This presentation provides an overview of plant tissue culture lab

1. By – Dr. Mafatlal M. Kher
Unit: 2 Plant tissue culture: :Lab
Date & Time : Monday, 02 August 2021
Semester : VI
Program : B.Sc. Biotechnology
School : School of Science
Subject : BSC5BT04

2. Plant tissue culture lab
Infrastructure, instruments, and chemicals
2

3. Infrastructure
 Washing Room
 Media Preparation Room
 Media Storage Room (s) maintained under positive pressure
 Inoculation Room (s) maintained under positive pressure
 Growth Room (s) maintained under positive pressure
 Transfer/ grading Room (s)
 Insect proof greenhouse/ poly house with double door entry fitted with
humidity control for primary hardening area
 Insect proof Nursery/Shade house Area (s) with double door entry covered
with appropriate mesh to provide partial shade for secondary hardening
area
3

4. Storage facility
4
Refrigerator
Chemical storage cabinet
• Properly labelled chemicals
• Stock records maintained
• Plant growth regulators and
stock solution of plant tissue
culture medium stored in
refrigerator.
• Refrigerator/freezer; capable
of maintaining a refrigerator
temperature of 0-50 C with a
freezer temperature of
approximately –200 C

5. Glassware's
5
Reagent Bottle Screw Cap Amber Colour
Beakers
• Amber coloured screw cap bottles are utilized for the storage of light sensitive
Conical flasks

6. Measurement
6
Measuring cylinders Variable range of Micropipettes
with stands
Digital Balance
• One for mg range: Plant Growth Regulators
• Gm range macronutrient/micronutrient/ sucrose

7. pH meter and Magnetic stirrer with hotplate
7
Magnetic stirrer
with hotplate
pH Meter
Hot Plate/Stirrer (7” x 7”
ceramic top; variable heating
range from ambient to 4000
C; variable stirring speed
from 50-150 rpm; chemically
resistant)
pH meter (range 0-14 +/- 0.01;
automatic temperature
compensation 0-600 C; one or two
point calibration)
Utilized to remove
magnetic bar

8. Forceps Blunt end and pointed end
8
Forceps 15 cm plus for inoculation of explant
Forceps ~10 cm for inoculation and
handling of small materials like
anther or zygotic embryo

9. Surgical blades and Scalpel blade holder
9
Surgical blades
Scalpel Blade Holder

10. Microwave oven and autoclave
10
Autoclave
Microwave Oven

11. Laminar Flow Cabinets
11
Laminar Flow Transfer Hood; incoming air should be
HEPA filtered to remove 99.99% of particles larger that
0.3µm; should meet or exceed the Class 100 Clean
Standard 209D; maintain a flow of 90 fpm +/- 20% at
static pressures of 0.6-1.2”

12. Flame and glass bead sterilizers
12
Glass Bead Sterilizer
Flame sterilizer
Flame sterilizer

13. Glassware's
13
Plant tissue
culture bottles Wired tray
For incubation of
plant tissue
culture bottle in
PTC lab

14. Borosilicate Test tubes 25 x 150 nm
14
Cotton plugs wrapped with
muslin cloth
Test tube cap
Test tubes with cotton plug Test tubes with Cap Metal wired test-tube stand

15. Test tube stand
15

16. Plant Growth Chambers
16

17. Plant Growth Chambers
17
Specification
• Light intensity controller 30-300 µMole
• Photoperiod controller: Variable range like 16Hrs,
12Hrs, 14Hrs etc
• Humidity controller: 75-90 % range
• Temperature control: 22 to 35 °C

18. Tissue Culture Rack
18
Specification
• Photoperiod adjustment 12/14/16
Hrs light.
• Cool white LED lights used to
reduced electricity cost.
• Lights are horizontal.
• Light intensity 50-70 µMole or 2000
to 3000 lux
• Permanent AC is required.
• Thermometer
• Lux meter/Light intensity meter

19. Water: RO Water v/s Distilled water v/s Milli Q (Ultra pure)
19
RO Water system
Distillation unit Distillation unit
Double Distillation unit
Milli Q (Ultra pure water)

20. Ready to use Medium
20

21. Surface sterilization: Carbendazim
21
Carbendazim is a widely used, systemic, broad-spectrum
benzimidazole fungicide and a metabolite of benomyl.
Carbendazim products are used for the control of a wide
range of fungal diseases such as mould, spot, mildew,
scorch, rot and blight in a variety of crops.

