Effect of plant growth regulator concentrations on callus induction and shoot regeneration of Andaliman

Efeito das concentrações de reguladores de crescimento vegetal na indução de calo e na regeneração de rebentos do Andaliman

Abstract

Andaliman (Zanthoxylum acanthopodium DC.) is an important spice-producing plant native to North Sumatra, particularly valued by the Batak community. However, the limited availability of uniform planting materials remains a constraint to its cultivation and sustainable utilization. This study aimed to determine the effects of NAA × BAP combinations on callus induction and growth characteristics and to evaluate the subsequent response of induced callus to IAA × TDZ treatments for shoot regeneration. A factorial Completely Randomized Design was used to evaluate callus weight, diameter, height, texture, structure, and colour, followed by descriptive assessment of shoot regeneration. Data were analysed using factorial analysis of variance followed by Duncan’s Multiple Range Test at the 5% significance level. NAA × BAP combinations significantly affected callus weight, diameter, and height. The highest callus fresh weight (96 mg) and height (11.33 ± 2.08 mm) were obtained with 5 mg/L NAA + 10 mg/L BAP, whereas the largest diameter (16.33 ± 1.53 mm) occurred with 1 mg/L NAA + 5 mg/L BAP. Shoot formation occurred in only 5 of 27 cultures (18.5%), with a maximum of three shoots per culture; the highest observed shoot response occurred at 1 mg/L IAA + 1.5 mg/L TDZ. These findings demonstrate regeneration potential rather than an efficient propagation protocol and provide a basis for further optimization for conservation and future commercial propagation of Andaliman.

Keywords:
Zanthoxylumacanthopodium DC.; in vitro propagation; callus induction; shoot regeneration; plant growth regulators

Resumo

O andaliman (Zanthoxylum acanthopodium DC.) é uma importante planta produtora de especiarias, nativa do norte de Sumatra, particularmente valorizada pela comunidade Batak. Contudo, a disponibilidade limitada de materiais de plantio uniformes permanece um obstáculo ao seu cultivo e à sua utilização sustentável. Este estudo teve como objetivo avaliar os efeitos das combinações de NAA × BAP na indução de calo e nas características de crescimento, bem como analisar a resposta subsequente do calo induzido aos tratamentos com IAA × TDZ para a regeneração de brotos. Utilizou-se um delineamento fatorial completamente aleatório para avaliar o peso, o diâmetro, a altura, a textura, a estrutura e a cor do calo, seguido de análise descritiva da regeneração de brotos. Os dados foram submetidos à análise de variância fatorial, seguida do Teste de Intervalo Múltiplo de Duncan, ao nível de significância de 5%. As combinações de NAA × BAP afetaram significativamente o peso, o diâmetro e a altura do calo. O maior peso fresco (96 mg) e a maior altura (11,33 ± 2,08 mm) foram obtidos com 5 mg/L de NAA + 10 mg/L de BAP, enquanto o maior diâmetro (16,33 ± 1,53 mm) ocorreu com 1 mg/L de NAA + 5 mg/L de BAP. A formação de brotos ocorreu em apenas 5 das 27 culturas (18,5%), com um máximo de três brotos por cultura; a maior resposta foi com 1 mg/L de IAA + 1,5 mg/L de TDZ. Esses resultados demonstram potencial de regeneração, mas não configuram ainda um protocolo eficiente de propagação eficiente, fornecendo uma base para otimizações futuras voltadas à conservação e à propagação comercial do andaliman.

Palavras-chave:
Zanthoxylum acanthopodium DC.; propagação in vitro; indução de calo; regeneração de brotos; reguladores de crescimento vegetal

1. Introduction

Andaliman (Zanthoxylum acanthopodium DC.) is a spice-producing plant belonging to the family Rutaceae and is indigenous to the highlands of North Sumatra, Indonesia, particularly the Lake Toba region (Wijaya and Napitupulu, 2019). It is traditionally used by the Batak community as a culinary spice and has also attracted scientific interest because of its phytochemical and pharmacological properties. Andaliman contains diverse bioactive compounds, with terpenoids and their derivatives being among the major constituents, together with alkaloids, flavonoids, glycosides, tannins, and saponins (Adrian et al., 2023; Agnihotria et al., 2022; Hutapea et al., 2024). Several terpenoid compounds, including limonene, linalool, citronellol, and geranyl acetate, have been identified in Andaliman and are associated with various biological activities (Asbur and Khairunnisyah, 2018; Kholibrina and Aswandi, 2021). The combination of its cultural, culinary, and potential pharmacological value highlights the importance of developing reliable propagation methods for this indigenous spice plant (Pardede and Manik, 2019).

