Influence of Bavistin, Cefotoxime, Kanamycin and Silver Thiosulphate on Plant Regeneration of Solanum viarum (Dunal)—An Important Anticancer Medicinal Plant ()
2. Materials and Methods
2.1. Plant Material
Axillary buds were excised from the in vitro seed germinated of four week old S. viarum. The seeds were inoculated on MS medium [9] containing 0.8% (w/v) agar supplemented with various concentrations of cytokinins (BAP and Kinetin) and auxins (IAA, NAA and IBA).
2.2. Culture Medium and Culture Conditions
Axillary bud explants (1.0 - 2.0 cm) from in vitro seed germinated of four week old S. viarum were inoculated on MS medium containing different concentrations (10 - 120 mg/l) of bavistin singly and in combination of BAP and TDZ to induce multiple shoots. In the same way, axillary bud explants were also inoculated on MS medium containing different concentrations of cefotoxime and kanamycin (10 - 150 µM/L) and silver thosulphate (5.0 - 100 µM/L). Both proliferation and rooting media containing (3%) sucrose gelled with (0.8%) agar (Himedia, India). The PH of the medium was adjusted to 5.8 using 0.1 N NaOH and 0.1 N HCl and gelled with addition of agar and autoclaved at 121˚C, 15 lbs. pressure for 15 minutes. The culture room conditions maintained for in vitro cultures were 26˚C ± 2˚C and 60% to 70% relative humidity. Light intensity was 3000 lux with a photoperiod of 18 hours day light and 6 hours in dark.
2.3. Sub Culturing
Sub culturing was carried out at regular intervals of thirty days visual observations of the cultures were taken for every transfer and the effects of different treatments were quantified on the basis of percentage of cultures showing response on shoot proliferation, shoot development, number of shoots per explant, length of the regenerated shoots, number of roots per shoot and root length.
2.4. Data Analysis
The variables such as shoot regeneration frequency, number of shoots per explant, shoot length, number of roots per shoot and root length were recorded.
2.5. Statistical Analysis
All experiments were conducted with a minimum of 20 explants. All assays were repeated at least three times. The experimental data were statistically analyzed by one-way ANOVA using the DMRT (Duncan’s Multiple Range Test) (P < 0.05) and were presented as the average ± standard error (SE).
3. Results and Discussion
3.1. Effect of Bavistin on the in vitro Plant Regeneration from Axillary Bud Explants
In the present investigation, axillary bud explants responded well with bavistin. Maximum frequency of regeneration (85%), maximum mean shoot number (4.21 ± 0.83) and shoot length (4.9 ± 0.80 cm) was obtained in bavistin (100 mg/L). Bavistin in combination with BAP (2.0 mg/L) showed increased regeneration frequency (90%), maximum mean shoot number (5.55 ± 0.28) with decreased shoot length (4.5 ± 0.38 cm) when compared with bavistin alone supplemented MS medium. Table 1 (Figure 1(A)). Bavistin with TDZ also showed less regeneration frequency (83%), mean shoot number (4.13 ± 0.44) and shoot length (3.9 ± 0.29 cm) compared with the cultures having only bavistin. Bavistin is a systemic fungicide that belongs to benzimidazole family. Benzimidazole is group of organic fungicides with systemic action that are extensively used in agriculture [10]. It has been reported that the molecular structure of methyl benzimidazole carbonate has some semblance to cytokinins based on adenine [11]. One of the most effective and extensively used benzimidazole has a cytokinin activity in soy and radish [12,13]. Bavistin was found to be the least toxic to plant cells and has a broad-spectrum fungicidal activity [14]. It has also been demonstrated that these compounds can have beneficial effects on the physiology of the plant [15]. In present study it has been known that the usage of bavistin to control the fungal contamination does not show any negative effect on S. viarum cultures and further promotes the shoot regeneration.
3.2. Effect of Cefotoxime on the in vitro Plant Regeneration from Axillary Bud Explants
The obtained results reveal the influence of cefotoxime on the regeneration of axillary bud cultures of S. viarum. Maximum number of shoot formation (4.38 ± 0.45) with a maximum shoot length (4.2 ± 1.35 cm) and high frequency of regeneration (88%) at a concentration of (80 µM/L). At highest concentration, cefotoxime (150 µM/L) showed decreased frequency of regeneration (50%), shoot number (1.5 ± 0.64) and shoot length (2.5 ± 0.37 cm). A concentration of (80 µM/L) of cefotoxime was chosen as the optimum concentration for S. viarum cultures Table 2 (Figure 1(B)). Similar results reported as

