In the growth condition(s) of plants, numerous secondary metabolites (SMs) are produced by them to serve variety of cellular functions essential for physiological processes, and recent increasing evidences have implicated stress and defense response signaling in their production. The type and concentration(s) of secondary molecule(s) produced by a plant are determined by the species, genotype, physiology, developmental stage and environmental factors during growth. This suggests the physiological adaptive responses employed by various plant taxonomic groups in coping with the stress and defensive stimuli. The past recent decades had witnessed renewed interest to study abiotic factors that influence secondary metabolism during in vitro and in vivo growth of plants. Application of molecular biology tools and techniques are facilitating understanding the signaling processes and pathways involved in the SMs production at subcellular, cellular, organ and whole plant systems during in vivo and in vitro growth, with application in metabolic engineering of biosynthetic pathways intermediates.

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Stress and defense responses in plant secondary metabolites production

Plant cell and organ cultures have emerged as potential sources of secondary metabolites, which are used as pharmaceuticals, agrochemicals, flavors, fragrances, coloring agents, biopesticides, and food additives. In recent years, various strategies have been developed to assess biomass accumulation and synthesis of secondary compounds in cultures. Biomass accumulation and metabolite biosynthesis are two-stage events, and the parameters that control the growth and multiplication of cultured cells/organs and biomass accumulation are controlled in the first stage. Parameters that assist with the biosynthesis of metabolites are controlled in the second stage. The selection of high-producing cells or organ clones; optimization of medium parameters such as suitable medium, salt, sugar, nitrogen, phosphate, and plant growth regulator levels; and physical factors such as temperature, illumination, light quality, medium pH, agitation, aeration, and environmental gas (e.g., oxygen, carbon dioxide, and ethylene) are controlled in the first stage of the culture process. Elicitation, replenishment of nutrient and precursor feeding, permeabilization, and immobilization strategies assist with the accumulation of metabolites and can be applied in the second stage of the culture process. By following stage-specific strategies, it is possible to produce large amounts of biomass with an increase in the accumulation of secondary compounds.

Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation

Improving production of plant secondary metabolites through biotic and abiotic elicitation

Gymnema sylvestre (Asclepiadaceae), popularly known as “gurmar” for its distinct property as sugar destroyer, is a reputed herb in the Ayurvedic system of medicine. The phytoconstituents responsible for sweet suppression activity includes triterpene saponins known as gymnemic acids, gymnemasaponins, and a polypeptide, gurmarin. The herb exhibits a broad range of therapeutic effects as an effective natural remedy for diabetes, besides being used for arthritis, diuretic, anemia, osteoporosis, hypercholesterolemia, cardiopathy, asthma, constipation, microbial infections, indigestion, and anti-inflammatory. G. sylvestre has good prospects in the treatment of diabetes as it shows positive effects on blood sugar homeostasis, controls sugar cravings, and promotes regeneration of pancreas. The herbal extract is used in dietary supplements since it reduces body weight, blood cholesterol, and triglyceride levels and holds great prospects in dietary as well as pharmacological applications. This review explores the transition of a traditional therapeutic to a modern contemporary medication with an overview of phytochemistry and pharmacological activities of the herb and its phytoconstituents.

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Phytochemical and Pharmacological Properties of Gymnema sylvestre: An Important Medicinal Plant

Biotechnology is playing a vital alternative role in the production of pharmaceutical plant secondary metabolites to support industrial production and mitigate over-exploitation of natural sources. High-value pharmaceuticals that include alkaloids, flavonoids, terpenes, steroids, among others, are biosynthesized as a defensive strategy by plants in response to perturbations under natural environmental conditions. However, they can also be produced using plant cell, tissue, and organ culture techniques through the application of various in vitro approaches and strategies. In the past decades, efforts were on the clonal propagation, biomass and secondary metabolites production in the in vitro cultures of medicinally important plants that produce these molecules. In recent years, the effort has shifted towards optimizing culture conditions for their production through the application of cell line selection, elicitation, precursor feeding, two-phase co-culture among cell, tissue, and organ culture approaches. The efforts are made with the possibility to scale-up the production, meet pharmaceutical industry demand and conserve natural sources of the molecules. Applications of metabolic engineering and production from endophytes are also getting increasing attention but, the approaches are far from practical application in their industrial production.

