Plant tissue culture

Plant tissue culture
B-Pharmacy
4th sem
Pharmacognosy-1
Unit-03

1. PLANT TISSUE CULTURE PRESENTED BY Gourav singh B-pharmacy Presented by- Gourav singh 1

2. PLANT TISSUE CULTURE A method practiced for plant propagation under sterile condition to produce clones of a plant is called Plant tissue culture. Advantage Disadvantage It can be used to study biogenesis of secondary metabolite. This procedure are very variable The cells of any plants can be multiplied to obtain the specific metabolite they produce The procedure need special attention and diligently done observation Cultured cell are maintained such that they remain free from microbial contamination and insect attack There may be error in the identity of the organism after culture Presented by- Gourav singh 2

3. HISTORY Year Author Plant species Result 1902 Haberlandt Tradeseantia First cultivation experiment with isolated plant cell, cell growth, but no cell division obtained 1904 Hannig Cochleria Saphanus Establishment of embryo culture from several cruciferous species. 1892 Klercher ------------- First attempt to isolate protoplast 1922 Kolte, Robbins Zea In vitro-cultivation of root tips, no permanent culture obtained 1924 Dieterich luium Embryo rescue –”artificial premature birth” 1934 White Lycopersicum First permanent root culture which terminated in 1988 1934 Gautheret Naucus, Nicotina First permanent callus culture using B-vitamins Presented by- Gourav singh 3

4. HISTORY Year Author Plant species Result 1946 Ball (Father of micro- propagation) Lupinus and Trapaeolum Development of transplantable whole plants from leaf primoda 1954 Muiretal Jagetes Nicotiana First suspension culture of single cell or cell aggregates nurse culture 1957 Skoog, Miller Nicotiana Demonstrated the role of auxin and cytokinin on root and shoot formation 1959 Julecke, Nickess lolium First report of large scale (1341) culture of plant cell, carboy system 1960 Bergmann Nicotiana Cells clone obtained from single culture cell placed in an agar medium Presented by- Gourav singh 4

5. HISTORY Year Author Plant species Result 1962 Murashige, skoog Nicotiana Introduced the medium 1965 Morel lycopersicon Clonal multiplication of horticulture through tissue culture, protocorn formation 1966 Kohlenbach Macleaya First cell division and culture of differentiated mesophyll cells 1970 Carlson Nicotiana Isolation of auicotrophic mutant from cultured cells 1971 Nagata, Takabe Nicotiana Regeneration of plant cells from cultured protoplast 1974 Reinhard _________ Biotransformation in plant tissue culture Presented by- Gourav singh 5

6. History Year Author Plant species Result 1978 Meichers ___________ Production of somatic hybrid pomato 1983 Mitsui petrochemical Industry Limited Lithosperman First industrial production of secondary plant products by suspension culture Presented by- Gourav singh 6

7. TERMS USED IN TISSUE CULTURE Terms Description Explants An excised piece of differentiated tissue or organ is regarded as an explant. The explant may be taken from any part of the plant body . e.g. leaf, stem, root etc. Callus The unorganized, autonomus, uncontrolled and undifferentiated mass of plant cell is known as callus Differentiation It is the process by which meristem cells are converted into two or more types of cells or tissues that are different from each other. Dedifferentiation The phenomenon of mature cells reverting to meristematic state to produce callus, means the process of formation of unorganized tissues from the highly organized tissues. Re-Differentiation It m eans that the dedifferentiated cells lose the capacity of division and become mature to form specific functions Presented by- Gourav singh 7

8. TERMS USED IN TISSUE CULTURE Terms Description Totipotency The ability of the callus cells to differentiate into a plant organ or a whole plant is referred to as cellular totipotency. Plasticity It is the condition of the adaptability of a plant species to change in its environments or differences between its various habitats. Organogenesis The development of adventitious organ or primordia from undifferentiated cell mass in tissue culture by the process of differentiation. Synthetic seed Where artificial encapsulation of somatic embryos shoot bud or aggregates of cell of any tissues done by a hydrogel which have the ability to form a plant in In-Vitro Somaclonal variation The genetic variations found in the In-vitro cultured cells are known as somaclonal variation Micropropagation The production of a large number of individual plants from a small piece of plant tissue cultured without formation of callus in an aseptic nutrient medium Presented by- Gourav singh 8

