Differential Gene Expression and Withanolides Biosynthesis During in vitro and ex vitro Growth of Withania somnifera (L.) Dunal

Abstract

Ashwagandha (Withania somnifera L. Dunal) is a medicinally important plant with withanolides as its major bioactive compounds, abundant in the roots and leaves. We examined the influence of plant growth regulators (PGRs) on direct organogenesis, adventitious root development, withanolide biosynthetic pathway gene expression, withanolide contents, and metabolites during vegetative and reproductive growth phases under in vitro and ex vitro conditions. The highest shooting responses were observed with 6-benzylaminopurine (BAP) (2.0 mg L^-1) + Kinetin (KIN) (1.5 mg L^-1) supplementation. Furthermore, BAP (2.0 mg L^-1) + KIN (1.5 mg L^-1) + gibberellic acid (GA₃) (0.5 mg L^-1) exhibited better elongation responses with in vitro flowering. Half-strength MS medium with indole-3-butyric acid (IBA) (1.5 mg L^-1) exhibited the highest rooting responses and IBA (1.0 mg L^-1) with highest fruits, and overall biomass. Higher contents of withaferin A (WFA) [∼8.2 mg g^-1 dry weight (DW)] were detected in the reproductive phase, whereas substantially lower WFA contents (∼1.10 mg g^-1 DW) were detected in the vegetative phase. Cycloartenol synthase (CAS) (P = 0.0025), sterol methyltransferase (SMT) (P = 0.0059), and 1-deoxy-D-xylulose-5-phosphate reductase (DXR) (P = 0.0375) genes resulted in a significant fold change in expression during the reproductive phase. The liquid chromatography-mass spectrometry (LC-MS) analysis revealed metabolites that were common (177) and distinct in reproductive (218) and vegetative (167) phases. Adventitious roots cultured using varying concentrations of indole-3-acetic acid (IAA) (0.5 mg L^-1) + IBA (1.0 mg L^-1) + GA₃ (0.2 mg L^-1) exhibited the highest biomass, and IAA (0.5 mg L^-1) + IBA (1.0 mg L^-1) exhibited the highest withanolides content. Overall, our findings demonstrate the peculiarity of withanolide biosynthesis during distinct growth phases, which is relevant for the large-scale production of withanolides.

Keywords: Withania somnifera; adventitious roots; differential gene expression; ex vitro; in vitro; metabolites; organogenesis; withanolide.

FIGURE 1

The in vitro culture of Withania somnifera. (A) Shoot multiplication (scale = 2.5 cm), (B) shoot elongation (scale = 3.6 cm), (C) shoot elongation and rooting (scale = 5.8 cm), and (D) hardening of plantlets (scale = 7.7 cm).

FIGURE 2

Multiple shoot induction in Withania somnifera. (A) Induction of multiple shoots from nodal explant with 6-benzylaminopurine (BAP) and Kinetin (KIN) (scale bar = 0.5–1.3 cm), (B) multiplication of shoots with BAP and TDZ (scale bar = 1.00–3.05 cm), (C) elongation of shoots using BAP and KIN (scale bar = 2–5.3 cm), (D) induction of roots from elongated shoots [indole-3-butyric acid (IBA) 0.3 mg/L] (scale bar = 6.5 cm), (E) well-developed plantlets (scale bar = 18 cm), and (F) hardening of plantlets at 25 ± 2 °C.

FIGURE 3

In vitro flowering of Withania somnifera. (A) In vitro flower formation from second node (scale bar = 0.7 cm), (B) first and second nodes (scale bar = 0.6 and 0.5 cm); (C,D) second node (scale bar = 0.6 and 0.4 cm), (E) sixth node (scale bar = 0.5 cm), and (F) third, fourth, and fifth nodes (scale bar = 0.4, 0.3, and 0.7 cm).

FIGURE 4

Ex vitro flowering in 30-day-old plantlets of Withania somnifera. Flower formation from (A) 6th node (scale bar = 13.7 cm), (B) 7th node (scale bar = 14.4 cm), (C) 11th node (scale bar = 17.5 cm), (D) 10th node (scale bar = 12.8 cm), (E) 10th node (scale bar = 12.5 cm), and (F) 12th node (scale bar = 17.6 cm).

FIGURE 5

Induction and culture of adventitious roots. (A) Profuse growth of thick ramified roots after 25 days, (B) active root growth observed at 25 days, (C) thin, brittle roots, turned brown after 30 days, (D) thick roots, showing a hint of callogenesis at midrib after 40 days, (E) adventitious roots in MS basal media, (F) roots in MS + 0.5 mg L^–1 indole-3-acetic acid (IAA) + 1.0 mg L^–1 IBA, (G) MS + 0.5 mg L^–1 IAA + 1.0 m gL^–1 IBA + 0.20 mg L^–1 gibberellic acid (GA₃), and (H) MS media + 0.5 mg L^–1 IAA + 2.0 mg L^–1 IBA.

FIGURE 6

Expression of selected withanolide biosynthetic pathway genes analyzed by qRT-PCR in the leaves of Withania somnifera from vegetative (VP) and reproductive (RP) growth phases. The data were represented as mean ± SEM of fold change with respect to VP phase (n = 3) and multiple t-test were performed. Significant difference at *P > 0.05; **P > 0.01.

FIGURE 7

Withanolide content (mg g^–1 DW) of Withania somnifera. (A) RP and (B) VP. Mean values within a column having the same alphabet are not significantly different (p = 0.05) according to Duncan’s multiple range test (DMRT) (n = 3).

FIGURE 8

Withanolide A and withanone content (mg g^–1 DW) from root cultures of Withania somnifera. (1) MS + 0.5 mg L^–1 IAA + 1.0 mg L^–1 IBA, (2) MS + 0.5 mg L^–1 IAA + 2 mg L^–1 IBA, (3) MS + 0.5 mg L^–1 IAA + 2 mg L^–1 IBA + 0.2 mg L^–1 GA₃, and (4) MS + 0.5 mg L^–1 IAA + 1 mg L^–1 IBA + 0.2 mg L^–1 GA₃. Mean values within a column having the same alphabet are not significantly different (p = 0.05) according to DMRT (n = 3).

FIGURE 9

Comparative analysis and distribution of untargeted LC-MS hits of metabolites from VP and RP from whole plant extract of Withania somnifera. The hits were noted and analyzed for its distribution in distinct growth phases.