Despite its importance, the availability of uniform Andaliman planting materials remains limited (Fransiska and Sujarwati, 2023). Generative propagation is constrained by poor and irregular seed germination. Previous studies reported that Andaliman seeds germinate slowly and at relatively low percentages, with germination extending over several weeks (Siregar, 2013). More recent research has confirmed the difficulty of obtaining high and uniform germination, with physiological maturity and seed treatment influencing germination performance; even the most effective treatment in a recent study produced a germination percentage of only 20% (Fahmi et al., 2026). These constraints can limit the availability of seedlings for cultivation and may hinder the expansion and sustainable utilization of Andaliman (Siregar, 2003). Therefore, alternative propagation techniques that can produce more uniform planting materials are required (Phuyal et al., 2018).

In vitro propagation through plant tissue culture provides an alternative approach for overcoming limitations associated with conventional propagation (Kulus and Tymoszuk, 2024; Basri, 2016). Tissue culture is performed under controlled and aseptic conditions using a nutrient medium, commonly Murashige and Skoog (MS) medium, which provides essential macro- and micronutrients required for explant growth (Neumann et al., 2009; Smith, 2006). The response of explants in vitro is strongly influenced by the type and concentration of plant growth regulators (PGRs), particularly auxins and cytokinins. Their relative balance regulates cell division, cell differentiation, callus formation, and organ development (Fauziah et al., 2019). For callus induction, NAA and BAP are frequently used in combination because NAA provides auxin activity that promotes dedifferentiation and callus formation, while BAP, a synthetic cytokinin, promotes cell division and proliferation (Maninggolang et al., 2018; Lestari et al., 2013). Thus, the interaction between NAA and BAP concentrations may determine the quality and growth characteristics of the resulting callus (Nicole et al., 2023).

However, successful callus induction does not necessarily result in efficient plant regeneration. The induced callus must subsequently undergo organogenic development to produce shoots. Therefore, a second stage involving a different PGR combination is required to stimulate shoot formation. IAA is an endogenous auxin that can regulate cell division, differentiation, and organ development, whereas TDZ is a potent cytokinin-like compound that can stimulate cell division and adventitious shoot formation (Kasutjianingati et al., 2011; Nurmaningrum et al., 2017). The combination of IAA and TDZ has been reported to promote shoot induction in several plant species (Tefera and Wannakrairoj, 2006). In the present study, these regulators were therefore applied sequentially after callus induction to evaluate whether the induced callus could be converted into shoots. This two-stage approach distinguishes the requirements for callus formation from those for subsequent shoot regeneration (Timburas et al., 2023; Yusnita et al., 2018; Fauziah et al., 2021).

Although various PGRs, including 2,4-dichlorophenoxyacetic acid (2,4-D) (Munawar et al., 2024; Khoirunnisa and Mercuriani, 2022; Satria and Jasminarni, 2019) and indole-3-butyric acid (IBA) (Alizadeh and Dumanoğlu, 2022; Dreger and Szalata, 2022; Harahap et al., 2021), have been widely used in plant tissue culture, their responses are highly dependent on plant species, explant type, and concentration (Samanhudi, 2010). The present study focused on NAA and BAP for callus induction because of their complementary auxin–cytokinin functions, followed by IAA and TDZ for shoot regeneration (Wardana et al., 2024; Hwang and Hwang, 2003). Previous studies on Andaliman have demonstrated the potential of tissue culture for shoot initiation and multiplication; however, systematic evaluation of the interaction between NAA and BAP during callus induction and the subsequent response of the induced callus to IAA and TDZ for shoot regeneration remains limited. Addressing this gap is important for developing a reproducible regeneration system for Andaliman (Carsono et al., 2021).

