Basic techniques of plant tissue culture

Techniques of Plant tissue Culture andPlant cell Immobilization

1. BASIC TECHNIQUES OF PLANT TISSUE CULTURE AND PLANT CELL IMMOBILIZATION BY NIVETHA.B M .PHARMACY (PHARMACOGNOSY) I YEAR 1

2. WHAT IS TISSUE CULTURE ? Tissue culture is the term used for “the process of growing cells artificially in the laboratory ”. Tissue culture is invitro cultivation of plant cell or tissue under aseptic and controlled environmental conditions, 2

3. in liquid or on semisolid well defined nutrient medium for the production of primary and secondary metabolites or to regenerate plant. Tissue culture involves both plant and animal cells. 3

4. BASIC TECHNIQUES OT PLANT TISSUE CULTURE The technique of plant tissue and cell culture has evolved over decades. This technique combine with recent advances in developmental, cellular, molecular genetic, metabolic engineering, genetic transformation and using conventional plant breeding have turned plant biotechnology into an exciting research field with a significant impact on pharmaceutical industries, agriculture, horticulture and forestry. 4

5. TYPES OF TECHNIQUES : There are mainly two major techniques in plant tissue culture. They are explained as follows, STATIC CULTURE SUSPENSION CULTURE 5

6. STATIC CULTURE Static culture is otherwise known as callus culture or solid culture. In this technique i solated piece of plant tissue is cultured on a nutrient medium. Plant growth regulators such as auxins , cytokinins , and gibberllins are supplemented into the medium to initiate callus formation. . 6

7. Finally, an unorganized mass of cell appears and it is called as callus. 7

8. 8 This callus is transferred on to different media to regenerate plants. This callus culture technique is easier and convenient for initial maintanence of cell lines and also for carrying out the investigation studies related to organogenesis.

9. CALLUS GROWTH 9

10. SUSPENSION CULTURE Suspension culture is also known as liquid culture. It is the culture of isolated cells or very small cell aggregates dispersed in liquid medium. The cell suspension is obtained by agitating pieces of callus in liquid medium on gyrating shaker. 10

11. Growing cells in a liquid medium under controlled conditions. Transfer of an established callus culture into the liquid medium in Erlenmeyer flask. Medium with high auxin content, appropriate concentration of auxin and yeast or auxin vs kinetin helps to maintain suspension. Incubated at 25 ˚ C in darkness or with less light intensity fluorescent light. 11

12. It contains a uniform suspension of separate cells in a liquid medium callus liquid medium agitated continuously finally cells separated sub-culture the cells This can be achieved by rotary shaker attached within the incubator at a rate of 50-150 rpm . 12

13. TYPES OF SUSPENSION CULTURE There are three types of suspension culture. 1.Batch suspension culture 2. Continuous culture (I)Open type (II)Close type 3.Semi-continuous culture 13

14. BATCH CULTURE These cultures are maintained continously by propagating a small aliquot of inoculum in the moving liquid and transferring it to fresh medium at regular intervals. Generally cell suspension are grown in flasks(100-250 ml) containing 25-27 ml of the culture medium. 14

15. When the cell number in suspension culture is plotted against the time of incubation , a growth curve is obtained. 15

16. CONTINUOUS CULTURE In this system , the liquid medium is continuously replaced by the fresh liquid medium to stabilize the physiological stage of growing cells. Cell proliferation takes place under constant conditions 16

17. As a result ,the active growth phase of the cell declines the depletion of nutrient. The cells passing through out flowing medium are separated mechanically. The cells are always kept in exponential growth phase. 17

18. MEASUREMENT OF GROWTH OF CELL CULTURES Methods used to determine are.. CELL NUMBER By counting the cell number in haemocytometer under a microscope. Suspension culture is preferable . 18

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20. PACKED CELL VOLUME Cell suspension is transfer to graduated centrifuge. Centrifuged at 2000 rpm for 5mints. Cell will form pellets called biomass volume, expressed by ml -1 20

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23. FRESH CELL WEIGHT When cells increase in number, the liquid will be turbid. As a result optical density altered, detected by colorimeter. 23

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25. VIABLE CELL TEST The staining method such as fluorescein di -acetate is used for accessing the cell viability. Dead cells appear as fluorescein red. 25

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27. PLANT CELL IMMOBILIZATION Immobilization is the newest culture of plant cell and considered has to be the most ‘natural’. It has been defined as a technique, which confines to a catalytically active enzyme or to a cell within a reactor system and prevents its entry into mobile phase, which carries the substrate and product. 27

28. ADVANTAGES: Retention of biomass enables its continuous reutilization as a product system, a definite advantage with slow growing plant cells e.g. Papaver somniferum have remained stable and active for up to six months. Continuous process : Immobilization allows a continuous process ,which increase volumetric productivity and allows the removal of metabolic inhibitors. 28

29. Separation of cells from medium :The immobilization separates cells from medium and the desired product in extra cellular, which will simplify down stream processing compared to extraction from tissue. Decoupling of growth and product formation : Immobilization is compatible with non growth associated product formation. 29

30. Reduces problems such as aggegrate,growth and foaming. High biomass levels. 30

31. DISADVANTAGES: Secretion of secondary metabolites requires cellular transport or a artificially altered membrane permeability’ The efficiency of the production process depends on the rate of release of products rather than actual raye of biosynthesis. 31

32. The immobiliztion process may reduce biosynthetic capacity. Products must be released from the cell into medium. Relese of single cells from cell aggregate may make processing of the product more difficult. 32

33. TYPES OF IMMOBILIZATION Direct intracellular binding due to natural affinity(adsorption , adhesion and agglutination). Covalent coupling Intracellular connection via bi or poly functional reagent(cross-linking) 33

34. Mixing with suitable materials,changing their consistency with temperature(embedding). Physical retention within the framework of diverse pore size and permeability (entrapment , micro encapsulation). 34

35. METHOD OF IMMOBILIZATION Adsorption Covalent attachment Entrapment a) Natural polymer Alginate Agar Agarose 35

36. b)Synthetic polymer Polyacrylamide c)Porous structure Polyurethane foam d)Membranes Hollow fiber Flat plate 36

37. ENTRAPMENT: GEL ENTRAPMENT BY POLYMERIZATION : A monomer or a mixture of monomers is polymerized in the presence of cell suspension, which is entrapped inside the lattice of the polymer. The most common example is polyacrylamide . 37

38. GEL ENTRAPMENT FORMATION BY PRECIPITATION: Gels may be formed by precipitation of some natural and synthetic polymers by changing one or more parameters in the solution, such as temperature, salinity or pH of solvent . Several methods can be used for entrapment. 38

39. Gel entrapment by precipitation SPECIES GEL Catharanthus roseus Agarose , Agrose , Agar Silybum marianum Agar 39

40. SURFACE IMMOBILIZATION: Surface immobilization may occur on both natural and other matrices. Eg for natural matrices are deeper callus layers and cellulose. For synthetic steel and nylon are used. 40

41. REFERENCE: Medicinal Plant Biotechnology by Ciddi Veeresham . Pharmaceutical Biotechnology by S S Kori . Trease and evans pharmacognosy by W.C evans . A textbook of industrial pharmacognosy by A.N.Kalia . Pharmacognosy and photochemistry-part 2 by Vinod.D.Rangari . 41

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