Tissue culture media preparation for plants

1. PLANTTISSUE
CULTURE MEDIUM

2. PlantTissue Culture medium
• Media used in plant tissue culture contain nutritional components.
• Nutritional components are essential for growth and development
of cultured tissue
• The success of the tissue culture depends very much on the type of
culture media used.

3. • Each plant tissue culture medium must contain the following essential
components to support invitro plant growth.
• These are as follows.
1) Macro inorganic nutrients.
2) Micro inorganic nutrients
3) Iron (as chelating agent)
4) Vitamins
5) Carbon sources
6) Organic nitrogen
7) Plant growth regulators
8) Agar (as gelling substance)

4. Nutrients
Inorganic nutrients Organic nutrients
macro nutrients micro nutrients
1. vitamins
2. hexitols
3. amino acids
4. carbohydrate
Nitrogen
Phosphorus

5. 1. Macro nutrients
• Need of macro nutrients is higher.
• It is present in milli molar (mM) quantities, more than 30ppm
• Macro nutrients provide both anions and cations for the plant cells.

6. • Macro nutrients are nitrogen (as NO3 and
NH4),phosphorus(PO4),potassium(K),sulphur(as
SO4),magnesium(Mg),and calcium(Ca).
• Macro nutrients have structural and functional role in protein synthesis, cell
wall synthesis enzyme Co-factors and membrane integrity.

7. Nitrogen
• In organic form used as amino acids, different organic acids
and casein hydrolysate.
• In inorganic form used as Nitrate or ammonia.
• Nitrogen is major component of all plant tissue culture
media.
• Nitrogen helps to synthesis complex organic molecule.

8. Potassium
• K ion is present in high concentration in the cytoplasm (100-200 mM)
and in chloroplast(20-200 mM).
• K+ is essential for mentaining the ion balancing, activation of many
enzymes. Maintaining osmotic pressure and osmotic regulation of
cells.

9. Calcium
• Calcium functions with different enzymes as Co-
factor and bound to the cell wall and cell
membrane.
• It gives strength to cell wall.
• It helps in the regulation of the the cell
membrane structure.
• Deficiency causes disintegration of the membrane
and shoot tip necrosis.
• Important in cell and root multiplication.
• Supplied as calcium chloride and calcium nitrate.

10. Phosphorus
• Phosphorus
• Very important for energy metabolism.
• Essential element for DNA & RNA.
• Deficiency may cause delayed growth and dark green coloure
of leaves.
• Supplied as sodium hydrogen phosphate or potassium
hydrogen phosphate.

11. Magnesium
• Essential for enzymatic reactions, energy metabolism(ATP
synthesis).
• Part of chlorophyll
• Supplied as magnesium sulphate.

12. • Sulphur
• Important substance.
• Deficiency of Sulphur inhibits protein synthesis and decreases
Chlorophyll in leaves.
• Supplied as magnesium Sulphate and Potassium Sulphate.

13. • Micro Nutrients
• Boron(B),
Manganese(Mn),Zinc(Zn),Molybdenum(Mo),Copper(Cu),Cobal
t(Co).
• Used in less amount less than 30ppm.(mg/l).
• Concentration is always in uM.

14. • Zinc
• Zn plays an active role in protein synthesis and in the synthesis
of tryptophan.
• Supplied as Zinc Sulphate.

15. Manganes
• Plays an important role in the Hill reaction of photasynthesis.
• Required in many enzymatic activities.
• Supplied as Manganese Sulphate.

16. Copper
• Copper plays important role in photosynthesis.
• Intermediate of the electron transport chain between photo
system 1 & 2
• Deficiency leads to decrease in photosynthesis.
• Supplied asCopper Sulphate.

17. Molybdenum
• Essential for conversation of Nitrate to Ammonium.
• Supplied as Sodium molybdate.

18. Boron
• Involves in different enzymatic activities.
• Supplied as Boric acid.

19. Iron
• Important Enzyme Co-factor
• Supplied in uM quantities.
• It is supplemented with chelators and Complex compounds due to
its solubility problem.
• Supplied as Na2FeEDTA
• Iron deficiency have severe effects on the growth and development
plant cells.

20. Organic Nutrients
1. Vitamins
• Plant synthesis required vitamins.
• Essential for many biochemical reaction.
• Cultured cell are capable to produle vitamins at
some level.
• They require an exogenous supply of different
vitamins for optimum growth.
• Most usable vitamins areThiamine, Pyridoxine
nicotinic acidVitamin B Complex.

21. Hexitols
• Most tissue culture media have this compound.
• Essential for seed germination, sugar transport, carbohydrate
metabolism, membrane structure and cell wall formation.
• Mannitol and sorbital are hexitols.

22. Amino Acids
• Glycine is the most common AminoAcid used in different
culture media.
• It is not essential but Nitrogen containingAminoAcid enhance
growth and plant regeneration.

23. Carbohydrate
• Cells and tissue reduires exogeneous supply of carbohydrates
to replace the carbon
which the plant normally fixes from the atmosphere by
photosyntheis.
• Supplied by adding sucrose.
• Concentration is 20-30 gm/l.

24. Gelling Agent
• Agar – Agar
• Agar is a natural product of seaweeds.
• Since 1658 agar-agar is obtain from red algae (Gelidium Gracilaria)
• With water it melts at 100’C and solidify at 45’C

25. Agarose
• It is highly purified agar prepared from Gelidium sp. Of
seaweed.
• Agarose melt and gel at temperatures below 30’C and dissolve
through boiling.
• Agarose is much more expensive agar-agar

26. Gelrite or Phytagel
• Gelrite is a naturally derived polymer and produced by the
microbial termentation of a bacterium Pseudomonas elodea.
• It is low cost gelling agent.
• 0.1-0.2 % concentration per litre required.

