This technique has already been successfully used to rapidly and sustainably propagate several other traditional medicinal plants including Polygonum multiflorum (Kang et al., 2018), Codonopsis pilosula (Gang et al., 2023), Asparagus racemosus Wild (Bopana and Saxena, 2008), Rhodiolo dumulosa (Lu et al., 2023), among others. The application of in vitro propagation to A. macrocephala would ensure mass production of the plant’s raw materials, and steady supply for herbal medicine production. However, Pence (2011) emphasize that the success of in vitro propagation relies on the efficiency and scalability of the protocol. This paper presents a highly efficient and scalable in vitro propagation protocol of A. macrocephala, offering a potential strategy for the mass production and conservation of the plant. Although a few studies have explored the micropropagation of A. macrocephala (Mao et al., 2009; Tai et al., 2023), these studies have primarily focused on propagation using a narrow range of media and a limited evaluation of alternative plant growth regulators (PGRs). Additionally, no studies to date have compared in vitro-regenerated plants with their maternal counterparts in terms of genetic stability, physiological performance, FT-NIR spectra, and related traits. Such comparisons are important in validating the reliability of in vitro propagation, maintaining the genetic fidelity, and assessing potential physiological or metabolic variations that may affect large-scale cultivation or conservation programs. This gap is particularly notable following the observations of Duta-Cornescu et al. (2023), who highlighted the potential of micropropagation to induce somaclonal variations between maternal plants and tissue culture-regenerated plants. Additionally, Polivanova and Bedarev (2022) and Hazarika (2006) reported that tissue culture techniques can induce physiological disorder in in vitro-regenerated plants. Their observation underscore the need for more comprehensive research on in vitro-regenerated plants, as an important tissue culture technique. Therefore, The limited scope of the previous studies has hindered a comprehensive understanding of the plant's full in vitro propagation potential.

To fill these gaps, the current study provides a thorough and comprehensive analysis of the effects of various media and a range of PGRs at varying concentrations on shoot and root proliferation, aiming at optimizing shoot and root number and length in the in vitro propagation protocol for A. macrocephala. Additionally, a comparative analysis was conducted between in vitro-regenerated plants and maternal plants to evaluate their physiological parameters using Soil Plant Analysis Development (SPAD) readings, FluorPen FP110 measurements, and FT-NIR spectrometry to asses chlorophyll pigment content, photosynthetic efficiency, and chemical composition respectively. To examine potential somaclonal variations, genomic stability of the in vitro-regenerated A. macrocephala was assed using flow cytometry.

This research will play a critical role in optimizing A. macrocephala propagation techniques, ensuring genetic consistency, and providing insights that can support the sustainable cultivation and conservation of this homologus resource of medicine and food. Additionally, the findings of this study contribute significantly to the efficient mass propagation of A. macrocephala for commercial production.

Seed germination and Explant Preparation

A. macrocephala seeds used in this study were donated by Chungbuk Agricultural Research and Extension Services in Chungju, Korea. Seeds coats were gently and carefully removed, and the seeds were treated with detergent Tween 20 (0.1%) for 15 minutes and again washed with tap water until the detergents washed clearly. After that, the seeds were surface sterilized by dipping them in 70% alcohol for 2 minutes followed by 1% sodium hypochlorite solution for 15 minutes and were subsequently rinsed in sterilized water at least three times. The sterilized seeds were spread over a few sterile papers under a laminar flow hood to be dried for 5 minutes. For germination, Murashige and Skoogs (MS) basal medium (Murashige and Skoog, 1962), plus 30.0 gL^− 1 sucrose was used without any growth regulator. The pH of the medium was adjusted to 5.7–5.8 before adding 3.0 g/L gelrite prior to autoclaving at 121°C for 15 minutes Afterwards, the medium was poured into 90 x 15 mm disposable plastic petri dishes (25 mL into each of them). For efficient germination, the parafilm-sealed plates were kept in a growth chamber at constant temperature of 22 ± 2°C (16/8h photo period) under radiation of 35 µmol m^− 2s^− 1 provided by cool white fluorescent tubes (Master TL-D 840, Philips, Pila, Poland) at 70 ± 10% relative humidity. The seeds were thinly spread over the surface of MS basal medium under aseptic condition as seed in (Fig. 1A). Seedlings started to emerge after 2 weeks of culture and subsequently developed into a plantlet (Fig. 1B). After six weeks of growth, the plantlet derived from the seeds was selected as a primary explants, serving as the source of the shoot tip used for in vitro propagation.

