lec2 a. In vitro culture environment 2023-1.ppt

In vitro culture environment required for tissue culture

1. Lec 2 a. IN VITROCULTURE ENVIRONMENT
PLANT TISSUE CULTURE MEDIA

2. Types of Culture Media
•Solid Media
•Liquid Media

3. Composition of Culture Media
–Essential elements
•Complex mixture of salts
–An organic supplement
•Vitamins/amino acids
–A source of fixed carbon
•Sucrose

4. Essential elements
•Macroelements
–N, S, P, Ca, Mg, K
•Microelements
–Fe, B, Co, I, Cu, Mn, Mo, Zn
•An iron source
–Fe EDTA

5. Macro elements
N, S, P, Ca, Mg, K
•Nitrogen (N):
Integral constituent of amino acids, proteins,
nucleic acids and chlorophyll
Supplied in two forms (NO
3 andNH
4)
Nitrates conc. 25-20 mM
Ammonium conc. 2 -20 mM. conc. in excess
of 8 mM may be deleterious to cell growth

6. •Sulpher (S)
Vital component of proteins in AA
residues of Cystein & methionine
Present in iron-sulphur proteins
Usual conc. 1-3 mM
Supplied in the form of SO
4in
combination with magnesium

7. •Phosphorus (P)
Constituent of nucleic acids DNA and RNA
Present in phospholipids of biomembranes &
phosphate esters like ATP
Important substrate in metabolic reactions
Usual conc. 1-3 mM
Supplied as PO
4
(Sodium or Potassium dihydrogen phosphate)

8. •Magnesium (Mg)
Central atom in the aggregation of
ribosomal subunits
Essential factor for enzymatic reactions
Keeps cation-anion balance
Usual conc. 1-3 mM
Supplied as magnesium sulphate

9. •Calcium (Ca)
Important modulator of enzyme action
Required for cell wall synthesis and
stabilization
Usual conc. 1-3 mM
Supplied as chloride or nitrate salt

10. •Potassium (K)
Functions in osmoregulation, cation-anion
balance & pH stabilization
Activator for many enzyme-catalyzed reactions
Involved in translation process of protein
synthesis
Usual conc. 20-30 mM
Supplied as potassium nitrate

11. TheoptimumconcentrationsofP,Mg,S,andCa
rangefrom1-3mMwhenallotherrequirements
forcellgrowtharesatisfied.Higher
concentrationsofthesenutrientsmaybe
requiredifdeficienciesinothernutrientsexist.

12. Micro Elements
Iron (Fe), Manganese (Mn), Zinc (Zn), Boron (B),
Copper (Cu), Molybdenum (Mo)
•Iron (Fe)
Required for chlorophyll synthesis
Functions in redox reactions (as a constituent of
cytochromes and iron-sulphuer proteins)
EDTA essential to keep Ferric iron in solution
Ferricironisinsoluble,needschelators.Ironmaybethemost
criticalofallthemicronutrients.Ironcitrateandtartratemaybe
usedinculturemedia,butthesecompoundsaredifficultto
dissolveandfrequentlyprecipitateaftermediaareprepared.
MurashigeandSkoogusedanethylenediaminetetraaceticacid
(EDTA)-ironchelatetobypassthisproblem.

13. •Boron (B)
Part of structural complexes of cell wall
Required for cell division in apical
meristems of roots
Supplied as boric acid
•Cobalt (Co)
No direct function in higher plants
Usual concentration 0.1 µM

14. •Copper (Cu)
Function as protein bound copper in redox
reactions
Usual conc. 0.1 µM
Supplied as Copper sulphate
•Iodine (I)
Beneficial for root & callus growth

15. •Manganese (Mn)
Found in metalloprotein
Usual conc. 5-30 µM
Supplied as manganese sulphate
•Molybdenum (Mo)
Cofactor of a few redox enzymes
Supplied as sodium molybdate at 0.1 µM
•Zinc (Zn)
Required for activity of Hydrogenases and DNA
and RNA polymerases
Supplied as zinc sulphate at 5-30 µM

16. •Organic supplements
–Vitamins
•The most frequently used in plant tissue culture media
are thiamine, nicotinic acid, pyridoxine and myo-
inositol
•Thiamine (Vit. B1, aneurine) in the form of thiamine
pyrophosphate, is an essential cofactor in
carbohydrate metabolism and is directly involved in
the biosynthesis of some amino acids.
•Myoinositol important for cell wall and membrane
development especially in monocots and some dictos
biotin, Pantothenic acid, ascorbic acid, parabenzoic
acid, Riboflavin, D2 and D3, vitamin E

