Introduction

The technique of plant tissue culture has proven to be highly effective, serving as a means of multiplying plants at high rates under controlled phytosanitary conditions (1). Moreover, the tools of plant tissue culture are being applied to a wide range of biotechnological endeavors, particularly to the clonal propagation and genetic improvement of crop and medicinal plants (2-3). Solanum nigrum, belonging to the family Solanaceae, contains several medicinally critical secondary metabolites such as solanine, sapogenin, diosgenin, tigogenin, solanidine, solamargine, etc (4). In traditional folk medicine, the berries are widely valued and have been used as an emetic, antispasmodic, and diuretic, as well as a remedy for fever, diarrhea, and different eye ailments (5-6). Apart from its medicinal uses, the leaves and berries of this herb are often eaten as food or cooked and served as a vegetable (7). In vitro adventitious shoot development is crucial for the propagation and conservation of medicinally and economically important plants, serving as a key tool in genetic transformation for the production of transgenic plants. Shoot tip culture in particular is extensively applied due to its high regeneration potential, attributed to the dome of totipotent cells that functions as a central hub for diverse developmental processes (8-9). Despite its importance, relatively few studies have focused on plant regeneration using shoot tip explants of S. nigrum. To date, reports on in vitro direct regeneration through adventitious shoot induction remain limited. However, this approach has been highlighted as a promising method for achieving mass propagation and long-term conservation of the species (10-15). Hence, the present study aims to establish a rapid and efficient protocol for direct adventitious shoot regeneration using shoot tip explants of S. nigrum with applications in conservation, sustainable production of elite clones, and genetic transformation studies.

Materials and Methods

Shoot tip segments were used as explants. The explants were initially washed with tap water, followed by the Tween 20 solution. After that, the explants were surface-sterilized by dipping in 70% alcohol for approximately 30 s, followed by treatment with 0.1% HgCl₂ for 3 to 4 min, and then washed several times with sterile double-distilled water. The explants were inoculated into the culture medium. The entire above process was performed aseptically under a laminar airflow cabinet. Murashige and Skoog media (16) were used in the study, with a carbohydrate content of 3% sucrose. The pH of the media was adjusted to 5.8 before autoclaving at 15 pounds pressure and temperature at 121 °C for 15-20 min. Gelling of the press was done with 0.8% agar-agar. The cultures were maintained under a cool fluorescent light intensity of approximately 3000lux for 16 h, with a temperature of 25 ± 2 °C. Each treatment consisted of 10 replicates and was repeated twice.

The effect of cytokinins (BAP and TDZ) on the induction of adventitious shoot proliferation was observed singly or in combination with indole-3-acetic acid (IAA). Six different concentrations of BAP and TDZ (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/L) were tested singly for shoot induction, and combinations with IAA (0.5 mg/L) were also tested for the same purpose. To initiate rooting, the shoots were transferred to MS medium supplemented with IBA (indole-3-butyric acid) (0.5-4.0 mg/L).

The rooted plantlets were transferred to the pots containing a mixture of loam soil and cow dung manure (2:1), watered, and covered with transparent polypropylene bags to maintain high humidity conditions. The pots were kept in daylight for seven days, after which the polypropylene cover was removed and the potted plants were left to preserve in the open environment.

All experiments were conducted in triplicate, with 10 replicates per treatment in each run. Data were recorded for percentage response, the number of shoots per explant, the number of roots per shoot, and the root length per root. The mean and standard error (SE) were calculated for each treatment.