Effect of growth media on the growth of A. macrocephala.

To evaluate the effect of The effects of six basal media on shoot and root development of A. macrocephala, excised shoot tip (0.8–1.3 cm) were cultured in 100 ml of gelled media (MS, DJ, LS, QL, WPM, and CHU) contained in polystyrene culture vessels (125 × 110 mm, Gaooze 1011C, Gyeonggi-do, South Korea). At inoculation, the initial shoot length was recorded. Each treatment consisted of 10 replicates with two explants per vessel as shown in (Fig. 1C1). The experiment was repeated three times. Cultures were maintained at 24–25°C under 500–1000 lux illumination with a 16/18 h light/dark photoperiod, using cool white fluorescent lamps providing a light intensity of 33.73 µmol m² s⁻¹ at 80% relative humidity. All media were supplemented with 30 g/L sucrose and vitamins, adjusted to pH 5.7–5.8 with 0.1 N NaOH or HCl, and solidified with 3 g/L gelrite before autoclaving at 121°C for ~ 20 minutes. Explants were inoculated in the solidified media without roots (Fig. 1C1). After six weeks of shoot tip culture (Fig. 1C2), data were collected from the plants focusing on mean shoot length and root number. Based on the collected data, MS produced the highest mean shoot length and root number and was therefore selected for subsequent experiments.

MS medium supplemented with vitamins was independently enriched with four different auxins naphthaleneacetic acid (NAA), Indole-3-butyric acid (IBA), Indole-3-Acetic Acid (IAA), and 2,4-Dichlorophenoxyacetic acid (2-4D) at concentrations of 0.1, 0.5, 1.0, 1.5, and 2.0 mg/L. Each medium was also supplemented with 30 g/L sucrose, and the pH was adjusted to 5.7 ± 5.8 with 0.1 NaOH or HCl before adding 3 g/L gelrite. All medium hormone combinations were autoclaved at 121°C for 20 minutes. After sterilization, 100 mL of the prepared medium was dispensed into polystyrene culture vessels (125 × 110 mm; Gaooze 1011C, Gyeonggi-do, South Korea). Shoot tips measuring 20–40 mm were excised from previously proliferated plants. Shoot tips were placed vertically into the cooled, solidified MS media containing different auxin at varying concentrations. Each test tube contained one shoot apex (Fig. 1E1), with 10 replicates per treatment. After six weeks of inoculation (Fig. 1E2), root number, root length, and root surface were recorded for all treatments. The root surface area was obtained using the WinRHIZO Pro software (Reagent Instruments, Queben, Canada) after acquiring 2D root images (Fig. 1E3) by scanning the roots using an expression 12000XL scanner (Epson, Sueas, Nagano, Japan) developed per explant was recorded.

The experiment was conducted in a completely randomized design (CRB) with 10 replications. Data were recorded as centimeters for shoot length and root length, number of components for each plant, square centimeter for roots surface area, grams for fresh, dry weight of leaves, leaf powder and root powder. The cultures were periodically monitored to record the percentage response for shoot bud regeneration, multiple shoots development and rooting. The total number of shoots and roots were determined by visual observation, root surface area was assed using WinRHIZOR, weight of leaves was measured using weighing scale.

After six weeks of culturing, well-rooted A. macrocephala plantlets were taken out of test tubes and carefully washed with sterile water to remove any remaining medium adhering to the roots (Fig. 1F). The cleaned plantlets were transplanted into plastic pots (7 cm in diameter, 8 cm in height) containing a sterile horticultural soil and perlite mixture (2:1, pH 6.5). Each pot was covered with a transparent plastic bag to maintain adequate humidity during the hardening process (Fig. 1H). Watering was carried out twice weekly for the first three weeks and then reduced to once weekly in the following weeks. After 15 days, the plastic covers were gradually removed, allowing the plantlets to adapt to natural conditions. The survival rate of plantlets was evaluated after eight weeks of acclimatization (Fig. 1H), the survival rate of the plants was determined.

