Sample propagation was performed through subculture techniques. Explants were subcultured on Murashige and Skoog (MS) medium supplemented with 2 mg L⁻¹ benzyladenine (BA). In vitro shoots were removed from the culture bottles and excised approximately 0.5 cm above a Petri dish containing distilled water supplemented with betadine. Subsequently, the explants were transferred onto culture media under sterile conditions. The explant initiation process was conducted in a Laminar Air Flow (LAF) cabinet. Following initiation, the culture bottles were covered with plastic wrap, secured with rubber bands, and incubated at 21 °C under a light intensity of approximately 1000 lux for four months.

Ethyl methanesulfonate (EMS) induction on porang shoots was carried out using semi-solid MS medium sterilized at 121 °C for 30 minutes. Under sterile conditions in LAF cabinet, the medium was allowed to reach a temperature of approximately 50-60 °C, after which EMS was added at concentrations of 0% (control), 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% through a 0.22 µm Millipore filter. The explants were transferred to media containing EMS and incubated at 21°C with a light intensity of ±1000 lux for 7 days. After 7 days of EMS treatment, the explants were transferred to MS media with 2 mg/L BA incubated at a temperature of 21°C with a light intensity of ±1000 lux for 35 days.

About 100 mg of porang leaf tissue was used for DNA extraction. Genomic DNA was extracted using the Tiangen Genomic DNA kit following the manufacturer’s instructions. The quality of the extracted DNA was qualitatively verified by electrophoresis using a blueGel^TM electrophoresis system on a 1% agarose gel stained with 2 µg mL⁻¹ DNA stain. NEXmark 100 bp Plus Blue DNA ladder was used as the standard.

DNA amplification was performed using a Thermal Cycler ELVE-32G with 20 OPA primers (Operon Technologies) (Table 1). The PCR reaction was prepared in a total volume of 10 µL, consisting of 1 µL of DNA template (20 ng µL), 1 µL of forward primer (10 pmol), 1 µL of reverse primer (10 pmol), 3 µL of nuclease-free water, and 5 µL of GoTaq Master Mix. RAPD-PCR amplification was performed with an initial denaturation at 94 °C for 5 minutes, followed by 45 cycles of denaturation at 94 °C for 30 seconds, annealing temperatures were varied each primer for 60 sec (Table 1), the extension for 90 seconds at 72 °C and terminated by final extension for 7 minutes at 72 ℃ (Wahyudi et al., 2020). The amplification products were analyzed by electrophoresis on a 1.5% agarose gel in 20 mL 1× TBE buffer containing 2 µg/ml nucleic acid stain (NEX View) and visualized using a blueGel^TM electrophoresis system with a 100 bp DNA ladder (NEXmark Ladder).

Induction of mutations using EMS has been proven to increase the genetic diversity of porang. The increased genetic diversity is indicated by the rise in the number of alleles (Na) and the number of effective alleles (Ne) in the treatment group compared to the control group (Table 2). The value of Na increased from 1.2381 to 1.8571. Similarly, the number of effective alleles (Ne) also increased from 1.2019 to 1.5893.

The heterozygosity value (h) also increased from 0.1072 to 0.3242. According to Nei’s classification (1973), the heterozygosity value in the control sample was categorized very low (h < 0.30), reflecting a relatively homozygous population condition. The heterozygosity value after mutation induction using EMS remains in the low category (0.3242), however it indicates an increase in genetic diversity. The Shannon index (I) value also increased from 0.1524 to 0.4739. According to the Shannon and Weaver (1997) classification, the Shannon index value in the control sample falls into the low category (< 1.00), indicating the dominance of certain alleles and a very limited level of genetic diversity. The Shannon index value after EMS-induced mutation increased, although it is still classified as low (0.4739), demonstrating an increase in allelic diversity.

Overall, genetic diversity among populations also increased. he analysis of genetic diversity showed that the total genetic diversity (Ht) was 0.2589 ± 0.0267, while the average heterozygosity within populations (Hs) reached 0.2157 ± 0.0217 (Table 3). The relatively small difference between Ht and Hs indicates that most of the genetic diversity is maintained within populations. Approximately 83.03% of the total genetic diversity is distributed within populations, whereas only 16.97% occurs among populations.

An increase in genetic diversity was also reflected by the coefficient of genetic differentiation among populations (Gst), which was 0.1669, indicating that 16.69% of the variation occurs among populations, while the remaining 83.31% is found within populations. The relatively high Gst value indicates substantial genetic differentiation among populations. The gene flow value (Nm) was 2.4964, indicating a relatively high level of gene flow among populations.

Ethyl methanesulfonate (EMS) is an effective mutagen that induces point mutations by altering DNA bases from G/C to A/T ((Sikora et al., 2011); (Savitri & Fauziah, 2018)). Therefore, it can cause complementary base mispairing and increase the frequency of gene mutations, thereby enhancing genetic diversity (Sonsan et al., 2023). A similar phenomenon was reported by (Wahyudi et al., 2020), where EMS was able to increase the genetic diversity of Glycine max (L.) Merr., as detected using RAPD molecular markers. In addition, EMS has been widely used as an agent for inducing genetic variability in several other plant species, such as Capsicum frutescens L. (Dwinianti et al., 2019), Neolamarckia cadamba (Roxb.) Bosser, and Leucaena leucocephala (Lam.) de Wit (ZakyZayed et al., 2014). Overall, these findings highlight the potential of EMS as an effective mutagenic agent for enhancing genetic diversity.

