The document discusses plant tissue culture. It defines plant tissue culture as the technique of growing plant cells, tissues, or organs in an artificial nutrient medium under sterile conditions. The key applications of plant tissue culture include commercial plant production, conservation of endangered species, plant breeding, production of valuable compounds, and crossing distantly related plant species. The document then provides details on preparing Murashige and Skoog medium, including composition, sterilization techniques, and procedures for inoculation of plant materials like seeds and rose buds.

1. PLANT TISSUE CULTURE
BY :-
HIMANSI SURA
Msc student(BIOTECHNOLOGY)
Chandigarh Group of Colleges, Landran

2. PLANT TISSUE CULTURE

3. PLANT TISSUE CULTURE
• Plant tissue culture is the technique of growing plant cells
tissue , organs in an artificial prepared nutrient medium
static or liquid under aseptic conditions. The term tissue
culture is commonly used in a very wide sense to include
in vitro culture of plant cells, tissue as well as organs. But
in a strict sense, tissue culture denotes the in vitro
cultivation of plant cells in an unorganised mass, e.g callus
cultures. Another term, cell culture is used for in vitro
culture of single or relatively small groups of plant cells,
e.g suspension cultures and cell culture is often used for
callus cultures as well. When organised structures like root
tips, shoot tips, embryos, etc. are cultured in vitro to
obtain their development as organised structures, it is
called organ cultures.

4. • The technique of in vitro cultivation of plant cells or
organs is primarily devoted to solve two basic
problems : first, to keep the plant cells and organs
free from microbes i.e, bacteria and fungi, and second
to ensure the desired development in the cells and
organs by providing suitable nutrient media and other
environmental conditions. Plant tissue culture can be
initiated from almost any part of a plant however, for
micro propagation or direct shoot regeneration,
meristemetic tissue such as shoot tip is ideal. The
mother plant must be healthy and free from obvious
signs of diseases or pest.

5. APPLICATIONS
Plant tissue culture is used widely in the plant sciences,
forestry, and in horticulture .
It includes :-
1. The commercial production of plants used as
potting, landscape, and florist subjects, which uses
meristem and shoot culture to produce large numbers
of identical individuals.
2. To conserve rare or endangered plant species.
3. A plant breeder may use tissue culture to screen
cells rather than plants for advantageous characters,
e.g. herbicide resistance/tolerance.
4. Large scale growth of plant cells in liquid culture in
bioreactors for the production of valuable compounds
like plant- derived secondary metabolites and
recombinant protein used as biopharmaceuticals.
.

6. 5. to cross distantly related species by protoplast fusion
and regeneration of the novel hybrid.
6. to rapidly study the molecular basis for physiology,
biochemical and reproductive mechanism in plants for
examples in vitro selection for stress tolerant plants and
in vitro flowering studies.
7. To cross pollinate distantly related species and then
tissue culture the resulting embryo which would
otherwise normally die (embryo rescue).
8. For chromosomes doubling and induction of
polyploidy , for example doubled haploids, tetraploids
and other forms of polyploidy

7. TO PREPARE MS MEDIUM
MS - Murashige and skoog (1962)
OBJECTIVE :- Nutritional requirement for optimal growth of a tissue in vitro may
very with the species . Even tissue from different parts of a plant may have
different restrictive requirements for satisfactory growth. A defined nutrient
medium consists of inorganic salts, a carbon sources, vitamins and growth
regulators. A gelling agent is also used in static medium.
1. Murashige-Skoog medium or MS medium(1962)
2. White medium- White (1963)
3. Eriksson- Eriksson (1965)
4. Bs medium-Gamborg et at (1968),
are well designed medium in plant tissue culture.
MATERIALS :-
a. Distilled water
b. Beaker
c. Magnetic stirrer
d. Acid and base for adjustment of pH
e. Weighing balance

8. COMPOSITION OF NUTRIENT MEDIUM
1. INORGANIC NUTRIENTS :- For normal growth of plants many inorganic nutrients like
carbon, hydrogen, oxygen, nitrogen, calcium, potassium, phosphorous, sulphur,
magnesium, iron, manganese, copper, boron, zinc, molybednum are required.
In addition to C, H, O ….N, P, K,Ca,S,Mg are in large amount and known as macronutrients
(conc. More than 0.05m mol1-1) and rest Fe,Zn,Mn,Cu,B,Mo are micronutrients and are
required in conc.<0.05m mol1-1. In White medium iron is added in the form of Fe(SO)4
but in most media’s iron is provided in the form of FeEDTA.
2.ORGANIC NUTRIENTS :- organic nutrients like amino acids and vitamins like thiamine
are added to culture medium to get better results. Sucrose is added as a source of
carbohydrates to provide carbon. Some synthetic substances like coconut milk, yeast
extract, tomato juice, casein hydrolase etc. can also be added to promote growth of callus
tissue.
3.GROWTH HORMONE :- Culture medium is supplemented with growth regulators like
auxins, gibberllins and cytokinins. The commonly used auxins are Indole-3-acetic acid
(IAA), Indole-3-butyric acid(IBA), Napthalene acetic acid(NAA), 2,4-D(2,4-Dichlorophenoxy
acetic acid) which is used to support cell division and callus growth, somatic embroy(SE),
induction , rooting etc. Cytokinins areKinetin, BAP( benzylamino purine), zeatin, etc are
employed to promote cell division, regeneration of shoots, and to enhance proliferation
and growth of axillary buds. Abscisic acid(ABA) promotes SE and shoot bud regeneration
and improves SE maturation. GA3 is one of the most common gibberllins (out of 23) for
its stimulating effect for development of plantlets.

