3. What is Plant Biotechnology?
• Biotechnology is the use of
biological processes,
organisms, or systems to
manufacture products intended
to improve the quality of
human life.
3
4. Why Plant Biotechnology ?
• As the world population, demand for medicinal plants is
increasing and it leads to endangering some species.
• Advances in biotechnology particularly methods for
culturing plant cell cultures, should provide new
strategies for the commercial processing.
4
6. Plant Cell Culture
• Plant cell culture systems represent a potential
renewable source of valuable medicinal compounds
which cannot be produced by microbial cells or chemical
synthesis.
• Cell culture systems could be used for the large scale
culturing of plant cells from which secondary metabolites
can be extracted.
6
7. Plant Cell Culture
• The major advantages of cell cultures includes:
1. Synthesis of bioactive secondary metabolites is running
in controlled environment.
2. Negative biological influences in the nature are
eliminated (microorganisms and insects).
3. Selection of cultivars with higher production of secondary
metabolites.
4. Automatization of cell growth control and metabolic
processes regulation, cost price can decrease and
production increase.
7
8. Plant Cell Culture
• The maximization of the production
and accumulation of secondary
metabolites requires:
1. Manipulating the parameters of the
environment and medium,
2. Selecting high yielding cell clones,
3. Precursor feeding, and
4. Elicitation.
8
10. Media Components
• Plant cell growth and corresponding product
formation are strongly influenced by
modifications in media components.
Therefore optimizing the medium is common
by modifying the basal culture media (MS,
B5) or by varying phytohormone levels.
10
11. Plant Callus
• Plants generate unorganized cell masses, such as callus
or tumors, in response to stresses.
• Callus cells are dedifferentiated, being able to regenerate
the whole plant body.
11
Taxus media
Nicotiana tabacum
Corylus avellena
12. 12
ell and Organ Culture Types
Cultures
Hairy Root CulturesProtoplast Culture
ue Culture
Plant Cell Suspension Cultures
13. 1. Callus Cultures
• The callus culture and its appearance depend on the
donor tissue, the surface sterilization method, the culture
conditions, the age of the callus, and the medium.
• Properties of good callus:
• Diameter of 5–10 mm
• 20–100 mg fresh weight
• Rapid growth with doubling times 7 - 10 days
• White, light yellow, green, or red color
13
14. 1. Callus Cultures
• In general process, sterile organs or pieces of tissue are
used.
• Achieving sterility of the plant material requires surface
sterilization.
• Process for sterilization in our cultures:
• Pre-sterilization – water/ethanol
• Sterilization – hypochloride (5-10% 5-30 minute), HgCl2 mixture
(30 minute)
• Post-sterilization – washing three times with distilled water.
• For lignified plant material, additional ultrasonic
14
17. 1. Callus Cultures
• Callus cultures are predisposed to genetic instability and
loss of their morphogenetic characteristics during long
passage (subculture) periods.
• Therefore, it is recommended that callus are subcultured
every 3–4 weeks depending on the species and the
growth rate.
17
19. 2. Plant Cell Suspension Cultures
• To initiate plant cell suspension cultures, the callus is
dispersed by inoculating it into a liquid culture medium.
• After the initial passage,the callus and a suspension
beginning to form, the culture is usually filtered with a
sieve to remove larger aggregates and is finally
transferred into the fresh medium. The whole procedure
called homogenization.
19
20. 2. Plant Cell Suspension Cultures
• It is performed every 7–10 days for fast-growing cells,
and every 14–21 days for slower growing cells. It is
obvious that plant cell suspension cultures grow faster
than their callus cultures.
• Suspension cultures are the most used plant cell culture
type in the research and production of secondary
metabolites.
20
21. 2. Plant Cell Suspension Cultures
• Plant suspensions cells very rarely grow as single cells.
They form a aggregates. The aggregation is based on
cell adhesion.
21
22. 3. Hairy Root Cultures
• Hairy roots (or transformed roots) are generated by the
transformation of plants or explants with agropine- and
mannopine-type strains (A4, ATCC, 15834, TR7, TR101,
etc.) of Agrobacterium rhizogenes, a gram-negative soil
bacterium.
• When the bacterium infects the plant or explant, the DNA
from root-inducing plasmid, is transferred and integrated
into the genome of the host plant. This transformation
process produces hairy roots.
22
24. 3. Hairy Root Cultures
• In most cases, wounding a sterile leaf (midrib and major
veins) or stem tissue is carried out with the sterile tip of a
needle attached to a syringe before infection takes place.
In our laboratory infection is done with needle while
wounding the plant.
24
After 2 weeks After 3 weeks Grow in MS media
25. 3. Hairy Root Cultures
• Cocultivation follows, in which plant cells divide and
dedifferentiate, and bacteria divide and infect the
wounded plant tissue.
• The result; transformed cells acquire the capacity to
develop into a tiny multiple hairy roots at the infection
site.