22. Surface sterilization: Mancozeb
22
Mancozeb is a dithiocarbamate non-systemic agricultural
fungicide with multi-site, protective action on contact. It is a
combination of two other dithiocarbamates: maneb and zineb.
The mixture controls many fungal diseases in a wide range of
field crops, fruits, nuts, vegetables, and ornamentals

23. Surface sterilization: Carbendazim + Mancozeb
 Carbendazim 12% + Mencozeb
63% WP (Wettable Powder) is a
very effective, protective and
curative fungicide.
23

24. Surface sterilization: Mercuric chloride and ethanol
24
0.1 % to 0.5 % Mercuric chloride 1 to 5 mins 70% Ethanol for 30 second to 5 mins.

25. Why 70% ethanol
Pure Ethanol Prevents Cell Death
 Testing has been done to show that when pure ethanol (at 100%) is poured
onto a single celled organism, it will coagulate (clot) its protein.
 The ethanol penetrates its cellular wall in all directions.
 The protein located just within the cell wall is what coagulates. It’s much
like a defense mechanism. This ring of coagulated protein actually prevents
the ethanol from penetrating deeper into the cell wall of the organism. No
more coagulation takes place. Basically, this renders the organism
dormant, but doesn’t kill it. If the ethanol were to be washed away, then it’s
possible the organism would come back to life.
 This process defeats the purpose of using ethanol to kill microbes. Instead,
scientists have found a way to trick these microbes with a lower percentage
of ethanol
25

26. Why 70% ethanol
 How 70% Ethanol Causes Cell Death
 In the same study, when the 70% ethanol was poured onto a single celled
organism, the ethanol also caused its protein to coagulate, but this
occurred at a much slower rate.
 This actually allowed the ethanol to penetrate the entire cell before it had a
chance for its coagulation to block it. The entire cell is then coagulated,
causing the cell to die.
26

27. Plant Tissue Culture Media
27

28. Logical Basis
 For healthy and vigorous growth, intact plants need to take up from soil of an
essential elements.
Essential elements (Epstein, 1971):
1. A plant grown in a medium adequately purged of that elements, failed to grow
properly or to complete its life cycle
2. It is a constituent of a molecule that is known to be an essential metabolite
28

29. Media components
 Inorganic salts: Macronutrients and Micronutrients
 Organic compounds: Vitamins, amino acids, carbohydrates and plant
growth regulators
 Gelling agents?: Agar-agar, Gellan gum (Gelrite, phytagel, Clerigel), or
liquid culture (Suspension culture/ Bioreactor/MicroRocker system/
temporary immersion system).
29

30. Essential element
 Macro element/major plant nutrition: Relatively large amount required
a. Carbon (C) d. Nitrogen (N) g. Potassium (K)
b. Hydrogen (H) e. Calcium (Ca) h. Phosphorus (P)
c. Oxygen (O) f. Magnesium (Mg) i. Sulphur (S)
 Micro element/ minor plant nutrient/trace elements: Small quantities required
a. Iron (Fe) f. Sodium (Na)
b. Chlorine (Cl) g. Manganese (Mn)
c. Zinc (Zn) h. Boron (B)
d. Copper (Cu) i. Molybdenum (Mo)
e. Nickel (Ni)
30

31. Why plant in vitro culture needs media?
Functions of media
Provide water
Provide mineral nutritional needs
Provide vitamins
Provide growth regulators
Provide amino acids
Provide sugars
Access to atmosphere for gas exchange
Removal of plant metabolite waste
31

32. Plant tissue culture media
1. Macronutrients (always employed)
2. Micronutrients (nearly always employed, although sometimes just one element,
iron, has been used)
3. Vitamins (generally incorporated , although the actual number of compounds
added, varies greatly)
4. Amino acids and other nitrogen supplements (usually omitted, but sometimes
used with advantage)
5. Sugar (nearly always added, but omitted for some special purposes)
6. Undefined supplements (which, when used, contribute some above components,
and also plant growth substances or regulants)
7. Buffers (have seldom been used in the past, but recently suggest that the
additions of organic acids or buffers could be beneficial in some circumstances)
8. A solidifying agent (used when a semi solid medium is required)
32