9. BAISC REQUIREMENT FOR TISSUES CULTURE LABORATORY A tissues culture laboratory should have the general basic facilities:- Sr. no Requirement Description 01 Apparatus For growing culture different kind of vessel are used. Callus culture can be grown successfully in large test tube (25 x 150mm) or wide mouth flask. Glassware such as graduated pipettes, measuring cylinder, beaker, filters, funnel and petridishes are also required for making preparation. All the glassware should be cleaning 02 Equipment Sprit burner, An Autoclave, Hot air oven, Scissors, Seallpels and forceps, pH meter, shaker, weighing balance, laminar air flow, and BOD incubator. Presented by- Gourav singh 9

10. Sr. no Requirement Description 03 Washing and storage facilities First requirement of tissue culture laboratory is provision for fresh water supply and disposal of the waste water. Acid and Alkali resistant wash basin for apparatus washing, It is compulsory to maintain cleanliness in the area of washing, drying and storage 04 Media preparation room It should have sufficient space to accommodate , chemical, glassware, culture vessel and equipment required for weighing and mixing, pH meter, hot plate. Burner with gas supply, autoclave, microwave oven and freezer for storage for prepared media and stock solution. 05 Aseptic chamber for culture Aseptic chamber requires an ordinary type of small wooden hood, have a glass door, fitted with U.V. tubes. The air is coming out of the filter is ultra clean and having adequate velocity to prevent micro contamination of working area by worker sitling in the front of the cabinet. Inside the cabinet, there is arranged for bunsen burner and a UV tube fitted on the ceiling of the cabinet. Presented by- Gourav singh 10

11. Sr no. Requirement Description 06 Culture room Environmental have a great effect on the growth and differentiation of cultured tissues. Therefore it is very much essential to incubate all types of culture in well controlled. Environmental condition like temperature, air circulation and humidity. 07 Data collection and recording of the observation The growth and maintenance of the tissue culture in the incubator should be observed and recorded at regular interval. All the observation should be done in the laminar air flow. For microscopic examination separate dust free space should be marked for microscopic work. All the recorded data shou;d be feeded in computer. Presented by- Gourav singh 11

12. GENERAL PROCEDURE USED FOR PLANT TISSUE CULTURE Presented by- Gourav singh 12

13. TYPES OF TISSUE CULTURE Presented by- Gourav singh 13

14. MERISTEM CULTURE: Shoot apex (apical meristematic dome with or without one or two leaf primordia) Give one single shoot. Generally, meristem tips, between 0.2-0.5 mm. It help in production of virus free plants. It help in rapid clonal multiplication. The method is successful in case of herbaceous plants than woody plants. It help in a culture of potato, Banana, Cardamom, Sugarcane, Sweet potato etc. Presented by- Gourav singh 14

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16. SHOOT CULTURE: Shoot tips, or buds (larger than the shoot apices having several leaf primordia). Usually produce multiple shoots. In which the terminal end (0.1 -1.0 mm) of a shoot carrying the meristem (0.05 -0.1 mm) Primordial and developing leaf and adjacent stem tissues is cultured. Presented by- Gourav singh 16

17. NODE CULTURE: Stem piece carrying either single or multiple nodes. Each bud is grown to provide a single shoot. Presented by- Gourav singh 17

18. ROOT CULTURE Can be established from root tips taken from primary or lateral roots of many plants. It is a process in which the radical tips of seed germinated aseptically are cut out and cultured in a liquid medium under controlled condition to facilitates their growth. Presented by- Gourav singh 18

19. EMBRYO AND OVULE CULTURES: Embryos are dissected from seeds, individually isolated and germinated in vitro to provide one plant per explant. In some plant, It has been possible to excise and culture pollinated ovaries and immature ovules. Presented by- Gourav singh 19

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21. CALLUS CULTURE: An amorphous mass of loosely arranged thin walled parenchyma cells arising from the proliferating cells of the parents tissues cultured on agar medium under aseptic condition is known as callus culture. This method is the source of Tissue for cell S uspension Culture. Several biochemical assays are performed from callus culture. Chromosomal variation occurs genetically in the cells of callus tissue. Presented by- Gourav singh 21

22. SUSPENSION CULTURES: Tissue and cells cultured in a agitated liquid medium produce a suspension of single cells and cells clumps of few to may cell, these are called suspension cultures Shorter duration and continuous process. It help for induction in somatic embryos and shoots. No toxic products are formed with this culture techniques. Batch culture: A batch culture is a cells suspension culture growth in a fixed volume of nutrient culture medium. Continuous cultures: Open: Both cells and the used medium are taken out from open continuously culture and replaced by equal volume of fresh medium. Closed: The cells separated from used medium taken out for replacement and added back to the suspension culture, so that the cell biomass keeps on increasing. Presented by- Gourav singh 22

