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.
Types of secondary metabolites
Flavonoids and allied phenolic compoundsTerpenoidsNitrogen-containing alkaloids and sulphur-
containing compounds
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
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 cm3 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
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
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
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) + GA3 (1.0 µM) 0.0l
9 MS + TDZ (5.0 µM) + GA3 (1.0 µM) 10.5k
10 MS + 2,4-D (5.0 µM) + GA3 (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
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
Bioreactor
Bioreactor • 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
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Air driven bioreactor
Rotating drum bioreactor
Spin filter bioreactor
Gaseous phase bioreactor
Light introducing bioreactor(Photo bioreactor)
BIOREACTOR CONSIDERATION
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
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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
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SCHEMATIC DIAGRAM:BUBBLE COLUMN
Gas is sparged at the base
Movement of the liquid is caused by the
density differences
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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
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This 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
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