Cluster analysis of porang after EMS mutation induction indicates that EMS treatment can increase the genetic diversity of porang. The clustering analysis generated six clusters (Figure 1), with treatments E1 (0.4%) as well as B2 and B1 (0.1%) forming clusters I, II, and III, which were separated from other concentration groups with a similarity value of approximately 0.56, indicating that EMS induction effectively increased the genetic diversity of porang. A similar pattern was observed in the 0.2% EMS treatment (C1 and C2), which was separated from the other clusters into clusters IV and VI with a similarity value of approximately 0.60–0.67, indicating a high level of genetic diversity. However, several treatments at 0.3% (D1, D2, and D3), 0.4% (E2), and 0.5% (F1 and F2) were grouped in the same cluster as the control with a similarity value of approximately 0.77, indicating a high level of genetic similarity.

The similarity values between the control group (A1–A3) and the 0.1% EMS treatment group (B1–B2) ranged from 0.31 to 0.63, indicating very high genetic diversity in porang. Meanwhile, the EMS 0.2% treatment group (C1–C2) showed higher similarity values, ranging from 0.50 to 0.69. At an EMS concentration of 0.3% (D1–D3), the similarity values further increased, ranging from 0.63 to 0.82. This Jaccard similarity pattern indicates that an increase in EMS concentration is inversely proportional to the level of induced genetic change. In the 0.4% EMS treatment group (E1–E2), the similarity values varied widely, ranging from 0.33 to 0.86. The similarity values increased again in the 0.5% EMS treatment group (F1–F2), indicating a high level of genetic similarity compared to other treatment groups, with values ranging from 0.73 to 0.91. Overall, similarity values tended to increase with increasing EMS concentration. This pattern may be attributed to the use of RAPD markers, which employ random primers; therefore, amplification can occur at multiple genomic locations simultaneously and is dominant, resulting in polymorphic DNA band patterns that may be inconsistent (Probojati et al., 2019). However, the 0.2% EMS treatment group (C1 and C2) was considered the most optimal treatment, as it showed clear cluster separation from the control group without inducing excessively extreme genetic changes.

Six of the 20 primers were found to successfully amplify polymorphic DNA bands (Table 5). OPA-2, OPA-9, OPA-11, and OPA-13 produced the highest number of DNA bands (four bands each), while OPA-3 produced the lowest number of DNA bands (two bands). The total number of bands consisted of 17 polymorphic bands (80.95%) and 4 monomorphic bands (19.05%). Analysis of several primers indicated that primer effectiveness in detecting mutations is not determined only by the number of bands produced, but also by its ability to show differences in DNA band patterns between control and treatment samples. OPA-11 showed a high total number of bands, a high level of polymorphism, and high informative parameter values, including PIC, EMR, and MI (Table 5 and Figure 2C). However, the DNA band patterns produced by OPA-11 could not clearly distinguish between control and treatment samples, unlike those produced by OPA-2 and OPA-9 (Figures 2A and 2B).

OPA-9 and OPA-2 produced clearer and more consistent DNA band patterns. These primers showed differences in band profiles, where the DNA band at the 2000 bp locus was absent in all control samples and in several treatment samples (B1, E2, F1, and F2). Meanwhile, other treatment samples (B2, C1, C2, D1, D2, D3, and E1) showed diversitys in DNA band patterns (Figure 2B). A similar trend was also observed with the OPA-2, in which the DNA band at the 1000 bp locus was not amplified in all control samples (A1–A3) and several treatment samples (C2, D1, E2, F1, and F2), but appeared in other treatment samples (B1, B2, C1, D2, D3, and E1). The appearance or loss of DNA bands following EMS mutation induction is thought to result from random point mutations in nucleotide sequences, leading to changes in amplification patterns and generating diversity in DNA bands detected by the marker (Shah et al., 2016). In addition, EMS induction may influence primer annealing efficiency, resulting in the disappearance of existing bands or the emergence of new bands due to altered amplification patterns (Poerba et al., 2009)(Suteja et al., 2019). Therefore, primers OPA-2 and OPA-9 are recommended as potential biomarkers for evaluating the success of EMS mutation induction in porang.

Mutation induction using EMS in porang results in the formation of new alleles and alterations in DNA banding patterns, reflecting an increase in genetic diversity at the molecular level. This result is consistent with the findings of (Wahyudi et al., 2020) in soybean, which reported that EMS effectively increase genetic diversity as detected by RAPD molecular markers. Diversitys in DNA banding patterns observed in mutant plants indicate that random point mutations induced by EMS can affect genome structure through changes in nucleotide sequences, leading to the loss or emergence of specific alleles. At the molecular level, EMS is known to induce G/C to A/T base substitutions, causing errors in complementary base pairing and increasing the frequency of gene mutations ((Sikora et al., 2011); (Savitri & Fauziah, 2018)), thereby confirming the potential of EMS as an effective mutagen.