Results and Discussion

The shoot tips of S. nigrum were cultured on twenty-five media. The results of the experiment were evaluated for various tissue culture responses at regular intervals. Among the 25 types of media, the maximum shoot induction rate (98.5 ± 0.38%) and the maximum average shoot number per shoot tip (142.2 ± 0.33) were obtained on MS medium supplemented with TDZ (2 mg/L) and IAA (0.5 mg/L) after eight weeks of culture (see Table 1). The explants did not exhibit any shoot formation on MS medium without plant growth regulators (PGRs). In contrast, shoot induction was observed in all PGR-supplemented treatments; however, the response was scanty. The supplementation of MS media with different concentrations of TDZ, with or without IAA combination, resulted in effective adventitious shoot induction from shoot tip explants, compared to media fortified with BAP alone or in combination with IAA. The results indicated that the combination of cytokinin with IAA stimulated the rapid induction of adventitious shoots, likely due to their synergistic effect (17), a phenomenon previously reported in the in vitro multiplication of several plant species (10, 18). The in vitro response has been progressive from 0.5 to 2.0 mg/L cytokinin (BAP/TDZ) singly or in combination with IAA (0.5 mg/L). At the same time, beyond the concentration of (2 mg/L) BAP/TDZ alone or combined with IAA (0.5 mg/L), the percentage shooting and average shoot number per shoot tip were found to decrease in the present study. Kiran et al. (19) reported similar findings in Phyllanthus amarus regeneration studies. Earlier researchers who worked on S. nigrum’s shoot tip explants shoot multiplication reported 2 to 24.8 shoots per shoot tip on MS medium supplemented with BAP/KIN/TDZ alone or combined with IAA (10-15). The present study investigated the in vitro response to the medium MS+TDZ (2 mg/L) + IAA (0.5 mg/L), which was optimal for shoot multiplication and exhibited a superior outcome compared to earlier in vitro responses on Solanum nigrum shoot tip explants (see Figure 1). Handy number roots were developed from the base of the shoot after 2 weeks of culture on MS medium supplemented with different concentrations of IBA (see Table 2). The rooting response varied with IBA concentrations. MS medium supplemented["Table", "Table 1. Direct induction of adventitious shoots from shoot tip explants of Solanum nigrum L. using different PGRs after 8 weeks of culture.", "8pt", "2", "false"] ["Table", "Table 2. In vitro rhizogenesis of regenerated S. nigrum shoots using the PGR (IBA) after 2 weeks of culture.", "8pt", "2", "false"]with IBA (2.5 mg/L) produced the maximum rhizogenesis, i.e., rooting frequency (100%), root number (19.0 ± 0.18 per shoot), and root length (6.9 ± 0.09 cm per root). These results align with the findings of Kolar et al. (20).

No.	MS media with PGR (mg/L)	Shooting (%)	Shoot no./ shoot tip
1.	MS	00.0 ± 0.00	00.0 ± 0.00
2.	BAP (0.5)	42.0 ± 0.06	08.8 ± 0.11
3.	BAP (1.0)	55.0 ± 0.14	10.2 ± 0.06
4.	BAP (1.5)	62.5 ± 0.11	13.8 ± 0.16
5.	BAP (2.0)	82.0 ± 0.24	26.3 ± 0.14
6.	BAP (2.5)	75.0 ± 0.19	22.4 ± 0.13
7.	BAP (3.0)	63.0 ± 0.12	15.0 ± 0.02
8.	TDZ (0.5)	45.2 ± 0.23	15.2 ± 0.17
9	TDZ (1.0)	60.0 ± 0.19	24.5 ± 0.20
10.	TDZ (1.5)	62.3 ± 0.22	31.3 ± 0.18
11.	TDZ (2.0)	75.5 ± 0.32	53.0 ± 0.33
12.	TDZ (2.5)	68.2 ± 0.30	44.5 ± 0.15
13.	TDZ (3.0)	66.0 ± 0.22	39.2 ± 0.24
14.	BAP (0.5) ± IAA (0.5)	68.7 ± 0.19	34.6 ± 0.18
15.	BAP (1.0) ± IAA (0.5)	74.0 ± 0.30	29.7 ± 0.38
16.	BAP (1.5) ± IAA (0.5)	78.7 ± 0.19	30.5 ± 0.69
17.	BAP (2.0) ± IAA (0.5)	82.1 ± 0.34	47.2 ± 0.69
18.	BAP (2.5) ± IAA (0.5)	66.6 ± 0.18	24.2 ± 0.76
19.	BAP (3.0) ± IAA (0.5)	58.7 ± 0.38	17.6 ± 0.52
20.	TDZ (0.5) ± IAA (0.5)	80.4 ± 0.19	60.2 ± 0.29
21.	TDZ (1.0) ± IAA (0.5)	85.2 ± 0.10	85.5 ± 0.20
22.	TDZ (1.5) ± IAA (0.5)	89.2 ± 0.16	110.6 ± 0.19
23.	TDZ (2.0) ± IAA (0.5)	98.5 ± 0.38	142.2 ± 0.33
24.	TDZ (2.5) ± IAA (0.5)	92.4 ± 0.22	121.3 ± 0.18
25.	TDZ (3.0) ± IAA (0.5)	80.3 ± 0.26	96.9 ± 0.22

