Seconadry metabolites

secondary metabolites

1. Secondary metabolite production and Bioreactor Ritasree Sarma

2. Secondary metabolites Secondary metabolites Chemicals produced by plants for which no role in growth, photosynthesis, reproduction, or other "primary" functions.   Secondary metabolism plays a pinnacle role in keeping all the of plants' systems working properly . 

3. Types of secondary metabolites Flavonoids and allied phenolic compounds Terpenoids Nitrogen-containing alkaloids and sulphur -containing compounds

4. Why tissue culture??

5. Culture system To regulate maximized production of useful compounds, culture system should be established first Here, establishment of three main culture systems will be introduced: Cell suspension culture Hairy root culture and Adventitious root culture Three kinds of system are all liquid-form culture systems

6. Establishment of cell culture system Establishment of cell suspension culture A) Callus induction During the callus induction, explants of plant origin should be surface sterilized and sliced into pieces about 0.5 cm 3 in clean bench Inoculated on autoclaved solid basic media (MS, B5, N6, White ) supplemented with sucrose, hormones and agar After the callus was induced, it should be sub-cultured

7. After sub-culture, usually, various types of callus can be found with different texture and color Callus of various types should be introduced into liquid media (most of the time, after removal of agar, the formula of solid media can be used for liquid media preparation) Contents determination of active compounds should be carried out for cell line selection

8. Screening of cell lines

9. Sustainable production of azadirachtin from differentiated  in vitro  cell lines of neem ( Azadirachta indica ) (A) Flower buds of 4 mm size used for ovary culture (B) An excised ovary from 4 mm flower buds (C) A 2-week-old ovary slice culture on MS + 2,4-D (0.5 µM) + kinetin (4.5 µM) (D) Same as (C), after 4 weeks, where the entire explant is covered with the cream, friable and fast-growing callus (E) A 4-week-old callus subculture on MS + BAP (5.0 µM) + IAA (0.5 µM), showing shoot proliferation (F) A 4-week-old bright green, compact callus (g), 4 weeks after subculture to the same medium, showing differentiation of shoots from dark green, compact nodular regions (H) Histological section of a regenerating ovary callus, showing well-developed tracheids Mithilesh Singh, and Rakhi Chaturvedi AoB PLANTS 2013;5:plt034

10. Sl. no. Media Per cent callusing response 1 MS basal medium 0.0l 2 MS + BAP (5.0 µM) 35.2g 3 MS + TDZ (5.0 µM) 13.4j 4 MS + 2,4-D (5.0 µM) 0.0l 5 MS + BAP (5.0 µM) + ABA (1.0 µM) 18.9i 6 MS + TDZ (5.0 µM) + ABA (1.0 µM) 35.8g 7 MS + 2,4-D (5.0 µM) + ABA (1.0 µM) 0.0l 8 MS + BAP (5.0 µM) + GA 3  (1.0 µM) 0.0l 9 MS + TDZ (5.0 µM) + GA 3  (1.0 µM) 10.5k 10 MS + 2,4-D (5.0 µM) + GA 3  (1.0 µM) 0.0l 11 MS + BAP (5.0 µM) + 2,4-D (1.0 µM) 55.6e 12 MS + BAP (5.0 µM) + 2,4-D (1.0 µM) + NAA (1.0 µM) 100a 13 MS + BAP (5.0 µM) + 2,4-D (1.0 µM) + CH (500 mg L −1 ) 54.3e

11. Identification and Quantification of azadirachtin by HPLC Source Medium Culture Azadirech tin content(mg/g DW) Ovary 1.38 ± 0.02eMS + BAP (9.0 µM) + IAA (5.0 µM) + CH (500 mg L −1 Redifferentiated 1.28 ± 0.02 MS + 2,4-D (0.5 µM) + kinetin (4.5 µM) Dedifferentiated 1.03 ± 0.01

12. Bioreactor   B ioreactor   It refers to any   manufactured  or  engineered   device or system that supports a biologically active environment Process   where   organisms  or  biochemically  active  substances   are used to essential produce product or biomass

13. Air driven bioreactor Rotating drum bioreactor Spin filter bioreactor Gaseous phase bioreactor Light introducing bioreactor(Photo bioreactor) 15 4/19/2016 BIOREACTOR CONSIDERATION

14. The simplest design is the air-driven bioreactor equipped with sparger at the bottom of the vessel It is widely used for plant cell, tissue, and organ cultures. In cases where the cells grow rapidly and the cell mass occupies 40-60% of the reactor volume, the flow characteristics become non-Newtonian and the culture medium can no longer be agitated by simple aeration AIR DRIVEN BIOREACTORS

15. Alternative designs to the airlift bioreactor have been used in the cultivation of plant cells where mixing or aeration is achieved at low shear rates. A bioreactor based on 2 concentric rotating cylinders as been used to grow Beta vulguris cells Aeration is provided by inner cylinder which was gas permeable. Mixing by vortices produced by Taylor- Couette Flow. TAYLOR-COUETTE FLOW 17 4/19/2016

16. SCHEMATIC DIAGRAM:BUBBLE COLUMN 18 Gas is sparged at the base Movement of the liquid is caused by the density differences

17. Another bioreactor is designed to provide bubble-free aeration via rotating coil of gas permeable membranes . It turns on rollers and the oxygen supply mechanism is entirely different from either the mechanically agitated or the air-lift bioreactor It is suitable not only for the growth of plant cell, tissue, and organs but also for the production of metabolites under high viscosity and high density cultures . ROTATING DRUM BIOREACTOR 19 4/19/2016

18. T his type of bioreactor is equipped with filters on which the culture is supported and with a shower nozzle for spraying on the medium. Seed cultures are inoculated on the filters and the medium is supplied to the culture by spraying from a shower nozzle . The drained medium is collected on the bottom of the bioreactor. This type of bioreactor is excellent for plant cell, tissue, and organ cultures because there is no mechanical agitation (e.g., driven impeller, aerator) and, therefore, the growth rate and the secondary metabolite production are enhanced. GASEOUS PHASE BIOREACTOR 20

19. LIGHT INTRODUCING BIOREACTOR/PHOTO BIOREACTOR A photo bioreactor is a bioreactor that incorporates a light source to provide photonic energy input into the reactor The light source was a sunlight collector system which operated automatically by computer control and the collected light was introduced into the bioreactor through the optical fibers Activation of specific enzymes such as phenylalanine ammonia lyase (PAL) and to induce the production of flavonoids or anthocyanins Photomorphogenesis such as development of leaves

20. THANK YOU