The result showed that increasing Clorox concentration increases decontamination, but decreases viability of the seeds. The percentage of commercial Clorox needs to be varied depending on the species and environment [15]. On the other hand, the work is in disagreement with that of Srivastava et al. [16] who found that sterilization with NaOCl did not give acceptable sterilization even on increasing concentration over Aconitum heterphyllum (medicinal herb) seeds. According to our results, the highest percentage of decontaminated seeds was observed at 15% sodium hypochlorite (Clorox), but low germination percentage. Guanih et al. [15] found that sterilization of de-coated seeds of a medicinal plant, Dryobalanops lanceolata with 30% Clorox concentration for 5 min and cultured on MS medium showed less than 20% contamination, but sterilization of coated seeds with 50% Clorox concentration for more than 20 min broke the seed coat and reduced seed viability.
Among those Clorox concentrations, 10% Clorox resulted in 85% decontaminated seeds and the highest germination percentage (80%). Talei et al. [17] reported on medicinal herb, Andrographis pancuatanee, absence of significant correlation between contamination and germination percentages. Ghareeb and Taha [18] obtained 100% of decontamination for cultures and survival of Antigonon leptopus explants using 10% Clorox for 3 min. In general, the lower the Clorox concentration, the higher the contamination and germination percentage.
Our result showed that the de-coated seeds showed better germination than coated seeds. As Zulu et al. [19] reported that removing the seed coat could reduce contamination sources while improving the imbibitions of water and subsequent germination in S. longipedunculata. In addition, Negash [20] reported successful germination (91 ± 2.7%) of de-coated seeds of a tree plant, Prunus africana, in a laboratory.
In the present study, the germination on full-strength MS medium was best among other half-strength germination media. According to Pandey et al. [21], the highest percentage of seed germination (95%) from an important medicinal plant, Psoralea corylifolia, was recorded on full-strength MS medium. Maślanka and Bach [22] obtained higher percentage of germinating seeds on half-strength than full-strength MS medium after 6 months of culture. However, after 10 months, the highest germination percentage was observed on full-strength MS medium. Our result showed that none of the coated seeds germinated in all media. The reason may be due to the presence of the seed coat hampered the imbibitions of water. Pandey et al. [21] suggested the presence of seed coat challenges the entry of water and oxygen into the embryo. In contrast, Zulu et al. [19] obtained 45% germination rate of intact seeds of S. longipedunculata using GA3.
Among all media, the highest germination percentage (100%) of seeds was observed within 7–10 days on MS medium, when the seeds were de-coated and transversally cut at the tip. This is partly due to the removing of the seed coat facilitated the uptake of water and cutting at the tip reduces the size to use the limited amount of moisture on the MS medium. Similarly, Ganaie et al. [23] reported maximum germination percentage (96.66%) of Arnebia benthamii by removing the seed coat within mean germination time of around 4 days.
In this study, the effect of plant growth regulators on shoot induction from shoot explants was investigated. Sixteen different treatments including the control were used to induce shoots from S. longipedunculata shoot explants. These treatments, with various combinations of BAP and TDZ resulted in shoot initiation. The percentage response of explants for shoot induction, shoot number, and shoot length varies according to the type and concentration of cytokinins used [24]. The response of plants to different types and concentrations of growth regulators varies because of differences in endogenous level of growth regulators. In the present study, the shoots induced microshoots within a week on full-strength MS medium.
The highest percentage (87%) of explants survived on MS medium containing 1.0 mg/l BAP. Tiwari et al. [25] reported that MS medium containing 1.0 mg/l BAP was better for establishment of Trichosanthes dioica from nodal explants. The microshoots that resulted from MS medium containing 1.0 mg/l BAP were higher in number, but shorter in length than the shoots obtained from 1.0 mg/l BAP in combination with 0.5 mg/l TDZ. Ahmed and Anis [26] reported exposure of culture to TDZ had an adverse effect.
In general, the shoots were initiated better on MS medium containing BAP alone than MS medium containing TDZ alone or in combination with BAP. According to Kumar and Singh [27], BAP was found to perform better in shoot initiation of the multipurpose desert tree, Prosopis cineraria. Similarly, Malik and Saxena [28] pointed out the advantageous outcome of BAP for shoot induction as they observed on tissue culture of grain legume, Phaseolus vulgaris.
