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Internship of plant physiology department in universitat de


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Internship of plant physiology department in universitat de

  2. 2. 2 I. Plant Cell Cultures in Plant Biotechnology
  3. 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. 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
  5. 5. Plant Cell Culture 5 Culture Chamber
  6. 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. 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. 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
  9. 9. Media 9
  10. 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. 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. 12 ell and Organ Culture Types Cultures Hairy Root CulturesProtoplast Culture ue Culture Plant Cell Suspension Cultures
  13. 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. 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
  15. 15. 1. Callus Cultures 15 v
  16. 16. 1. Callus Cultures 16 Stages for Formation Callus
  17. 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
  18. 18. 18
  19. 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. 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. 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. 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
  23. 23. 3. Hairy Root Cultures 23
  24. 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. 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. 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. 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. 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
  29. 29. 29 4. Plant Tissue Culture
  30. 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. 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. 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.
  33. 33. 33 II. Analysis of Key Genes Involved in Taxane Metabolic Pathway
  34. 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 enlarge=0000+0000+0612+1886 psychoaktive-gattung-2/ LatinName=Taxus+baccata
  35. 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
  36. 36. 36Croteau et al. Phytochemistry Reviews. 2006, 5:75-97 GGPP s TXS T5αOH TAT T13αOH T10βOH T14βOH DBTNBT T2’αOH BAPT PAM Transferase DBAT Oxomutase Epoxidase T2αOH T9αOH T1βOH T7βOH TBT C9 oxidase Taxol Biosynthesis
  37. 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. 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. 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. 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
  41. 41. In vivo 41 TB506-N. tabacum pEarlyGate203- TB506 (His-tag) His TB506 Nicotiana tabacum TB506 protein
  42. 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. 43. 1. Construction of the plasmid • The result of PCR to check the size of fragments, after adding two digestive enzymes. 43
  44. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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