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24. PROTOPLAST CULTURES: Isolated protoplasts have been described as “naked” cells because the cell wall has been removed by either a mechanical or an enzymatic process. Protoplast can be induced to reform a cell and divide if placed in a suitable nutrient than form callus. It develop novel hybrid plants through protoplast fusion Protoplast cells also can regenerate into whole plants. It help in gene transfer. Presented by- Gourav singh 24

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26. EMBRYO CULTURE: Embryo culture is usually done from the need to rescue embryo from wide crosses where fertilization occurred, but not the embryo developments. Production of haploid plants. A common explant for the initiation of somatic embryogenetic cultures. Overcoming abortion of embryos of wide hybrids at very early stages of development due to incompatibility barriers. In vitro fertilization for the production of distant hybrids avoiding style and stigmatic incompatibility that inhibits pollen germination and pollen tube growth. Fig:-different step in ovule culture of spathiphyllum: flower(leaf), ovary (middle left),ovule (middle right), secondary embryogenesis Presented by- Gourav singh 26

27. ANTHER CULTURE Anther culture is a technique by which the developing anthers from unopened flower bud are cultured on a nutrient medium where the microspores within the cultured anther develop into callus tissue or embryoids that give rise to haploid plants. Production of haploid plants Uncovering mutations or recessive phenotypes. It is used for mutation studies It is used for formation of double haploid that are homozygous and fertile. It is used to study of factor controlling pollen embryogenesis of higher plants. It is used to study genetic recombination in higher plants. Presented by- Gourav singh 27

28. ESTABLISHMENT AND MAINTENANCE OF VARIOUS CULTURE There are three main culture system, selected on the basic of the objective. Growth of callus masses on solidified media. Growth in lipid media consist of mixture of single cell or cell aggregates. Protoplast culture. Presented by- Gourav singh 28

29. A.CALLUS CULTURE Callus is an amorphous aggregates of loosely arranged parenchyma cell, which proliferate from mother cell. Cultivation of callus usually on a solidified nutrient medium under as condition is known as callus culture. Maintenance of callus culture:- After sufficient time of callus growth of same medium following change will occur such as:- Depletion of nutrient in the medium Gradually losses of water Accumulation of metabolic toxins Callus tissues is transferred under aseptic condition to fresh medium. Sub-culturing should be repeated after of 4 -5 weeks. Many callus culture remain healthy and continue to grow at slow rate for much longer period without sub-culturing. Presented by- Gourav singh 29

30. A.CALLUS CULTURE If the incubation to be carried out at low temperature 5 – 10 degree Celsius below the normal temperature (16 – 18 0C). Normally, total depletion take about 28 days. Principal:- Aseptic preparation of plant material. Incubation of culture under controlled physical condition. Selection of suitable nutrient medium supplemented with appropriate ratio of plant growth regulators such as auxin and cytokinins or only appropriate auxin . Presented by- Gourav singh 30

31. Step involved:- Presented by- Gourav singh 31

32. B. SUSPENSION CULTURE Maintenance of suspension culture:- Presented by- Gourav singh 32

33. Suspension culture contains a uniform suspension of separate cells in liquid medium Flow diagram :- Illustrating the method of cell suspension culture and regeneration of plant through embryogenesis. Presented by- Gourav singh 33

34. CULTURE MEDIUM Defined medium for the growth of cell cultures consists of the following components. 1. Inorganic cells Concentration of potassium and nitrate at least 50-25mM and phosphate, sulphate 1-3mM and magnesium appear to be adequate, Ammonium is essential 8Mm, and micronutrient like iodide, boric acid, zinc, manganese, copper, cobalt, iron. 2. Vitamins Thaimine, pyridoxine, myo-inositol, nicotinic acid improves cell growth. 3. Carbon source Sucrose, 2-4% 4. Growth regulators It induce cell division most commonly used regulator is NAA naphthalene acetic acid, and 2,4-dichlrophenoxy acetic acid(2,4-D) 5 Organic supplements Protein hydrolyzates, yeast, malt, extract and coconut milk used for enhancement in growth rate. Presented by- Gourav singh 34

35. 06 Whites medium used for root culture 07 MS medium used for organogenesis 08 B5 medium used for callus culture, cell suspension and Protoplast culture 09 N6 medium used for cereal anther culture 10 Nitsch’s medium used for anther culture Presented by- Gourav singh 35

