Callus culture and suspension culture can be used to produce secondary metabolites. Callus is formed from explants on semi-solid media with auxins and cytokinins. Suspension cultures use broken up callus in liquid media with agitation. Secondary metabolite production can be improved by adding precursors, elicitors, mutagenesis, or immobilization. Important products from plant cell cultures include dyes, pharmaceuticals like vinblastine and taxol, and precursors like diosgenin. Vinblastine is produced from coupling catharanthine and vindoline in Catharanthus roseus cultures.

1. Callus culture, suspension
culture and secondary metabolite
production

2. Callus culture

3. CALLUS
A callus consists of an amorphous mass of loosely
arranged thin-walled parenchyma cells arising from the
proliferating cells of the parent tissue

4. Callus
• Dedifferentiation of explant
– Loosely arranged thinned walled,
outgrowths from explant
• Often can be maintained indefinitely by
subculture, but may lose ability to
redifferentiate

5. The phenomenon of a mature cell
reverting back to meristematic state and
forming an undifferentiated mass of cells
(Callus).
Dedifferentiation
The ability of dedifferentiated cell to form a
whole plant / plant organs.
Redifferentiation

6. Callus culture
• Auxin + cytokinin: Intermediate ratios
of 1:1 favor callus growth
• Explants
– Plant parts including hypocotyls, cotyledons,
leaves, petioles, roots, flower stalks, anthers,
embryos and seeds.
• Compact vs friable

7. Habituation in callus
• Hormone-autonomous callus which did not
require plant growth regulators for growth
and subsequent regeneration is known as
habituation

8. Three stages of callus culture
• Induction: Cells in explant dedifferentiate and begin
to divide
• Proliferative Stage: Rapid cell division
• Differentiation stage (sometimes): metabolic
pathway or organogenesis

10. Suspension Culture

11. Suspension cultures
• Can be initiated from any
part of the plant.
• Usually initiated from friable
callus already growing in
culture.
• Transferred into liquid
medium.

12. Cell suspension culture
• When callus pieces are agitated in a liquid medium, they tend
to break up.
• Suspensions are much easier to bulk up than callus
• Large scale (50,000l) commercial fermentations for Shikonin
and Berberine.

13. Introduction of callus into suspension
• ‘Friable’ callus goes easily
into suspension
– 2,4-D
– low cytokinin
– semi-solid medium
– enzymic digestion with
pectinase
• Removal of large cell
aggregates by sieving
• Plating of single cells and
small cell aggregates - only
viable cells will grow and
can be re-introduced into
suspension

14. Agitation
• Breakdown of cell aggregates into smaller clumps of
cells
• Maintains a uniform distribution of cells and cell
clumps in the medium
• Provides gas exchange

15. Synchronization
• Cold treatment: 4oC
• Starvation: deprivation of an essential growth compound, e.g.
N →accumulation in G1
• Use of DNA synthesis inhibitors: thymidine, 5-
fluorodeoxyuridine, hydroxyurea
• Colchicine method: arresting the cells in metaphase stage,

17. Growth Curve
E. Sutton, UC Davis

18. Batch Cultures
• A certain number of cells is used to inoculate the
culture, in a given volume
• Erlenmeyer flask: volume should be about 20% of
flask capacity for aeration.

20. Continuous Culture
• Bioreactors
• Closed continuous cultures: Remove some of the media and
replace with fresh. Continuous removal or periodic. Terminate
growth at harvest. Start over.
• Open continuous culture: Not only remove some of media, but
cells too. Maintain cell density at optimal level. Can be grown
for years.

22. Choice of bioreactor
• If secondary products are produced at the end of
the growth phase, it is logical to consider a two-step
process where a first reactor is used for building up
the biomass, and a second one for metabolite
production.
• On the other hand, when the production of a given
metabolite is growth-associated,a single-step
reactor is sufficient to grow the cells and recover the
molecules at the same time

23. Choice of bioreactor
If the metabolites remain intracellular, it is usually
necessary to kill the biomass, so that the chemicals
can be extracted from the cells. This leads to a
batch or fed-batch process. Conversely,
extracellular production avoids destruction of the
biomass for the extraction of the compounds as
they can be directly recovered from the medium.

24. 1. Packed cell volume (PCV)
2. Cell Number:
3. Wet and Dry weight :
4. Protein and DNA content
5. Medium conductivity:
6. Cell viability:
7. Mitotic Index
Growth of cells in suspension cultures

26. Secondary metabolite
production

27. Cell suspension cultures
• it is necessary to screen the different callus lines to provide
an efficient metabolite production.
•
.