33. Macronutrients
1. Macronutrients for plant tissue culture are provided from salt, however
plant absorb entirely as ions
2. Nitrogen is mainly absorbed in the form of ammonium or nitrate
3. Phosphorus as the phosphate ions
4. Sulphur as sulphate ions
5. The most important step in deriving medium is the selection of
macronutrient ions in the correct concentration and balanced
6. The salts normally used to provide macroelements also provide sodium
and chlorine, however, plant cell tolerate high concentration of both ions
without injury, these ions are frequently given little importance when
contemplating media changes
33

34. Quantity of macronutrients
34

35. Nitrogen
1. It is essential to plant life
2. Both growth and morphogenesis is markedly influenced by the availability of
nitrogen and the form in which it is presented
3. Most media contain more nitrate than ammonium ions. Most intact plants,
tissues and organ taken up nitrogen effectively, and grow more rapidly on
nutrient solutions containing both nitrate and ammonium ions
4. Nitrate has to be reduced to ammonium before being utilized biosynthetically
5. Ammonium in high concentration is latent toxic
6. For most type of culture, nitrate needs to be presented together with the
reduced form of nitrogen and tissue will usually fail to grow on a medium with
nitrate as the only nitrogen source
35

36. NH4
+ and NO3
- Regulate Medium pH and Root Morphogenesis of
Rose Shoots
36

37. Amino acids
 Amino acids can be added to satisfy the requirement for reduced nitrogen,
but as they are expensive to purchase, they will only be used on media for
mass propagation where this results in improved result.
 A casein hydrolysate, yeast extract which mainly consist of a mixture of
amino acids substantially increased the yield of callus.
 Organic supplements have been especially beneficial for growth or
morphogenesis when cells were cultured on media which do not contain
ammonium ions.
 Glycine is a common ingredient of many media. It is difficult to find hard
evidence that glycine is really essential for so many tissue culture, but
possible it helps to protect cell membranes from osmotic and temperature
stress
37

38. Amino acids
 The most common sources of organic nitrogen used in culture media are
amino acid mixtures.
 Its uptake more rapidly than in organic amino acids. (e.g., casein
hydrolysate), L-glutamine, L-asparagine, and adenine.
 When amino acids are added alone, they can be inhibitory to cell growth. .
38

39. Beneficial effects of Amino acids
 Rapid growth
 Protoplast cell division
 Conservation of ATP
 AS chelating agent
 Enhanced nitrogen assimilation
 Not toxic as ammonium
 As buffer
39

40. Phosphorous
 It is a vital element in plant biochemistry
 It occurs in numerous macromolecules such as nucleic acids,
phospholipids and co-enzymes
 It functions in energy transfer via pyrophosphate bond I ATP
 Phosphate groups attached to different sugar provide energy in respiration
and photosynthesis and phosphate bound to proteins regulate their activity
 Phosphorous is absorbed into plants in the form of the primary or
secondary orthophosphate anions by an active process which requires the
expenditure of respiratory energy
 Phosphate in not reduced in plants, but it remains in the oxydised form
 It is used in plant as the fully oxydised orthophosphate form
40

41. Potassium
 It is not metabolized
 It is a major cation within the plants
 It contributes significantly to the osmotic potential of cells
 It is transported quickly across cell membrane and two of its major role is
regulating the pH and osmotic environment within the cells
 Many protein show a high specificity for potassium which acting as a
cofactor, alters their configuration so that it become active enzyme
 It is also neutralize organic anions produce in the cytoplasm and so
stabilize the pH and osmotic potential of the cells
41

42. Sodium
 It is taken up into plant but in most cases it is not required for growth and
development
 Many plants actively secret it from their roots to maintain a low internal
concentration
 It is only appeared to be essential to salt tolerance plant
42

43. Magnesium
 It is an essential component of the chlorophyll molecules
 It is also required non-specifically for the activity of many enzymes,
especially in the transfer of phosphate
 ATP synthesis has an absolute requirement for magnesium and it is a
bridging element in the aggregation of ribosome sub-unit
 It is the central atom in the phorphyrin structure of the chlorophyll
molecules
43

44. Sulfur
 It is mainly absorbed as sulfate
 Its uptake is coupled to nitrogen assimilation
 It is incorporated into chemical compounds mainly as reduced –SH, -S_ or
–S-S groups
 It is used in lipid synthesis and in regulating the structure of proline through
the formation of S-S bridges
 It acts as a ligand joining ion of iron, zinc, copper to metalloportein and
enzymes
44