Therefore, this study aimed to determine the effects of NAA × BAP interactions on callus induction and growth characteristics and to evaluate the effects of IAA × TDZ combinations on subsequent shoot regeneration from the induced callus of Andaliman (Zanthoxylum acanthopodium DC.) (Sualang et al., 2023; Nursandi et al., 2024). The hypotheses were that (1) the interaction between NAA and BAP concentrations significantly affects callus weight, diameter, height, and morphological characteristics, and (2) the interaction between IAA and TDZ concentrations significantly affects shoot regeneration from the induced callus. The findings are expected to contribute to the development of a preliminary and reproducible in vitro propagation approach for producing more uniform Andaliman planting materials and to support future conservation and commercial propagation efforts.

2. Materials and Methods

2.1. Study site and experimental design

The study was conducted from January to December 2025 at the Tissue Culture Laboratory of the Technical Management Unit (UPT) of the Central Horticulture Center (BIH), Johor Building, Department of Agriculture, North Sumatra, Indonesia.

This study employed a quantitative experimental design to evaluate the effects of plant growth regulators (PGRs) on the in vitro propagation of Andaliman (Zanthoxylum acanthopodium DC.). The study employed a factorial Completely Randomized Design (CRD) to evaluate the effects of NAA and BAP on in vitro callus induction of Andaliman (Zanthoxylum acanthopodium DC.). NAA was tested at 1, 5, and 10 mg/L, while BAP was tested at 5, 10, and 15 mg/L, resulting in nine treatment combinations with three replicates each (27 culture bottles). No PGR-free control (0 mg/L NAA+0 mg/L BAP) was included; therefore, the treatment effects cannot be compared with a PGR-free baseline and are interpreted only within the tested concentration range. The use of three replicates may also reduce statistical power and is acknowledged as a limitation.

The selected concentrations were based on preliminary optimization and the relatively slow callus induction response of Andaliman. However, the relatively wide concentration intervals may not fully identify the optimum concentration. Following callus induction, healthy and actively growing calli were transferred to MS medium supplemented with IAA at 1, 2, and 3 mg/L in combination with TDZ at 0.5, 1.0, and 1.5 mg/L. The experiment consisted of nine IAA × TDZ combinations with three replicates. Shoot regeneration was evaluated based on shoot formation and the number of shoots produced.

2.2. Plant material and explant source

The explants consisted of healthy in vitro-grown Andaliman nodal segments maintained at the BIH Tissue Culture Laboratory. Selected nodal segments were aseptically transferred from stock cultures to the respective treatment media. After two months of callus induction, healthy, actively growing callus with relatively uniform size and morphology was selected for subculture. Callus showing contamination, necrosis, browning, or poor growth was excluded.

2.3. Culture conditions and procedures

Murashige and Skoog (1962) (MS) medium was used as the basal medium. The medium contained 30 g/L sucrose, vitamins, myo-inositol, amino acids, and 0.25% Gelrite (2.5 g/L). PGRs were added according to the treatment. The medium pH was adjusted to 5.8 using 0.1 N NaOH or HCl before Gelrite addition. Approximately 25 mL of medium was dispensed into each culture bottle and autoclaved at 121°C and 20 psi for 15 min.

Explants were inoculated aseptically in a laminar air flow cabinet and incubated for two months under a 16 h light/8 h dark photoperiod, light intensity of 1,000–4,000 lux, and temperature of 26 ± 2 °C. Callus was subsequently transferred to the IAA- and TDZ-supplemented medium for two months. Regenerated shoots were subsequently transferred to rooting medium for approximately three months, followed by acclimatization and greenhouse cultivation for two months.

2.4. Data collection and statistical analysis

Callus fresh weight, height, diameter, colour, and morphology were recorded after the callus induction period, while shoot formation and shoot height were recorded during the regeneration stage. Callus weight, height, and diameter were analysed using factorial ANOVA at the 5% significance level, followed by Duncan’s Multiple Range Test (DMRT) when significant differences were detected. Analyses were performed using SPSS version 26. All quantitative results are presented as mean ± standard deviation (SD), based on three replicates (n = 3). Normality and homogeneity of variance were not formally assessed; therefore, the ANOVA results were interpreted cautiously. The absence of formal assessment of normality and homogeneity of variance represents an additional statistical limitation and should be addressed in future experiments using larger sample sizes. When zero values occurred, the transformation √(x + 0.5) was applied before analysis.

