Shoots of Anubias barteri var. nana Petite were subjected to surface sterilization using soap for 15 minutes, followed by exposure to an antifungal agent (Mancozeb, India) for 10 minutes, washed with running water, and further rinsed with 70% alcohol for 30 seconds. Subsequently, explants were soaked in a commercial bleach solution (5% sodium hypochlorite) at concentrations of 10%, 20%, 30%, and 40% (v/v) for 15 minutes, followed by a 30-minute soak with an antibiotic solution (1 mg/mL). After disinfection, 3-5 mm shoot tip explants were isolated and placed on MS (Murashige and Skoog, 1962) basal medium.

Shoot tips free of microbial infection were cultured in MS media supplemented with (0, 1.0, 2.0, 3.0, 4.0 mg/L) BAP for 6 weeks for shoot induction. In a subsequent experiment, a single shoot (approximately 0.8 cm in height) was transferred to MS medium supplemented with various BAP concentrations (2.0, 3.0, 4.0 mg/L) for multiplication over 4 weeks. Shoot multiplication parameters, including the number of shoots, shoot height, number of leaves per shoot, and leaf length were recorded.

For PLB induction, in vitro stems were separated from the cluster, leaves were removed, and the stem was cut into 1-2 mm transverse slices. These slices were placed on MS medium containing 10% (v/v) coconut water (CW), 2% (w/v) sucrose, and complemented with (0.5-3.0 mg/L) BAP along with (0.5-2.0 mg/L) NAA. The percentage of PLB induction and the number of PLBs per explant were recorded after 6 weeks of dark culturing. PLB morphology and counting were performed at 2X to 4X magnification under a stereo microscope (Olympus SZ51, Japan).

For PLB proliferation, PLB clusters were separated into individuals and placed on MS medium containing 10% (v/v) CW, 2% (w/v) sucrose, added (0.5-1.5 mg/L) BA and (0.2-0.5 mg/L) IAA. After 3 weeks, the secondary PLB number and diameter, indicating PLB growth speed, were recorded.

To regenerate shoots from PLBs, tuber-shaped PLBs were separated and transferred to MS medium containing 10% (v/v) CW, 2% (w/v) sucrose, 1.0 g/L activated charcoal, 2 g/L peptone, and 1-3 mg/L BA and 0.5 mg/L IBA. After 3 weeks, shoot height was measured, and shoot quality was determined by leaf color, stem diameter, and leaf length.

To assess the impact of potato extract on shoot proliferation, PLBs were cultured on MS medium complemented with 1.5 mg/L BAP and 0.5 mg/L NAA ranging from 10 g/L to 50 g/L of PE. Data on shoot number and individual shoot height were collected after the designated culture period.

In vitro plantlets (1-1.5 cm) regenerated from PLBs were cultured on MS medium supplemented with 0.1% (w/v) activated carbon, 10% (v/v) CW, and Indole-3-Butyric Acid (IBA) at different concentrations (0.5, 1.0, 1.5, 2.0 mg/L). After 4 weeks, the number of roots per shoot, root length, shoot height, and number of leaves per shoot were recorded.

All experiments were conducted in a Completely Randomized Design (CRD) with three repetitions for each treatment, and each repetition included five jars, each containing at least one explant. The data underwent basic statistical analysis and were examined using ANOVA. Mean values were compared using Duncan’s method at 5% probability level. The data were presented as the average followed by the standard deviation (M±SD).

To prepare explants, decontamination was performed using various concentrations of commercial bleach (5% sodium hypochlorite) for a 15-minute disinfection. Explants were crucially transferred to clean water for at least one week before sterilization to avoid a high infection rate. A 10% bleach solution yielded a 40% disinfection rate, but higher concentrations (up to 40%) were necessary to achieve a 100% sterilization rate, ensuring successful decontamination. The shoot tips, retaining their green color, initiated new shoots.

In this experiment, varying concentrations of BAP were added to the MS medium to promote shoot formation and multiplication (Table 1). During the shoot induction stage, shoot tips showed distinct responses to different BAP concentrations after 10 days of culture. PGR-free MS media resulted in minimal shoot formation (1.13 shoots/explant), significantly increasing with higher BAP concentrations. The maximum shoot yield (5.33 shoots/explant) was observed after 6 weeks on a medium with 3 mg/L BA, showcasing superior quality in terms of average shoot height (1.01 cm), leaves per shoot (4.92), and leaf length (1.03 cm) (; Figure 1a-c). Subsequent stages confirmed the efficiency of 3 mg/L BAP in shoot multiplication, with clusters displaying large, green leaves, and long roots (Figure 1d-f).