Figure 1

After nine weeks of acclimatization, the leaf chlorophyll content of in vitro-regenerated and maternal A. macrocephala plants was assessed using a calibrated Soil Plant Analysis Development (SPAD) chlorophyll meter (SPAD-502 Plus, Konica Minolta, Inc., Japan) as shown in (Fig. 2A). For each plant, eight leaves were randomly selected: One pair from the apical region, two pairs from the mid-stem, and one pair from the lower stem. SPAD measurements were taken at four different points per leaf as shown in (Fig. 2B), and the average value was calculated for each leaf. The mean of the eight leaf averages was considered the overall chlorophyll content for that plant. Measurements were recorded weekly over eight weeks for a total of 20 plants (10 in vitro-regenerated and 10 maternal plants) and the results are as shown in (Fig. 2C).

Figure 2

To assess the relative water content (RWC) of the leaves from in vitro regenerated and A. macrocephala maternal plants, the technique was modified from (Hewlett and Kramer, 1963). The measurement was conducted after 10 weeks of acclimatization. Fully expanded leaves that had received light were selected for the experiment. During preparation, empty sample tubes were numbered and weighed. Five fully expanded leaves were selected and cut from randomly chosen plants in each plant together with their leaf stalk as shown in (Fig. 4A). The top and bottom parts of each leaf were removed, and any dead or dying tissue was discarded as illustrated in (Fig. 4B). The prepared leaves were immediately placed into the centrifuge tube and sealed with lid to prevent moisture loss or gain. The sample tubes were weighed (tubeW + FW) as illustrated in (Fig. 4C). Next, 1 cm of distilled water was added to each tube as shown in (Fig. 4D), and the tubes were placed in a refrigerator at 4°C in darkness for 24 hours, allowing the leaves to reach full turgidity. After 24 hours, the leaves were carefully removed from the tubes, blotted dried with a paper towel as illustrated in (Fig. 4E), and weighed (TW; turgid weight). The leaf samples were then placed in labeled envelopes and dried at 60°C for 24 hours or until they reached a constant mass as shown in (Fig. 4F), after which the dry weight (DW) was recorded.

To calculate the fresh weight (FW) of the leaf samples, the formula used was:

FW = tubeW + FW - tubeW

Finally, the leaf RWC was calculated using the formula:

Where: FW = Fresh weight, TW = Turgid weight, DW = Dry weight., tube W = tube weight

Figure 4

The genomic stability and ploidy level of in vitro regenerated A. macrocephala plants were assessed using flow cytometry, following the protocol described by Abdolinejad et al. (2020) with slight modifications. In brief, leaf samples were randomly collected from both in vitro regenerated and maternal plants. Secale cereale, with a known 2C DNA content of 16.19 pg, (Zwyrtková et al., 2020) was used as an internal standard. Approximately 0.5 cm² of leaf tissue from each sample and the internal standard were placed in a plastic petri dish. To each dish, 500 µL of nuclei extraction buffer (CyStain UV Precise P Nuclei Extraction Buffer; Sysmex Partec, Germany) was added, and the tissues were finely chopped with a sharp razor blade for 40 seconds. The resulting suspension was incubated for 30–90 seconds and subsequently filtered through a 50 µm mesh filter (CellTrics, Sysmex Partec, Germany) into sample tubes. Each filtrate was then supplemented with 2000 µL of staining solution, consisting of staining buffer (CyStain UV Precise P Staining Buffer), propidium iodide, and RNAse A stock solution at a ratio of 333.333:2:1. Samples were shielded from light and incubated at room temperature (25°C) for 60 minutes. Fluorescence intensity of the isolated nuclei was measured using a flow cytometer (BD FACSCalibur, USA). The ploidy levels of both in-vitro regenerated and maternal A. macrocephala plants were determined by comparing their relative fluorescence intensities against that of the internal standard, according to the following formula:

2C DNA content of A. macrocephala (pg) =

Where:

In vitro propagation, a key component of plant biotechnology, has proven to be a reliable method for the rapid mass production of high-quality, disease-free medicinal plants. It also supports the propagation of species that are challenging to reproduce through conventional methods (Gaurav et al., 2018; Anuruddi et al., 2023). Additionally, In vitro propagation, is known for maintaining the genetic stability of regenerated plants without exhibiting somaclonal variations while supporting the enhancement of desirable traits such as high secondary metabolites content (Bidabadi and Jain, 2020; Duta-Cornescu et al., 2023). Within the food-medicine continuum, this is particularly important, as maintaining both the medicinal efficacy and nutritional quality of species like A. macrocephala is essential for their dual role in healthcare and diet. Thus, in vitro propagation provides a sustainable approach for mass production, and conservation or medicinal plants including homologous food-medicine resources .