Because shoot formation occurred in only 5 of 27 culture bottles (18.5%) and the dataset contained numerous zero observations, the shoot regeneration data were considered too sparse for reliable parametric ANOVA with the available replication. Therefore, shoot regeneration was analysed descriptively, and the observed treatment response was interpreted as an indication of regeneration potential rather than a statistically confirmed optimum.

3. Results

3.1. Callus fresh weight

Analysis of variance showed a significant effect of the NAA × BAP treatments on the fresh weight of Andaliman (Zanthoxylum acanthopodium DC.) callus. DMRT at the 5% significance level further indicated differences among treatment combinations (Table 1). The response of callus biomass was not proportional to increasing NAA or BAP concentrations, indicating that callus growth depended on the specific combination of the two PGRs. The highest mean fresh weight was obtained with 5 mg/L NAA + 10 mg/L BAP (96 mg), whereas the lowest was observed with 1 mg/L NAA + 5 mg/L BAP.

Table 1
Effect of NAA and BAP concentrations on fresh callus weight of Andaliman.

Concentration of ZPT BAP (mg/L)	Concentration of ZPT NAA (mg/l)
	1	5	10
5	51 ± 1.0 a	79 ± 2.0 d	80 ± 6.0 d
10	62 ± 1.0 b	96 ± 3.0 e	72 ± 3.0 c
15	64 ± 2.6 b	83 ± 3.0 d	56 ± 2.0 a

• Different lowercase letters indicate significant differences according to DMRT at α = 0.05. Description: Numbers followed by lowercase letters in the same column and row are not significantly different in the 5% DMRT test. Values are presented as mean ± standard deviation (SD), n = 3.

The fresh callus weights recorded after eight weeks ranged from approximately 51 to 96 mg. These values indicate relatively low biomass accumulation during the induction period. The low biomass may be associated with the inherently slow growth of Andaliman callus, differences in callus viability, and the occurrence of browning and tissue death in some cultures. Thus, the observed biomass reflects the growth response under the specific culture conditions and PGR concentrations tested.

Figure 1 shows an interaction between the plant growth regulators NAA and BAP on the increase and decrease of Andaliman callus weight (Zanthoxylum acanthopodium DC.), indicated by intersecting lines. Increasing the concentration of NAA from 1 mg/l to 5 mg/l can increase the average callus weight of Andaliman up to 96 mg, but increasing the NAA concentration from 5 mg/l to 10 mg/l decreases the average callus weight of Andaliman to 56 mg. In contrast, BAP shows fluctuations in the average callus weight of Andaliman with respect to its concentration; increasing the BAP concentration from 5 mg/l to 10 mg/l decreases the average callus weight from 80 mg to 62 mg, but the average callus weight increases again, reaching a maximum of 96 mg with the addition of 5 mg/l NAA concentration. Furthermore, increasing the BAP concentration to 15 mg/l reduces the average callus weight of Andaliman to 56 mg.

Figure 1
Interaction graph of NAA and BAP concentration treatments on the callus weight of Andaliman (Zanthoxylum acanthopodium DC.).

3.2. Callus height

NAA and BAP treatments significantly affected callus height (Table 2). The highest mean height was observed in the 5 mg/L NAA + 10 mg/L BAP treatment, reaching 11.33 ± 2.08 mm, while the lowest value was 5.33 ± 0.58 mm. DMRT grouping showed that treatment responses were distributed into statistically distinguishable groups, indicating that some NAA × BAP combinations produced significantly different callus heights.

Table 2
Effect of NAA and BAP concentrations on callus height of Andaliman.