9. COMPOSITION OF NUTRIENT MEDIUM

10. 4.VITAMINS :- For optimum growth. The following vitamins are required; inositol,
thiamine, pyridoxine and nicotinic acid of which thiamine is essential rest are
promotory.
5.CARBON SOURCE :- Sucrose is the most commonly used carbon source for all
cultured plant materials. Autoclaving hydrolysis sucrose , which enhances its
availability to plant cells. In some systems e.g , monocots, Glucose may be superior
to sucrose.
6. AGAR :- It is used to solidify the medium . It is a polysaccaride obtained from
seaweeds belonging to red algae. Higher concentration of agar makes the medium
hard and diffusion of nutrients in plant tissue may be checked. Difco noble
bacteriological agar is the purest form which is used for std work.

11. STERILIZATION
 AUTOCLAVING :- Culture vessels , etc are generally sterilized by heating in an
autoclave or a pressure cooker to 121degree C at 15 p.s.i for 15 to 40 mins, the
time being longer for larger medium volumes. Sterilization during autoclaving
depends mainly on temperature. Certain types of plasticware and some
instruments e.g., micropipettes, etc. are also autoclavable. Care should be
taken to properly stopper all the vessels and to open the autoclave only when
its pressure gauge indicates zero pressure.
 FILTER STERILIZATION :- Some growth regulators, e.g., GA3, zeatin, ABA, urea,
certain vitamins and enzymes are heat labile. Such compounds are filter
sterilized by passing through a membrane filter of 0.45u or lower pore size. The
membrane filter is held in a suitable assembly ; the assembly together with the
filter is sterilized by autoclaving before use.
Laminar flow cabinets are used to create an aseptic working space by blowing
filter- sterilized air through an enclosed space. The air is first filtered through a
coarse prefilter to remove large particles; it is then passed through a HEPA (high
efficiency particulate air) filter, which filter out all particles larger than 0.3um .
This sterilized air blow through the cabinet at 1.8km/hr, which is sufficient to
keep the enclosed working area aseptic.

12. STERILIZATION

13.  DRY HEAT :- Glassware and teflon plasticware , and instrument may be sterilized by
dry heat in an oven at 160-180degreeC for 3 hrs.
ETHANOL :- The surface that can not be sterilized by other techniques e.g,. Platform
of laminar air flow cabinet, hands of the operator, etc are sterilized by wiping them
thoroughly with 70% ethyl alcohol and the alcohol is allowed to dry.
SURFACE STERILIZATION :- All the plant material to be used for culture are treated
with an appropriate sterilizing agent to inactivate the microbes present on their
surface.
Sterilizing agents are calcium hypochlorite(9-10%), sodium hypochlorite(2%), mercuric
chloride(0.1-1%), silver nitrate(1%), bromine water(1-2%), H2O2(10-12%) and
antibiotics(4-50mg/l) The treatment is given for 15-30 minutes.

14. MEDIA ROOM
The washing area in the media room should have ;
1. Brushes of various sizes and shapes.
2. A washing machine
3. A large sink
4. Running hot and cold water.
The usual facilities required for the preparation of culture medium include :-
1. Benches at a height suitable to work while standing.
2. A deep freeze for strong the stock solution, enzyme solution etc.
3. A refrigerator to store various chemicals, plant materials, short term storage
of stock solution, etc.
4. Weighing balance.
5. Hot plate aim-magnetic stirrer for dissolving chemicals.
6. A pH meter.
7. An aspirator vacuum pump to facilitate filter sterilization.
8. A plastic carboys for storing distilled water.
9. An autoclave for media

15. PREPARATION OF SOLUTIONS
1. MS MAJOR SALTS (mg/l) (500ML) (20X)
* NH4NO3 -> 1650mg 16.5 gm
* KNO3 -> 1900mg 19 gm
* CaCl2.7H2O -> 440mg 4.4 gm
* MgSO4.7H2O -> 370mg 3.7 gm
* KH2PO4 -> 170mg 1.7 gm
2. MS MINOR SALTS (mg/l) (500ML) (20X)
*H3BO3 -> 6.2mg 620mg
*MnSO4.4H2O -> 22.3mg 2230mg
*ZnSO4.4H2O ->8.6mg 860mg
*KI ->0.83mg 83mg
*Na2MoO4.2H2O ->0.25mg 25mg
*CoCl2.6H2O ->0.025mg 2.5mg
*CuSO4.5H2O ->0.025mg 2.5mg