Effects of culture media, cytokinin, and auxins on A. macrocephala seed germination, shoot, and root growth

Following sterilization process of the seeds, they were carefully placed in MS basal medium (Murashige and Skoog, 1962) for germination. Under aseptic conditions, the seeds were evenly spread on the surface of the medium as indicated in (Fig. 1A). Germination began approximately two weeks after culture initiation, and zygotic plantlets aged 21 days were selected as primary explants for subsequent in vitro experiments. A similar study that used MS free from regulators for the germination of seeds is the germination of Polygonum multiflorum (Kang et al., 2018). To identify the optimal medium for A. macrocephala growth, explants from germinated seeds were transferred to and tested across different media. Plant tissue culture media serve as the primary source of mineral nutrients and water, both essential for supporting plant growth and development under in vitro conditions. Their composition act as key factors that influencing the success of in vitro propagation, as they play a vital role in directing morphogenesis by enabling plant tissues and cells to develop into whole organs or complete plants (Monfort et al., 2018; Jayusman et al., 2022). However, different culture media formulations vary in their composition, particularly in macronutrient content and total ion concentrations, which significantly influence plant morphogenesis (Okello et al., 2021a; Gang et al., 2023). For instance, MS medium is characterized by high levels of nitrate (39.4 mM) and ammonium (20.6 mM) ions but contains a low concentration of sulfate (1.7 mM). In contrast, QL medium has a lower ammonium content (5.0 mM), while Woody Plant Medium (WPM) features both low ammonium (5.0 mM) and nitrate (9.7 mM) levels, but relatively high sulfate content (7.5 mM) (Okello et al., 2021a). Because of the significant variation in the mineral composition of the media, this study observed notable differences in individual growth parameters of A. macrocephala across different culture media. Nevertheless, all media contributed to the plant's growth, as each provided a distinct nutrient profile, suggesting that while nutrient availability varied, all tested media were capable of supporting plant development to varying degrees.

The present study indicates that MS exhibited the best growth parameters (height of plant and number of roots) on A. macrocephala. Increase in shoot length in MS medium was (1.47 ± 10.12%), however, the percentage increase in shoot length in MS medium did not significantly differ from the percentage increase recorded in LS (1.36 ± 13.03%) and WPM (1.35 ± 12.78%) media as indicated in (Fig. 6A). The smallest percentage increase in shoot length (0.71 ± 40.92%) was observed in CHU medium. Regarding root development, MS medium yielded the greatest root formation, with an average of (7.8 ± 1.06) roots. This was significantly different compared to other media as illustrated in (Fig. 6B). The CHU medium recorded the lowest root formation, with an average of (1 ± 0.80) root. The height of the plant and the number of roots are important parameters for evaluating the effectiveness of growth media in in vitro propagation. These indicators reflect the overall vigor and developmental success of plantlets in response to the nutrient composition of the medium. Taller plants typically signify enhanced shoot proliferation and nutrient uptake, while a greater number of roots indicate efficient root induction and adaptation to the culture environment (Hussain et al., 2022). Together, these traits provide a reliable measure of the medium’s ability to support balanced morphogenesis and healthy plantlet development. In addition to plant height and length or roots, plants cultured in MS medium produced dark green, healthier leaves compared to plants culture in other media. According to Okello et al. (2021a), leaf color serves as a visual indicator of a plants’ nutritional status and physiological health. These colors reflect availability and balance of essential nutrients within the culture medium. In this context, MS medium, known for its high concentrations of macronutrients, particularly nitrate and ammonium, often supports vigorous growth and the development of healthy, deep green foliage in many plant species (Alamgir, 2017; Isah and Umar, 2020). The richness of MS medium in essential elements contributes to enhanced chlorophyll synthesis, which is directly linked to the dark green colour of the leaves (Razavizadeh et al., 2023). Thus, the presence of vibrant green leaves in plantlets grown on MS medium can be attributed to its nutrient-rich composition, which promotes overall plant health and robust physiological performance. Successful use of MS in the propagation of in the same family “Asteracea” have been recorded in Sausurea esthonica (Gailīte et al., 2010), Susurea medusa Maxim (Liu and Saxena, 2009), Tripleurospermum insularum (Inceer et al., 2022), Calendula maritima Guss (Catalano et al., 2022), Tripleurospermum ziganaese (Cuce and Inceer, 2024). However, other studies have demonstrated that certain Asteracea species respond better to alternative media WPM and LS media. For instance, WPM has been effectively used in the propagation of Rhaponticoides mykalea (Hayta et al., 2017), and Achyrocline satureioides (Guariniello et al., 2018), while Stevia rebaudiana Bertoni exhibited enhanced shoot elongation in LS medium (Miladinova-Georgieva et al., 2023). These findings align with observations in the present study, where no significant differences in shoot length were recorded for the lengths of A. macrocephala across MS, WPM, and LS media, suggesting that all three media are capable of supporting comparable growth performance in this species.