Concentration of ZPT BAP (mg/L)	Concentration of ZPT NAA (mg/l)
	1	5	10
5	5.33 ± 0.58 a	10.33 ± 1.53 d	6.67 ± 1.53 b
10	10.00 ± 1.00 d	11.33 ± 2.08 e	7.00 ± 1.00 b
15	6.33 ± 0.58 b	8.33 ± 1.15 c	9.00 ± 1.73 c

• Different lowercase letters indicate significant differences according to DMRT at α = 0.05. Values followed by the same lowercase letter are not significantly different, whereas values followed by different lowercase letters are significantly different. Values are presented as mean ± standard deviation (SD), n = 3.

The highest response at the intermediate NAA and BAP concentrations suggests that increasing PGR concentrations beyond the favourable combination did not necessarily promote further vertical growth. The interaction pattern shown in Figure 2 further indicates that the effect of one PGR depended on the concentration of the other.

Figure 2
Graph of the interaction of NAA and BAP concentration treatments on the height of Andaliman (Zanthoxylum acanthopodium DC.) callus clumps.

3.3. Callus diameter

Significant variation in callus diameter was observed among the NAA × BAP combinations (Table 3). The largest mean diameter was recorded with 1 mg/L NAA + 5 mg/L BAP (16.33 mm), whereas the smallest was obtained with 5 mg/L NAA + 10 mg/L BAP (6.67 mm). The DMRT results separated the treatment means into four significantly different groups, indicating clear differences in callus expansion among treatments.

Table 3
Effect of NAA and BAP concentrations on callus diameter of Andaliman.

Concentration of ZPT BAP (mg/L)	Concentration of ZPT NAA (mg/l)
	1	5	10
5	16.33 ± 1.53 d	9.00 ± 2.65 ab	14.00 ± 2.65 bcd
10	10.00 ± 2.65 abc	6.67 ± 1.53 a	11.67 ± 1.53 bcd
15	14.33 ± 2.52 bcd	15.00 ± 2.00 cd	10.00 ± 2.00 abc

• Different lowercase letters indicate significant differences according to DMRT at α = 0.05. Values followed by the same lowercase letter are not significantly different, whereas values followed by different lowercase letters are significantly different. Values are presented as mean ± standard deviation (SD), n = 3.

Based on the notation, there are four treatment value groups that differ significantly, namely: 1) notation a for the group with mean values of 6.67–10; 2) notation b for the group with values of 9.0–14.33; 3) notation c for the group with mean values of 10–15; 4) notation d for the group with values ranging from 11.67 to 16.33 mm. Figure 3, shown below, is a bar graph illustrating the three parameters described above.

Figure 3
Bar chart comparing the average weight, height of callus stacks, and diameter of Andaliman callus (Zanthoxylum acanthopodi um DC.) using NAA and BAP concentration treatments.

An important finding was the inverse pattern between callus diameter and callus biomass and height. The treatment producing the greatest callus diameter (NAA1BAP5) did not produce the highest biomass or height, whereas NAA5BAP10 produced the highest fresh weight and height but the smallest diameter. This contrasting growth pattern suggests that callus expansion was not necessarily accompanied by proportional biomass accumulation or vertical growth.

The observed pattern may be related to differences in callus morphology. Compact callus tends to form dense aggregated masses, which may result in greater biomass and height within a relatively restricted area. In contrast, friable callus tends to spread laterally across the culture medium, resulting in a larger apparent diameter but potentially lower biomass density. This morphological variation provides a possible explanation for the contrasting responses among the measured callus parameters.

3.4. Callus texture, structure, and colour

Variation in callus morphology was observed among the treatments (Figure 4). The calli exhibited whitish-brown, whitish-yellow, and white coloration, with some cultures subsequently developing greenish areas. Differences were also observed in callus texture and structure, ranging from compact to relatively friable forms.

Figure 4
Various callus expressions from in vitro culture of Andaliman (Zanthoxylum acanthopodium DC.). (A, B, C) Compact callus; (D, E, F) Friable callus, ec123 = embryogenic callus changed colour, nec123 = non-embryogenic callus unchanged colour.

These morphological differences were associated with the subsequent regeneration response. Most compact calli did not produce shoots after transfer to the shoot induction medium, whereas shoot formation was observed in only a limited number of cultures. However, the relationship between callus morphology and regeneration competence requires further investigation because embryogenic potential was not directly quantified in this study.