Secondary metabolism of pharmaceuticals in the plant in vitro cultures: strategies, approaches, and limitations to achieving higher yield

Brahmi (Bacopa monnieri) is an important medicinal plant mainly used for the treatment of neurological disorders and depression. Recent investigations revealed that bacoside A is major chemical component shown to be responsible for memory facilitating action of brahmi. The current investigation was carried out to assess the potential for increasing biomass and the concentration of bacoside A in the in vitro regenerated shoots by varying sucrose and pH levels of shoot regeneration medium. The leaf explants were cultured on the Murashige and Skoog (MS) medium supplemented with 2 mg l−1 kinetin (KN) and with varying concentrations of sucrose (0, 1, 2, 3, 4, 5 and 6% at pH 5.8) and pH (4.5, 5.0, 5.5, 6.0 and 6.5 with 2% sucrose) with the objective of verifying the effects of sucrose and pH level on shoot regeneration and to verify the accumulation of bacoside A in the regenerated shoots. The shoot biomass increased (150.50 ± 2.84 shoots per explant, fresh wt 6.31 ± 0.12 g and dry wt 250 ± 5.00 mg) on the medium supplemented with 2% sucrose and pH which was set at 4.5. The results of HPLC analysis indicate that increase in sucrose concentration (0, 1, 2, 3, 4, 5 and 6% at pH 5.8) lead to decrease in the bacoside A content (39.51, 22.43, 13.05, 12.17, 10.73, 9.56 and 8.93 mg g−1 dry wt, respectively) in regenerated shoots. These findings provide evidence that stressful condition of inadequate supply of carbon elevated synthesis of bacoside A in brahmi shoots. However, 2% sucrose is found suitable for biomass accumulation. Therefore, medium supplemented with 2% sucrose and pH set at 4.5 was found suitable for both biomass (6.31 ± 0.12 g fresh wt and 250 ± 5.00 mg dry wt) and bacoside A accumulation (13.09 mg g−1 dry wt).

Effects of sucrose and pH levels on in vitro shoot regeneration from leaf explants of Bacopa monnieri and accumulation of bacoside A in regenerated shoots

The major objectives of this study were to investigate an efficient rapid protocol for mass propagation of adventitious shoots of brahmi using semisolid and liquid cultures; and to assess the amount of bacoside A accumulated in the regenerated shoots. Leaf explants were grown in vitro on Murashige and Skoog semisolid medium supplemented with 0.5, 1.0, 1.5, 2.0 and 2.5 mg l−1 6-benzyladenine or kinetin (KN) or thidiazuron (TDZ) for 4 weeks. Adventitious shoots developed from leaf explants on all cytokinin supplemented media. After 4 weeks of incubation, leaf explants were split into two batches and one set was subcultured on semisolid medium and another set in liquid medium containing same concentration of cytokinins where they have come from. Highest rate of shoot regeneration was observed for explants cultured on medium with 2 mg l−1 KN. The fresh and dry weight of shoots was also highest with this treatment. Liquid cultures were found suitable for proliferation of shoots (155.6 shoots per explant) and they also favored highest biomass accumulation (8.60 g fresh and 0.35 g dry biomass). The bacoside A contents were determined in shoots using HPLC. Analysis revealed that, the contents were highest with shoots regenerated on medium supplemented with 2 mg l−1 KN. The amount of bacoside A was highest in the shoots regenerated in liquid medium (11.92 mg g−1 DW) and it was 2.2-fold higher compared to shoots grown on semisolid cultures.

