Phytochemical Composition and Detection of Novel Bioactives in Anther Callus of Catharanthus roseus L

Abstract

Catharanthus roseus L. (G.) Don is the most widely studied plant because of its high pharmacological value. In vitro culture uses various plant parts such as leaves, nodes, internodes and roots for inducing callus and subsequent plant regeneration in C. roseus. However, till now, little work has been conducted on anther tissue using plant tissue culture techniques. Therefore, the aim of this work is to establish a protocol for in vitro induction of callus by utilizing anthers as explants in MS (Murashige and Skoog) medium fortified with different concentrations and combinations of PGRs. The best callusing medium contains high α-naphthalene acetic acid (NAA) and low kinetin (Kn) concentrations showing a callusing frequency of 86.6%. SEM-EDX analysis was carried out to compare the elemental distribution on the surfaces of anther and anther-derived calli, and the two were noted to be nearly identical in their elemental composition. Gas chromatography-mass spectrometry (GC-MS) analysis of methanol extracts of anther and anther-derived calli was conducted, which revealed the presence of a wide range of phytocompounds. Some of them are ajmalicine, vindolinine, coronaridine, squalene, pleiocarpamine, stigmasterol, etc. More importantly, about 17 compounds are exclusively present in anther-derived callus (not in anther) of Catharanthus. The ploidy status of anther-derived callus was examined via flow cytometry (FCM), and it was estimated to be 0.76 pg, showing the haploid nature of callus. The present work therefore represents an efficient way to produce high-value medicinal compounds from anther callus in a lesser period of time on a larger scale.

Keywords: GC–MS; SEM–EDX; anther culture; flow cytometry; phytochemical profiling; ploidy level; secondary metabolites.

Figure 1

In vitro callus induction, proliferation and scanning electron microscopic (SEM) images of anther and anther-derived callus of C. roseus. (A,B): callus initiation (bars = 0.5 cm); (C,D): callus proliferation after 6 and 9 weeks, respectively (bars (C) = 1.0 cm, (D) = 0.5 cm); (E): side view of anther (bar = 200 µm); (F): a portion of anther-derived callus (bar = 20 µm).

Figure 1

In vitro callus induction, proliferation and scanning electron microscopic (SEM) images of anther and anther-derived callus of C. roseus. (A,B): callus initiation (bars = 0.5 cm); (C,D): callus proliferation after 6 and 9 weeks, respectively (bars (C) = 1.0 cm, (D) = 0.5 cm); (E): side view of anther (bar = 200 µm); (F): a portion of anther-derived callus (bar = 20 µm).

Figure 2

SEM–EDX analysis micrographs showing elemental composition of C. roseus. (A): field grown anther; (B): anther-derived callus.

Figure 3

Extract preparation for GC–MS analysis of C. roseus. (A): dried powder of anther-derived callus; (B): dried powder of field-grown anther; (C): methanolic extracts of the samples (A,B).

Figure 4

(A): GC–MS chromatogram (total ionic chromatogram) of methanolic extract of anthers of C. roseus; (B): GC–MS chromatogram (total ionic chromatogram) of methanolic extract of anther-derived callus of C. roseus.

Figure 4

(A): GC–MS chromatogram (total ionic chromatogram) of methanolic extract of anthers of C. roseus; (B): GC–MS chromatogram (total ionic chromatogram) of methanolic extract of anther-derived callus of C. roseus.

Figure 5

Flow cytometric histograms revealing ploidy level of (A) diploid leaves of C. roseus (standard) and (B) anther-derived callus of C. roseus.