Cytokinins, a group of essential plant hormones are classified into two main types: purine-type cytokinins and phenyl urea-type cytokinins (Grzegorczyk-Karolak and Hnatuszko-Konka, 2021). Both types play a central role in in vitro propagation by regulating key processes involved in plant growth and development. Their primary function is to promote cell division and shoot formation, making them essential components in most plant tissue culture protocols (Schoene and Yeager, 2005; Asghar et al., 2023). In the present study, both types were used. The purine-type cytokinins applied included BAP, TDZ, and 2iP, while TDZ, a phenyl urea-type cytokinins was also applied. Despite their structural differences, both types effectively promote shoot proliferation and multiplication in the in vitro propagation of medicinal plants (Kapchina-Toteva et al., 2000). Effective shoot proliferation and multiplication largely depends on culture medium enriched with cytokinins, which serve as key regulators in tissue differentiation and morphogenesis (Cavallaro et al., 2022). According to (Kumari et al., 2018), Plant growth regulators (cytokinins and auxins) affect various physiological functions in plants, particularly the initiation and multiplication of shoots and roots.

In this study, addition of various cytokinins at different concentrations in MS medium promoted shoot proliferation and multiplication. Considering the mean number of shoot per explants and the length of initiated shoot the best results were achieved in BAP under the concentration of 1.5 mg/L was the most effective in promoting shoot initiation and multiplication from A. macrocephala shoot with 17.5 ± 1.21 shoots generated from 1 shoot apex as indicated in (Fig. 7A). The same concentration (MS medium supplemented with BAP 1.5 mg/L) exhibited the greatest increase in shoot length (320.0 ± 11.93%), making it significantly different from other MS-cytokinin combinations. Additionally, plants in MS medium-BAP 1.5 mg/L combination developed dark-green leaves-an indicator of good physiological health, making them more suitable for further development. Su et al. (2023) indicates that leaf colour is an indicator of healthy plant as dark-green leaves exhibit higher expression of photosynthesis-related genes and greater chlorophyll content, which correlated with improved photosynthetic capacity and potential crop yield, while (Dutta Gupta et al., 2013) illustrates that dark green leaves typically reflect adequate or high levels of essential nutrients like nitrogen and magnesium, key components for chlorophyll synthesis whereas pale or yellowing leaves signal nutrient deficiencies. Overall, MS medium supplemented with 1.5 mg/L BAP was identified as the optimal medium-hormone combination for enhancing shoot proliferation, multiplication, and elongation in A. macrocephala. Plants grown under this treatment developed dark-green leaves-an indicator of good physiological health and consistently outperformed those in all other treatments across all evaluated parameters. A similar observation of BAP producing healthy plants with dark-green leaves were observed in the propagation of Ficus benjamina (Benedetto et al., 2020). Studies that indicate the superiority BAP concentrations over other cytokinins include the propagation of; Aspilia Africana (Okello et al., 2021a), Hedyyotis biflora (Linn.) Lam (Revathi et al., 2019), Ipomoea batatas (L.) Lam. (Dewir et al., 2020), and Crataeva nurvala (Buch Ham) (Kher and Nataraj, 2020). The results of this study indicate that the mean number of shoots increased as BAP concentrations rose, reaching an optimum at 1.5 mg/L. Beyond this level, additional increases in BAP led to a reduction in shoot proliferation. This trend aligns with the findings of Wang et al. (2018) who reported that exceeding the optimum concentration can hinder shoot regeneration.