Table 1. Effect of Bavistin alone and in combination with cytokinins on shoot organogenesis from axillary bud explants of in vitro grown plants of S. viarum. Data represent treatment means ± SE fallowed by different letter(s) within column indicate significant differences according to ANOVA and DMRT test (P < 0.05).
the cefotoxime alone stimulated the shoot organogenesis in the axillary bud explants of Mentha piperita whereas in case of apricot cefotoxime alone did not induce the shoot organogenesis but in combination with the other antibiotics such as timentin or vancomycin, stimulated the shoot organogenesis [16]. The activity of cefotoxime in culture is that the molecule mimics a plant growth regulator. The chance that cell metabolism converts cefotoxime to a compound with growth regulator activity has been reported in embryogenesis and regeneration in Triticum aestivum [17]. The addition of cefotoxime in the culture medium also boosted up the photosynthetic machinery of sugarcane plants [18]. Several reports concluded that cefotoxime might be killing endophytic bacteria that finally result in disease-free and vigorously growing plant cultures As a matter of fact; cefotoxime is a β-lactam antibiotic that inhibits cell wall synthesis in dividing bacterial cells and results in cell lysis [19].
3.3. Effect of Kanamycin on the in vitro Plant Regeneration from Axillary Bud Explants
The influence of kanamycin on regeneration of S. viarum was evaluated at different concentrations. Shoot initiation was observed after one week of inoculation. MS medium supplemented with 50 µM/L kanamycin induced maximum number of shoots (3.4 ± 0.48) with maximum shoot length (5.2 ± 0.12 cm) and highest mean shoot regeneration frequency (95%). The regeneration frequency gradually decreased with the increase in concentration up to (150 µM/L). Higher concentrations above (150 µM/L) tested completely inhibited regeneration Table 3 (Figure 1(C)). Kanamycin above (50 μM/L) concentration, the regeneration frequencies gradually decreased with increase in concentrations up to (150 μM/L). Higher concentrations above (150 μM/L) tested completely inhibited regeneration. Similar results were also found with quite tolerant species such as pear [20], olive [21]. The use of kanamycin along with the nutrient media is also useful to inhibit the growth of a wide range of unwanted bacteria in tissue culture vessels. The possible mechanism of stimulatory effect of antibiotics on regeneration may involve generation of stress that makes cells competent for regeneration. Alternatively, the antibiotics may mimic plant growth regulators [22]. Earlier reports demonstrated that certain antibiotics like chloramphenicol exhibited stimulatory effect on shoot organogenesis in callus cultures of Hyoscyamus muticus [23].
3.4. Effect of Ethylene Inhibitor Silver Thiosulphate on Axillary Bud Explants
In in vitro conditions the axillary bud explants bud break was observed one week after inoculation on MS medium supplemented with different concentrations of silver thosulphate. At any concentration tested with silver thiosulphate had a positive effect and showed shoot formation. Highest regeneration frequency (90%) was observed at (40 µM/L) STS and maximum number of shoots (4.75 ± 0.43) and highest shoot length (5.5 ± 0.35 cm). Lower

Table 2. Influence of cefotoxime added to MS medium supplemented with BAP (2.0 mg/l) on regeneration of plantlets from axillary bud explants of Solanum viarum. Data represent treatment means ± SE fallowed by different letter(s) within column indicate significant differences according to ANOVA and DMRT test (P < 0.05).