The response to BAP concentration observed in this study aligns with previous research on Araceae species, such as A. barteri var nana (Kanchanapoom et al., 2012) (Rittirat et al., 2021), Cryptocoryne wendtii (Rittirat et al., 2019), and Anubias heterophylla (Rittirat et al., 2021). These findings highlight the significant impact of BAP concentration on shoot regeneration in A. barteri from shoot tips. However, adaptation to high PGR concentrations is not universal. For successful shoot regeneration from basal buds of A. barteri var. nana, it is necessary to use a minimal concentration of BAP (0.2 mg/L), and shoot elongation was obtained on PGR-free MS basal medium (Sheeja et al., 2015). Other studies within the Araceae family, utilizing lower BAP concentrations, resulted in fewer shoots (George et al., 2015).

concentrations after 6 weeks

In general, BA, with or without auxin supplementation, serves as the primary plant growth regulator in shoot induction and multiplication. MS medium supplemented with 3 mg/L BAP has proven suitable for maintaining long-term in vitro cultures. Shoot quality remained stable after four subculture cycles.

After 10 days of culture, transverse slices exhibited callus formation. After four weeks of cultivation, PLBs began to emerge, initially appearing opaque white and assuming a tuber-like shape (Table 2; Figure 2). The ANOVA and subsequent post hoc analysis demonstrated a highly significant influence of BAP and NAA, and their interaction, on explant responses. For BA, three distinct groups revealed concentrations that induced significant PLB formation. Similarly, specific NAA concentrations significantly impacted explant responses, particularly in PLB induction.

After 6 weeks of culture in media containing either 0.5 or 1.0 mg/L BAP combined with 0.5 mg/L NAA (P1, P3) resulted in underdeveloped buds on the browned callus, whereas 1.5 mg/L BAP with 1.0 or 1.5 mg/L NAA (P9, P15) induced callogenesis without PLBs (Figure 2a-b). Conversely, treatments P2, P5, P8, P11, P12, P14, P19, P20, P21, and P23 led to significant PLB induction, showing variations in leafy plantlet, globular callus, and adventitious roots. Treatments P4, P6, P10, P13, P16, P17, P18, P22, P24 resulted in tiny PLB germination (Figure 2c-e). Specific combinations of BAP and NAA concentrations (1.0 mg/L BAP + 0.5 mg/L NAA; 3.0 mg/L BAP + 1.5 mg/L NAA; 1.0 mg/L BAP + 0.5 mg/L NAA) significantly enhanced PLB formation and leafy plantlet development (Figure 2 f-h). However, concentration-dependent effects were observed, leading to callusing or limited PLB growth as certain concentrations. Notably, a BAP concentration of 1.5 mg/L, combined with 0.5 mg/L NAA, showed a tendency to promote a robust PLB induction rate.

Research has showcased the efficacy of utilizing both NAA and BAP in stimulating PLB induction across different species within the Araceae family. (Rittirat et al., 2021) demonstrated the number of shoots per explants (3.60±0.24) was formed in the culture medium containing 1.0 mg/L NAA in combination with 1.0 mg/L BAP, followed by culture medium with 3.0 mg/L BAP (2.40±0.24). (Mondal et al., 2014) found that a combination of 2 mg/L NAA and 2 mg/L BAP was most effective for PLB induction in Doritis pulcherrima Lindl. (Sarma & Tanti, 2017) further supported these findings, showing that a combination of 3.0 mg/L BAP and 1.0 mg/L NAA was most successful in inducing shoot formation in Aristolochia saccate. These studies collectively demonstrated that a combination of NAA and BAP is a popular approach for promoting PLB induction and plantlet regeneration in Araceae species.

PLBs were subcultured on MS media with varying BAP and IAA concentrations, leading to secondary PLB (sPLB) formation (Table 3). At 0.5 mg/L BAP and 0.2 mg/L IAA, a 25.0% PLB induction rate was observed, with a mean diameter of 0.61 cm. However, the quantity of sPLB generated by each explant was 0.33, indicating moderate development. Increasing BAP to 1.0 mg/L whereas maintaining 0.2 mg/L IAA resulted in a notable decline in the PLB induction rate (0.00%), along with a decrease in both the number of secondary PLBs and their diameter, suggesting a dose-dependent negative effect of BAP on PLB formation at this concentration. Interestingly, at 1.0 mg/L BAP with an elevated IAA concentration of 0.5 mg/L, the PLB induction rate rebounded to 33.3%, with an increase in both the number of sPLB and their diameter, suggesting a potential synergistic effect between BAP and IAA. At the highest concentrations of both BAP (1.5 mg/L) and IAA (0.5 mg/L), the PLB induction rate reached 68.1%, demonstrating substantial improvement. The number of sPLBs increased to 1.55, and the diameter of the PLBs reached 0.67 cm, indicating a robust response to the combined higher concentrations of BAP and IAA. These results highlight the significant influence of the interplay between BAP and IAA concentrations on PLB induction and subsequent development. The optimal combination for enhanced PLB formation appears to be 1.5 mg/L BAP and 0.5 mg/L IAA, offering valuable insights for optimizing micropropagation protocols for this specific plant.