45. Calcium
 It helps to balance anion within the plant
 It is not readily mobile
 It is involved in the structure and physiologically properties of cell
membranes and the middle lamella of the cell walls (Calcium pectate)
 The enzyme -(1-3)-glucan synthase depends on calcium ions
 It is a cofactor in the enzymes responsible for the hydrolisis of ATP
45

46. Chlorine
 It has been found to be essential for plant growth
 It is sometimes considered as micro nutrient, because it is required in a
small amount
 It is required for water – splitting protein complex of photosystem II
 It can function in osmoregulation in particular stomata guard cell
46

47. Micronutrients
 Plant requirement for microelement have only been elucidated in the 19th
century
 In the early of 20th century, uncertainty still existed over the nature of the
essential microelements
 many tissue undoubtedly grown successfully because they were cultured
on media prepared from impure chemicals or solidified with agar which
acted as a micronutrient source
 In the first instance, the advantage of adding micronutrients was mainly
evaluated by their capability to improve the callus growth or root culture
 Knudson (1922) incorporated Fe and Mn on very successful orchid seed
media
 Heller (1953) was first well demonstrated the advantages of microelement
on tissue culture media
47

48. Why in the first development many
tissue were undoubtedly grown
successfully in tissue culture media
without micronutrient?
• Media is solidified with agar which acted as a micronutrient source
• Plant cells are more demanding for micronutrients when undergoing
morphogenesis
48

49. Quantity of micronutrients
49
MS medium was formulated from the ash content of tobacco callus. The higher
concentration of salts substantially enhanced cell division

50. Boron (B)
 It is involved in plasma membrane integrity and function, probably by influencing
membrane protein and cell wall intactness.
 It is required for the metabolism of phenolic acids, and for lignin biosynthesis.
 It is probably a component, or co-factor of the enzyme which converts p-coumaric
acid to 5-hydroxyferulate.
 It is necessary for the maintenance of meristematic activity, most likely because it
is involved in the synthesis of N-bases.
 It is also thought to be involved in the maintenance of membrane structure and
function, possibly by stabilizing natural metal chelates, which are important in wall
and membrane structure and function
50

51. Manganese (Mn)
 It is the most important micro nutrients
 It has similar properties to Magnesium, it is apparently able to replace
magnesium is some enzyme systems
 It is involved in respiration and photosynthesis as metalloprotein structure
 It is known to be required for the activity of several enzymes
 It is necessary for the maintenance of chloroplast ultra structure
 It is involved in regulation of enzymes and growth hormones.
 It assists in photosynthesis and respiration.
51

52. Zinc (Zn)
 It is a component of stable metallo enzymes with many diverse function.
 It is required in more than 300 enzymes.
 Its deficient plants will suffer from reduced enzyme activities and as a
consequent will diminute in protein, nucleic acid and chlorophyl synthesis.
 There is a close relationship between zinc concentration of plants and their
auxin content
52

53. Copper (Cu)
 Plant only contains a few part of million of Cu
 It becomes attached to enzymes, many of which bind to and reach with
oxygen
 It occurs in plastocynain, a pigment participating in electron transport
 Highly concentration of Cu can be toxic
53

54. Molybdenum (Mo)
 It is utilized in the form of hexavalent Mo
 It is absorbed as the molybdate ions
 It is a component of several plant enzymes, two being nitrate reductase and
nitrogenase, in which it is a cofactor together with iron
54

55. Cobalt (Co)
 It is sometimes not regarded as an essential elements
 It might have a role in regulating morhogenesis of higher plants
 It is the metal component of vitamin B12 which is concerned with nucleic
acid synthesis, though evidence that it has any marked stimulatory effect
on growth and morphogenesis is hard to find
 It can have a protective action against metal chelate toxicity and it is able to
inhibit oxidative reaction catalyzed by copper and iron
 Cobalt can inhibit ethylene biosynthesis
55

56. Nickel (Ni)
 It is a component of urease enzyme which convert urea to ammonia
 It has been shown to be an essential micronutrient for some legumes
 The presence of Ni strongly stimulate the cell growth in a medium
containing urea as a nitrogen source
 Agar contains relatively high levels of nickel and the possibility of urea
toxicity may have been avoided because in tissue culture media, urea
diffuses into the medium
56

