2. PLANT TISSUE CULTURE
Plant tissue culture is a collection of techniques used to maintain or
grow plant cells, tissues, or organs under sterile condition on a
nutrient culture medium.
• It is responsible for invitro growth and
morphogenesis of plant tissue.
• Depends on 2 parameters
1. The particular species of plant
2. The type of material used for culture
3. TISSUE CULTURE MEDIA TYPES
• WHITE’S MEDIA – ROOT CULTURE
• MS MEDIA – INDUCE ORGANOGENESIS AND
REGENERATION
• B5 MEDIA – CELL SUSPENSION AND CALLUS CULTURE
• N6 MEDIA – CEREAL ANTHER CULTURE
• NITSCH’S MEDIA – ANTHER CULTURE
5. INORGANIC NUTRIENTS
(MACRO AND MICRO NUTRIENTS)
Macto nutrients
• Nitrogen – Essential component of protein, Nuclei acids and some
coenzyme
• Phosphorus – Involved in energy transfer
• Pottasium – regulate osmatic potential
• Calcium – synthesis of cell wall , membrane function and cell
signalling
• Magnesium – component of chlorophyll and cofactor
• Sulfur – component of certain amino acids ( methionine, cysteine and
some cofactor)
Micro nutrients
• Iron – electron transfer
• Manganese – cofactor
• Zinc – required for chlorophyll biosynthesis
• Copper – electron transfer reaction
• Molybdenum – component of certain enzyme (nitrate reductase)
6. CARBON AND ENERGY SOURCES
• PLANT CELLS AND TISSUES IN CULTURE MEDIUM ARE HETERO TROPHIC,
THEY ARE DEPENDENT ON THE EXTERNAL CARBON FOR ENERGY
• SUCROSE IS PREFERRED
• DURING AUTOCLAVE SUCROSE GETS HYDROLYSED TO GLUCOSE TO
FRUCTOSE
• PLANT CELL UTILIZE FIRST GLUCOSE AND FRUCTOSE
7. ORGANIC SUPPLEMENTS
VITAMINS ( TO ACHIEVE GOOD GROWTH OF CELLS )
E.G THIAMINE, RIBOFLAVIN, NIACIN, PYRIDOXINE, FOLIC ACID, PANTHOTHENIC
ACID, BIOTIN, ASCORBIC ACID, MYOINOSITOL, VITAMIN E
AMINO ACIDS (STIMULATE CELL GROWTH)
E.G L-GLUTAMINE, L-ASPARAGINE, L-ARGININE, L-CYSTEINE
ORGANIC ACIDS (ENHANCE THE GROWTH)
E.G. CITRATE, MALATE, SUCCINATE OR FUMERATE , PYRUVATE (MOSTLY USED)
8. • ORGANIC EXTRACT ( AVOID THE USE OF NATURAL EXTRACT)
SUPPLEMENT CULTURE MEDIA WITH ORGANIC EXTRACT YEAST, CASEIN
HYDROLYATE, COCONUT MILK, ORANGE POTATO JUICE.
• REPLACEMENT OF YEAST BY L- ASPARAGINE
• REPLACEMENT OF FRUIT BY L-GLUTAMINE
ACTIVATED CHARCOAL
REMOVE THE TOXIC OR INHIBITORY COMPOUND (PHENOL) PRODUCED BY CULTURED
PLANTS.
ANTIBIOTICS
TO PREVENT THE GROWTH OF MICRO ORGANISM
E.G STREPTOMYCIN / KANAMYCIN
9. PLANT GROWTH HORMONE
• GROUP OF NATURAL ORGANIC COMPOUNDS THAT PROMOTE GROWTH AND
DIFFERENTIATION OF PLANTS
1. AUXIN –INDUCE CELL DIVISION, CELL ELONGATION, FORMATION OF CALLUS
INDUCTION
• LOW CONCENTRATION OF AUXIN – ROOT FORMATION
• HIGH CONCENTRATION OF AUXIN – CALLUS FORMATION
E.G. 2,4 – DICHLOROPHENOXY ACETIC ACID , IAA, NAA
2. CYTOKININ – DERIVATIVE OF PURINE NAMELY ADENINE
• INVOLVED IN CELL DIVISION
• SHOOT DIFFERENTION
STIMULATE PROTEIN AND ENZYME ACTIVITY
E.G KINETIN, BENZYL / AMINO PURINE , ZEATIN, BAP
11. SOLIDIFYING AGENTS
• SUPPORT TO TISSUEA GROWING IN THE STATIC CONDITIONS.
AGAR – DOES NOT REACT WITH MEDIA CONSTITUENTS AND IT DOES NOT DIVESTED BY PLANT
ENZYMES AND STABLE AT CULTURE TEMPERATURE
• CONCENTRATION 0.5 – 1%
PH – 5-6 (OPTIMUM )
• AFTER AUTOCLAVE 0.3 – 0.5
• IF THE PH IS HIGHER THAN 7 OR LOWER THAN 4.5 YE PLANT CELLS STOP GROWING IN CULTURES
STERILIZATION TECHNIQUE ( TO KILL THE MICRO ORGANISM)
• 121°C – 20 MINUTES
12. PLANT TISSUE CULTURE MEDIA
PROTOCOL
Mix a powdered medium with distilled water
Add 30 g sucrose – pH 5.8
Add agar – 8 g
Add growth regulator (auxin, cytokinin)
Autoclave the media at 121°C
Pour the media in aterilied petriplate
