1. Practical 4 : To prepare composition of various tissue culture media
BIOTECHNOLOGY OF HORTICULTURAL CROPS
HRT 552
2. Course Code : HRT 552
Course Title : BIOTECHNOLOGY OF
HORTICULTURAL CROPS
3. Macronutrients, Micronutrients, Vitamins, Amino acids, Nitrogen
supplements, Organic supplements, Growth regulators and Solidifying
agents.
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4.
5.
6. Types of medium
Chemically defined nutrient medium
Chemically undefined nutrient medium:
Complex additives viz. coconut milk, Casein hydrolysate, yeast
extract, water melon juice, etc. are added in the medium.
1.Solid medium: 6-8% agar-agar
2.Semi solid medium: Less amount of agar
3.Liquid medium: Agar is not added. It is used for cell suspension
culture.
8. Contd.
● Plant tissues/organs are grown in vitro on a suitable artificially
prepared nutrient medium/culture medium.
● Single medium can not be used for the all types of plants and
organs.
● Commonly used medium are Murashige and Skoog (MS), Nitsch,
Gamborg, White, etc.
● MS medium is the most commonly used for plant tissue culture.
● Medium is composed of inorganic salts, iron, vitamins, amino
acids, plant hormones and a carbohydrate supply.
10. ● Salts are supplied in the form of macronutrients viz. N, Mg, K, Ca,
P
● Micronutrients Cu, Ni, Mn, Co, etc.
● Iron is supplied in the chelated, Fe-EDTA (Ferric-Sodium
Ethylene-Amine Tetra Acetate) form.
● Vitamins viz. meso-inositol, thiamin (B1), nicotinic acid (B3),
pyridoxine (B6), etc.
● Aminoacids, mostly glycine is used.
● Carbohydrate is supplied usually in the form of sucrose.
11. ● Phytohormones (auxins and cytokinins), their chemical
form, concentration and ratio may vary from plant to
plant.
● In general Auxins, such as IAA (Indole Acetic Acid)
NAA (Naphthalene Acetic Acid), IBA (Indole Butyric
acid); Cytokinins viz. Kinetin (6-furfuryl amino purine)
6-BAP (6, Benzyl Amino Purine) and Zeatin are used
in nutrient medium.
16. Antibiotics :
Stertomycin,kanamycin Activated charcoal
Other organic supplements :
Protein, coconut milk,yest,malt extract, orange juice, and tomato juice
Growth regulators :
Auxins,cytokinins
Water :
Demineralized or distilled water
Solidifying agents :
Agar, gelatin.
pH adjusters :
5 - 6 it is considered to be optimum. 1
6
17. 1
7
PREPARATION OF CULTURE MEDIA:
Stock solution 1 : MgSo4,KH2Po4,KNO3,NH4No3,CaCl2
Stock solution 2 : H3Bo3,MnSo4,ZnSo4,CuSo4,Cocl2
Stock solution 3 : FeSo4,sodium EDTA
Stock solution 4 : ionositol,thiamine,pyridamine,nicotinic acid,glycine
To prepare 1 liter of medium:
Take 50 ml of stock solution 1 + 5ml of stock solution 2 & 4 in a beaker. The
stock solution 3 prepared separately in a other 450ml flask by adding double
distilled water and heat with constant stirring.
Mix two solutions and adjust PH to 5.5.
18.
19.
20. Contamination Issues
• Sterile Incubator
• Thawing Step Source of Contamination
• Spray with 70% Ethanol, gloves
• Sterile Hood
• Sterile Hands
• Minimize Exposure to Open Air
21. STERILISATION OF MEDIA
• The prepared media should be sterilized by ISI mark Autoclave(
for large amounts) at 121º Domestic pressure cookers( for small
amounts)
• For the sterilization of glassware and metallic equipments Hot air
oven with adjustable tray is required.
9
Incubator Hot air oven
22. Sterilization
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Culture medium supports the growth of microbes e.g bacteria, fungi, etc. these
grow fast and kills the plant cells.
Microbes may come from glass vials, instruments, nutrient medium and also from
the plant material.
Therefore, the surface of plant tissue and all non-living articles including nutrient
medium must be sterilized.
Sterilization of non-living articles: The non-living articles viz. Nutrient medium,
glassware, distilled water, instruments (wrapped with brown paper) are sterilized
by autoclaving under steam at a 15 lb/inc2 and temperature 121oC for 15 min. The
glassware can also be sterilized by heating in oven at 150oC for 3-4 hrs. The
thermoilabile compounds are sterilized by passing through the bacterial filters.
23. Sterilization of the plant material
(Surface) sterilization
● The plant material should be surface sterilized to remove the surface
borne micro-organisms.
Water
10% v/v solution of liquid detergent (Teepol) for 10-15 min.
70% ethyl alcohol for 1 min. in front of laminar air flow.
Treatment with 0.1% HgCl2 (W/V) or 5-10% sodium hypochlorite.
24. SURFACE STERILISATION OF EXPLANT
For surface sterilization chromic acid, Hgcl(0.11%),calcium
hypochlorite, sodium hypochlorite(1-2%),alcohal(70%) are used.
Process depends on the type of explant.
SEED : absolute ethyl alcohol calcium hypochlorite bromine
water sterile water
FRUIT : ethyl alcohol
STEM : running water
LEAF : surface clean
sodium hypochlorite
sodium hypochlorite
Hgcl2 sterile water
sterile water
sterile water
dried
explant