17. •Undefined Organic Supplements
•Fruitjuices,coconutmilk,yeastormaltextracts
andhydrolysedcasein
•Usedinearlyexperimentstofulfillthe
requirementsfortraceamountsofcertainorganic
substances
•Thesecontributevitamins,aminoacidsand
growthregulatorstoaculturemedium
•Theuseofundefinedsupplementsis
declinedastheneedforspecificorganic
compoundshasbeendefined,andthesehave
becomelistedincataloguesaspurechemicals.

18. •Carbon Source
•Carbohydrates play an important role in in vitro cultures as an
energy and carbon source, as well as an osmotic agent.
•The preferred carbohydrate in plant cell culture media is
sucrose. Glucose and fructose may be substituted in some
cases, glucose being as effective as sucrose and fructose
being somewhat less effective.
Sucrose is cheap, easily available, relatively stable. Refined
white domestic sugar is sufficiently pure for most practical
purposes
•Sucrose hydrolysedtotally, or partially, into the component
monosaccharidesglucose and fructose, inversion is due to
invertaseenzyme located in plant cell walls
Balances osmotic potential of culture medium
Supplied at 2-3% w/v

19. •Plant Growth Regulators
Auxins
Cytokinins
Gibberellins
Abscic acid
Ethylene

20. Natural Auxins (IAA)
•Synthesized in stem and root apices
•Stimulates elongation growth of cells
•Stimulates cell division and differentiation
•Together with Cytokinin regulates several
developmental processes
•Causes parthenocarpic fruit formation
•Inhibits senescence and abscission of leaves, fruits
and flowers
Synthetic Auxins
•2,4-D, NAA, IBA, pCPA
•used with cytokinins to control differentiation and
morphogenesis
•Higher conc. causes growth abnormalities
Auxins

21. Cytokinins
Natural (Zeatin)
Synthetic (Kinetin, BAP, 2iP)
Stimulates cell division
Stimulates morphogenesis (shoot initiation/bud
formation) in tissue culture
Stimulates the growth of lateral buds-release of apical
dominance
Stimulates leaf expansion resulting from cell enlargement
May enhance stomatal opening in some species
Promotes the conversion of etioplasts into chloroplasts via
stimulation of chlorophyll synthesis.

22. The illustration above shows the effect of cytokinin and auxin
concentration on tissue culture experiments (Mauseth, 1991)

23. Named GA
1....GA
nin order of discovery
GA
3first gibberellin to be structurally characterised
Stimulates stem elongation
Breaks seed dormancy in some plants which require
stratification or light to induce germination
Stimulates enzyme production (a-amylase) in germinating
cereal grains for mobilization of seed reserves
Can cause parthenocarpic (seedless) fruit development
Can delay senescence in leaves and citrus fruits
Gibberellins

24. Abscisic Acid
•plays mostly inhibitory roles
•Stimulates the closure of stomata (water stress brings about an
increase in ABA synthesis)
•Inhibits shoot growth but will not have as much effect on roots or
may even promote growth of roots
•Induces seeds to synthesize storage proteins
•Inhibits the effect of gibberellins on stimulating de novo synthesis
of α-amylase
•Has some effect on induction and maintenance of dormancy
•Induces gene transcription especially for proteinase inhibitors in
response to wounding which may explain an apparent role in
pathogen defense

25. Ethylene
•Gaseous hormone; the only member of its
class.
•Stimulates the release of dormancy
•Stimulates shoot and root growth and
differentiation (triple response)
•Stimulates leaf and fruit abscission.
•Stimulates flower opening and senescence.
•Stimulates fruit ripening.