57. Iodine (I)
 It is not recognized as a essential element for plants, although it may be
necessary for the growth of some algae and small amount was
accumulated in higher plant
 It has been added to many tissue culture media
 In improve the in vitro root growth It prevent the explant browning
 It enhance the destruction and/or the lateral transport of auxin
57

58. Iron (Fe)
 A key properties of iron is its capacity to be oxidized easily from the ferrous
(Fe(II)) to the ferric (Fe(III)) state and for ferric compounds to be readily reduced
back to the ferrous form.
 Iron is primarily used in the chloroplasts, mitochondria and peroxisomes for
effecting oxidation/reduction reaction.
 It is a component of ferredoxin proteins which function as electron carriers in
photosynthesis.
 Iron is an essential micronutrient for plant tissue culture and can be taken up as
either ferrous or ferric ions.
 Iron may not be available to plant cells, unless the pH falls sufficiently to bring
free ions back to solutions.
 Iron can be chelated with EDTA
 The addition of Fe-EDTA chelate greatly improved the availability of the element
58

59. Chelating agent
 Some organic compounds are capable of forming complexes with metal
cations, in which the metal is held with fairly tight chemical bonds
 Metal can be bound (sequestered) by a chelating agent and held in solution
under conditions where free ions would react with anions to form insoluble
compounds, and some complexes can be more chemically reactive than
the metals themselves
 Chelating agents vary in their sequestering capacity according to chemical
structure and their degree of ionisation, which changes with pH of the
solution
 Naturally –occurring compounds can act as chelating agents such as
proteins, peptides, carboxylic acids and amino acids
 There are also synthetic chelating agents with high avidity for divalent and
trivalent ions
59

60. Chelating agent
60
No. Chelating agents Chemical names
1. EDTA Ethylenediamine tetra acetic acid
2. EGTA Ethyleneglycol-bis(2-
aminoethylether) tetra acetic acid
3. EDDHA Ethylenediamine-di(o-
hydroxyphenyl) acetic acid
4. DTPA Diethylenetriaminepentaacetic acid
5. DHPTA 1,3 diamino-2-hydroxypropane-
tetra acetic acid

61. Carbon source
 Most plant tissue cultures are not highly autotrophic due to limitation of CO2.
 Therefore, sugar is added to the medium as an energy source. Sucrose is the
most common sugar added, although glucose, fructose, manitol and sorbitol are
also used in certain instances.
 The concentration of sugars in nutrient media generally ranges from 20 to 40 g/l.
 Sugars also contribute to the osmotic potential in the culture The presence of
sucrose specifically inhibits chlorophyll formation and photosynthesis, making
autotrophic growth less feasible
 Sucrose in the culture media is usually hydrolyzed totally or partially into the
component monosaccharides glucose and fructose
 The general superiority of sucrose over glucose may be on account of the more
effective translocation of sucrose to apical meristems.
 Why Sucrose?: Because, sucrose is only transportable form of sugar in plant.
Therefore, sucrose is utilized in plant tissue culture medium.
61

62. Organic supplements
 Vitamins: Only thiamine (vitamin B1) is essential for most plant cultures, it
is required for carbohydrate metabolism and the biosynthesis of some
amino acids
 Thiamine (vitamin B1) Essential as a coenzyme in the citric acid cycle
 Nicotinic acid (niacin) and pyridoxine (B6)
62

63. Organic supplements
 Myo-inositol: Although it is not essential for growth of many plant species,
its effect on growth is significant. Part of the B complex, in phosphate form
is part of cell membranes, organelles and is not essential to growth but
beneficial
 Complex organics: Such as coconut milk, coconut water, yeast extract,
fruit juices and fruit pulps.
63

64. Physical supporting agents
 Gelling agents: When semi-solid or solid culture media are required,
gelling agents are used. An example: Agar, agarose, gelrite, phytagel
 Structural supports: Filter paper bridges, liquid permeable membrane
support systems, Rockwool
64

65. Agar
 Agar is the most commonly used gelling agent
 It is a natural product extracted from species of red algae, especially Gelidium
amansii
 It is synthetic polysaccharide gelling agents
 Agar tertiary structure is a double helix the central cavity of which can
accommodate water molecules
 Agar consists of 2 components
 Agarose is an alternating D-galactose and 3,6-anhydro-L-galactose with side
chains of 6-methyl-D-galactose residues (50 -90%).
 Agaropectin is like agarose but additionally contains sulfate ester side chains
and D-glucuronic acid.
65

