Hosakatte Niranjana Murthy

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

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.

The effect of light quality on the growth and development of in vitro cultured Doritaenopsis plants

The influence of light quality on growth and development of in vitro grown Doritaenopsis hort. (Orchidaceae) plants was investigated. Growth parameters like leaf and root fresh/dry mass and leaf area were highest with plants grown under red plus blue light emitting diodes (LEDs). Leaf length was greater with the plants grown under red LED. Carbohydrate (starch, sucrose, glucose and fructose) and leaf pigment (chlorophylls and carotenoids) biosynthesis of the plants was significantly increased in plants grown under red plus blue LEDs compared to red or blue LED and fluorescent light treatments. This study suggests that the production of quality Doritaenopsis plants is possible by culturing the plants in vitro under a mixture of blue plus red light sources.

Rapid propagation of Phalaenopsis from floral stalk-derived leaves

SummaryAn efficient and rapid in vitro method was developed for regeneration of Phalaenopsis using leaf segments derived in vitro from flower stalk nodes. Leaf segments of four cultivars Tinny Sunshine ‘Annie’, ‘Taisuco Hatarot’, Teipei Gold ‘Golden Star’, Tinny Galaxy ‘Annie’ cultured on Murashige and Skoog medium supplemented with N6-benzyladenine (BA; 88,8 μM) and α-naphthaleneacetic acid (NAA; 5,4 μM) produced an average of 10–13 protocorm-like bodies (PLBs) after 12 wk. PLB proliferation was achieved on a modified Hyponex medium (1 gl−1 6.5N−4.5P−19K+20N−20P−20K+2gl−1 peplone +3% (w/v) potato homogenate +0.05% activated 1 gl−1 charcoal) and an optimal number of 13–18 PLBs developed from single PLB sections of different cultivars. Plantlet development was also achieved on a modified Hyponex medium. By repeated subculture of PLBs on a proliferation medium, and culturing them in the plantlet regeneration medium, plantlets could be produced continuously. Approximately 6 mo, were required from the initiation of culture to the production of plantlets for transplant to community pots.

Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of Panax ginseng in 5-l balloon type bubble bioreactors

The effects of methyl jasmonate (MJ) elicitation on the cell growth and accumulation of ginsenoside in 5-l bioreactor suspension cultures of Panax ginseng were investigated. Ginsenoside accumulation was enhanced by elicitation by MJ (in the range 50–400 μM); however, fresh weight, dry weight and growth ratio of the cells was strongly inhibited by increasing MJ concentration. The highest ginsenoside yield was obtained at 200 μM MJ. In the second experiment, 200 μM MJ was added on day 15 during the cultivation. The ginsenoside, Rb group, and Rg group ginsenoside content increased 2.9, 3.7, and 1.6 times, respectively, after 8 days of MJ treatment. Rb group gisnsenosides accumulated more than Rg group ginsenosides. Among Rb group ginsenosides, Rb1 content increased significantly by four times but the contents of Rb2, Rc and Rd increased only slightly. Among Rg group ginsenosides, Rg1 and Re showed 2.3-fold and 3.0-fold increments, respectively, whereas there was only a slight increment in Rf group ginsenosides. These results suggest that MJ elicitation is beneficial for ginsenoside production using 5-l bioreactor cell suspension cultures.

Adventitious roots and secondary metabolism.

Plants are a rich source of valuable secondary metabolites and in the recent years plant cell, tissue and organ cultures have been developed as an important alternative sources for the production of these compounds. Adventitious roots have been successfully induced in many plant species and cultured for the production of high value secondary metabolites of pharmaceutical, nutraceutical and industrial importance. Adoption of elicitation methods have shown improved synthesis of secondary metabolites in adventitious root cultures. Development of large-scale culture methods using bioreactors has opened up feasibilities of production of secondary metabolites at the industrial levels. In the present review we summarize the progress made in recent past in the area of adventitious root cultures for the production of secondary metabolites.