25
26. 3. Hairy Root Cultures
• After cocultivation, the infected explants are cleared of
excessively growing Agrobacteria by transferring them to
a culture medium, which also contains antibiotics to
eliminate Agrobacterium.
• Repetition of the transfer to fresh medium with antibiotics
at intervals of 2–4 days is stringently necessary because
of the appearance of bacterial infections.
26
27. 3. Hairy Root Cultures
• A significant advantage of hairy roots is that they are
generally easy to isolate and grow in a selective medium.
• Furthermore, hairy roots have been found to synthesize
secondary metabolites at similar or higher levels to those
found in whole plants.
27
28. 4. Plant Tissue Culture
• Plant tissue culture is the manipulation of cells and
organs in aseptic and under controlled conditions of light,
humidity and temperature.
• The controlled production system allows the
standardization of the extracts, such as the concentration
of the desired compounds, maintaining the same genetic
characteristics of the highest production clones.
28
30. • The cell wall blocks the passage of DNA into the cell in
that case protoplasts are widely used for DNA
transformation because protoplasts are cells which have
had their cell wall removed, usually by digestion with
enzymes.
30
5. Protoplast Culture
31. 5. Protoplast Culture
• Protoplast cultures will be one of the most effective
source materials when considering the supply of
precursor (10-deacetylbaccatin III) is still a problem.
31
32. 5. Protoplast Culture
Procedure of Protoplast Production
• The protocol contains preplasmolisis and washing solution.
Both of them contains same chemicals but the difference is
pH.
• Media conditions affect the protoplast production because
protoplast cells are very fragile and affected from sugar
balance. For example, less amount of sugar may lead to
explosion of protoplast cells.
32
Preplasmolisis
pH 5.4
Activation of enzymes which leads to breaking of cell wall.
Washing solution
pH 7
Inactivation of enzymes.
34. Taxus species
• Taxus is a genus of small coniferous trees or shrubs in
the yew family Taxaceae. They are relatively slow-
growing and can be very long-lived.
34
Taxus baccata Taxus brevifolia
http://calphotos.berkeley.edu/cgi/img_query?
enlarge=0000+0000+0612+1886
http://psychotropicon.info/taxus-spp-eine-
psychoaktive-gattung-2/
http://www.pfaf.org/user/Plant.aspx?
LatinName=Taxus+baccata
35. Taxol
• Taxol is a successful anti-cancer drug which synthesized
from Taxus spp.
• Taxol is largely produced by Taxus cell culture methods or
by semi-synthetic means from advanced precursors that
are more readily available from the needles of various
yew species as a renewable resource.
35
37. Taxol Biosynthesis
• The biosynthesis of taxol is completed by 19 steps from
the universal diterpenoid progenitor geranylgeranyl
diphosphate.
37
• There are some of
enzymes in the
biosynthesis are not
known. To work in the
enhancement of Taxol
and derivatives
production, the
understanding of the
pathway is important step.
38. Taxol Biosynthesis
• There is one unknown enzyme seen in that part of
biosynthesis, which can be use in semisynthesis of taxol.
• TB506 is a gene which found in Taxus spp. is one of the
candidate gene that synthesize the unknown enzyme.
• The unknown enzyme produce 30-N-debenzoyl-2-
deoxytaxol by hydroxylation from intermediate compound
which is combination of baccatin III and phenylisoserine.
38
39. In vitro
Study of Hydroxylase Activity of
TB506
• The aim of the experiment is to determine if our
candidate is the unknown enzyme in biosynthesis of
afterwards steps of Baccatin III.
• There are two parts of the functional study of the
hydroxylase enzyme. For this purpose an in vitro and an
in vivo assay were done.
39
In vivo
40. In vivo
• For obtaining taxol production from baccatin III (percursor
molecule), BAPT, the enzyme candidate and DBTNBT
have to be inserted in transgenic N. tabacum plant.
• In order to check the candidate enzymes activity, Taxus
species are not used.
Steps of Experiment
40
Construction of the plasmidCloning Transformation Plant Regeneration
42. 1. Construction of the plasmid
i. pDONR221 named plasmid which includes TB506 gene
without stop codon is extracted from E. Coli.
ii. The restriction enzymes is used to control the size of
fragments for understanding the presence of TB506
gene.
iii. The Gateway LR clonase assay is done by mixing the
plasmid and another commercial plasmid that has
histidine tail pJCV52 which will be used to purify the
protein.
42
43. 1. Construction of the plasmid
• The result of PCR to check the size of fragments, after
adding two digestive enzymes.
43
44. 2. Cloning
• The mixed plasmid is get inside to the E. coli’s cells by
thermal shock for cloning. The colonies are checked by
PCR.
• Protocol of thermal shock for E. coli:
• Put the mixture which obtained from Gateway LR clonase
assay to ice for 30 minute.
• Wait 30 second in 42 degree without shaking then put in ice.
