In plant tissue culture, media preparation is a crucial step because the medium provides the nutrients and hormones necessary for the growth and development of plant tissues (explants). The media composition needs to be carefully designed depending on the plant species, tissue type, and the specific stage of tissue growth (e.g., callus formation, shoot or root development).
Here’s a breakdown of the components of tissue culture media and how they are prepared.
1. Components of Tissue Culture Media
Most plant tissue culture media are based on Murashige and Skoog (MS) medium, which is widely used, but other media like B5, Gamborg’s, or White’s medium may be used depending on the plant species. The general components of culture media include:
a. Macronutrients
Macronutrients are essential nutrients required in large quantities for plant growth. They provide the basic elements that plants need for their metabolism.
Nitrogen (N): Typically supplied as ammonium nitrate (NH₄NO₃) or potassium nitrate (KNO₃). Important for protein synthesis and overall growth.
Phosphorus (P): Usually in the form of phosphates like KH₂PO₄. It’s essential for energy transfer and nucleic acid synthesis.
Potassium (K): Potassium chloride (KCl) or potassium nitrate (KNO₃) is used. It helps in enzyme activation and osmoregulation.
Calcium (Ca): Supplied as calcium chloride (CaCl₂). It plays a role in cell wall structure and signaling.
Magnesium (Mg): Magnesium sulfate (MgSO₄) is commonly used, and it’s important for chlorophyll production.
Sulfur (S): Supplied as sulfate (SO₄²⁻), involved in amino acid and protein synthesis.
b. Micronutrients
Micronutrients are required in smaller amounts but are still essential for enzymatic functions and metabolic processes.
Iron (Fe): Usually provided as ferrous sulfate (FeSO₄) and often combined with a chelator like EDTA to enhance availability.
Zinc (Zn), Manganese (Mn), Copper (Cu), Boron (B), Molybdenum (Mo): Supplied as zinc sulfate, manganese sulfate, copper sulfate, boric acid, and sodium molybdate, respectively.
c. Vitamins
Vitamins are important coenzymes in plant tissue culture, required in small quantities.
Thiamine (Vitamin B1): Essential for carbohydrate metabolism.
Pyridoxine (Vitamin B6) and Nicotinic Acid (Vitamin B3): Help in various biochemical processes like amino acid and protein synthesis.
Inositol: A sugar alcohol, it is involved in cell wall synthesis and cell division.
d. Carbohydrates (Energy Source)
Since plant tissue cultures cannot photosynthesize in the early stages of development, an external carbon source (sugar) is required.
Sucrose (2-5%): The most common sugar used as an energy source. Glucose or fructose can also be used.
e. Gelling Agent (for Solid Media)
If the medium needs to be solidified for explant growth, a gelling agent is added.
Agar (0.6-0.8%): A polysaccharide derived from seaweed, widely used to solidify the medium. Other options include Gelrite or Phytagel.
f. Plant Growth Regulators (PGRs)
Plant growth regulators (hormones) are essential for directing the growth and development of tissues. The type and concentration of PGRs in the medium determine whether the tissue forms shoots, roots, or undifferentiated callus.
Auxins (e.g., IAA, IBA, NAA, 2,4-D): Promote root formation and cell division. For callus formation, auxins like 2,4-D (2,4-dichlorophenoxyacetic acid) are commonly used.
Cytokinins (e.g., BAP, Kinetin): Stimulate shoot proliferation and delay senescence. Used in high concentrations for shoot regeneration.
Gibberellins (e.g., GA₃): Promote stem elongation and seed germination, although less commonly used.
Abscisic Acid (ABA): Used to induce dormancy or stress tolerance in cultures.
g. pH Adjusting Agents
Hydrochloric Acid (HCl) or Sodium Hydroxide (NaOH): The pH of the medium is usually adjusted to around 5.6-5.8 before autoclaving, as this is optimal for plant tissue growth.
2. Media Preparation Steps
a. Preparing the Stock Solutions
To make media preparation easier, solutions for the macronutrients, micronutrients, vitamins, and hormones are often prepared as stock solutions. These concentrated solutions can be stored and used to prepare fresh medium when needed.
Weigh the Chemicals: Use an analytical balance to weigh out the correct amounts of each component (macronutrients, micronutrients, vitamins, etc.).
Dissolve in Distilled Water: Dissolve the chemicals in distilled water in separate beakers for each stock solution.
Store Stock Solutions: Once prepared, stock solutions can be stored in sterilized glass bottles and kept in a refrigerator for several weeks.
b. Making the Medium
Mix Stock Solutions:
Start by adding the macronutrient stock solution into a large beaker containing distilled water (around 70% of the final required volume).
Then, add the micronutrient, vitamin, and other required stock solutions.
Add Sucrose (Sugar): Weigh and add the appropriate amount of sucrose (usually 30 g/L or 3%).
Add Gelling Agent (If Solid Medium):
If you are preparing a solid medium, add agar (6-8 g/L) and stir until dissolved.
Add Plant Growth Regulators (Hormones): Based on the specific requirements, add the desired concentrations of auxins, cytokinins, or other PGRs to the medium.
Adjust the pH: Use a pH meter to adjust the pH of the solution to 5.6-5.8 by adding small amounts of either HCl or NaOH.
c. Sterilization and Dispensing
Autoclave: Pour the prepared medium into culture bottles, flasks, or test tubes, and sterilize them in an autoclave at 121°C for 15-20 minutes at 15 psi.
Cooling and Dispensing: Once autoclaved, allow the media to cool slightly. If necessary, pour it into sterile Petri dishes or tubes for solid media or store it in sterile bottles for future use.
Sealing: Seal the vessels with caps or wrap them with parafilm to avoid contamination.
3. Composition Examples for Different Stages
Callus Induction Medium:
MS salts, vitamins
Sucrose (3%)
2,4-D (1-2 mg/L)
Agar (if solid)
Shoot Induction Medium:
MS salts, vitamins
Sucrose (3%)
BAP (0.5-2 mg/L)
Agar (if solid)
Rooting Medium:
MS salts, vitamins
Sucrose (2%)
IBA (0.5-1 mg/L)
Agar (if solid)
Summary
Media preparation involves selecting the right combination of macronutrients, micronutrients, vitamins, carbohydrates, plant growth regulators, and adjusting the pH, depending on the plant species and the growth stage of the explants. Proper sterilization and handling ensure the medium remains free from contamination for successful plant tissue culture