Plant tissue culture Practice Lec 6 Preparing Plant Tissue Culture Medium

Preparing Plant Tissue Culture Medium

• • In the plant tissue culture process, plant parts are cut into small pieces that will give rise to many more individual plants. Soil or hydroponically grown plants only need fertilizer (a source of K, N and P plus trace minerals), water, air and light to grow, because they can make their own sugar, amino acids, etc.

Plant tissue cultures need an outside source of sucrose because the tissue culture cells go into shock when the explants are made. It helps to add vitamins in addition to the fertilizer and sucrose.

Preparing Plant Tissue Culture Medium

Tissue culture media were first developed from nutrient solutions used for culturing whole plants e.g. root culture medium of White include and callus culture medium of Gautheret. White’s medium was based on Uspenski and Uspenska’s medium for algae, Gautheret’s medium was based on Knop’s salt solution. Basic media that are frequently used Murashige and Skoog (MS) medium , Linsmaier and Skoog (LS) medium , (B5) medium medium.

and Gamborg Nitsch and Nitsch (NN)

Preparing Plant Tissue Culture Medium Media composition

Plant tissue culture media should generally contain some or all of the following components: 1-Macronutrients 2-Micronutrients 3- Vitamins, 4- Amino acids or nitrogen supplements 5- Source(s) of carbon 6- Undefined organic supplements 7- Growth regulators 8- Solidifying agents

Preparing Plant Tissue Culture Medium Macronutrients

The essential elements in plant cell or tissue culture media include, besides C, H and O, macro elements: nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulphur (S)

Preparing Plant Tissue Culture Medium Macronutrients

The essential elements in plant cell or tissue culture media include, besides C, H and O, macro elements: nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulphur (S)

Preparing Plant Tissue Culture Medium

Micronutrients

The essential micronutrients (minor elements) for plant cell and tissue growth include iron (Fe), manganese (Mn), zinc (Zn), boron (B), copper (Cu) and molybdenum (Mo). Iron is usually the most critical of all the micronutrients.

Preparing Plant Tissue Culture Medium

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Carbon and energy sources

Sucrose Glucose Galactose Fructose Lactose Maltose Starch

Preparing Plant Tissue Culture Medium Vitamins and myo-inositol

• • • • Inositol (B8) Thiamine (B1) Pyridoxine and Nicotinic acid (B6) Biotin, folic acid, ascorbic acid, pantothenic acid, tocopherol (vitamin E)

Preparing Plant Tissue Culture Medium Amino acids

Glycine Glutamine Asparagine L-arginine Cysteine L-arginine and cysteine

Preparing Plant Tissue Culture Medium

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Undefined organic supplements

Charcoal coconut milk yeast extract malt extract ground banana orange juice tomato juice

Preparing Plant Tissue Culture Medium

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Solidifying agents

There are a number of gelling agents such as agar, agarose and gellan gum .

Preparing Plant Tissue Culture Medium Growth regulators

Plant growth regulators are important in plant tissue culture since they play vital roles in stem elongation, tropism, and apical dominance. They are generally classified into the following groups; auxins, cytokinins , gibberellins and abscisic acid . Moreover, proportion of auxins to cytokinins determines the type and extent of organogenesis in plant cell cultures

Preparing Plant Tissue Culture Medium

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Media preparation procedure

Measure out approximately 90% of the final required volume of tissue culture grade water e.g. 900 ml for a final volume of 1000 ml. Select a container twice the size of the final volume.

While stirring the water add the powdered medium and stir until completely dissolved. Heating may be required to bring powders into solution.

Rinse the original container with a small volume of tissue culture grade water to remove traces of the powder. Add to the solution in Step 2.

Preparing Plant Tissue Culture Medium

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Media preparation procedure

Add desired heat stable supplements (e.g. sucrose, gelling agent, vitamins, auxins, cytokinins, etc.) Add additional tissue culture grade water to bring the medium to the final volume.

While stirring, adjust medium to desired pH using NaOH, HCl or KOH.

If a gelling agent is used, heat until the solution is clear.

Preparing Plant Tissue Culture Medium

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Media preparation procedure

Dispense the medium into the culture vessels before (or after) autoclaving according to your application. Add heat labile constituents after autoclaving.

Sterilize the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121 °C, for the time period described under Sterilization of Media Protocol.

Allow medium to cool prior to use.

Preparing Plant Tissue Culture Medium Media Sterilization

Plant tissue culture media are generally sterilized by autoclaving at 121 °C and 1.05 kg/cm 2 (15-20 psi)