3.5. Shoot regeneration

Following callus induction, selected calli were transferred to the shoot induction medium containing IAA and TDZ (Table 4). Shoot formation occurred in only 5 of 27 culture bottles (18.5%), with a maximum of three shoots per culture bottle. Thus, the observed regeneration frequency was low, and the results do not support describing the protocol as an efficient shoot regeneration system.

Table 4
Effect of Plant Growth Regulators NAA and BAP on the texture, structure, color of callus and callus sprouts of Andaliman (Zanthoxylum acanthopodium DC.).

No.	IAA (mg/L)	TDZ (mg/L)	Cultures	Callus texture/structure	Colour	Shoot-producing cultures	No. of shoots
1	1.0	0.5	3	Compact / nec	BY	0/3	0
2	1.0	1.0	3	Compact / nec	BY	0/3	0
3	1.0	1.5	3	Friable / ec	WY	2/3	3
4	2.0	0.5	3	Compact / nec	BY	0/3	0
5	2.0	1.0	3	Compact / nec	BY	0/3	0
6	2.0	1.5	3	Friable / ec	WY	1/3	1
7	3.0	0.5	3	Compact / nec	BY	0/3	0
8	3.0	1.0	3	Friable / ec	WY	2/3	2
9	3.0	1.5	3	Compact / nec	BY	0/3	0

• Description: IAA = Indole Acetic Acid, TDZ = Thidiazuron, by = brownish yellow, wy = whitish yellow, nec = non-embryonic callus, ec = embryonic callus.

Because shoot formation occurred in only a small proportion of cultures and the dataset contained numerous zero observations, the shoot response was presented descriptively rather than subjected to parametric ANOVA. Therefore, the observed response at the IAA and TDZ treatments should be interpreted as an indication of regeneration potential rather than a statistically confirmed optimum.

Most cultures that failed to regenerate shoots contained compact callus. This observation suggests that callus morphology may be associated with regeneration competence; however, further experiments with larger sample sizes and appropriate statistical analysis are required to establish this relationship.

3.6. Relationship among callus parameters

The relationship between callus fresh weight, height, and diameter is presented in Figure 5. The graphical pattern indicates that the three parameters did not increase proportionally. In particular, the treatment with the largest callus diameter differed from the treatment producing the highest fresh weight and height.

Figure 5
Relationship between weight and height of callus pile (A), relationship between pile height and callus diameter (B).

3.7 Shoot-forming callus

The induction of callus into shoots uses a combination of plant growth regulators IAA and TDZ. Experimental results show that only friable callus can form shoots, as shown in Figure 6.

Figure 6
In vitro shoot induction process from Andaliman plant (Zanthoxylum acanthopodium DC.), ec = embryogenic callus, fc = friable callus, bc = browning callus, si = shoot initiation, s = shoot, bs = branched shoot.

Figure 6 shows friable callus texture that can form shoots (Negi et al., 2024), callus color (yellow, white and green spots) (Abas et al., 2025) and callus surface appearance that is smooth, nodular, and granular (Safitri and Nurhidayah, 2023). Friable callus appears loosely arranged with larger intercellular spaces and lighter color, while compact callus shows tightly arranged cells with more intense coloration. Friable callus is considered morphogenetically competent, whereas color changes to brown or necrosis are associated with decreased viability. The characteristics of the callus in Figure 6 are embryogenic or embryogenic callus because it is capable of inducing shoots (Pasternak and Steinmacher, 2024).

4. Discussion

The present study demonstrated that NAA × BAP combinations significantly affected callus fresh weight, height, and diameter of Andaliman. The highest fresh weight and callus height were obtained with 5 mg/L NAA + 10 mg/L BAP, whereas the largest diameter occurred with 1 mg/L NAA + 5 mg/L BAP. Thus, biomass accumulation and spatial expansion responded differently to the auxin–cytokinin balance rather than increasing linearly with PGR concentration (Farikha and Habibah, 2025).