36. MEDIA PREPARATION Chemical are dissolved in distilled water. The stock solution of vitamins, micro-nutrient and growth regulators and hormones are added and pH adjusted to 5.5-6.5. The solution is made to volume 50 to 100 ml quantities distributed in to 250 ml Erlenmeyer flask. Flasks are supported with cotton plug and autoclaved at 120 0C 15 min, all media are stored at 10 0C. Presented by- Gourav singh 36

37. COMPOSITION OF PLANT TISSUE CULTURE MEDIUM Macronutrients Micronutrients Iron source Carbon and energy source Vitamin Amino acid Other complex organic supplement Plant growth regulators Gelling or solidifying agents pH regulators. Presented by- Gourav singh 37

38. Macroelements Potassium (K) 20-30 mM Phosphorous (P) 1-3 mM Calcium (Ca) 1-3 mM Magnesium (Mg)1-3 mM Sulfur (S) 1-3 mM Micronutrients 1. Iron (Fe) 1 mM 2. Manganese (Mn) 5-30 mM 3. Zinc (Zn) 4. Boron (B) 5. Copper (Cu) 0.1 mM 6. Molybdenum (Mo) 1 mM 7. Cobalt (Co) 0.1 mM CULTURE MEDIUM Vitamins 1. Thiamine (vitamin B1) 2. Nicotinic acid 3. Pyridoxine (B6) 4. Myo-inosital Sugar Sucrose Others 20 to 40 g/l Support system Agar Agarose Gelrite (Phytagel) Plant growth regulator Amino acid Tyrosine Proline Glutamic acid Alanine Aspartic acid Casein hydrolysate Plant growth regulators Auxins 4. Ethylene Cytokinins 5. Activated charcoal Gibberellins 6. Abscisic acid Presented by- Gourav singh 38

39. Other complex organic supplement Coconut milk Peptone Yeast extract Ground banana Watermelon juice Potato extract CULTURE MEDIUM Iron Source Ferric sulphate (FeSO 4 , 7H 2 O) Chelate irons (Fe-EDTA) Carbon and energy source Fructose Maltose Galactose Raffinose Sorbitol Gelling agent Gelrite Gellan Phytagel Agargel Presented by- Gourav singh 39

40. Element Function Nitrogen (N) Compound of protein s, nucleic acid and some coenzymes Element required in greatest amount. Potassium (P) Regulates osmotic potential, principal inorganic cation. Calcium (Ca) Cell wall synthesis, membrane function, cell signaling. Magnesium (Mg) Enzyme cofactor, component of chlorophyll. Phosphorous (P) Component of nucleic acids, energy transfer, component of intermediates in respiration and photosynthesis. Sulphur (S) Component of some amino acid (methionine, cysteine) and some cofactors. Chlorine (Cl) Required for photosynthesis. Iron (Fe) Electron transfer as a component of cytochromes. Manganese (Mn) Enzyme cofactor. Cobalt (Co) Component of some vitamins. Copper (Cu) Enzyme cofactor, electron-transfer reaction. Zinc (Zn) Enzyme cofactor, component of nitrate reductase. Molybdenum (Mo) Enzyme cofactor, component of nitrate reductase. Presented by- Gourav singh 40

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42. GENERAL STEP INVOLVES IN PLANT TISSUE CULTURE Presented by- Gourav singh 42

43. APPLICATION OF PLANT TISSUE CULTURE It help in rapid multiplication of plants. A large number of plantlets are obtained within a short period. To study Respiration and Metabolism. Production of secondary Metabolites Production of haploids Micropropagation Cloning Development of Transgenic Plants. Genetically similar plants are formed by this method. It is an easy, safe and economical method for plant propagation. Presented by- Gourav singh 43

44. APPLICATION OF PLANT TISSUE CULTURE Production of artificial seeds. Single cell culture of higher plants. Germplasm Storage. Plant breeding Plant physiology Presented by- Gourav singh 44

45. EDIBLE VACCINES In the edible vaccine, Transgenic plants are used as vaccine production system. Definition: Edible vaccine is define as the vaccination is a disease preventive measure, where the immune system of a person is boosted against a particular disease. Edible vaccine is a new approach to oral immunization certain food under investigation for use in edible vaccine such as banana, potato, tomato, spice etc. Edible vaccine are act by Stimulating the mucosal as well as systemic immunity Transformation: Edible vaccine are develop by introducing the selected desirable genes into the plants, and then allowing the production of encoded protein by these altered plants the process is called transformation Presented by- Gourav singh 45