27. Natural Media
• Endosperm fluid / coconut
milk
• Fruit materials
• Potato extract
• Extracts of malts, yeast
• Animal extracts
• Protein hydrolysates
• Coconut fruit
• Orange juice,
Tomato juice
Banana pulp
• potato
• Malt,Yeast
• Fish emulsion
• Casein hydrolysate
Peptone

28. Plant growth regulators
• A plant hormone can be defined as a small organic molecule that
elicits a physiological response at very low conc.
• PGR plays an important role in the phenotype.
• Act as messenger between environment and the genome.

29. Auxins
• Essential for cell division, cell elongation, cell differentiation,
organogenesis and embryogenesis, callus formation
• Natural form auxins are IAA, IBA, PAA
• Synthetic form of auxins are NAA, 2, 4-D.

30. Cytokinins
• Cytokinins promote cell division, shoot proliferation and influence the cell
cycle.
• Embryogenesis and inhibit roof formation.
• Synthetic form is 2-ip which is most active cytokinins.
• Snthetic forms is BAP and natural form Zeatin

31. Gibberellins
• It promotes stem elongation, bulb corm formation and embryo
maturation but can inhibit callus growth and root induction.
• GA3 is most common gibberellins.

32. Abscisic acid
• It inhibits shoot growth and germination of embryo.
• It is thermostable but light sensitive

33. pH of tissue culturemedia
• pH is adjusted between 5 & 5.8 before gelling and sterilization with
the help of dilute NaOH, KOH or HCL.
• pH below 5 will not gel properly.
• pH above 6 may be too hard.

34. Murashige and Skoog medium or (MSO or MS0 (MS-
zero)) is a plant growth medium used in the laboratories
for cultivation of plant cell culture. MSO was invented by
plant scientists Toshio Murashige and Folke K. Skoog in
1962 during Murashige's search for a new plant growth
regulator.

35. Constituents Working concentration
(mg l-1)
Stock solutions
(mg l-1)
1. Macronutrients
NH4NO3 1650 16,500
KNO3 1900 19,000
CaCl2.2H2O 440 4,400
MgSO4.7H2O 370 3,700
KH2PO4.H2O 170 1,700
2. Micronutrients
KI 0.83 83.0
H3BO3 6.2 620
MnSO4.H2O 22.3 2,230
ZnSO4.7H2O 8.6 860
Na2MoO4.2H2O 0.25 25.0
CuSO4.5H2O 0.025 2.5
CoCl2.6H2O 0.025 2.5
3. Iron-EDTA
FeSO4.7H2O 27.8 2780
Na2EDTA.2H2O 37.3 2730
4. MS Vitamins
Myo-inositol 100.0 10,000
Nicotinic acid 0.5 50
Pyridoxine HCl 0.5 50
Thiamine HCl 0.1 10
5. Carbon source
Sucrose 30,000 -
6. Gelling agent
Agar 8,000 -
Composition of MS basal medium.

36. Plant growth
regulators
Mol. wt. Solvent Diluent Storage of stock
solutions
Sterilization
Auxins
IAA 175.2 50% EtOH H2O -0 oC F
IBA 203.2 1 N NaOH H2O -0 oC CA/F
NAA 186.2 1 N NaOH H2O 2–8 oC CA
Cytokinins
BAP 225.3 1 N NaOH H2O 2–8 oC CA
KIN 215.2 1 N NaOH H2O -0 oC CA/F
ZET 219.2 1 N NaOH H2O -0 oC F
TDZ 220.2 1 N NaOH H2O -0 oC CA/F
2-iP 203.2 1 N NaOH H2O -0 oC F
Giberellins
GA3 346.4 50% EtOH H2O 2–8 oC CA/F
CA – Co-autoclave with media components
F – Filter sterilized with 0.22  nylon membrane syringe filter (Millipore, USA)
Preparation and storage of growth regulators used in tissue culture.

37. Antibiotics Concentration Storage
Stock Working
Ampicillin 100 mg ml-1 in H2O 100 μg ml-1 –20 oC
Kanamycin 50 mg ml-1 in H2O 50 μg ml-1 4 oC
Streptomycin 50 mg ml-1 in H2O 50 μg ml-1 –20 oC
Cefotaxime 250 mg ml-1 in H2O 500 μg ml-1 4 oC
Rifampicin 50 mg ml-1 in DMSO 50 μg ml-1 4 oC
Preparation and storage of antibiotic stock solutions

38. Media preparation for plant tissue culture
MS basal medium for plant tissue culture was prepared in deionized water was used and the various
components were added according to the sequence given below:
Desired volumes of macronutrients, micronutrients, Fe-EDTA and vitamin stock solutions were
pipetted out in the desired volumes in a beaker.
Appropriate amounts of plant growth regulators (PGR’s) were added and desired amount of sucrose or
maltose was weighed, added and deionized water was added to make up the desired final volume of the
medium.
The components were mixed thoroughly on a magnetic stirrer; the pH was adjusted to 5.8 using 1 N
NaOH or 1 N HCl.
Semi-solid medium was prepared with required amount of agar depending on the final concentration.
Medium was steam-sterilized in autoclave at 15 psi (1.06 kg/cm2) for 20 min at 121oC.
Thermo-labile plant growth regulators and antibiotics were filter-sterilized and added into the
autoclaved media when cooled to below 50 oC and mixed thoroughly in laminar flow hood. Media were
dispensed in autoclaved culture vessels or sterile Petri dishes as per requirement.