Understanding Wetherell and Dougall (WD) Medium in Plant Tissue Culture: Formulation and Applications
In the fascinating world of plant tissue culture, growth media are the backbone for success. They provide the essential nutrients and conditions for plant tissue, cells, and organs to thrive in an in vitro environment. One such specific media formulation that has been extensively used for the culture of various plant tissues, particularly in studying certain plant embryogenesis and morphogenesis, is Wetherell and Dougall (WD) Medium.
What is Wetherell and Dougall (WD) Medium?
Wetherell and Dougall (WD) medium was initially developed by D.F. Wetherell and D.K. Dougall (1980) for the culture of plant cells and tissues, particularly for studying plant protoplasts, callus growth, and somatic embryogenesis. Later on, it became a key tool in tissue culture research for studying developmental processes, including organogenesis and embryogenesis, in a controlled environment.
The medium is supplemented with various macronutrients, micronutrients, vitamins, and organic compounds to provide the right balance of nutrients that sustain plant cells, promote growth, and eventually help with differentiation into plants. Unlike standard plant culture media like Murashige and Skoog (MS) medium, WD medium is more specialized and applied for specific tissue culture scenarios, especially somatic embryos of species like Cleome.
Applications of Wetherell and Dougall (WD) Medium
WD medium finds significant application in various areas of plant tissue culture, including:
Somatic Embryogenesis and Organogenesis:
The WD medium is particularly useful in inducing somatic embryos from plant tissues or callus cultures. Somatic embryogenesis is the process by which non-reproductive cells can form embryos capable of developing into plants. This is vital in large-scale plant propagation, conservation of endangered species, and in genetic transformation studies.Plant Regeneration from Protoplasts:
Plant protoplasts are plant cells that have had their cell wall removed. WD medium supports protoplast regeneration into complete plants, especially in dicotyledonous species.Callus Culture:
WD medium is effective in callus formation or the production of undifferentiated plant cells. Calluses are often further used for plant regeneration, somatic embryogenesis, or as a material for biotechnology applications, such as genetic engineering.- Studying Plant Metabolism:
The precise nutrient combinations in the WD medium allow researchers to study the effects of various compounds in metabolic pathways and investigate how plants synthesize certain metabolites under specific in vitro conditions.
The Formulation of Wetherell and Dougall (WD) Medium (Per Liter)
The formulation of WD medium is carefully balanced to supply key nutrients for plant tissue growth. Below is the detailed composition of the major components in WD medium:
1. Macronutrients (mg/L):
- KNO₃ (Potassium nitrate): 2020 mg
- NH₄NO₃ (Ammonium nitrate): 1180 mg
- KH₂PO₄ (Potassium dihydrogen phosphate): 170 mg
- MgSO₄·7H₂O (Magnesium sulfate heptahydrate): 160 mg
- CaCl₂·2H₂O (Calcium chloride dihydrate): 440 mg
2. Micronutrients (mg/L):
- MnSO₄·H₂O (Manganese sulfate monohydrate): 22.3 mg
- ZnSO₄·7H₂O (Zinc sulfate heptahydrate): 8.6 mg
- H₃BO₃ (Boric acid): 6.2 mg
- Na₂MoO₄·2H₂O (Sodium molybdate dihydrate): 0.25 mg
- KI (Potassium iodide): 0.83 mg
- CuSO₄·5H₂O (Copper sulfate pentahydrate): 0.025 mg
- CoCl₂·6H₂O (Cobalt chloride hexahydrate): 0.025 mg
3. Iron Source (Chelated Iron Solution):
- Na₂EDTA (Disodium ethylenediaminetetraacetate): 37.3 mg
- FeSO₄·7H₂O (Ferrous sulfate heptahydrate): 27.8 mg
4. Vitamins and Organic Compounds (mg/L):
- Myo-Inositol: 100 mg
- Nicotinic acid (Niacin): 0.5 mg
- Pyridoxine HCl (Vitamin B6): 0.5 mg
- Thiamine HCl (Vitamin B1): 0.1 mg
5. Carbon Source:
- Sucrose: 30 g (3%)
6. Growth Regulators (Optional and Subject to Experimental Requirement):
Different growth regulators may be added to the medium based on the type of tissue you are working with and the desired developmental response. These can include:
- Auxins (such as 2,4-D, IAA): Promote cell division and root formation.
- Cytokinins (such as BAP, Kinetin): Stimulate shoot formation and the differentiation of cells.
7. Gelling Agent:
- Agar (optional, depending on solid or liquid culture): 7.5 g – 10 g for solidified medium.
pH Adjustment:
Make sure to adjust the pH of the final solution to 5.7 using either NaOH or HCl before autoclaving the medium for sterilization.
Sterilization:
The medium should be autoclaved at 121°C for 15-20 minutes to ensure sterile conditions before plant tissue inoculation.
Conclusion
Wetherell and Dougall (WD) Medium stands as a robust and specialized medium tailored for a range of plant tissue culture applications, most notably somatic embryogenesis and protoplast regeneration. While it shares some basic similarities with other tissue culture media (in terms of providing nutrients and vitamins), WD medium has unique features that enable it to support delicate plant cells in processes like callus formation and embryo development. Researchers rely on WD media in the lab to efficiently investigate specific growth behaviors of plants, with particular emphasis on dicotyledonous species.
If you’re setting up plant culture experiments focused on somatic embryo induction or protoplast culture, the WD medium is certainly one you should consider. Its balanced formulation of macronutrients, micronutrients, vitamins, and carbon sources offers a well-rounded support system for plant regeneration and in vitro morphogenesis.
In the constantly evolving field of plant biotechnology, fine-tuned media like Wetherell and Dougall (WD) Medium will continue to play a critical role in pushing boundaries and developing valuable plant tissue culture techniques.
References:
- Wetherell, D.F., and Dougall, D.K., 1980. "Reduction of Genotypic Limitation in Cultured Wild Carrot Cells via Nutritional Manipulations."