No.	MS media with PGR (mg/L)	Rhizogenesis
No.	MS media with PGR (mg/L)	Root induction (%)	Root no./ shoot	Root length (cm)
1.	MS	00.0 ± 0.00	0.0 ± 0.00	0.0 ± 0.00
2.	IBA (0.5)	55.0 ± 0.21	5.0 ± 0.19	4.0 ± 0.11
3.	IBA (1.0)	60.0 ± 0.11	5.2 ± 0.22	4.3 ± 0.15
4.	IBA (1.5)	68.0 ± 0.14	8.6 ± 0.11	5.2 ± 0.20
5.	IBA (2.0)	75.0 ± 0.16	10.3 ± 0.20	5.8 ± 0.10
6.	IBA (2.5)	100 ± 0.00	19.0 ± 0.18	6.9 ± 0.09
7.	IBA (3.0)	85.0 ± 0.12	14.5 ± 0.24	6.5 ± 0.14
8.	IBA (3.5)	80.0 ± 0.17	8.1 ± 0.15	5.0 ± 0.23
9.	IBA (4.0)	55.0 ± 0.26	5.0 ± 0.13	4.6 ± 0.26

Subsequently, these plantlets were removed from the["Figure", "https://etflin.com/file/figure/202508240856551902729719.png", "Figure 1. Direct adventitious shoot induction and Plant regeneration using shoot tip explants of Solanum nigrum L. (A) shoot tip showing shoot projection initials, (B) adventitious shoots, (C) multiple shoot cluster, (D) rhizogenesis of plantlet, (E) plantlet showing roots, and (F) acclimatized plants.", "", "100%", "1"]cultures, washed free of agar and transferred to pots containing a mixture of loam soil and cow dung manure (2:1). The plants were kept covered with a polythene bag for 10 days to check excessive transpiration. The plants were then successfully established in field conditions. The acclimatized plantlets exhibited a 90% survival rate. Thus, the protocol standardized through this study demonstrates the possibility of developing an efficient in vitro propagation system for the successful mass propagation of S. nigrum L.

Conclusion

The present experiment successfully standardized a highly efficient and reproducible protocol for the in vitro regeneration of the medicinal herb Solanum nigrum. Among the various PGR combinations tested, Murashige and Skoog (MS) medium supplemented with 2 mg/L TDZ and 0.5 mg/L IAA was identified as the most effective for inducing profuse adventitious shoot proliferation from shoot tip explants, yielding an average of 142.2 ± 0.33 shoots per explant — a remarkable improvement over previously reported methods. Furthermore, rhizogenesis was optimally achieved on MS medium supplemented with 2.5 mg/L indole-3-butyric acid (IBA), resulting in robust root formation and healthy plantlet development.

The established regeneration system not only offers a rapid and reliable approach for large-scale propagation of S. nigrum but also provides a valuable platform for future applications in genetic improvement, secondary metabolite production, and conservation of elite germplasm. In addition, this standardized protocol may facilitate in vitro mutagenesis and transformation studies aimed at enhancing the pharmacological properties of this important medicinal plant. Further optimization of acclimatization conditions and metabolite profiling of regenerated plants could strengthen the potential use of this system in commercial and pharmaceutical contexts.

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