In vitro mass propagation and production of bioactive metabolites from Valeriana jatamansi Jones and Tinospora cordifolia (Wild.) Hook.F. & Thoms

Abstract

Valeriana jatamansi Jones (Sugandhawal) is a perennial herb, belonging to the Caprifoliaceae family known for its medicinal and ethnobotanical values. The species is threatened in its natural habitats as its rhizomes and roots are used for the production of medicines in the herbal and pharmaceutical industries due to the presence of valepotriates. Similarly, Tinospora cordifolia (Wild.) Hook. f. & Thoms. (Gurjo) is a climbing shrub belonging to the Menispermaceace family and has been shown to have a variety of ethnomedicinal, pharmacological and medicinal activities. T. cordifolia is also one of the most commercially used plants in pharmacy. The phytochemical and pharmacological properties of these two species have been extensively studied worldwide. However, in the context of Nepal, they are yet to be explored. In the case of V. jatamansi Jones, research has been carried out to optimize an efficient plant tissue culture system for mass propagation. The nodal segments taken from the in vitro plants developed on BAP (2 mg/L) with different PGRs at different concentrations and 10% coconut water produced the maximum shoot length (6 cm), shoot number (13.1), root length (7.5 cm), and root number (19.6). RAPD and ISSR markers were used to confirm the genetic integrity of the in vitro plants, acclimatised plants and wild plants. The effects of sodium nitroprusside (SNP), a nitric oxide (NO) donor, and various growth regulators on callus induction and shoot organogenesis of V. jatamansi were investigated. 1.5 mg/L of 1-Naphthaleneacetic acid (NAA)+15 µM SNP induced the highest callus frequency (91.18%). Maximum in vitro shoot multiplication was observed with 10% CW+15 μM SNP (89.32%). In addition, IAA, IBA, NAA and the elicitors methyl jasmonate (MeJA) and salicylic acid were added to adventitious root cultures of V. jatamansi Jones to analyse the production and growth kinetics (SA). Among the plant growth regulators studied, NAA (2.0 mg/L) induced maximum biomass (PGRs). MeJA, increased the biomass of the root cultures as compared to SA. Root cultures were analysed for their phytoconstituents and antioxidant potential. Methanolic in vitro extracts of the roots of V. jatamansi in NAA (2 mg/L) gave the highest content of phenols (55±1.00 mg GAE/g), flavonoids (219±1.00 mg QE/g) and DPPH activity (84.33±0.577%) as compared to the wild plants. Compared to NAA, the elicitor MeJA produced more biomass but less phytoconstituents. SA did not produce significant biomass. The GC-MS analysis showed that the roots treated with NAA accumulated 50 bioactive

compounds, similar to the roots of the wild plants, which contained 48 compounds. Valepotriates such as valeric anhydride, valeric acid, and derivatives were found in areas of the treated roots that had higher concentrations than those of the wild plants. The MeJA-treated cultures accumulated 21 compounds while the SA elicitation accumulated 23 compounds, including derivatives of valeric acid. The examination of gene expression using the sesquiterpene synthase genes revealed that the TPS2 gene expression was significantly higher in both samples (more than five fold increase) while TPS5 had the highest gene expression (16 fold) and TPS6 had a four-fold increase in the NAA treated in vitro root cultures compared to the in vivo root cultures. In T. cordifolia, the explants of nodal segments were first treated in MS with different doses of BAP and/or KIN, with MS+BAP (2.0 mg/L) and the nodal segments were treated with various concentrations of BAP, BAP+KIN, and coconut water for proliferation of which, 5 mg/L induced significant shoot induction (9.0 shoots per explant), and mean shoot length (8.0 cm). Nodal segments proliferation increased when the media were supplemented with nodal segments at 5% and 10%. However, the addition of MS BAP (5mg/L)+10% coconut water induced maximum response with shoot number (24.0) and mean shoot length (12 cm). The genetic fidelity of these plants was also confirmed by RAPD and ISSR marker analyses in wild and in vitro cultures. Total phenolic content (TPC) was highest in wild stem followed by in vitro plant extract and wild leaves. The TFC content, and antioxidant potential were highest in wild stems followed by the wild leaves and the in vitro plant. (TPC; wild stem 128.55±0.491 mg/g, in vitro plant 107.33±0.985, wild leaves 105.6847±1.060) (TFC wild stem; 83.61±0.2773 mg/g, in vitro plant 58.034±0.148, wild leaves 62.50±0.329). Antioxidant activity; wild stem with (IC50 193.229±0.013), in vitro plant with (IC50 219.83±0.468), wild leaves with (IC50 204.386±0.165). GC-MS Analysis of the wild stem revealed the presence of 40 compounds and 31 compounds in the in vitro plant detecting various bioactive compounds including palmitic acid. In the present study mass propagation through in vitro cultures of V. jatamansi and T. cordifolia, and phytochemical screening of both V. jatamansi and T. cordifolia were standardised which is the first of its kind with respect to these species in Nepal. Root cultures establishment, characterization of valepotriates in adventitious root cultures of V. jatamansi and expression of the sesquiterpene synthase gene was successfully developed