26. Support Matrices
•Gelling agents (Agar, agarose, gelrite, gelatin, gellan gums)
•Alternativemethodsofsupport:perforatedcellophane,
filterpaperbridges,filterpaperwicks,polyurethanefoam,
andpolyesterfleece
•Agar
complex polysaccharide; comprises of agarose (70%) and
agaropectin (30%)
Melts at 100°C, gels at 35°C
Stable, doesn't react with media constituents
•Agar contains Ca, Mg and trace elements on comparing
different agar brands (Bacto, Noble and purified agar)
•supplied at 0.8 to 1% w/v

27. •Agarose
•Agarose is a purified extract of agar without the agaropectin
fraction and sulphate groups.
•required in lesser quantities since it has a higher gelling
strength.
•used for more demanding procedures like culturing
protoplasts.
•Gellan gums (Phytagel, Gelrite)
Polymers of glucose, glucuronic acid and rhamnose
withstand 120 °C heat, making it especially useful in
culturing thermophilic organisms.
Clear gel helps in visual screening for contamination

28. Additional requirements
•Antioxidants
Citric acid, ascorbic acid, pyrogallol, L-cysteine
Reduce excessive browning
Adsorbents (PVP, activated charcoal) check
browning
Water
Use de-mineralized or double distilled water

29. Commonly used Media
Medium Description
MS medium (Murashigeand Skoog, 1962) developed for
culture of tobacco
LS medium (Linsmaierand Skoog, 1965) a version of
MS medium with modified organic
constituents
White's medium (White, 1963) developed for the culture of
tomato roots.
Gamborg's B5 medium (Gamborget al., 1968) developed for
soybean callus culture.
SH medium (Schenk and Hildebrandt, 1972) developed
for monocot and dicotcallus culture.
N6 medium (Nitschand Nitsch, 1969) developed for
anther culture
WPM medium (Lloyd and McCown, 1981) developed for
tree species.

30. MS , SH and B-5 medium are all high in macronutrients

31. Safety Data Sheet
(SDS) Safety Data Sheet (SDS), also referred to as
Material Safety Data Sheet (MSDS),
is a form containing information regarding the properties
of a particular substance. The SDS includes physical data
such as melting point, boiling point, and flash point,
information
on the substance’s toxicity, reactivity, health effects,
storage, and disposal, as well as recommended protective
equipment and procedures for handling spills.
The SDSs for all commercial in vitro media ingredients are
mostly available online. Check SDS information of a
particular chemical in advance before using it for the first
time

32. Important observations about these media:
•All of these are fully defined (no complex organics).
•MS and SH are high salt media.
•MS and SH use both ammonium and nitrate ions as nitrogen sources.
•SH contains a very high level of myo-inositol.
•White's is low salt media
•Sucrose is the carbon source
•A chelateis a chemical compound composed of a metal ion and a
chelating agent. A chelating agent is a substance whose molecules can
form several bonds to a single metal ion. In other words, a chelating
agent is a multidentate ligand. An example of a simple chelating agent is
ethylenediamine.
•Osmoticum: An agent, such as PEG, mannitol, glucose or sucrose,
employed to maintain the osmotic potential of a nutrient medium
equivalent to that of the cultured cells (isotonic). Because of this
osmotic equilibrium, cells are not damaged in vitro.

33. v.Imp vitamins
•Experimentation often shows that some vitamins
•can be omitted from recommended media. Although
•four vitamins were used in MS medium, later work at
•Professor Skoog’s laboratory showed that the
•optimum rate of growth of tobacco callus tissue on
•MS salts required the addition of only myo-inositol
•and thiamine. The level of thiamine was increased
•four-fold over that used by Murashige and Skoog
•(1962), but nicotinic acid, pyridoxine and glycine(amino acid) were
unnecessary (Linsmaier and
•Skoog, 1965). A similar simplification of the MS
•vitamins was made by Earle and Torrey (1965) for
•the culture of Convolvulus callus.

34. •Nitrogen is most commonly supplied as a mixture of nitrate ions (from
the
•KNO3) and ammonium ions (from the NH4NO3). Theoretically, there is
an
•advantage in supplying nitrogen in the form of ammonium ions, as
nitrogen
•must be in the reduced form to be incorporated into macromolecules.
Nitrate
•ions therefore need to be reduced before incorporation. However, at
high concentrations,
•ammonium ions can be toxic to plant cell cultures and uptake of
•ammonium ions from the medium causes acidification of the medium. In
•order to use ammonium ions as the sole nitrogen source, the medium
needs to
•be buffered. High concentrations of ammonium ions can also cause
culture
•Redox reactions, or oxidation-reduction reactions,primarily involve the
transfer of electrons between two chemical species. The compound that
loses an electron is said to be oxidized, the one that gains an electron is
said to be reduced. There are also specific terms that describe the
specific chemical species. A compound that is oxidized is referred to as a
reducing agent, while a compound that is reduced is referred to as the
oxidizing agent.