Tissue culture basic techniques

1. PLANT TISSUE CULTURE
BASIC REQUIREMENT AND
TECHNIQUES
Source:plant growth ... agrocares.com, What is the Tissue Culture Process and ... plantcelltechnology.com,, Tissue Culture - GRIPP | The Gosling ...gripp.ca
Prepared by:
Dr. E. Gayathiri
Department of Plant Biology and Plant Biotechnology,
Guru Nanak college, Chennai

2. •Concepts of Plant Tissue Culture
•Instruments in Plant Tissue Culture
•Sterilization technique
•for Glassware, Media and Explant
•Major techniques involved in Plant Tissue
Culture

3. CONCEPTS OF PLANT TISSUE CULTURE
•Plant tissue culture is a technique
that uses an artificial medium to
grow and maintain plant cells and
tissue , under controlled
environmental conditions.
Source: What is plant tissue culture? – Mysuru ...citytoday.news, The Importance of Plant Tissue Culture ...
plantcelltechnology.com

4. EXPLANTS FOR PLANT TISSUE
CULTURE
• Apical meristem
• Leaves
• Shoot tips
• Root tips
• Growth buds and
• Flower parts
Parts of Plant used as
Explants
Source:irrecenvhort.ifas.ufl.edu

5. TOTIPOTENCY
•Totipotency (Lat. totipotentia, "ability for
all [things]") is the ability of a single cell to
divide and produce all of the
differentiated cells in an organism.
Source :Doubtnut

6. LAMINAR AIR FLOW
(LAF)
Source: Alibaba.com, SlideShare

7. AUTOCLA
VE
Source: Alibaba.com, SlideShare

8. CULTURE
VESSELS
Source: Culture tubes for PTC and seed labs ... labassociates.com, TPP.ch
Test tube /Bottles
Wide – Mouthed
Troughs

9. INSTRUMENT TO HANDLE
EXPLANTS
Source: Indiamart, Shutterstock
Scissors Scapels Bend Forceps Sharp
Forceps

10. TISSUE CULTURE
RACK
Source: IndiaMART
• Fluorescent tubes,
• Bulbs
• U.V. germicidal lights.
• ElectronicChokes
• Timer to control light and dark
period

11. HARDENING
CHAMBERS
Source: Polyhouse Mist Chamber, indiamart.com
Green chamber
Mist chamber
Green Mist
chamber

12. HARDENING
CHAMBERS
Green Netted sheet
Source: Amazon.in, Neha Shade Ne

13. PLANT TISSUE CULTURE
MEDIUM
• Plant tissue culture media should generally contain some or all of the
following components:
•Macronutrients
•Micronutrients
•Vitamins
•Amino Acids Or Nitrogen Supplements
• Source(s) Of Carbon, Undefined Organic
Supplements Growth Regulators
•Solidifying agents.

14. MAJOR SALTS
• The essential elements in plant cell or tissue culture media
include, besides C, H and O.
•Nitrogen (N)
• Phosphorus (P)
• Potassium (K)
• Calcium (Ca)
• Magnesium (Mg)
•Sulphur (S)

15. •The essential micronutrients (minor elements)
for plant cell and tissue growth include
• Iron (Fe)
• Manganese (Mn)
• Zinc (Zn)
• Boron (B)
• Copper (Cu)
•Molybdenum (Mo)
MINOR
SALTS

16. •In plant cell culture media, besides the sucrose,
frequently used
•Lactose
•Galactose
•Maltose
•Raffinose
•Trehalose
•Cellobiose
CARBON AND ENERGY
SOURCE

17. •They are generally classified into the following
groups
•Auxins
• Cytokinins
• Gibberellins
• Abscisic acid
PLANT
HORMONES

18. Plant hormones- auxin
• IAA is the only natural auxin
• Indole-3- acetic acid (IAA)
• Indole-3- butric acid (IBA)
• 2,4-dichlorophenoxy-acetic acid (2,4-D)
• Naphthalene- acetic acid (NAA).
Source: https://www.intechopen.com/books/recent-advances-in-plant-in-vitro-culture/plant-tissue-culture-media

19. Plant hormones- Cytokinins
•Cytokinins commonly used in
culture media include
• BAP (6-benzyloaminopurine)
• 2iP (6-dimethylaminopurine)
• kinetin (N-2-furanylmethyl-1H-
purine-6-amine)
Source: https://www.intechopen.com/books/recent-advances-in-plant-in-vitro-culture/plant-tissue-culture-media

20. GROWTH REGULATORS
Ratio of the growth regulators vary among
•Morphogenesis
•Embryogenesis
•Callus initiation
•Root initiation
•Shoot proliferation
Callus culture
normally require
auxin for root
induction and
Cytokinin for shoot
for induction