16. PREPARATION OF SOLUTIONS

17. 3. IRON SOLUTIONS (200X) g/l
*FeSO4.7H2O -> 5.56
*Na2EDTA.2H2O -> 7.46
OR
For 500 ml dissolve 3.725gm of Na2EDTA in 250ml of distilled water and
2.785gm of FeSO4.7H2O in 250 ml of distilled water, and now boil Na2EDTA
solution and add to it FeSO4 solution by stirring .
4. VITAMIN’S SOLUTION (400X) g/l
*Inositol -> 20
*Nicotinic acid -> 0.1
*Pyridoxine -> 0.1
*Thiamine HCl ->0.02
*Glycerine ->0.4
5. GROWTH HORMONES STOCK SOLUTION 1N
*IAA(indole acetic acid) ->0.2mg/ml NaOH/KOH
*BAP(Benzylamino purine) ->0.2mg/ml NaOH/KOH
6. OTHERS
*Sucrose -> 30gm/l
*Agar -> 8gm/l

19. PROCEDURE FOR MS MEDIUM
1) Prepare macronutrients solution in 100 ml of distilled water.
2) Prepare micronutrients solution in 100ml of distilled water.
3) Add macronutrients and micronutrients solutions while stirring into 100
ml distilled water.
4) Add the other heat stable constituents (sucrose, vitamins and
hormones) and agar powder.
5) Make the final volume of the medium by the addition of more distilled
water.
6) Adjust the pH of medium from pH meter to 5.7 while stirring using 0.1
NaOH or 0.1 HCl.
7) If solid medium is desired i.e agar is used , heat the solution while
stirring until agar dissolved.
8) Pour the medium into desired culture vessel.
9) Plug the culture vessels with non-absorbent cotton wool wrapped in
cheese cloth or in with any suitable cloruls.
10) Transfer the culture vessels to appropriate wetting of plugs during
autoclaving.
11) Sterilize the medium upon the volume of the medium in the vessels.
12) The medium is always allowed to cool at low temperature.

22. TO PREPARE WORKING MS MEDIUM
1) First we will take approximately 600 ml of water and boil it for some
time.
2) Now add 0.44 g of calcium chloride and them mix it well with the help
of stirrer.
3) Measured agar powdered 8gm is added to the solution and mix it well
with stirrer.
4) To the solution now add 100 ml of major salt solution , 2.5 ml of minor
salt solution and 2.5 ml of vitamin solution and 5 ml of iron solution
and mix them all with stirrer
5) Now make up the volume of the solution to 1litre.
6) Add 40 ml of IBA and 20 ml of butyricamino purine (BAP) to the solution
7) Now adjust the pH of solution by pH meter to the 5.8 by adding HCl or
NaOH to the solution
8) Cover the mouth of solution flask with cotton plug and wrap it with foil
paper before transferring it to autoclave.
9) Autoclave the media at 15 p.s.i for the 20-25 minutes.

24. i

25. INOCULATION OF SEEDS IN MS MEDIUM
1. After autoclaving the media and vessels in autoclave , transfer
them all in laminar cabinet for some time.
2. From the large vessel of media, transfer 20-20 ml in small flasks or
test tube .
3. Keep the test tubes at an angle of 45 degree and let it to become
little solidify.
4. First we will inoculate the musturd seeds , keep the seeds in HgCl2
for 2-10 minutes.
5. After this, transfer the seeds in distilled water.
6. Now with the help of forceps transfer seeds in test tubes having ms
media and plug the tube with cotton.
7. Keep the test tubes in tube stand for the growth of plant tissue in
laminar cabinet.
8. After a couple of day observe weather there is any growth or not in
the test tubes containing seeds.

26. i

27. i

28. Inoculated mustard seeds in Petri plate in
ms media

29. INOCULATION OF ROSE BUDS IN MS MEDIA
1. Same procedure will be done for making agar medium and
autoclaving it in autoclave.
2. After autoclaving the medium and vessels and rose buds, we will
transfer them all in laminar air flow cabinet.
3. Wash the surface and your hands with 70% alcohol and burn the
burner.
4. With the help of forceps transfer the rose buds in a solution of
HgCl2 for 2-10minutes. And then transfer them in distilled water
containing flask .
5. Then sterilize again the forceps on burner and with the help of it
transfer the buds into test tubes containing semi solidify media.
6. Put the cotton plugs on each test tubes containing media and
plantlet.
7. Now let the test tubes for 48 hours for the growth of plant tissue.
8. After 48 hours observe the growth of plant(rose) bud.

30. w
Cut the bud part of the rose stem and wash it and
place the all pieces of stem in the solution of HgCl2
and let it keep undisturbed in laminar air cabinet
for some time till the media get autoclaved .

31. r

33. Regeneration of Plantlet

34. OBSERVATIONS