Application of BAP in combination with IBA with different concentrations induced the highest shoot number and length per explant. From our result, the MS medium containing 1.5 mg/l BAP in combination with 0.1 mg/l IBA produced the highest mean number of shoots (8.50 ± 0.69). Medium supplemented with auxin at low concentrations in combination with cytokinin promote the growth and formation of new shoots, consequently, increasing multiplication rate [29]. Adsul et al. [30] reported that nodal buds cultured on MS medium supplemented with 2.0 mg/l BAP in combination with 0.5 mg/l IBA resulted in production of maximum number of shoots (17.1 ± 1.2) of Ceropegia mohanramii. In contrast, Fraternale et al. [31] reported high concentration of IBA with BAP (1.5 mg/l) in MS medium was suitable for shoot multiplication of Bupleurum fruticosum. According to Askari-Khorasgani et al. [32], in comparison to the application of BAP alone, the combination of BAP along with IBA, led to more shoot formation. In addition, Ahmad et al. [33] stated that the response of the explants is a communal result of endogenous and exogenous plant growth regulators concentrations. Inclusion of a low concentration of auxin along with cytokinin activates much higher rate of shoot multiplication [34, 35]. Bramhanapalli et al. [36] reported high frequency shoot bud induction from different explants on MS medium containing 2.0 mg/l BAP as compared to other tested media indicating the high influence of BAP on shoot multiplication.
The highest mean shoot length (2.11 ± 0.45 cm) was obtained on 2.5 mg/l BAP in combination with 0.1 mg/l IBA whereas the lowest shoot length (0.96 ± 0.40 cm) was recorded on MS medium containing 0.5 mg/l BAP in combination with 0.5 mg/l IBA. This is surprising as the concentration of BAP increased, shorter shoots are expected because of apical dominance indicating the present finding requires further investigation.
On medium containing TDZ alone or in combinations with IBA, the highest mean number of shoots per explant (6.20 ± 0.43) was produced on MS medium containing 0.5 mg/l TDZ alone. Mirici [37] reported that TDZ alone prompted reasonable shoot multiplication in Astragalus polemoniacus.
In terms of length, the highest mean shoot length (1.55 ± 0.09 cm) was obtained on MS medium containing 0.5 mg/l TDZ. Aasim et al. [38] showed that the maximum number of shoots was recorded on MS medium containing 0.4 mg/l TDZ with highest shoot length of 1.20 cm. On the other hand, the shortest mean shoot length (0.31 ± 0.02 cm) was produced on MS medium containing 1.0 mg/l TDZ in combination with 0.5 mg/l IBA. Bisht et al. [39] who worked on Polygonatum verticillatum, suggested that the promoter effect of TDZ regarding growth is due to its own biological activities similar to N-substituted cytokinin or it may induce the synthesis or accumulation of an endogenous cytokinin. In case of TDZ and NAA combinations, the highest mean number of shoots (4.70 ± 0.32) was produced on MS medium containing 1.5 mg/l TDZ in combination with 0.5 mg/l NAA
Generally, among all growth regulators used in shoot multiplication, the highest mean shoot number (8.5 ± 0.69) was obtained on MS medium containing 1.5 mg/l BAP in combination with 0.1 mg/l IBA. On the other hand, the lowest mean shoot number per explant (2.10 ± 0.12) was obtained on basal MS medium. MS medium containing 2.5 mg/l BAP in combination with 0.1 mg/l IBA produced the highest mean shoot length (2.11 ± 0.45 cm) while the shortest mean shoot length (0.16 ± 0.01 cm) was produced on MS medium containing 2.5 mg/l TDZ in combination with 0.01 mg/l NAA and this short shoot production could be due to higher inhibition of apical dominance by TDZ than BAP.
Interestingly, full-strength MS medium containing 2.0 mg/l IAA, resulted in better rooting response (3.73 ± 0.69) as compared to IBA and NAA contrary to the most common half-strength MS medium used for rooting. IAA was found to be effective on rooting of Malus pumila [40]. In addition, Alagesaboopathi [41] reported 55.14% of rooting from Andrographis macrobotrys using IAA. Contrary to the results of our study, Shen et al. [42] pointed out that IAA failed to induce root formation on a medicinal tree plant, Casuarina cunninghamiana. It is important to work more on in vitro production of root and also root culture as the roots are used for medicinal purposes.
In this study, 60% plants survived in glasshouse after a month. This low survival percentage might be due to small root number or hairs, fungal contamination, and rotting. According to Bohidar et al. [43] who worked on medicinal plant, Ruta graveolens, the dead plantlets were due to improper development of root system in the culture. None of the plantlets survived in pots which were not covered with polyethylene bags.