Seed germination in P. zeylanica
In the present study, ½-MS + GA3 showed seed germination and seedling length in the range of 76.67 – 88.33 % and 6.50 – 10.53 cm respectively with higher response at 2.89 µM of GA3 (Table 1, Figure 1 a – c). Whereas, significantly higher seed germination (86.67 – 98.33 %) and seedling growth (9.27 – 13.57 cm) was observed on different concentrations of ½-MS + SLEs (Table 1). Maximum seed germination (98.33 %) and seedling length (13.57 cm) was recorded for 2 % SLE which showed 1.11 and 1.28 fold increase for studied parameters as compared to the GA3 (2.89 µM) (Table 1, Figure 1 d – f). SLEs can be a new low-cost alternative to GA3 that can aid to overcome the production cost of GA3 (Camara et al. 2018) and its toxic effects in animals and humans (Boğa et al. 2009; Hosseinchi et al. 2013) which can limit its use for large scale seed germination and in vitro applications. The results in this study revealed that seed germination and development of seedling length in P. zeylanica is dependent on the concentrations of GA3 and SLEs, as at higher concentration there was a substantial decrease in response (Table 1). Similar response was reported in Lycopersicon esculentum (Vinoth et al. 2012) and Solanum melongena (Satish et al. 2015). Researchers have reviewed the chemical constituents of SLEs obtained from green and brown algae and have stated presence of GA3 (Nabti et al. 2016; Stirk et al. 2020), which could be the reason for higher seed germination and seedling development in P. zeylanica. In the present investigation, the in vitro seed germination protocol was not only established for rapid regeneration of seedlings, but also served as a source of sterile explants for in vitro studies that will eliminate the contamination in cultures.
Effect of PGRs on multiple shoots and root induction
Effect of PGRs on shoot multiplication has been reported in P. zeylanica earlier (Chaplot et al. 2006; Kanungo et al. 2012; Ceasar et al. 2013; Roy and Bharadvaja, 2017). In the present investigation two-stage culture system was used, where in the first stage MS media was fortified with cytokinins alone, while in the second stage the responsive media was used in combination with auxins. In first stage, MS media fortified with BAP showed a substantial response as compared to Kin and Zea (Table 2). MS + BAP (3.33 µM) showed 81.67 % shoot proliferation, 75.51 % explants with multiple shoot, 8.67 shoots (Table 2, Figure 2 a). These results are in accordance with the previous reports on in vitro regeneration of P. zeylanica which stated that BAP exhibited significant response for multiple shoot induction (Sahoo and Debata, 1998; Rout et al. 1999; Sivanesan and Jeong, 2009). Further this responsive media was used in combination with auxins like IAA (1.43 – 7.14 µM), NAA (1.34 – 6.70 µM) and IBA (1.23 – 6.15 µM) to enhance the number of multiple shoots (Table 2). Maximum response of shoot proliferation (96.67 %), shoots per explant (14.33), and cultures with multiple shoots (96.55 %) were observed on BAP + IAA (3.33 + 4.28 µM) after 4 weeks (Figure 2 b). Similar results are reported by Chaplot et al. (2006) where MS medium supplemented with different combinations of BAP and IAA developed multiple shoots. Thus, our results imply that two-stage culture system is superior to obtain high multiple shoot induction in P. zeylanica. Present study showed better response as compared to previous studies (Rout et al. 1999; Chaplot et al. 2006; Kanungo et al. 2012; Ceasar et al. 2013; Roy and Bharadvaja, 2017). Further, the multiple shoots grown on MS + BAP (3.33 µM) + IAA (4.28 µM) was excised and cultured on ½–MS medium having IBA, IAA, and NAA alone for rooting (Table 3). Rooting was successfully achieved on all the concentrations of studied PGRs where significantly higher response was recorded on IBA (6.15 µM) for shoots rooted (83.33 %), roots per shoot (26.33), and average root length (9.17 cm) (Table 3, Figure 2 c). This rooting response showed agreement with the previous studies in P. zeylanica (Chaplot et al. 2006; Caesar et al. 2013). The percentage of shoots rooted and average root length obtained on IBA (6.15 µM) was approximately 2.5 times higher, while the number of roots per shoot were 7 times higher as compared to the control (Table 3). Though the MS media supplemented with PGRs is found to be effective for micropropagation of P. zeylanica, its production on large scale can be hindered due to high cost of chemicals and longer culture duration which is 4 weeks for every stage of culturing. However, these limitations with micropropagation can be overcome by the employment of cost effective SLEs (Sharma et al. 2015).
