In Vitro Calli Production Resulted in Different Profiles of Plant-Derived Medicinal Compounds in Phyllanthus amarus

Abstract

The efficient production of plant-derived medicinal compounds (PDMCs) from in vitro plants requires improvements in knowledge about control of plant or organ development and factors affecting the biosynthesis pathway of specific PDMCs under in vitro conditions, leading to a realistic large-scale tool for in vitro secondary metabolite production. Thus, this study aimed to develop an in vitro technique, through the induction and proliferation of calli, for production of plant fresh weight, and to compare the PDMC profile obtained from the plants versus in vitro calli of Phyllanthus amarus. It was successfully possible to obtain and proliferate two types of calli, one with a beige color and a friable appearance, obtained in the dark using Murashige and Skoog (MS) medium plus 2,4-dichlorophenoxyacetic acid (2,4-D), and a second type with a green color, rigid consistency, and nonfriable appearance obtained under light conditions and MS medium plus 6-benzyladenine (6-BA). In vitro micropropagated plants that gave rise to calli were also acclimatized in a greenhouse and cultivated until obtaining the mass for PDMC analysis and used as a control. While the micropropagated-derived plants concentrated the lignans niranthin, nirtetralin, and phyllanthin, the Phyllanthus amarus calli proliferated in vitro concentrated a completely different biochemical profile and synthesis of compounds, such as betulone, squalene, stigmasterol, and β-sitosterol, in addition to others not identified by GC-MS database. These results demonstrate the possibility of applying the calli in vitro from Phyllanthus amarus for production of important PDMCs unlike those obtained in cultures of differentiated tissues from field plants.

Keywords: Phyllanthus amarus; callus; hypophyllanthin; micropropagation; phyllanthin; secondary metabolites.

Figure 1

Differential responses of nodal segments of Phyllanthus amarus obtained with individual or mixed treatments with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) (mg/L): T1, free of phytoregulators (control); T2, BA 1.0; T3, BA 2.0; 0.25 mg/L 2,4-D (T4) + BA 1.0 (T5) or 2.0 (T6); 0.50 mg/L 2,4-D (T7) + BA 1.0 (T8) or 2.0 (T9); 0.75 mg/L 2,4-D (T10) + BA 1.0 (T11) or 2.0 (T12). White bar = 1.0 cm.

Figure 2

Green callus obtained with BA and lighting conditions (A); micropropagated plantlets under in vitro (B) and acclimatized under greenhouse conditions (C); effects of salicylic acid and chitosan on callus elicitation obtained with 2,4-D (0.50 mg/L) and darkness conditions (D) (bars = 1.0 cm).