The response can be interpreted through auxin–cytokinin balance theory. Auxin-rich conditions generally favour cellular dedifferentiation and callus formation, whereas a relatively stronger cytokinin response can promote cell division and regenerative competence (Pandiangan and Nainggolan, 2006; Pasternak and Steinmacher, 2024). At the cellular level, auxin signalling through the TIR1/AFB–Aux/IAA–ARF module regulates transcriptional responses associated with cellular reprogramming, including LBD-related pathways implicated in callus formation (Pramono et al., 2021). Cytokinin signalling through histidine kinase receptors, AHPs, and ARR proteins contributes to cell proliferation and acquisition of regenerative competence. Consequently, the observed response likely reflects interactions among exogenous PGR concentration, endogenous hormone status, and tissue competence rather than the absolute concentration of a single regulator (Ikeuchi et al., 2013; Walangadi et al., 2025).

The superior biomass response at 5 mg/L NAA + 10 mg/L BAP suggests that this combination provided a favourable hormonal balance for proliferation under the tested conditions. The decline in biomass at some higher concentrations indicates that increasing PGR concentration does not necessarily enhance callus growth and may instead alter differentiation or impose physiological stress (Muliati et al., 2017). This interpretation is consistent with reports that NAA and BAP combinations can strongly affect callus development in other species, although the optimum ratio is species- and explant-dependent (Pramono et al., 2021; Zahara et al., 2013; Wardana et al., 2024).

The response of Andaliman is broadly consistent with the strong PGR dependence reported in other Zanthoxylum species, although the optimal combinations differ among species and explant types. In Z. piperitum, shoot proliferation from shoot-tip explants was optimized using 0.5 mg/L BA, producing approximately 23 microshoots per explant after 40 days; combinations of BA with NAA or IBA have also supported regeneration in Z. piperitum f. inerme (Hwang and Hwang, 2003; Anjani et al., 2022). Z. armatum has demonstrated indirect regeneration through callus, with a reported maximum of 10.4 ± 0.74 shoots per callus under a BA + IAA + GA3 regime (Negi et al., 2024). Z. zanthoxyloides has likewise shown higher multiplication responses under species-specific BA and IBA conditions (Khaya et al., 2026; Ahmad and Spoor, 1999). The lower response observed in Andaliman therefore appears to be specific to the species and protocol tested rather than a general characteristic of the genus.

An important finding was the contrasting pattern among callus diameter, fresh weight, and height. The treatment with the greatest diameter (1 mg/L NAA + 5 mg/L BAP) did not produce the greatest biomass or height, whereas 5 mg/L NAA + 10 mg/L BAP produced the highest weight and height but the smallest diameter. This pattern does not support the assumption that increasing NAA directly increases callus diameter. Instead, the lower PGR combination may have favoured lateral expansion, whereas the intermediate combination favoured biomass accumulation and vertical development. Differences in cell packing, water content, cell division, and spatial organization may contribute to this pattern (Cai et al., 2013; Toharah et al., 2015). Compact callus tends to contain densely aggregated cells, whereas friable callus has a looser cellular arrangement and can spread laterally across the culture surface (Mosoh et al., 2024; Nurussakinah et al., 2022).

The correlation analysis supports this multidimensional growth pattern. Fresh weight and height showed a moderate positive association, but it was not statistically significant (r = 0.523, p = 0.149). In contrast, height and diameter were strongly and negatively correlated (r = −0.896, p = 0.001), indicating that cultures with greater vertical accumulation tended to have smaller lateral expansion. This may reflect differences in tissue architecture and growth direction rather than a simple increase in total growth. The correlation should nevertheless be interpreted cautiously because it was calculated from only nine treatment means.

Callus morphology also differed among treatments. Compact and friable tissues and changes from brownish-yellow to whitish-yellow and greenish coloration were observed (Mahadi et al., 2016). Such traits can be useful morphological indicators of developmental status, but colour alone should not be considered definitive evidence of embryogenicity without histological or molecular confirmation (Rosmaina and Aryani, 2015). The predominance of compact callus among non-regenerating cultures may indicate low organogenic competence, while the limited shoot formation from friable/colour-changing callus suggests that tissue morphology may be associated with regenerative capacity (Abas et al., 2025; Safitri and Nurhidayah, 2023).