Large Scale Culture of Ginseng Adventitious Roots for Production of Ginsenosides

Ginseng (Panax ginseng C. A. Meyer) is one of the most famous oriental medicinal plants used as crude drugs in Asian countries, and now it is being used worldwide for preventive and therapeutic purposes. Among diverse constituents of ginseng, saponins (ginsenosides) have been found to be major components responsible for their biological and pharmacological actions. On the other hand, difficulties in the supply of pure ginsenosides in quantity prevent the development of ginseng for clinical medicines. Cultivation of ginseng in fields takes a long time, generally 5-7 years, and needs extensive effort regarding quality control since growth is susceptible to many environmental factors including soil, shade, climate, pathogens and pests. To solve the problems, cell and tissue cultures have been widely explored for more rapid and efficient production of ginseng biomass and ginsenosides. Recently, cell and adventitious root cultures of P. ginseng have been established in large scale bioreactors with a view to commercial application. Various physiological and engineering parameters affecting the biomass production and ginsenoside accumulation have been investigated. Advances in adventitious root cultures including factors for process scale-up are reviewed in this chapter. In addition, biosafety analyses of ginseng adventitious roots are also discussed for real application.

Establishment of cell suspension cultures of Withania somnifera for the production of withanolide A.

Cell suspension cultures of Withania somnifera were established in shake flasks and the effect of different growth regulators (auxins, combination of auxin and cytokinin), inoculum density (2.5-20 g L(-1)), different media (MS, B5, NN and N6), the strength of the MS medium (0.25-2.0 x), carbon source (sucrose, glucose, fructose, maltose), concentration of the sucrose (1-8% (w/v)) and the initial pH (4.0-6.5) of the medium were determined for the production of withanolide A. The optimized conditions for biomass accumulation and withanolide A production were found to be 10 g L(-1) of the inoculum on fresh weight basis, the full strength MS medium, 3% (w/v) sucrose, four weeks culture period and the initial medium pH of 5.8. The results of present study are useful for scale-up process.

Protocorm-like body induction and subsequent plant regeneration from root tip cultures of Doritaenopsis

Abstract The effect of cytokinins thidiazuron (TDZ), benzyladenine or zeatin on protocorm-like body (PLB) induction from root tips of Doritaenopsis grown in vitro was studied. Among the cytokinins tested, TDZ was found to be more effective cytokinin in the induction of PLBs than benzyladenine (BA) or zeatin. On modified Murashige and Skoog medium (MS) supplemented with 2.3 μM TDZ the highest percentage of PLB formation (47.2%) occurred and each explant produced two to six PLBs. Histological observations indicated the existence of two kinds of pathways during PLB formation: direct PLB induction from the root meristem and callus-mediated PLB induction from cortical cells. During callus-mediated PLB regeneration, the initial cell divisions occurred in the cortex region of root apices followed by accumulation of callus like masses in the cortex region. These cell masses developed globular structures that developed into PLBs after 5 weeks of culture. Upon subculturing to a modified Hyponex medium, PLBs developed into plantlets that were successfully acclimatized in green house conditions.

In vitro propagation of the endangered orchid, Geodorum densiflorum (Lam.) Schltr. through rhizome section culture

Micropropagation of the endangered terrestrial orchid, Geodorum densiflorum (Lam.) Schltr. was achieved through rhizome section culture from in vitro seed- derived rhizomes. Rhizome sections were cultured on Murashige and Skoog (MS) and Knudson C(KC) media supplemented with various growth regulators and 0.1% activated charcoal. The rhizome sections responded on MS medium. Naphthaleneacetic acid (NAA) at 2.0 μM stimulated rhizome growth. However, benzyladenine (BA) at 5.0 μM induced multiple shoots within four weeks of culture and inhibited rhizome growth. The regenerated shoots rooted on MS only or with NAA at 1.0 μM. Well-developed plantlets were transferred to community pots and then to a greenhouse where the plants have been acclimatised.

Production of adventitious root biomass and secondary metabolites of Hypericum perforatum L. in a balloon type airlift reactor

The effects of inoculum density, aeration volume and culture period on accumulation of biomass and secondary metabolites in adventitious roots of Hypericum perforatum in balloon type airlift bioreactors (3 l capacity) were investigated. The greatest increment of biomass as well as metabolite content occurred at an inoculum density of 3 g l(-1) and an aeration volume of 0.1 vvm. After 6 weeks of culture, an approximately 50-fold increase in biomass was recorded containing 60.11 mg g(-1) dry weight (DW) of phenolics, 42.7 mg g(-1) DW of flavonoids and 0.80 mg g(-1) DW of chlorogenic acid. Liquid chromatography-mass spectroscopy/mass spectroscopy demonstrated that the presence of quercetin and hyperoside in adventitious roots at a level of 1.33 and 14.01 μg g(-1) DW, respectively after 6 weeks of culture. The results suggest scale-up of adventitious root culture of H. perforatum for the production of chlorogenic acid, quercetin and hyperoside is feasible.