21. PLANT HORMONES- GIBBERELLINS AND ABSCISIC
ACID
Source: Biology Dictionary, The structure of abscisic acid researchgate.net
Gibberellins Abscisic acid

22. The vitamins most used in the cell
and tissue culture media include:
• Thiamin (B1),Riboflavin
• Niacin, Pyrodoxine
• Folic acid, Pantothenic acid
• Biotin, Ascorbic acid
• Tocopherol, Myoinositol
• Para amino benzoic acid
• Nicotinic acid , Pyridoxine (B6).
SUPPLEMENTS
Amino acid mixtures mostly
used are:
•Casein hydrolysate
• L-glutamine
• L-asparagine
•Adenine (organic nitrogen)
•Ammonium salts

23. The Organic acid most used in the
cell and tissue culture media
include:
• Pyruvate
• TCA cycle intermediates
SUPPLEMENTS
Complex mixture like
•Coconut milk
•Casein hydrolysate
•Yeast extract
•Tomato juicePyruvate

24. Sometimes activated
charcoal are added in
tissue culture media
Source: atomic structure of activated carbo core.ac.uk
SUPPLEMENTS
Antibiotics are added to
control the growth of
microorganisms

25. GELLING AGENTS
Agar
Gelatin
Phytogel
Agarose

26. STERILIZING AGENT FOR PLANT
EXPLANT
•Hypochlorite
•Mercuric chloride
•70% Ethanol
•Silver nitrate
•Hydrogen peroxide

27. MS MEDIUM
pH
of the
medium
5.6 to
5.8
Source:https://www.researchgate.net/publication/27797511_In_vitro_propagation_of_Stevia_rebaudiana_Bert_in_Bangladesh/figures?l

28. MEDIUM
PREPARATION
Agar added (Heated at about 60⁰C)
Mixed it still warm
Aliquoted into the culture vessel
20 ml medium is added to each culture tube
25 to 40 ml medium added to the culture
flask

29. MEDIUM
PREPARATION
Tubes are cotton plugged / Flasks are covered with
screw cap
Autoclaved
Cooled in room temperature
Medium is now used for initiating new cultures
as well as for transferring cultures for sub
culturing

30. EXPLANT
PREPARATION
Explant has to be thoroughly surface sterilized
Wetting agents are used to sterilize waxy coating
Commonly used wetting agent are tween 20 or Teepol
or 70% ethanol
It is immersed for 15- 20 minutes
Then to 70 % alcohol for 60 seconds

31. CULTURING
PROTOCOL
Combination of Mercuric chloride & Sodium Hypochlorite added to
Explant
Swirled frequently to enhance the effectiveness
Later to remove all the traces, explant is washed with several
change of distilled water

32. INOCULATIO
N
•Explants are carefully lifted, using sterile blunt forceps
•Transferred to the growth medium, partially immersed
•Explant has to partially exposed to air and medium

33. INCUBATIO
N
•Explants need to be exposed to adequate light for optimum
growth
•16 hour light and 8 hours dark photo cycle is provided
•4-10 X 103 lux light intensity is provided

34. INCUBATIO
N
•Incubation racks room temperature at 25 - 28⁰ C
•Plant culture take several weeks to develop
•If unhealthy culture appeared , they have
to removed.

35. SUBCULTURIN
G
Initiated culture grow first by shooting, which
will take 6 weeks time, at this time the medium
is depleted
The shoot are carefully prised apart carefully
and inoculated into fresh medium for further
growth
One single explant can give to hundreds and
thousands of plantlets by subculturing

36. SUBCULTURIN
G
Callus is cut into small pieces
Each can propagate into large mass of
callus
Whole plantlets can be developed and
mass propagated by inducing
differentiation and organogenesis

37. .
Higher Conc of cytokinin induce shoot formation ,
Higher Conc. of auxin is need for root induction
.
Medium level of both Cytokinin and auxin
induce both Shoot and Root induction
.
Medium level of Cytokinin ,with low level Auxin induces
dedifferentiation and results in callus formation
DIFFERENTIATION AND
ORGANOGENESIS
Differentiation
organogenesis

38. DIFFERENTIATION AND
ORGANOGENESIS
Source:images of stages in tissue culture ...researchgate.net

39. As plantlets produced in culture are
propagated in highly controlled condition,
they cannot be transferred directly into
field
Plantlets need to be prepared
for filed condition . This process
is called Hardening
Once acclimatize they are transferred to pots
containing soil, Kept in mist chamber and
transferred to Green house.
HARDENING

40. BENEFITS OF TISSUE CULTURE
• Mass production of plants
• Improved traits in plants
• Production of virus free and disease free plants
• Development of plant tissue banks
• Production of genetically identical plants by somatic
embryogenesis
• Mass production of secondary metabolites
• Year around production of plants and their products
• Preservation of endangered plants.

41. THANK YOU