66. Advantage and limitations of agar
Advantages:
 Agar is an inert component, form a gel in water that melt at 100 ° C and solidify at
nearly 45 ° C
 Concentrations commonly used in plant culture media range between 0.5% and
1%
 If necessary, agar can be washed to remove inhibitory organic and inorganic
impurities.
 Gels are not digested by plant enzymes
 Agar does not strongly react with media constituent (Inert)
Limitations:
 Agar does not gel well under acidic conditions (pH <4.5).
 The inclusion of activated charcoal in media may also inhibit gelling of agar.
66

67. Agarose
 It is extracted from agar leaving
behind agaropectin and its sulfate
groups.
 It is used when the impurities of
agar are a major disadvantage.
67

68. Gelrite™
 Gelrite consists of a polysaccharide
produced by the bacterium
Pseudomonas elodea.
 It gives clear-solidified medium that
leads to detection of contamination
at an early stage.
 Gelrite requires more stirring than
agar.
 Concentration of divalent cations
such as calcium and magnesium
must be within the range of 4-8
mM/L or the medium will not solidify
68

69. Phytagel™
 It is an agar substitute produced from a bacterial substrate composed of
glucuronic acid, rhamnose and glucose.
 It produces a clear, colorless, high-strength gel, which aids in detection of
microbial contamination.
 It is used at a concentration of 1.5-2.5 g/L.
 It should be prepared with rapid stirring to prevent clumping.
69

70. Commercial media formulations
 Murashige and Skoog (MS)
 Linsmaier and Skoog (LS)
 White Medium
 Gamborg medium
 Schenk and Hildebrandt medium
 Nitsch and Nitsch Medium
 Lloyd and McCown (Woody plant
medium)
 Knudson’s medium.
70

71. Plant hormones/ Phytohormones
 The Greek word hormaein, meaning "to excite".
 Small organic molecule that elicits a physiological response at very low
concentrations.
 Chemical signals that coordinate different parts of the organism.
 Internal and external signals that regulate growth are mediated, at least in
part, by growth-regulating substances, or hormones.
 A natural substance which produced by plant and acts to control plant
activities.
 Chemical messengers influencing many patterns of plant development.
 Naturally occurring or synthetic compounds that affect plant growth and
development.
71

72. Plant hormones differ from animal hormones in that:
 No evidence that the fundamental actions of plant and animal hormones
are the same.
 Unlike animal hormones, plant hormones are not made in tissues
specialized for hormone production. (e.g., sex hormones made in the
gonads, human growth hormone - pituitary gland).
 Unlike animal hormones, plant hormones do not have definite target areas
(e.g., auxins can stimulate adventitious root development in a cut shoot, or
shoot elongation or apical dominance, or differentiation of vascular tissue).
72

73. Plant hormones: Characteristics
 Synthesized by plants.
 Show specific activity at very low concentrations.
 Display multiple functions in plants.
 Play a role in regulating physiological phenomena in vivo in a dose-
dependent manner.
 They may interact, either synergistically or antagonistically, to produce a
particular effect.
73

74. Classification of
PGRs
Auxin
Cytokinin
Gibberelin
Abscisic acid
Ethylene
Other
Jasmonates
Salicylic acid
Brassinosteroids
74

75. Plant Hormones: As a chemical messengers
 Auxin
 Cytokinin
 Gibberelic acid
 Ethylene
75

76. Auxin
 Arpad Paál (1919) - Asymmetrical
placement of cut tips on coleoptiles resulted
in a bending of the coleoptile away from the
side onto which the tips were placed
(response mimicked the response seen in
phototropism).
 Frits Went (1926) determined auxin
enhanced cell elongation.
76

77. Auxin
 Absolutely essential (no mutants known)
 One compound: Indole-3-acetic acid.
 Many synthetic analogues: NAA, IBA, 2,4-D, 2,4,5-T, Picloram : Cheaper
& more stable
 Generally growth stimulatory.
 Promote rooting Stimulate cell elongation Increase the rate of transcription
 Mediate the response of bending in response to gravity or light
 Produced in meristems, especially shoot meristem and transported through
the plant in special cells in vascular bundles.
77

