Elicitation of gymnemic acid production in cell suspension cultures of Gymnema sylvestre R.Br. through endophytic fungi

Abstract

The enhancement of plant secondary metabolite production in cell suspension cultures through biotic or abiotic elicitation has become a potential biotechnological approach for commercialization or large-scale production of bioactive compounds. Gymnema sylvestre R.Br. is an important medicinal plant, rich in a group of oleanane triterpenoid saponins called gymnemic acid, well known for its anti-diabetic activity. Two endophytic fungal strains were isolated from the leaves of G. sylvestre and identified as Polyancora globosa and Xylaria sp. based on the PCR amplification and internal transcribed spacer (ITS 1-5.8S-ITS 2) sequencing of 18S rRNA gene. The process of elicitation of cell suspension cultures of G. sylvestre with dried powder of fungal mycelia (DPFM) and extracellular culture filtrate (ECF) of endophytic fungi consistently enhanced the accumulation of gymnemic acid and the DPFM was proved to be an effective elicitor when compared to the ECF. The DPFM elicited the gymnemic acid content in the range of 2.57-10.45-fold, while the ECF elicited the gymnemic acid content in the range of 2.39-7.8-fold. P. globosa, a novel and a rare endophytic fungal strain, has shown a great influence on the production of gymnemic acid. Cell suspension cultures elicited with DPFM of P. globosa produced higher amount of gymnemic acid content (124.23 mg/g dried cell weight) compared to the cultures elicited with DPFM of Xylaria sp. (102.24 mg/g DCW). But the cultures treated with consortium of DPFM of both fungi showed great influence on the production of gymnemic acid (139.98 mg/g DCW) than the cultures treated with DPFM alone. Similarly, cultures treated with consortium of ECF of both fungi produced more gymnemic acid content (94.86 mg/g DCW) compared with cultures treated with ECF of Xylaria sp. (77.93 mg/g DCW) and ECF of P. globosa (78.65 mg/g DCW) alone.

Keywords: 18S rRNA; Biotic elicitor; Cell suspension cultures; Endophytic fungi; Gymnema sylvestre; Gymnemic acid; Polyancora globosa.

Fig. 1

Callus cultures developed from the leaves of G. sylvestre on MS media containing 2.0 mg L⁻¹ 2,4-D and 1.0 mg L⁻¹ Kinetin

Fig. 2

Phylogenetic tree constructed using Neighbor-joining method by comparing the ITS region of 18S rRNA gene of Xylaria sp. VJ1 (Accession number KF598857) with closely related strains obtained from NCBI, Genbank. The above and underneath each knot indicating the boot strap values for 1000 replications

Fig. 3

Phylogenetic tree constructed using Neighbor-joining method by comparing the ITS region of 18S rRNA gene of P. globosa VJ17 (Accession number KJ638719) with closely related strains obtained from NCBI, Genbank. The above and underneath each knot indicating the boot strap values for 1000 replications

Fig. 4

HPLC chromatograms of a standard gymnemagenin, b gymnemagenin extracted from the elicited cultures with DPFM of P. globosa, c gymnemagenin extracted from the elicited cultures with DPFM of Xylaria sp., d Gymnemagenin extracted from the elicited cultures with consortium of DPFM, e Gymnemagenin extracted from the elicited cultures with consortium of ECF, f Gymnemagenin extracted from the elicited cultures with ECF of P. globosa

Fig. 5

Effect of DPFM of Xylaria sp. with elicitation time on the accumulation of gymnemic acid in the cell suspension cultures of G. sylvestre

Fig. 6

Effect of DPFM of P. globosa with elicitation time on the production of gymnemic acid in the cell suspension cultures of G. sylvestre

Fig. 7

Effect of consortium of DPFM with elicitation time on the production of gymnemic acid in the cell suspension cultures of G. sylvestre

Fig. 8

Effect of ECF of Xylaria sp. with elicitation time on the production of gymnemic acid in the cell suspension cultures of G. sylvestre

Fig. 9

Effect of ECF of P. globosa with elicitation time on the production of gymnemic acid in the cell suspension cultures of G. sylvestre

Fig. 10

Effect of consortium of ECF with elicitation time on the production of gymnemic acid in the cell suspension cultures of G. sylvestre

Fig. 11

Schematic representation of the elicitation of gymnemic acid through cell suspension cultures of G. sylvestre using endophytic fungi