• Add 1 ml of LB solution and wait 1 h at 37 degree
• Centrifuge the solution at 1 minute with 10000 rpm
• Remove 1 ml LB from the solution to obtain a pellet
• Put in a solid plate with antibiotic and wait to grow.44
45. 3. Transformation
• After cloning, the extraction of the plasmid is done by
using miniprep assay. Then thermal shock is used to gets
the plasmid inside to Agrobacterium rhizogenes.
• DBAT and DBTNBT enzyme´s producer genes are get
inside to Agrobacterium rhizogenes cells by thermal shock
too.
45
46. 3. Transformation
• Protocol of thermal shock for Agrobacterium AU:
• 1 µg plasmid is mixed with 100 µL of Agrobacterium.
• Wait 5 minute at 0 degree
• Wait 5 minutes in Nitrogen
• Wait 5 minute at 37 degree
• Add 1ml YEB to solution and waiting 2-4 h at 28 degree
• Centrifuge the solution at 1 minute with 14400 rpm.
• Remove 1 ml YEB from the solution to obtain a pellet.
• Put in a solid plate with antibiotic and wait to grow.
• In our experiment we used Rifampicin as antibiotic. 46
47. 3. Transformation
47
• After thermal shock process, transformed
Agrobacteriums are checked by PCR to control the
existence of genes.
• In the last step of transformation process, the three
different Agrobacterium solutions are mixed and
infected to N. tabacum plant by coinfection.
48. 3. Transformation
• Agrobacterium rhizogenes induces hairy roots in N. tabacum
plant. Hairy roots grow in a specific media which has
antibiotic and they will produce DBAT, DBTNBT and the
candidate enzyme.
48
49. 4.Plant Regeneration
• Plant regeneration is the last part of preparation of in vivo
experiment. It includes the growing of hairy roots to
obtain aerial part of the plant. The aim is that the
production of whole plant aids to control the existence of
TB506 gene. The growing period can be last one or two
months.
• Adding Baccatin III into the media of N. tabacum plant,
taxol production is expected to be observed with the aid
of these genes.
49
50. In vitro
• The aim of the in vitro part is to determine the
hydroxylase activity of TB506 gene in solution. The
enzyme is obtained by purification of TB506 from the
transgenic plants.
• Experiment is done by mixing β-phenylalanineCoA and
the enzyme in solution to obtain phenylisoserineCoA for
understanding the occurence of hydroxylation in the50
β-phenylalanineCoA
+
TB506
protein and
cofactors
?
PhenylisoserineC
oA
51. Conclusion
• Advances in biotechnology provide us enability to access
medicinal plants more easly with developing new
strategies. As increasing in the production of needed
demand provide us an high qualified products.
• Cell cultures are the most used techniques to obtain plant
products an easy way.
• In our internship we learned how to do in vitro cultures
and biomolecular work that are the basis of
biotechnology.
• In recent years the biosynthesis of Taxol is popular topic
on researches. Determination of unknown steps in the
pathway will lead to maximize Taxol production. In our
experiment we try to find the last unknown step of
biosynthesis if the unknown enzyme is the candidate
51
52. Anknowledge
• We wish to express our sincere thanks to Prof. Javier
Palazon Barandela who is one of Professor of plant
physiology department, for providing us with all the
necessary facilities
• We thank Rosa Mª Cusidó, Mercedes Bonfill and
Elisabeth Moyano.
• Our special thanks goes to Raul Sanchez who is one of
assistant for helping us in all steps of experiments both
understanding and practising parts with all his patience.
• We would like to thank Rafael Vidal and Diego Hidalgo
for answering our questions.
52
53. References
• Eibl R., Eibl D., (2009), Cell and Tissue Reaction Engineering: Principles and
Practice, 315, Springer-Verlag Berlin Heidelberg.
• Mulabagal V., Tsay H. S., 2004, Plant Cell Cultures – An Alternative and
Efficient Source for the Production of Biologically Important Secondary
Metabolites, Int. J. Appl. Sci. Eng., 2,1.
• Ikeuchi M., Sugimoto K., Iwas A., 2013, Plant Callus: Mechanisims of
induction and repression, The Plant Cell, Vol.25: 3159-3173.
• Diasa M.I., Sousaa M.J., Alvesb R.C., Ferreiraa I.C.F.R., Exploring plant
tissue culture to improve the production of phenolic compounds: A review.
• Luo J.P., Mu Q., Gu Y.H., Protoplast Culture and Paclitaxel Production by
Taxus yunnanensis, 1999, Plant Cell, Tissue and Organ Culture 59: 25-29.
• Croteau R., Ketchum R.E.B., Long R.M., Kaspera R., Wildung M.R., 2006,
Taxul Biosynthesis and Molecular Genetics, Phytochem Rev.; 5(1): 75-97.
doi: 10.1007/s11101-005-3748-2. 53
Editor's Notes
means initiation or improvement of the biosynthesis of specific compounds, after introduced elicitators in small concentrations to a living cell system
