plant tissue culture medium

INTRODUCTION
2. HISTORY
3. BASIC COMPONENT OF MEDIA
1. Inorganic nutrient
2. organic supplements
3. Carbon and energy source
4. Growth Regulators
5. Solidifying Agent
6. PH
4. TYPES OF MEDIA
5. MS MEDIA
6. IMPORTANCE
7. CONCLUSION
8. REFERANCE

1. By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. CONTENT
1. INTRODUCTION
2. HISTORY
3. BASIC COMPONENT OF MEDIA
1. Inorganic nutrient
2. organic supplements
3. Carbon and energy source
4. Growth Regulators
5. Solidifying Agent
6. PH
4. TYPES OF MEDIA
5. MS MEDIA
6. IMPORTANCE
7. CONCLUSION
8. REFERANCE

3. INTRODUCTION
What is plant tissue culture?
Plant tissue cultureis a collection of techniques used
to maintain or grow plant cells, tissues or organs under
sterile conditions on a nutrient culture medium of known
composition.
Plant tissue culture is widely used to produce clones of a
plant in a method known as micropropagation

4. TISSU CULTURE MEDIUM
Nutritional requirements for optimal growth of a tissue
in vitro may vary with the species.
Even tissues from different parts of a plant may have
different requirements for satisfactory growth
(Murashige and Skoog, 1962).
As such, no single medium can be suggested as being
entirely satisfactory for all types of plant tissues and
organs.
When starting with a new system, it is essential to work
out a medium that will fulfill the specific requirements of
that tissue.

5. HISTORY-
Some of the earliest plant tissue culture media, e.g.
root culture medium of White (1943) and callus culture
medium of Gautheret (1939), were developed from
nutrient solutions previously used for whole plant
culture.
White evolved the medium from Uspenski and
Uspenskaia's medium (1925) for algae, and
Gautheret's medium is based on Knop's (1865) salt
solution.
All subsequent media formulations are based on
White's and Gautheret's media.

6. TYPES OF MEDIUM
Heller's Medium
MS (Murashige and Skoog ) Medium
ER (Eriksson) Medium
B5 Medium
Nitsch's Medium
NT Medium
White medium

8. BASIC COMPONENTS OF MEDIA
INORGANIC NUTRIENTS -
In vitro growth of plants also requires combination of
macro and micronutrients like in vivo growth.
Macronutrientsare classified as those elements
which are required in concentration greater than 0.5
Mm.
Micronutrientsare those elements which are
required at a concentration less than 0.05mM.

9. MACRO NUTRIENTS -
The macro nutrients includes six major
elements-nitrogen, phosphorous, sulphur,
potassium, calcium, magnesium.
MICRO NUTRIENTS -
The micro nutrients includes iron, manganese,
zinc, boron, copper, molybdenum.

10. ORGANIC NUTRIENTS
1. VITAMINS-
organic substances required for metabolic processes as cofactors or parts
of enzymes.
optimum growth, medium should be supplemented with vitamins.
Thiamine (B1), nicotinic acid (B3), pyridoxine(B6), pantothenic acid(B5)
are commonly used vitamins of which thiamine (0.1 to 5mg/l) is essentially
added to medium as it is involved in carbohydrate metabolism.
Rest vitamins are promontory

11. 2.AMINO ACID-
.L-glutamine,L-asparagine,L-cystein,L-glycineare
commonlyusedaminoacidswhichareaddedtotheculture
mediuminformofmixturesasindividuallytheyinhibitcell
growth.
3. COMPLEX ORGANICS-
Are group of undefined supplements such as casein
hydrolysate, coconut milk, yeast extract, orange juice, tomato
juice etc.
Casein hydrolysate has given significant success in
tissue culture and potato extract also has been found
useful for anther culture.

12. ACTIVATED CHARCOAL
It absorbs brown-black pigments and
oxidized phenolics produced during culture
and thus reduce toxicity.
It also absorbs other organic compounds like
PGRs, vitamins etc which may cause the
inhibition of growth.
Another feature of activated charcoal is that it
causes darkening of medium and so helps
root formation and growth.

13. CARBON SOURCE
Sugar is very important part of nutrient medium as energy
source
The most preferred carbon or energy source is sucrose at a
concentration of 20-60g/l. While autoclaving the medium,
sucrose is hydrolysed to glucose and fructose which are then
used up for growth. Fructose, if autoclaved is toxic
Other mono or disaccharide and sugar alcohols like
glucose, sorbitol, raffinose etc may be used depending
upon plant species.