A similar optimized concentration of BAP 1.5 mg/L was reported by Khan et al. (2015) in a study on shoot regeneration in grapes (Vitis vinifera Vitaceae family), where the highest shoot regeneration frequency (53.33%) was achieved on MS medium supplemented with 1.5 mg/L BAP and 0.5 mg/L NAA. The regenerated shoots subsequently rooted successfully on a hormone-free half-strength MS medium. Similarly, Jafari et al. (2017) observed optimum shoot proliferation in Passiflora caerulea L. (Passifloraceae family), where the highest regeneration frequency (90%) and a maximum of 8.86 shoots per cotyledonary node explants were recorded on MS medium supplemented with BAP 1.5 mg/L. Although these species belong to different families other than A. macrocepha (Asteraceae), the consistent effectiveness of 1.5 mg/L BAP across these studies supports that this concentration could be broadly applicable for shoot induction, suggesting that optimal cytokinin levels may transcend family boundaries under certain culture conditions. However, the findings of the current study differ from several previous reports on members of the Asteraceae family, where different concentrations of BAP have been reported as the optimum for shoot proliferation. For instance Pandey et al. (2014) identified an optimal BAP concentration 1.15 mg/L for Spilanthes calva, (a members of Asteraceae family), producing up to 4.17 shoots per explant. Additionally, Okello et al. (2021a) found that on 1.0 mg/L resulted in the highest shoot proliferation in Aspilia africana, (a member of Asteraceae family), with an average of 13 shoots per nodal segment after six weeks of culture. This deviation in A. macrocephala suggests a species-specific response to cytokinins, even within the same family, emphasizing the need for empirical optimization rather than relying solely on trends within a taxonomic group.

Auxin hormones are essential for initiating meristematic competence in plant cells during in vitro propagation. They enable cells to regain the ability to divide and differentiate into root-forming cells, an important step for successful rooting (Pacurar et al., 2014; Ribeiro et al., 2022). By inducing the formation of root primordia and promoting the growth of functional roots, auxins are fundamental to the rooting process. Their careful manipulation in tissue culture media ensures efficient plant regeneration from small explants, making auxins key to the success of in vitro propagation and root development (Du and Scheres, 2018). These hormones promote root elongation, stimulate the formation of additional roots, and enhance the root surface area of the plant during in vitro propagation (Nibau et al., 2008; Komakech et al., 2020b). Several other studies have demonstrated varied rooting responses to different auxin hormones at varying concentrations in in vitro plant propagation, even within the same genus (Okello et al., 2021b).

In the current study, excised A. macrocephala shoot tips were rooted in MS medium with vitamins, supplemented with 30 g/L of sucrose and various auxin hormones at different concentrations. After six weeks of shoot tips induction, 100% rooting success was observed at all concentrations of NAA, IBA, IAA and MS as control, while no rooting occurred at any concentration of 2,4-D. Notably, root elongation, root number and root surface area varied across the different concentrations of auxin hormones. Results in this study indicated that the highest rooting mean number was observed in MS medium fortified with NAA2 with a maximum 25.8 ± 2.8 roots. This result was statistically different from all other treatments including the control (MS without auxins). The rooting mean number in NAA increased with increasing concentration. The second highest mean number of roots was observed with NAA 1.5 mg/L 22.4 ± 1.43, followed by MS medium fortified with IAA 1.5 mg/L with 21.2 ± 1.32, which was followed by NAA 1.0 mg/L 25 ± 1.58 (Fig. 8A). However, the highest root length recorded after six weeks of shoot tip culture was observed in MS medium fortified with IAA 1.5 mg/L measuring 8.76 cm ± 0.85. This root length was significantly different from all other medium-auxin combinations (Fig. 8B). This aligns with the findings by (Komakech et al., 2020b) that registered 1.5 mg/L in IAA with the longest roots. The significance of IAA concentrations to regenerate longest roots as compared to other auxin hormones have been recorded in the study about Polygonum multiflorum (Kang et al., 2018). In IAA treatments, root length initially increased with rising concentrations up to the optimum point, beyond which it declined as indicated in (Fig. 8B). The trend of increasing length of roots in IAA until the optimized concentration then start to decline was registered in studies Polygonum multiflorum (Kang et al., 2018), and Apios americana Medik (Gang et al., 2024b). Regrading root surface area, the highest value was observed in shoots treated with IAA 1.5 mg/L showing a surface area increase of 252.2 cm² ± 4.92 after six weeks of culture. This was significantly different from all other hormone concentrations. Similar findings identifying IAA as the optimal auxin for enhancing root surface area have also been reported in studies on Bellis perennis L. (Karakas and Turker, 2013), Stevia rebaudiana Bertoni (Ahmed et al., 2007).

