Introduction:
- Tissue
culture media provide essential nutrients and growth regulators
required for the in vitro growth of plant cells, tissues, and
organs.
- The
composition of the medium influences cell proliferation, organogenesis,
and somatic embryogenesis.
- Several
standardized media formulations exist, including Murashige and Skoog
(MS), Gamborg’s B5, White’s medium, and Nitsch &
Nitsch medium.
1. Basic Components of Tissue
Culture Media:
a. Macronutrients:
·
Required in large quantities for plant growth.
·
Examples:
o Nitrogen
(N): Provided as NH₄NO₃ and KNO₃.
o Phosphorus
(P): Provided as KH₂PO₄.
o Potassium
(K): Provided as KNO₃ and KH₂PO₄.
o Calcium
(Ca): Provided as CaCl₂.
o Magnesium
(Mg): Provided as MgSO₄.
b. Micronutrients:
·
Required in trace amounts for enzymatic
activities.
·
Examples:
o Iron
(Fe): Provided as Fe-EDTA.
o Manganese
(Mn): MnSO₄.
o Zinc
(Zn): ZnSO₄.
o Copper
(Cu): CuSO₄.
o Boron
(B): H₃BO₃.
c. Carbon Source:
·
Usually supplied as sucrose (2-3%).
·
Provides energy for cell division and growth.
d. Vitamins:
·
Essential for metabolic processes.
·
Examples:
o Thiamine
(B1)
o Pyridoxine
(B6)
o Nicotinic
acid
o Myo-inositol
e. Amino Acids and Organic
Supplements:
·
Enhance growth and development.
·
Examples:
o Glycine
o Casein
hydrolysate
o L-Glutamine
f. Plant Growth Regulators
(PGRs):
·
Control growth and differentiation.
·
Types:
o Auxins:
Promote cell division (e.g., IAA, 2,4-D).
o Cytokinins:
Promote shoot formation (e.g., BAP, kinetin).
o Gibberellins:
Stimulate elongation (e.g., GA₃).
g. Gelling Agents:
·
For solid media, usually agar (0.8-1.0%).
·
Alternatives: Gelrite.
2. Common Tissue Culture Media:
a. Murashige and Skoog (MS)
Medium:
·
Most widely used medium.
·
High concentration of macronutrients.
·
Ideal for a wide range of plant species.
·
Components:
o Macronutrients:
NH₄NO₃, KNO₃, KH₂PO₄, MgSO₄, CaCl₂.
o Micronutrients:
Fe-EDTA, MnSO₄, ZnSO₄, CuSO₄, H₃BO₃.
o Vitamins:
Thiamine, myo-inositol.
b. Gamborg’s B5 Medium:
·
Used for cell suspension cultures and legume
tissues.
·
Lower nitrate content than MS medium.
·
Components:
o Similar
to MS but with higher glycine and casein hydrolysate.
c. White’s Medium:
·
One of the oldest media.
·
Used for root cultures.
·
Lower nutrient concentration than MS.
d. Nitsch & Nitsch Medium:
·
Commonly used for anther cultures and pollen
culture.
3. Preparation of Tissue Culture
Media:
Steps:
1.
Calculate Required Quantities:
o Based
on the desired volume (e.g., 1 liter).
2.
Weigh and Dissolve Macronutrients:
o Dissolve
in distilled water in the order of least soluble first.
3.
Add Micronutrients and Vitamins:
o Prepare
as stock solutions and add to the macronutrient solution.
4.
Add Carbon Source (Sucrose):
o Mix
until fully dissolved.
5.
Add Plant Growth Regulators (PGRs):
o Add
in appropriate concentrations (e.g., 1 mg/L BAP).
6.
Adjust pH:
o Adjust
to 5.7-5.8 using NaOH or HCl.
7.
Add Gelling Agent (for Solid Media):
o Add
agar (0.8%) and heat until fully dissolved.
8.
Sterilization:
o Autoclave
at 121°C for 15-20 minutes.
9.
Pouring Media:
o Cool
to about 45-50°C and pour into sterile culture vessels.
4. Storage:
- Store
prepared media at 4°C for up to 2-3 weeks.
- Avoid
repeated freezing and thawing.
Conclusion:
- The
composition of tissue culture media plays a vital role in the successful
growth and development of plant tissues in vitro.
- Proper
preparation, sterilization, and maintenance of media ensure a contamination-free
environment, promoting healthy tissue culture outcomes.
Different media formulations like MS, B5, and White’s
are selected based on the specific requirements of the plant species and
culture objectives