14. GROWTH HORMONES
AUXIN
CYTOKININS
GIBBERELLINS & ABSCISIC ACID
1.AUXIN:-Induce cell division, cell elongation, apical
dominance, adventitious root formation, somatic embryogenesis
Commonly used synthetic auxins are-
1. IAA
2. IBA
3. NAA
4. 2,4D


15. CYTOKININS:-
promote celldivision and stimulate initiation and growth of shoots in vitro
1. ZEATIN
2. BAP
3. KINETIN
4. ADENINE
5. BENZYL ADENINE

16. • Gibbrellins and abscissic acid: -.
Gibbrellic acid (GA3) is mostly used for internode
elongation and meristem growth.
Abscissic acid (ABA) is used only for somatic
embryogenesis and for culturing woody species

17. SOLIDIFYING AGENT
used for preparing semisolid tissue culture media to enable explant to be
placed in right contact with nutrient media to provide aeration
Agar is high molecular weight polysaccharide obtained from sea weeds and
can bind water.
Agar is preferred over other gelling agents because it is inert, neither does it
react with media constituents nor digested by plant enzymes.
Agarose, a purified extract of agar is used for protoplast culture. Alternative
gelling compounds like gelrite etc form clear gels (unlike agar which is
translucent) and hence easier to detect contamination which might develop
during culture growth. Mechanical support for cell or tissue growth can also
be provided without using any gelling agent by filter paper bridge, perforated
cellophane and polyurethane foam etc.


18. PH
pH: pH affects absorption of ions and also solidification of
gelling agent.
Optimum pH for culture media is 5.8 before sterilization.
Values of pH lower than 4.5 or higher than 7.0 greatly inhibit
growth and development in vitro.
The pH of culture media generally drops by 0.3 to 0.5 units
after autoclaving and keeps changing through the period of
culture due to oxidation and also differential uptake and
secretion of substances by growing tissue.

19. PREPARATION OF MEDIA
This is a very crucial step for the experiment to be successful.
While making the media taking individual constituents, each ingredient
is separately weighed and dissolved before putting them together.
After making up volume by water, pH is adjusted and then medium is
autoclaved. Preferably, following four stock solutions are prepared.
• Major salts (20X concentration)
• Minor salts (200X concentration)
• Iron (200X concentration)
• Organic nutrients (200X concentration)
Separate stock solution for each growth regulator is prepared.
Appropriate quantities are taken from stocks and mixed to constitute
basal medium. Required quantity of agar, sucrose and organic
supplements if needed are added separately

20. PREPARATION OF TISSUE CULTURE MEDIUM
Growth and morphogenesis of plant tissue in vitroare largely governed
by the composition of the culture media
MS (Murashige and skoog ) medium is used commonly to induce
organogenasis and regeneration of plant in tissue culture.
The principle components of most plant tissue culture media are
inorganic nutrient (macronutrient) and micronutrient carbon source(s)
organic supplements ,growth regulators and a gelling agent
The simplest method of preparing media is to dissolve these powder
containing inorganic and organic nutrient in some quantity of distilled
water
After the contents have been thoroughly mixed in water , sugar and
agar (melted) other organic supplements are added finally the volume
is made up to one liter.
The PH is adjusted and the medium autoclaved.

21. Preparation of stock solution for MS-media
STOCK-1:-8.8gm of cacl2 (calcium chloride) in 100ml distilled
water.
STOCK-2:-H3BO3 -0.124 gm
 KH2P04 –3.4 gm
 KI -0.016 gm
 Na2MoO4 –0.0005 gm
 COCl2 –0.0005 gm
Dissolve above constitute in 100ml distill water.
STOCK 3:-Mnso4 –0.446 gm
 MgSO4 –7.4 gm
 Znso4 -0.172 gm
 Cuso4 –0.005 gm
Dissolve above constitute in 100ml distilled water.


22. STOCK-4:-Feso4 –0.5569 gm
 Na2EDTA-0.7469
STOCK-5:-Thiamine -3gm
 nicotinic acid –10gm
 pyridoxine HCL -10gm
 Glycine-40 gm
Dissolve above constituent in 20ml distilled water


23. PROTOCOL OF PREPARATION OF PTC MEDIUM
1. Dissolve 1.65gm NH4NO3 , 1.9 gm of KNO3 ,0.1gm inositol and 30g sucrose in 700ml
of distilled water.

2. After the content are throughly mixed in water, cytokinine BA (1ml) was
added.
3. 5ml of each stock I, II, III, and IV and was added in the preparing media
4. 1ml of vitamins was added and volume was made up to 1litre

5. PH of medium was adjusted to 5.7
6. 7gm of agar was added in the media and homogenized
7. The media was poured into different culture tubes and autoclave.

24. CONCLUSION
Growth and morphogenesis of plant tissue invitro are
largely governed by the composition of the culture
media. For plant tissue culture there are many types of
media which are used by many scientist like LS,B5,MS
etc medium. MS medium mostely used for this
technique which made by Murashige and Skoog. In
tissue culture medium all nutrients are present those
required for plant growth and development