Effect of SLEs on multiple shoots and root induction
SLEs contain several growth promoting hormones, nutrients, vitamins, amino acids, brassinosteroids, betains, sterols, polyamines and antibiotics which are responsible for growth development of plants by increasing the nutrient uptake and resistance to biotic and abiotic stresses (Stirk et al. 2014; Battacharyya et al. 2015; Nabti et al. 2016). Several reports bring to light use of SLEs for in vitro mass propagation in crop plants (Fakihi Kachkach et al. 2014; Satish et al. 2015; Vinoth et al. 2019). This study describes the effect of SLEs prepared from S. ilicifolium on the production of shoots and roots in P. zeylanica under in vitro condition. After 4 weeks’ study, maximum shoot proliferation of 98.33 % was observed in MS + SLE (5 and 6 %), whereas the highest cultures with multiple shoots (100 %) and number of multiple shoots (19.67) was noted on MS + 4 % SLEs (Table 4, Figure 3 a). The response of multiple shoots obtained on 4 % SLEs was 2.26 and 1.37 folds higher than BAP (3.33 µM) alone and in combination with IAA (4.28 µM) respectively (Table 2 and 4) and were used for root induction experiment. For root induction, explants were inoculated on ½–MS medium supplemented with SLEs (0.5 – 5 %) and the maximum rooting response for shoots rooted (88.33 %), roots per shoot (35.33), and root length (13.67) was recorded on 2.5 % SLE (Table 5 and Figure 3 b). The response observed on 2.5 % SLE was further compared with the rooting response of IBA (6.15 µM) which demonstrated 1.34 and 1.49 fold increase for the roots per shoots and average root length, respectively (Table 3 and 5). A similar study in Solanum melongena was carried out by Satish et al. (2015) who stated that SLEs played vital role in the in vitro multiple shoot induction and rooting response. Similar in vitro mass propagation of medicinal plants using SLEs is reported in Withania somnifera (Kannan et al. 2014), Ceropegia thwaitesii (Muthukrishnan et al. 2015), and Bacopa monnieri (Rency et al., 2016). Studies have reported the presence of cytokinins, auxins, and gibberellic acid from seaweed extracts (Khan et al. 2009; Battacharya et al. 2015), while brown algae are reported to be the most important source of PGRs due to the presence of high content of active compounds, continuous, and high availability all year round (Yalcin et al. 2019). Craigie (2011) enlisted the PGRs from a number of brown seaweeds, of which auxins, cytokinins and gibberellins have been reported from genus Sargassum. Elemental and hormone analyses of different Sargassum sp. revealed the presence of potassium, copper, manganese, zinc, iron, cobalt, magnesium, sodium, calcium, nitrogen, molybdenum, chloride, phosphate, sulphate and nitrate; and cytokinins, auxins, and gibberellins (Vijayanand et al. 2014; Ramya et al. 2015; Bharath et al. 2017; Uthirapandi et al. 2018). The in vitro response of P. zeylanica seed germination, multiple shoot induction and rooting can be because of the presence of these biostimulants. In the present study, all studied concentrations of SLEs showed exceedingly significant response for multiple shoots and root induction as compared to PGRs. These responses can perhaps be because of the presence of additional macro- and micro-elements, amino acids, and antibiotics in the SLEs (Nabti et al. 2016; Yalcin et al. 2019) that are essential for the growth and development of plants. We observed that the low concentration of SLEs are effective to attain optimum response which is similar to earlier studies in different plants (Vijayanand et al. 2014; Ramya et al. 2015; Bharath et al. 2017; Uthirapandi et al. 2018).
Effect of PGRs and SLEs on plumbagin accumulation
The 45 d old plantlets developed on ½–MS supplemented with auxins and SLEs were analyzed for the accumulation of plumbagin using HPTLC. For this analysis, various solvent systems were tested, of which n-hexane : ethyl acetate (7:3) showed better resolution and had reproducible peaks at Rf 0.60 representing plumbagin in the test samples (Figure 4). Significantly higher plumbagin content was observed on 2.5 % SLE (1588 µg/mg) which was 1.44 and 11 folds higher than that of 6.15 µM IBA (1101.33 µg/mg) and control (½–MS basal), respectively (Table 6, Figure 4 a – d). As compared to the PGRs and control, the plantlets cultured on SLEs showed higher accumulation of plumbagin, this is in concurrence with the previous study in Picrorhiza kurroa for the enhancement of picroside-I (Sharma et al. 2015). The increase in plumbagin content might be related to the higher availability of macro- and micro-elements from SLEs, which are used in media optimization and elicitation of cultures to enhance secondary metabolites in different plant systems (Battacharyya et al. 2015; Murthy et al. 2014). Also the SLEs contain different PGRs that modulate the accumulation of secondary metabolites in plant tissue culture (Jamwal et al. 2018). The present study elucidates that SLEs at lower concentrations are effective to accumulate plumbagin which are in concurrence with Ramya et al. (2015) who stated that lower concentrations of SLEs are more effective to alter biochemical responses.