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 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
include Murashige and Skoog (MS) medium,
Linsmaier and Skoog (LS) medium, Gamborg
(B5) medium and Nitsch and Nitsch (NN)
medium.
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
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
Undefined organic supplements
• Charcoal
• coconut milk
• yeast extract
• malt extract
• ground banana
• orange juice
• tomato juice
Preparing Plant Tissue Culture Medium
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
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
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
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/cm2 (15-20 psi)