The impact of different concentrations of BAP and IBA on shoot regeneration from PLBs is presented in Table 4. At 1.0 mg/L BAP and 0.5 mg/L IBA, shoots reached a height of 0.65 cm. Increasing BAP to 1.5 mg/L whereas maintaining IBA at 0.5 mg/L resulted in a slightly improved shoot length of 0.68 cm and enhanced shoot quality. Similarly, at BAP concentrations of 2.0 mg/L and 2.5 mg/L with 0.5 mg/L IBA, shoot length remained consistent at 0.68 cm. At higher BAP concentrations (3.0 mg/L) and 0.5 mg/L IBA, the shoot length increased to 0.83 cm. Notably, increasing IBA to 1.0 mg/L with BAP at 1.0 mg/L significantly improved shoot length to 1.23 cm, with good quality, indicating vigorous shoot regeneration. For BAP ranging from 1.5 mg/L to 3.0 mg/L with 1.0 mg/L IBA, shoot lengths varied from 0.94 cm to 1.57 cm, and the shoot quality was good.

These results emphasized the crucial role of the interaction between BAP and IBA concentrations in shoot regeneration potential from PLBs. The combination of BAP and IAA is effective in promoting shoot proliferation and elongation in Quercus suber L. (Romano et al., 1992). This combination also enhances shoot growth and proliferation in Aegle marmelos (Ajithkumar & Seeni, 1998). In the production of PLBs in orchids, BAP is the most effective growth regulator, followed by kinetin, NAA, IAA, 2,4-D, and gibberellic acid (GA3) (Saiprasad et al., 2002). In the case of pointed gourd, BAP has been found to induce callus formation, whereas the addition of IAA has been shown to enhance rooting (Komal, 2011). These findings suggested that the combination of BAP and IAA may have a positive effect on PLBs multiplication and shoot regeneration in Araceae, but further research is needed to confirm this.

The impact of varying potato extract (PE) concentrations on shoot regeneration from PLBs is shown in Table 5. Without PE, an average of 2.33 shoots was noted for each explant, accompanied by an average shoot length of 1.21 cm. However, at 10 g/L, 20 g/L, and 30 g/L PE, the number of shoots per explant nearly doubled to 4.19, 4.37, and 4.62, and the shoot height increased to 1.72, 2.08, and 2.36 cm. The most significant enhancement in shoot regeneration occurred at 40 g/L and 50 g/L PE concentrations. At 40 g/L, shoots per explant increased to 5.52, and the shoot height reached 2.74 cm. The highest concentration, 50 g/L, yielded the most favourable outcome, with 6.55 shoots per explant and a maximum shoot height of 2.80 cm (Figure 3). This highlighted a positive correlation between PE concentration and both the quantity and quality of shoots regenerated from PLBs, suggesting a potential role of specific compounds within the PE in promoting shoot development.

Studies indicate that plantlet regeneration from protocorm-like bodies in orchids is significantly enhanced by the application of PE. (Rahman et al., 2004) found that PE enhanced plantlet regeneration and growth, with the optimum concentration being 100 mL/L. This finding was further supported by (Islam et al., 2012), who reported that PE enhances the germination of seeds and the growth of seedlings of Vanda roxburgii orchids. The beneficial impact of PE on the growth and development of PLBs was also demonstrated by Lee (2003) in Cypripedium formosanum (Lee & Lee, 2003). Collectively, these studies suggest that PE can be a valuable supplement in the medium for regenerating of plantlets from PLBs in orchids.

The impact of IBA on the root and plantlet development of A. barteri after a 4-week culture period is shown in Table 6.

With IBA of 0.5 mg/L, all parameters showed significant improvement. The number of roots increased to 6.19, with a longer average root length of 2.50 cm. Shoot height and leaf number also increased to 4.72 cm and 6.81, respectively. At 1.0 mg/L IBA, whereas the number of roots slightly decreased to 4.81, both root length (2.14 cm) and shoot height (4.80 cm) remained relatively higher compared to the control, with a leaf number of 6.11. Further increases in IBA concentration to 1.5 mg/L and 2.0 mg/L led to a gradual decline in the number and length of roots, shoot height, and leaf number. The results indicated that IBA positively influences root and plantlet development in A. barteri var. nana Petite with an optimal response observed at 0.5 mg/L IBA. Higher concentrations, beyond 0.5 mg/L, showed diminishing returns, suggesting a dose-dependent effect.