In vitro regeneration of brahmi shoots using semisolid and liquid cultures and quantitative analysis of bacoside A

Adventitious roots were induced directly from leaf segments of Andrographis paniculata on Murashige and Skoog (MS) medium with 5.3 μM α-naphthaleneacetic acid (NAA) and 30 g/l sucrose. Adventitious roots cultured in flasks using MS liquid medium with 2.7 μM NAA and 30 g/l sucrose showed higher accumulation of biomass (fresh and dry weight) and andrographolide within four weeks. Seven-fold increment of fresh biomass was evident in suspension cultures along with 3.5-fold higher andrographolide compared to natural plants. These results showed a great potentiality of adventitious root cultures for the production of andrographolide.

Production of andrographolide from adventitious root cultures of Andrographis paniculata.

The present work deals with optimization of adventitious shoot culture of Bacopa monnieri for the production of biomass and bacoside A and has investigated the effects of macro elements (NH4NO3, KNO3, CaCl2, MgSO4 and KH2PO4) and nitrogen source [NH4+/NO3−] of Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (MS) on accumulation of biomass and bacoside A content. Optimum number of adventitious shoots (99.33 shoots explant−1), fresh weight (1.841 g) and dry weight (0.150 g) were obtained in the medium with 2.0× strength of NH4NO3. The highest production of bacoside A content was also recorded in the medium of 2.0× NH4NO3, which produced 17.935 mg g−1 DW. The number of adventitious shoot biomass and bacoside A content were optimum when the NO3− concentration was higher than that of NH4+. Maximum number of shoots (70.00 shoots explant−1), biomass (fresh weight 1.137 g and dry weight 0.080 g) and also bacoside A content (27.106 mg g−1 DW) were obtained at NH4+/NO3− ratio of 14.38/37.60 mM. Overall, MS medium supplemented with 2.0× NH4NO3 is recommended for most efficient bacoside A production.

Effects of macro elements and nitrogen source on biomass accumulation and bacoside A production from adventitious shoot cultures of Bacopa monnieri (L.)

The gymnemic acid contents in various organs of Gymnema sylvestre were investigated by High Performance Liquid Chromatography (HPLC) method. The results shows that the content of gymnemic acid in various organs, obviously different, was 54.29, 31.66, 28.82, 27.67, 25.39, 20.56 and 1.31 mg·g−1 DW in shoot tips, flowers, nodes, leaves, internodes, roots and seeds, respectively. The highest gymnemic acid content (54.29 mg·g−1 DW) was found in shoot tip, 1.96 fold higher than that in leaves (27.67 mg·g−1 DW). Maximum quantity of gymnemic acid (35.39 mg·g−1 DW) was observed in the young leaves, which was 1.52 times higher than that in old leaves (23.07 mg·g−1 DW). The content of gymnemic acid in young, middle and old internodes was 26.47, 25.77 and 23.94 mg·g−1 DW, respectively, all lower than that in leaves (27.67 mg·g−1 DW), whereas the content of gymnemic acid in young, middle and old nodes was 27.96, 28.81 and 29.66 mg·g−1 DW, respectively, all higher than that in leaves. The study provides the scientific evidences for the rational development and utilization of Gymnema sylvestre resources, since over exploitation of natural stands has caused depletion of these plants in nature.

S. H. Manohar

Papers

Effects of sucrose and pH levels on in vitro shoot regeneration from leaf explants of Bacopa monnieri and accumulation of bacoside A in regenerated shoots

In vitro regeneration of brahmi shoots using semisolid and liquid cultures and quantitative analysis of bacoside A

Production of andrographolide from adventitious root cultures of Andrographis paniculata.

Effects of macro elements and nitrogen source on biomass accumulation and bacoside A production from adventitious shoot cultures of Bacopa monnieri (L.)

Distribution of gymnemic acid in various organs of Gymnema sylvestre