Table 3. Influence of Kanamycin added to MS medium supplemented with BAP (2.0 mg/l) on regeneration of plantlets from axillary bud explants of Solanum viarum. Data represent treatment means ± SE fallowed by different letter(s) within column indicate significant differences according to ANOVA and DMRT test (P < 0.05).
concentrations of STS favoured the high frequency of generation and highest number of shoots. The most favorable range of STS concentrations recorded was 10 - 40 µM/L Table 4 (Figure 1(D)). The addition of STS to the regeneration media increased organogenesis in higher plants [24] were Ag+ ions inhibit ethylene action in a wide variety of ethylene induced responses in plants by reducing the receptor capacity to bind ethylene. Thus, silver thiosulphate might be useful as a media supplement to develop efficient protocols for in vitro propagation of S. viarum as it favors the shoot formation. The beneficial effects of the ethylene inhibitor STS on organogenesis have been widely reported regeneration in organogenesis of Asclepias curassavica [25]. The silver ions inhibit ethylene action in a wide variety of ethylene induced responses in plants. The ethylene inhibiting effect of silver is believed to be due to an interference with ethylene binding [26]. The positive effective of silver in shoot organogenesis suggests that ethylene produced by cultured explants inhibit shoot organogenesis of those explants.
3.5. In vitro Rooting
After regeneration and sufficient elongation of the micro shoots length (3.0 - 5.0 cm) were carefully excised and transferred on MS medium supplemented with different concentrations of auxins such as IBA, IAA and NAA (0.5 - 2.0 mg/l) separately. IBA (1.0 mg/l) produced the maximum number of roots (24.3 ± 0.31) with root length (5.7 ± 0.62 cm) and highest regeneration frequency (100%) whereas the least number of roots (7.4 ± 0.21) with root length (1.8 ± 0.23) were seen in IAA (2.0 mg/l). NAA (1.0 mg/l) gave the second highest number of roots (18.3 ± 0.31) with root length (4.7 ± 0.21 cm) was observed Table 5 (Figures 1(E) and (F)).
3.6. Acclimatization and Hardening
Well rooted plantlets were separated from the culture

Table 4. Effect of different concentrations of silver thiosulphate (STS) supplemented in MS medium containing BAP (2.0 mg/l) on regeneration of plantlets from axillary bud explants of Solanum viarum. Data represent treatment means ± SE fallowed by different letter(s) within column indicate significant differences according to ANOVA and DMRT test (P < 0.05).

Figure 1. Axillary bud explants of Solanum viarum cultured on MS medium supplemented with various concentrations of bavistin, cefotoxime, kanamycine and silver thiosulphate. A. Multiple shoots regenerated from axillary bud explant with MS + bavistin (100 mg/L) + BAP (2.0 mg /L); B. Shoot regeneration from axillary bud explant on MS + cefotaxime (80 μM/L); C. Maximum shoots formed on MS + Kanamycin (50 μM/L); D. MS + sliver thiosulphate (40 μM/L); E. Rooting from regenerated shoots on MS medium (IBA 1.0 mg/L); F. Well rooted plants ready for hardening; G & H. Hardened plants in polycups and pots containing sterile soil and vermiculate (1:1 ratio).
tubes, washed and transferred to polycups containing soil and vermiculate in (1:1) ratio for hardening. Lastly the hardened plantlets were transferred to field conditions. (Figures 1(G) and (H)). Rooted shoots showed the maximum percentage of survival.
4. Conclusion
In the present investigation, it is noticeable that the antimicrobial agents eliminate the contaminating microorganisms and do not show any negative effect on S. viarum cultures and further it promotes shoot regeneration. Hence the shoot promoting activities of these antimicrobial agents are very useful in micropropagation and conversion of this very important medicinal plant.

Table 5. Root organogenesis of in vitro derived shoot lets of Solanum viarum supplemented with various concentrations of IAA, NAA and IBA using MS medium. Data represent treatment means ± SE fallowed by different letter(s) within column indicate significant differences according to ANOVA and DMRT test (P < 0.05).
Acknowledgements
The authors are grateful to the UGC BSR (Non-SAP) New Delhi, India for providing financial assistance in the form of fellowship.
NOTES