78. Cytokinins
78
Zeatin
Adenine 6 benzyl aminopurine (BAP
Or Benzyle adenine (BA)
Kinetin or
6 furfyryl aminopurine

79. Discovery of cytokinin
 Gottlieb Haberlandt in 1913 reported an unknown compound that stimulated cellular
division.
 In the 1940s, Johannes van Overbeek, noted that plant embryos grew faster when they
were supplied with coconut milk (liquid endosperm), which is rich in nucleic acids.
 In the 1950s, Folke Skoog and Carlos Miller studying the influence of auxin on the
growth of tobacco in tissue culture.
 When auxin was added to artificial medium, the cells enlarged but did not divide. Miller
took herring-sperm DNA.
 Miller knew of Overbeek's work, and decided to add this to the culture medium, the
tobacco cells started dividing.
 He repeated this experiment with fresh herring-sperm DNA, but the results were not
repeated. Only old DNA seemed to work.
 Miller later discovered that adding the purine base of DNA (adenine) would cause the
cells to divide.
79

80. Discovery of cytokinin
 Adenine or adenine-like compounds induce cell division in plant tissue
culture.
 Miller, Skoog and their coworkers isolated the growth facto responsible for
cellular division from a DNA preparation calling it kinetin which belongs to a
class of compounds called cytokinins.
 In 1964, the first naturally occurring cytokinin was isolated from corn called
zeatin. Zeatin and zeatin riboside are found in coconut milk.
 All cytokinins (artificial or natural) are chemically similar to adenine.
 Cytokinins move nonpolarly in xylem, phloem, and parenchyma cells.
 Cytokinins are found in angiosperms, gymnosperms, mosses, and ferns. In
angiosperms, cytokinins are produced in the roots, seeds, fruits, and young
leaves
80

81. Cytokinins
 Absolutely essential (no mutants known)
 Natural compound: Zeatin, 2-isopentyl adenine (2iP)
 Synthetic analogues: Benyzladenine (BA), Kinetin.
 Stimulate cell division (with auxins).
 Promotes formation of adventitious shoots
 Stimulate cell division Stimulate dark germination
 Stimulate leaf expansion
 Produced in the root meristem and transported throughout the plant as the
Zeatin-riboside in the phloem.
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82. Basis for Plant Tissue Culture
 Two Hormones Affect Plant Differentiation:
 Auxin: Stimulates Root Development
 Cytokinin: Stimulates Shoot Development
 Generally, the ratio of these two hormones can determine plant
development:
  Auxin ↓Cytokinin = Root Development
  Cytokinin ↓Auxin = Shoot Development
 Auxin = Cytokinin = Callus Development
82

83. Auxin/ Cytokinin Ratio
83
Skoog, F., & Miller, C. (1957). Chemical regulation of
growth and organ formation in plant tissues cultured.
In Vitro Symp Soc Exp Biol.

84. Auxin/ Cytokinin Ratio
84
• In 1957 Skoog and Miller put forth the concept of
hormonal control of organ formation.
• In this classic paper, they showed that the
differentiation of roots and shoots in tobacco pith
tissue cultures was a function of the auxin-
cytokinin ratio, and that organ differentiation could
be regulated by changing the relative
concentrations of the two substances in the
medium; high concentrations of auxin promoted
rooting, whereas high levels of cytokinin supported
shoot formation.
• At equal concentrations of auxin and cytokinin the
tissue tended to grow in an unorganized fashion.
This concept of hormonal regulation of
organogenesis is now applicable to most plant
species.

85. Gibberellin(GA’s): discovery
 In 1930's, Ewiti Kurosawa and colleagues were
studying plants suffering from bakanae, or "foolish
seedling" disease in rice.
 Disease caused by fungus called, Gibberella fujikuroi,
which was stimulating cell elongation and division.
 Compound secreted by fungus could cause bakanae
disease in uninfected plants.
 Kurosawa named this compound gibberellin.
 Gibberella fujikuroi also causes stalk rot in corn,
sorghum and other plants.
 Secondary metabolites produced by the fungus include
mycotoxins, like fumonisin, which when ingested by
horses can cause equine leukoencephalomalacia -
necrotic brain or crazy horse or hole in the head
disease.
 Fumonisin is considered to be a carcinogen.
85

86. Gibberellin(GA’s)
 A family of over 70 related compounds, all forms of Gibberellic acid and
named as GA1, GA2.... GA110.
 Commercially, GA3 and GA4+9 available.
 Stimulate etiolation of stems. Help break bud and seed dormancy.
 Stimulate stem elongation by stimulation cell division and elongation
 Stimulate germination of pollen Produced in young leaves
86

