4.1 Callus multiplications: The callus of the Solanum villosum was successfully induced and multiplied in vitro using different plant growth regulators of cytokinins with 2,4-D as a constant supplement to the media. In our study, the most important growth regulator has played an important role in the callus induction was the Kinetin. Different studies had shown a positive impact of Kinetin on the callus culture when used in combination with 2,4-D. For example; callus cultures had been successfully produced using Kinetin and 2,4-D in Securinega suffruticosa (Raj et al. 2015), Viola uliginosa (Slazak et al. 2015), and Lantana camara Linn (Tahtamouni et al., 2020). This may be due to that Kinetin and 2,4-D has a major role in the plant tissue un-differentiation process (callus). On the other hand, our results using TDZ were not promising for callus multiplication. This may be that TDZ did not work well for producing friable callus in Solanum villosum and some herbaceous shrubs. On the contrary; Peganum harmala L. callus cultures were maximized at (1.5 mg/L) TDZ with 2.0 mg/L 2, 4-D) and gave (2.88 g) of callus fresh weight (Zatimeh et al., 2017). In this study, BA showed a moderate effect on callus culture. The callus remained friable and white in color, but the fresh weight reached up to 3.14 ± 0.16 at 2.0 mg/L of BA. In contrast, Veraplakorn (2016) found that BA was the best for callus multiplication in Lantana camara L. when used with NAA. We can notice from these results, that the plant differentiation process depends on both the type of the plant bgrwth regulator and the plant species. From previous studies, we can say that each plant species has its own growth pathway.
4.2 V-cryoplate cryopreservation: In our study Solanum villosum microshoots and callus were successfully preserved in liquid nitrogen using the V - cryoplate technique. The pre culture period of the plant material also affected significantly the preservation process. Previous studies reported that the preculture period could increase the survival rate of cryopreserved plant materials (Al abdallat et al., 2017; Shibli et al., 2016). V- cryoplate was successfully used to cryopreserve Foeniculum vulgar (Shibli et al., 2016). Also, Dianthus caryophyllus L. were successfully cryopreserved via V- cryoplate (Sekizawa, et al., 2011). The cryopresrved germplasm of the mint had shown a full regrowth when the v-Cryo plate method was used (Yamamoto, et al., 2012). Besides that, The mat rush (Juncus decipiens Nakai) lateral buds we cryopresrved successfully by v-cryoplate technique and the regrowth percentage reached 88% (Niino, et al. 2014). From previous studies, we had noticed that v-cryoplate technique had increased the success percentage of the cryopreservation process for different plant species rather than other ordinary methods such as vitrification and dehydration methods.
4.3 Seed cryopreservation experiment: Solanum villosum seeds cryopreservation was very efficient method. This because the viability and germination percentage is affected less by liquid nitrogen. Different plant species were tested for seed cryopreservation with different germination percentages. For example, Encholirium pedicellatum cryopreserved seeds showed high germination after cryopreservation (Tarré et al., 2007). Also (Solanum lycopersicum L.) cryopreserved seeds showed a high percentage of germination after cryopreservation (Al-Abdallat et al., 2017). So the seed cryopresrvation method could be used as a viable and quick method for the genetic resources preservation issue.
4.4 Antimicrobial activity: In this study, Solanum villosum methanol extracts have shown a wide range of antimicrobial activity against different microbes used. The methanol extract has been reported for it is activity against different bacterial strains (Al-Qudah, 2020). Different extract types were used in other plant species against microbes. For example; Tahtamouni (2018) found that Klebsiella pneumoniae inhibition zone (13.0 mm) was highly inhibited using Schinus molle leaves ethanolic extracts. On the other hand, Solanum nigrum ethyl acetate seed extracts were very efficient in prohibition of Pseudomonas, Proteous vulgaris, Klebsiella with the zones of inhibition (20.5–21.0 mm) (Sridhar et al., 2011). In our study the disk diffusion method was highly effected against Klebsiella pneumoniae and Staphylococcus epidermidis using the in vitro microshoots extracts. Two different methods of Agar disc diffusion and agar tube dilution were used in Solanum villosum to show the antimicrobial activity of their extracts (Abdel Gawwad et al., 2020). Abdel Gawwad et al., (2020) had used two extracts types (methanol and chloroform) and their activity reached up to 75% of prohibition against different bacteria species used. On the other hand, these two extracts gave about 50% of prohibition against fungi species used. Our results for the in vitro microshoots and callus extracts against microbes were recorded for the first time in Solanum villosum.
Furthermore the MIC method also was very promising using the extracts of the plant tissue culture materials of Solanum villosum against different used microbes. Previous studies had displayed different MIC effects from plant extracts on microbe’s species. Tahtamouni (2018) found that the fruit or leaf ethanol extracts of Schinus molle were very effective in prohibition of Micrococcus luteus at the level (6.25 mg/ml) using a microdilution assay method. On he other hand, the methanol extracts from different plant parts of Schinus mole did not express an inhibition effect against Micrococcus luteus (Tahtamouni, 2018). Solanum nigrum extracts also were effective using the MIC method against different microbes mainly; Pseudomonas putrida, Proteus vulgaris, and Klebsiella pneumonia (Sridhar et al., 2011). Also, methanolic extracts of S. torvum roots had exhibited promising results and antibacterial and antifungal effects on all organisms tested in comparison with that observed in the leaves, stems, and inflorescence extracts (Bari et al., 2010). From previous studies, we can see that the plant species and the plant material source have the major effect on antimicrobial activity. Also, we had noticed that for some microbes the plant tissue culture extracts were very promising more than wild types. This result can be used in future to depend on the in vitro sources of Solanum villosum to produce the extracts and other phytochemicals without depending on the wild source which could be not available in quantities and qualities all around the year. This is also, in order for commercial production for medicinal purposes from the Solanum villosum plant.