Tissue Culture Media Spotlight: De Fossard Medium
Tissue culture, commonly known as micropropagation, is a crucial technique in plant biotechnology that allows for the growth of plants in a controlled, nutrient-rich medium. One such specialized medium widely utilized in plant tissue culture, particularly for woody plants, is the De Fossard Medium. In this blog post, we will explore what the De Fossard Medium is, its applications, and provide the detailed formulation for preparing it on a per-liter basis.
What is De Fossard Medium?
De Fossard Medium, named after renowned plant tissue culturist Roger A. de Fossard, is a specialized nutrient medium designed for plant cell, tissue, and organ culture. It has been used extensively for the in vitro culture of several woody plant species, including fruit trees, ornamental plants, and forestry species. What makes De Fossard Medium particularly useful is its balanced nutrient profile, which accommodates the complex requirements of woody plants and allows for efficient callus formation, shoot regeneration, and rooting.
De Fossard Medium provides a range of essential macro- and micronutrients, vitamins, carbohydrates, amino acids, and plant growth regulators that are necessary for the growth and development of cultured plant tissues. It creates an optimal in vitro environment, promoting the growth of viable, healthy plantlets that can be later transferred to soil for acclimatization.
Applications of De Fossard Medium
Propagation of Woody Plants:
De Fossard Medium is often employed in the micropropagation of woody species, which are generally more difficult to culture than herbaceous plants. Species such as Eucalyptus, Prunus, Citrus, and Juglans (walnut) can be effectively propagated with this medium.Callus Induction and Regeneration:
The medium is highly efficient in callus induction – the process where plant cells begin to grow into an undifferentiated mass. This callus can later be induced to differentiate into organs, such as shoots and roots, aiding in plant regeneration.Rooting and Shoot Elongation:
De Fossard Medium can be specifically tailored for promoting shoot elongation and rooting. Auxins and cytokinins can be added to the medium to regulate these processes depending on the plant species and intended outcomes.- Genetic Engineering and Transformation Studies:
The medium serves as a base for tissue culturing in genetic modification efforts. Using this medium, scientists can propagate transgenic plants, study gene expression, and analyze plant responses to modifications.
De Fossard Medium: Formulation (Per Liter Basis)
The De Fossard Medium formulation is built on a foundation of essential salts, vitamins, and growth hormones. Below is the composition of this medium, per liter, along with component concentrations:
1. Macro Elements (g/L)
| Component | Concentration (mg/L) |
|---|---|
| Ammonium Nitrate (NH₄NO₃) | 1,000.0 |
| Potassium Phosphate (KH₂PO₄) | 400.0 |
| Calcium Chloride (CaCl₂·2H₂O) | 70.0 |
| Magnesium Sulfate (MgSO₄·7H₂O) | 370.0 |
| Potassium Sulfate (K₂SO₄) | 100.0 |
| Sodium Chloride (NaCl) | 100.0 |
2. Micro Elements
| Component | Concentration (mg/L) |
|---|---|
| Boric Acid (H₃BO₃) | 3.0 |
| Manganese Sulfate (MnSO₄·H₂O) | 10.0 |
| Zinc Sulfate (ZnSO₄·7H₂O) | 2.0 |
| Copper Sulfate (CuSO₄·5H₂O) | 0.1 |
| Potassium Iodide (KI) | 0.1 |
| Sodium Molybdate (Na₂MoO₄·2H₂O) | 0.1 |
| Ferrous Sulfate (FeSO₄·7H₂O) | 30.0 |
| Na₂EDTA (Disodium Ethylenediaminetetraacetate) | 37.3 |
3. Vitamins
| Component | Concentration (mg/L) |
|---|---|
| Thiamine HCl (Vitamin B₁) | 1.0 |
| Pyridoxine HCl (Vitamin B₆) | 1.0 |
| Nicotinic Acid (Niacin/Vitamin B₃) | 1.0 |
4. Amino Acid
| Component | Concentration (mg/L) |
|---|---|
| Glycine | 2.0 |
5. Carbohydrates
| Component | Concentration (g/L) |
|---|---|
| Sucrose | 20.0 (2%) |
6. Plant Growth Regulators (PGRs may vary by experiment)
| Example Hormones | Concentration |
|---|---|
| 6-Benzylaminopurine (BAP) | 1-5 mg/L (for shoot induction) |
| Naphthalene Acetic Acid (NAA) | 0.1-1 mg/L (for rooting) |
7. Gelling Agent (for semi-solid medium)
| Component | Concentration per Liter |
|---|---|
| Agar (optional) | 6-8 g/L |
| OR | |
| Phytagel | 2-4 g/L |
N.B.: The concentration of plant growth regulators (PGRs) like BAP, NAA, or IBA may need to be adjusted based on the specific requirements of the plant species and the developmental stage.
Preparation Notes
Dissolving Components: Dissolve the macro and micronutrients in distilled water in the proper order, adding the more soluble components first. Keep stirring to ensure that no precipitation occurs.
Adding Vitamins & Hormones: Add the vitamins and plant growth regulators (PGRs) once the salts are completely dissolved. Avoid overheating when adding heat-sensitive components like vitamins.
Adjusting pH: Adjust the pH of the solution to around 5.7-5.8 using either hydrochloric acid (HCl) or sodium hydroxide (NaOH).
Sterilization: Sterilize the medium by autoclaving at 121°C for 15-20 minutes to ensure it is free from contaminants.
- Solid or Liquid Medium: Depending on your specific needs, add agar if you need a solid or semi-solid medium. If you’re using it as a liquid medium, omitting agar is appropriate.
Conclusion
De Fossard Medium is a powerful tool in the hands of researchers and plant biotechnologists working on plant tissue culturing, especially for woody plants. Its balanced formulation, containing macro- and micronutrients, vitamins, and growth regulators, provides an ideal environment for the growth and development of plants in vitro. Whether you’re propagating endangered species, growing transgenic plants, or conducting research on callus formation and shoot development, De Fossard Medium offers flexibility and efficacy.
By leveraging the knowledge of appropriate nutrients and adjusting hormone concentrations, this medium can be tailored for a wide range of plant species and experimental objectives. With careful preparation, scientists can efficiently propagate healthy and robust plantlets that contribute to diverse fields of research and agricultural innovation.
Happy culturing!
References:
- De Fossard, R. A. (1981) "Tissue Culture for Plant Propagators."