46. Mechanism of Action of plants B ased E dible vaccines:- Intake of edible vaccine Peyer’s patches-rich source of Ig A Producing plasma cell. Edible vaccine break down at Peyer’s patches(contain 30-40 lymphoid nodules containing follicles for development of germinal center.) Antigen penetrate follicles accumulating antigen in lymphoid structure. Antigen contact M-cells which express MHC II molecule. Pocket formation occur which is filled with β cells, T cells & Macrophage. Mastication and degradation in intestine Presented by- Gourav singh 46

47. M-cell with antigen activates β cell with in the lymphoid follicles. Activated β cell leaves lymphoid follicles and reaches Mucosal Associated lymphoid tissue (MALT) Plasma cells are differentiated from β cell and IgA are produced. IgA are secreted into lumen where they interact with antigen. Presented by- Gourav singh 47

48. Advantage: Edible vaccine have efficient mode of action for immunization. They are comparatively cost effective, as they do not require cold chain storage. They are safe as they do not contain heat-killed pathogens. The production process is scaled up rapidly by breeding. They are affordable. They cause mucosal immunity. They have greater stability products. They do not need sophisticated equipment and machine. As they are easily grown on rich soils. They are widely accepted as they are orally administered unlike traditional vaccine they are injectable. Presented by- Gourav singh 48

49. Importance: It is used for cancer therapies like colon cancer and cervical cancer. It is used for autoimmune disease like Type-I diabetes and multiple sclerosis. It is applied for many infectious diseases like AIDS, tetanus, small pox, measles, plague, foot and mouth disease, tuberculosis, influenza etc. Limitation: Immune tolerance may develop in the individual for particular vaccine protein. Edible vaccine are dependent on plant stability. Edible vaccine are prone to get microbial infestation. Presented by- Gourav singh 49

50. IMPORTANT QUESTION What is the plant tissue culture technique and its type? What are the nutritional requirement for the development of plant tissue culture? Write a brief note on application of plant tissue culture in Pharmacognosy? Explain the detail history of plant tissue culture? Definition of PTC, it’s advantage and disadvantages Explain basic techniques of plant tissue culture Write in detailed about edible vaccine Presented by- Gourav singh 50

51. MCQ 1. Who is known as the Father of tissue culture? (a) Bonner (b ) Laibach (c) Haberlandt (d ) Gautheret Sol: (c) Haberlandt . 2. The production of secondary metabolites requires the use of ________. ( a) Meristem (b ) Protoplast ( c) Axillary buds (d ) Cell suspension Sol :(d) Cell suspension . 3. The pair of hormones required for a callus to differentiate are________. ( a) Ethylene and Auxin (b ) Auxin and cytokinin ( c) Auxin and Abscisic acid (d ) Cytokinin and gibberellin Sol : (b) Auxin and cytokinin. Presented by- Gourav singh 51

52. 4. Which of the following is the main application of embryo culture? ( a) Clonal propagation (b ) Production of embryoids ( c) Induction of somaclonal variations (d ) Overcoming hybridization barriers Sol : (d) Overcoming hybridization barriers .   5. Haploid plants can be obtained from________. (a) Anther culture (b ) Bud culture (c) Leaf culture (d ) Root culture Sol: (a) Anther culture 6.Which of the following growth hormones produces apical dominance? ( a) Ethylene (b ) Cytokinin ( c) Auxin (d ) Gibberellin Sol : (c) Auxin. Presented by- Gourav singh 52

53. 7. What is Callus? ( a) Tissues that grow to form an embryoid ( b) An unorganized actively dividing the mass of cells maintained in a culture ( c) An insoluble carbohydrate ( d) A tissue that grows from an embryo Sol : (b) An unorganized actively dividing mass of cells maintained in culture . 8. Growth of plant tissues in artificial media is called _______. ( a) cell hybridization (b ) plant tissue culture ( c) Transgenesis (d ) gene expression Sol: (b) Plant tissue culture 9. ___________ is the advantage of plant tissue culture over animal tissue culture a) Plant culture require less time b ) Plant tissues are easily available c ) Totipotency of the plant cell d ) Plant tissue culture can be easily maintained with minimum requirements Sol :(C ) Totipotency of the plant cell Presented by- Gourav singh 53

54. 10.___________ is the type of Cell culture a ) Organ culture b ) Protoplast culture c ) Callus culture d ) Explant culture Sol: (b) Protoplast culture Presented by- Gourav singh 54

55. THANK YOU Presented by- Gourav singh 55