• Ozlem Kocaagaoglu - Selin Yalcin

INTERNSHIP OF PLANT PHYSIOLOGY DEPARTMENT IN UNIVERSITAT DE

BARCELONA1

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I. Plant Cell Cultures in Plant Biotechnology

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.

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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.

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Plant Cell Culture

5Culture Chamber

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.

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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.

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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.

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Media

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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.

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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.

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Taxus media

Nicotiana tabacum

Corylus avellena

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Plant Cell and Organ Culture Types

Callus Cultures

Hairy Root CulturesProtoplast Culture

Plant Tissue Culture

Plant Cell Suspension Cultures

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

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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 treatment could be required.

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1. Callus Cultures

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v

1. Callus Cultures

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Stages for Formation Callus

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.

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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.

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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.

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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.

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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.

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3. Hairy Root Cultures

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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.

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After 2 weeks After 3 weeks Grow in MS media

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.

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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.

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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.

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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.

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4. Plant Tissue Culture

• 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.

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5. Protoplast Culture

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

5. Protoplast CultureProcedure 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.

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PreplasmolisispH 5.4

Activation of enzymes which leads to breaking of cell wall.

Washing solutionpH 7

Inactivation of enzymes.

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II. Analysis of Key Genes Involved in Taxane Metabolic Pathway

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.

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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

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.

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36Croteau et al. Phytochemistry Reviews. 2006, 5:75-97

GGPPs

TXS T5αOH TAT

T13αOHT10βOH

T14βOH

DBTNBT T2’αOH

BAPT

PAMTransferase

DBAT

Oxomutase Epoxidase

T2αOH

T9αOH

T1βOH

T7βOH

TBT

C9 oxidase

Taxol Biosynthesis

Taxol Biosynthesis

• The biosynthesis of taxol is completed by 19 steps from the universal diterpenoid progenitor geranylgeranyl diphosphate.

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• 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.

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.

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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.

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In vivo

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

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Construction of the plasmidCloning Transformation Plant Regeneration

In vivo

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TB506-N. tabacum

pEarlyGate203-TB506 (His-tag)

His

TB506

Nicotiana tabacum TB506 protein

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.

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1. Construction of the plasmid

• The result of PCR to check the size of fragments, after adding two digestive enzymes.

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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.

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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.

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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.

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3. Transformation

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• 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.

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.

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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.

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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 b-phenylalanineCoA and the enzyme in solution to obtain phenylisoserineCoA for understanding the occurence of hydroxylation in the correct position.

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b-phenylalanineCoA

+ TB506 protein and cofactors ?

PhenylisoserineCoA

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 TB506 gene, it will help to obtain production of Taxol by using Baccatin III as precursor molecule.

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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

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.

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