87. Abscisic acid (ABA)
87
Abscisic acid Xanthoxin
Xanthoxin is an intermediate in the biosynthesis of the
plant hormone abscisic acid

88. Abscisic acid (ABA): Discovery
 In 1940s, scientists started searching for hormones that would inhibit
growth and development, what Hemberg called dormins.
 In the early 1960s, Philip Wareing confirmed that application of a dormin to
a bud would induce dormancy.
 F.T. Addicott discovered that this substance stimulated abscission of cotton
fruit. he named this substance abscisin. (Subsequent research showed that
ethylene and not abscisin controls abscission).
 Abscisin is made from carotenoids and moves nonpolarly through plant
tissue.
88

89. Abscisic acid (ABA)
 Only one natural compound.
 Promotes leaf abscission and seed dormancy.
 Plays a dominant role in closing stomata in response to water stress
Involved in the abscission of buds, flower and fruits Inhibit cell division and
elongation
 Has an important role in embryogenesis in preparing embryos for
desiccation.
 Helps ensure ‘normal’ embryos.
89

90. Ethylene: Discovery
 In the 1800s, it was recognized that street lights
that burned gas, could cause neighboring plants
to develop short, thick stems and cause the
leaves to fall off.
 In 1901, Dimitry Neljubow identified that a
byproduct of gas combustion was ethylene gas
and that this gas could affect plant growth.
 R. Gane showed that this same gas was
naturally produced by plants and that it caused
faster ripening of many fruits.
 Synthesis of ethylene is inhibited by carbon
dioxide and requires oxygen.
90

91. Ethylene
 Gas - diffuses through tissues
 Stimulates abscission and fruit ripening
 Used in commercial ripening for bananas & green picked fruit Involved in
leaf abscission & flower senescence
 Primarily synthesized in response to stress Regulate cell death
programming
91

92. Brassinosteroids
 Promote shoot elongating
 Inhibit root growth
 Promote ethylene biosynthesis
 Enhance resistance to chilling, disease and herbicides
92

93. Salicylic acid
 Salicylic acid and its derivatives as one of the plant hormones produced by
the plant naturally belongs to the group of phenolic acids and consists of a
ring linked to the group of hydroxyl and carboxyl group, and the starting
ingredient to form is the cinnamic acid.
 It is mainly manufactured within the plant in cytoplasmic cell. This acid was
first discovered in Salix spp., which contains the salicin compound by 9.5–
11% and is present in the plant in the form of free phenolic acids or
associated with amino compounds.
 Promote flowering
 Stimulate plant pathogenesis protein production
93

94. Jasmonates
 Jasmonic acid (JA) and its precursors and dervatives, referred as
jasmonates (JAs).
 JAs are a class of plant hormones that play essential roles in response to
tissue wounding.
 They act on gene expression to slow down growth and to redirect
metabolism towards producing defense molecules and repairing damage
 Play an important role in plant defence mechanisms
94

95. Glassware Cleaning: Liquid detergent
95

96. Glassware Cleaning: Chromic acid
96
 The term chromic acid is usually used for a mixture made by adding
concentrated sulfuric acid to a dichromate.
 Chromic acid is a commonly used glassware cleaning reagent.
 It is prepared in a one liter container by dissolving 60 grams of potassium
dichromate in approximately 150 mls of warm distilled water and then slowly
adding concentrated sulfuric acid to produce a total volume of one liter Chromic
Acid solution.

97. Green House
97
Low Tunnel
Green House
Fan
Water fogger

98. Green House
98

99. NET House
99

100. Sterilization techniques
 USE OF CHEMICALS: Chemicals such as Chromic acid, Mercuric chloride,
Sodium hypochlorite, Calcium hypochlorite and alcohol are used for the
sterilization of glassware, work tables and source materials of explants.
 USE OF OVEN: A dry heat oven is used to sterilize glassware, metallic
instruments etc. by hot air (200-300C) for 1 hour.
 USE OF AUTOCLAVES: Autoclaving is done to sterilize nutrient media,
distilled water etc. with the help of steam (121C for 30 minutes)
 ULTRA FILTRATION: Vitamins, hormones etc. are unstable at high
temperatures. They are sterilized using millipore membrane filter etc.
 USE OF UV LIGHT: UV light is used in the incubation chamber to make it
germ-free.
100