Choosing the right tissue culture medium is vital for successfully culturing plant cells in vitro. Many are unfamiliar with the types of media available, their uses, and how to select the most appropriate one for specific plant cultures. Understanding your plant's needs is key to preparing a medium that supports its growth effectively.
Tissue culture media are nutrient-rich solutions formulated to foster the growth and development of plant cells, tissues, or organs in vitro. They contain essential elements, organic compounds, and growth regulators customized to meet the specific requirements of the plant material being cultured.
Macronutrients are essential for plant growth in tissue culture, including nitrogen (25-60 mM), potassium (20-30 mM), phosphorus, calcium, and magnesium (1-3 mM each), along with sulfur and carbon (added separately). These elements are critical for healthy plant development, with precise concentrations ensuring optimal growth.
Micronutrients, required in trace amounts, are vital for cell and tissue growth. These include iron (often chelated with EDTA for better availability), manganese, zinc, boron, copper, and molybdenum, with typical ranges like 0.1 µM for cobalt and iodine, 1.0 µM for iron and molybdenum, 5-30 µM for zinc, 25-100 µM for boron, and 20-90 µM for manganese. Other elements like cobalt or sodium may be included, though their roles are less clear.
Vitamins: Plants produce insufficient vitamins, so media are supplemented with thiamine (0.1-10 mg/L), nicotinic acid (0.5-5 mg/L), pyridoxine (0.1-10 mg/L), and others like riboflavin, folic acid, and myo-inositol to support growth and differentiation.
Myoinositol: This compound promotes cell division and growth, breaking down into ascorbic acid and pectin. It's typically used at 50-5000 mg/L, depending on the plant species.
Amino Acids: These provide a readily absorbed nitrogen source, enhancing cell growth. Common amino acids include casein hydrolysate (0.25-1 g/L), glycine (2.0 mg/L), glutamine (8 mM), and others like asparagine, tyrosine, arginine, and cysteine (10-100 mg/L).
Organic Extracts: Extracts like coconut milk, yeast extract, or fruit juices (e.g., orange, tomato) can boost growth, but their efficacy varies due to inconsistent active compounds. Activated charcoal may enhance growth in some plants (e.g., carrot, orchid) but can inhibit others (e.g., tobacco, soybean).
Agar: A seaweed-derived polysaccharide, agar (0.8-1.0%) is widely used for its stability and non-reactivity, providing a solid surface for cell growth.
Gelatin: Less common, gelatin loses its gelling properties at around 25°C, limiting its use compared to agar.
Auxins: Auxins like IAA, IBA, 2,4-D, and NAA drive cell division and elongation. High concentrations induce callus formation, while lower levels promote rooting. 2,4-D is particularly effective.
Cytokinins: Compounds like BAP, kinetin, and zeatin encourage shoot formation and proliferation while inhibiting roots. BAP and kinetin are commonly used for their reliability.
Auxin-to-Cytokinin Ratio: This ratio is critical—higher auxin levels favor embryogenesis and rooting, while higher cytokinin levels promote shoot growth. Adjustments depend on the plant and explant type.
Gibberellins: Gibberellin A3, among others, supports callus growth and elongation but may inhibit root or shoot formation, requiring careful use.
Abscisic Acid: Its effects vary by species, promoting or inhibiting callus growth and shoot proliferation while hindering late-stage embryogenesis.
To prevent contamination, antibiotics like kanamycin or streptomycin are sometimes used, but they may inhibit growth. Plant Preservative Mixture (PPM) is a preferred, heat-stable alternative that effectively controls microbial contamination without harming the culture.
Several media formulations are used in plant tissue culture, including Murashige and Skoog (MS), Gamborg (B5), Linsmaier and Skoog (LS), Nitsch and Nitsch (NN), and White's medium. Below is an overview of these key media.
Developed in 1962 by Murashige and Skoog, MS is the most widely used medium, featuring a balanced mix of salts, vitamins, and amino acids. It supports organogenesis, callus culture, micropropagation, and cell suspensions. Variants like MS0 (no sucrose) or MS10 (10 g/L sucrose) indicate sucrose levels.
Introduced in 1965, LS is similar to MS but optimized for tobacco culture with higher thiamine (0.4 mg/L) and inositol as the sole vitamin. It supports organogenesis, callus culture, and micropropagation.
Formulated in 1968 for soybean callus and cell suspensions, B5 has higher nitrate and potassium and lower ammonia levels. It's ideal for protoplast culture and root callus formation.
Developed in 1969 for Nicotiana anther culture, NN includes elevated thiamine, biotin, and folic acid to support anther callus development.
Created in 1963 for tomato root culture, White's medium has lower salts and higher magnesium sulfate, with nitrate levels 19% lower than MS. It suits shoot and callus cultures for species like banana and carrot.
Choosing the ideal medium requires experimentation, as no single formula suits all plants. Start by testing media with varying salt concentrations (high, medium, low) and adjusting the auxin-to-cytokinin ratio to achieve desired outcomes, such as shoot or root formation. Sucrose levels (2-6%) should also be optimized.
A structured approach, proposed by De Fossard, involves testing four component groups—minerals, auxins, cytokinins, and organic nutrients—at high, medium, and low concentrations. This creates 81 unique combinations, each coded (e.g., MLMH for medium salts, low auxin, medium cytokinin, high organic nutrients). After identifying the best combination, fine-tune auxin and cytokinin levels to perfect the medium for your specific plant culture.
By carefully tailoring nutrient levels and growth regulators, you can develop a medium that meets the unique needs of your plant culture, ensuring successful in vitro growth.