According to Palta and Watt (2009), a robust and extensive root system is essential for supporting resilient, healthy, and vigorous plant growth. Based on this principle, IAA 1.5 mg/L was selected as the most effective hormone for the subsequent experiments in A. macrocephala, as it produced the healthiest roots, with long roots and roots with high surface area. In contrast, MS medium supplemented with 2 mg/L NAA induced a higher number of roots, but these were shorter. As illustrated by (Loveys et al., 2003), numerous but short roots are less effective for plant acclimatization because they provide limited anchorage, reduced nutrient absorption capacity, and poorer water uptake during the transition to ex vitro conditions. For this reason, plants with longer, well-developed roots were preferred over those with many short roots.

Acclimatization of rooted in vitro-regenerated plants is an important step to ensure their successful transition from the in vitro environment to field conditions, requiring a period of adaptation (Sharma et al., 2023). The process helps the plants adjust to fluctuating external factors such as temperature, humidity, and light intensity, which they may not have encountered in the sterile culture environment. This adaptation phase is essential for reducing transplant shock and enhancing the plant’s ability to thrive and grow in the field, ensuring high survival rates and improved growth post-transplant (Shiwani et al., 2022; Sharma et al., 2023). In the current study, thirty well-rooted and healthy A. macrocephala plantlets, derived from the optimized conditions of the Auxin optimization experiments, were carefully removed from the culture medium. The roots were thoroughly washed to eliminate residual agar as illustrated in (Fig. 1F), and the plantlets were then transferred to sterile horticulture soil mixed with perlite at a ratio of 2:1, placed in plastic pots (22 cm diameter) for the acclimatization process as illustrated in (Fig. 1G). After two months, twenty eight out of thirty plants (93.33%) successfully survived as observed by plant representations in (Fig. 1H). The success of the acclimatization demonstrated the effectiveness of the acclimatization protocol. Furthermore, the plants remained healthy and robust after eight months of continuous growth, indicating their successful transition to ex vitro conditions. High survival rates have also been reported for other plant species within the same family, such as 100% survival for E. scaber (Abraham and Thomas, 2016), 95.7 for Aspilia Africana (Okello et al., 2021a), and 90–100% for E. alba (Singh et al., 2012).

The present study describes a standard protocol for the mass propagation of a valuable medicinal plant A. macrocephala from shoot tips as explants. MS containing vitamins supplemented with 30 g L^− 1 sucrose and 1.5 mg/L BAP regenerated the overall best shoot proliferation results with 17.5 ± 1.21 dark green, healthy shoots after six weeks of shoot tip induction, exceeding the 14.5 and 5.61 shoots reported in previous studies. While MS containing vitamins supplemented with 30 g L^− 1 sucrose and IAA 1.5 mg/L provided the optimum rate 21.2 ± 1.32 of in vitro root proliferation, facilitating root elongation of 8.76 cm ± 0.85, and root surface area of 252.2 ± 4.92 after 6 weeks explant induction. The observed similarity in the physiological and chemical composition between the in vitro regenerated and A. macrocephala maternal plants further confirms that this protocol produces genetically and physiologically stable plants. Importantly, this in vitro propagation method enables the mass production of high quality plantlets, offering an effective solution associated with seed-based propagation such as low seed germination rates, slow multiplication cycles, limited seed set even under manual pollination, sensitivity to environmental stress during germination, and the short viability period of A. macrocephala. By producing large numbers of uniform plants through in vitro propagation, this protocol reduces dependance on natural habitat as a seed source, thereby supporting both conservation efforts and large-scale commercial cultivation. Overall, the study presents a rapid, efficient, and replicable method for direct shoot and root multiplication from shoot tip explants. Its simplicity, high multiplication rate, and ability to produce uniform, disease-free plants make it suitable for commercial-scale propagation, germplasm preservation, and domestication of A. macrocephala, meeting the increasing demand in the herbal industry. Additionally, this protocol strengthens the food-medicine continuum, where A. macrocephala serves as both a therapeutic and dietary resource. This reliable in vitro propagation thus secures a consistent supply of high-quality raw material for herbal medicines and functional food products, meeting growing demand while safeguarding biodiversity and sustainability.