28. Methods to improve secondary
metabolite yield
• Addition of precursors: Addition to the culture media of
appropriate precursors or related compounds stimulates
secondary metabolite production.
• For example, amino acids have been added to cell
suspension culture media for production of tropane
alkaloids, indole alkaloids and Phenylalanine to salvia
cultures stimulated production of rosmarinic acid

29. Methods to improve secondary
metabolite yield
• This consists in applying chemical or physical stresses to
the cell suspension cultures that will trigger the production
of secondary metabolites that are normally not produced.
• This is currently done with biotic elicitors (chitosan,
autoclaved mycelium of pathogenic fungi, various protein
extracts) or abiotic factors (temperature, UV light, heavy
metal salts, pH, etc.).

30. Methods to improve secondary
metabolite yield
• Other methods than elicitation have been
developed with cells in liquid systems such as
immobilization .In this case plant cells or micro
aggregates are encapsulated in polymers(alginate,
carraghenans, etc.), and this usually enhances the
production of secondary metabolites The main
explanations for this come from a possible matrix
effect of the polymers around the cells which could
mimic a tissue organization between them

31. • Elictor treatment: Microbial infections of
infected plants often elicit the synthesis of
specific secondary metabolites. E.g Papaver
somniferum- accumulated sanguinarine with
Botryytis fungus, addition of yeast to
cultured cells of Lithosprmum increased
rosmarinic caid
Methods to improve secondary
metabolite yield

32. Methods to improve secondary
metabolite yield
• Phytochemical elicitation using simple organic
and inorganic molecules. E.g Sodium
orthovanadate and vanadyl sulphate induced
accumulation of isoflavone glucosides in Vigna
angularis and indole alkaloid in Catharanthus

33. Methods to improve secondary
metabolite yield
• Mutagenesis- Increase of metabolites using
regulatory mutants. X ray treatment of Anisodus
resulted in 30 per cent higher scopolamine than the
parent and X ray treatment to C.roseus increased
serpentine levels to 2 per cent.

34. Dyes
• Shikonin- Lithospermum erythrorhizon
• Anthraquinones-morinda citrifolia, Rubia tinctorum
• Anthocyanins-Daucus carota, Euphorbia milli, Vitis
vinifera, Aralia cordata
• Betalins-Chenopodium rubrum
• Betacyanin, betaxanthine- Beta vulgaris-
• Crocin and crocetins-Crocus sativus
• Safflower yellow, Carthamin -Carthamus tinctorius

35. Pharmaceutical compounds
• Andrographis- paniculid
• Catharanthus roseus—Catharanthine, serpentin,
vinblastine and vincristine
• Diocscorea deltoidea- Diosgenin
• Panax gingene- Gingenoside
• Atropa belladonna Tropane alkaloid
• Taxus brevifolia-taxol

36. Vinblastine & Vincristine
• –indole alkaloids – cell cultures
• The dimeric indole alkaloids, vinblastine and
vincristine have become highly valued drugs
in cancer chemotherapy due to their potent
antitumor activity against various leukemias,
and solid tumors.
• They are currently produced commercially by
extraction from Catharanthus roseus
(Apocyanaceae) plants, but concentration of
both vinblastine and vincristine was only
0.0005% as a dry weight basis.

37. Vinblastine
• The vinblastine molecule is derived from
two monomeric alkaloids, catharanthine
and vindoline.
• The concentration of vindoline in the intact
C. roseus plant is approximately 0.2% as a
dry weight basis, which is much a higher
level than catharanthine, and the cost of
vindoline is less expensive compared to
catharanthine and vinblastin

38. cell suspension culture
• process with a selected C. roseus cell line
induced from anthers on Gamborg's B5 medium
containing 2% sucrose, 1 mg/L 2,4-D and 0.1
mg/L kinetin.
• The cells were grown in 250 ml flasks containing
60 ml of MS liquid medium supplemented with
3% sucrose, 1 mg/L NAA and 0.1 mg/L kinetin
under continuous diffuse light on a rotary shaker
(250 r.p.m.) at 25 C.
•

39. catharanthine
• For optimization of catharanthine
production, they transferred 7 day old cells
to a test medium and subcultured for 3
passages. In the 4th passage, 60 ml
cultures were harvested in triplicate after 2
or 3 weeks growth, and the cell mass and
alkaloid content were determined.

40. Catharanthine
• Ferric ion catalyzed the coupling reaction
significantly in the absence of the enzyme.
Products of the chemical coupling were
not only anhydrovinblastine but also
vinblastine. The yields of both alkaloids
were 52.8% and 12.3%, respectively after
3 hours incubation at 30 C, pH 7.0.