The second stage of the experiment evaluated whether induced callus could regenerate shoots under IAA × TDZ conditions. Shoot formation occurred in only 5 of 27 cultures (18.5%), with a maximum of three shoots per culture. Therefore, the present protocol should be regarded as demonstrating shoot-regeneration potential rather than an efficient regeneration system. The highest observed response occurred at 1 mg/L IAA + 1.5 mg/L TDZ; however, because the number of responding cultures was small and many observations were zero, this treatment cannot be regarded as a statistically confirmed optimum (Nofanda et al., 2016; Lestari et al., 2013). The limited regeneration may be related to callus competence, the hormonal transition between callus induction and shoot induction, callus age, and species-specific recalcitrance.

The low shoot regeneration observed here contrasts with the higher responses reported in Z. armatum, Z. piperitum, and Z. zanthoxyloides, emphasizing that regeneration protocols cannot necessarily be transferred directly between Zanthoxylum species. Further optimization in Andaliman should evaluate narrower IAA and TDZ concentration intervals, alternative cytokinins, callus age, explant source, subculture interval, and culture conditions. Increasing the number of biological replicates would also improve the statistical basis for identifying regeneration-promoting treatments.

4.1. Limitations and implications

Several limitations should be considered. First, only three replicates were used per treatment, limiting statistical power and increasing uncertainty in treatment-effect estimates. Second, no PGR-free control was included; therefore, the contribution of NAA and BAP cannot be separated conclusively from the intrinsic callus-forming capacity of the explants. Third, the concentration intervals were relatively wide and may have missed intermediate optima. Fourth, shoot regeneration was low, with only 18.5% of cultures producing shoots (Waryastuti et al., 2017). Finally, embryogenic competence was classified morphologically and was not confirmed histologically or molecularly.

Despite these limitations, the study provides an initial two-stage framework for Andaliman propagation. The NAA5BAP10 combination is a useful starting point for further callus optimization, while the IAA×TDZ results identify conditions under which shoot formation was observed. Future studies should increase replication, include PGR-free controls, test narrower concentration ranges, and optimize shoot regeneration and acclimatization before large-scale conservation or commercial application.

5. Conclusion

NAA × BAP combinations significantly affected callus development in Andaliman. The combination of 5 mg/L NAA + 10 mg/L BAP produced the highest callus fresh weight (96 mg) and height (11.33 ± 2.08 mm), whereas 1 mg/L NAA + 5 mg/L BAP produced the largest callus diameter (16.33 ± 1.53 mm). Shoot regeneration was observed after transfer to IAA × TDZ media, but only 5 of 27 cultures (18.5%) produced shoots, with a maximum of three shoots per culture. Thus, the present study demonstrates the feasibility and regeneration potential of a two-stage NAA–BAP followed by IAA–TDZ approach rather than an efficient propagation protocol. The findings provide a preliminary basis for developing more reliable propagation methods that could contribute to Andaliman conservation and future commercial production of uniform planting materials. Further work should incorporate PGR-free controls, increase replication, refine PGR concentrations, and improve shoot regeneration and acclimatization before large-scale application. The approach may also provide a basis for developing in vitro propagation protocols for other species with similar propagation constraints.

Acknowledgements

Thank you to the Vice-Chancellor of the University of Medan and his team for providing research funding. We would also like to express our gratitude to the Head of the Tissue Culture Laboratory at the Technical Management Unit (UPT) of the Central Horticulture Centre (BIH) in the Johor Building, Department of Agriculture, North Sumatra, Indonesia. This research was funded by the Non-Tax State Revenue (PNBP) of the University of Medan, Indonesia, for the 2025 financial year, under the applied research scheme. Once again, we would like to express our sincere gratitude to the Vice-Chancellor of the University of Medan and his team for providing the research funding.

Data Availability Statement

The data generated and/or analyzed during this study are available from the corresponding author upon reasonable request.

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*

e-mail: [e-mail]

Editor:

Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  21 Sept 2026
• Date of issue
  2026

History

• Received
  06 Apr 2026
• Accepted
  17 Aug 2026