A General Account of Plant Tissue Culture

2. The term Biotechnology was introduced by Karl Ereky (1919)
 Term Biotechnology is derived from a fusion of ‘Biology’
and ‘Technology’
 Scientific art of using living organisms in industries to
produce valuable chemicals.

3. Definitions
 US National Science Foundation – Biotechnology consists of
controlled use of biological agents such as microorganisms or
cellular components for beneficial use.
 European Federation - Biotechnology – Integrated use of
biochemistry, microorganisms, cultured tissues or cells and
parts there of.
 Utilization of biological entities (micro organisms, cells of
higher organisms (either living or dead), their components or
constituents (eg. Enzymes) in such a way that some product or
service is generated which should enhance human welfare.

4. OLD BIOTECHNOLOGY:
Process which are based on the natural capabilities of micro organisms.
Eg. Use of microorganisms as early as 5000 BC for making wine, vinegar,
curd etc.
NEW BIOTECHNOLOGY :
 Use of microorganisms for the production of valuable chemicals of
commercial importance such as antibiotics, human insulin, vaccines,
interferons, organic acids, growth hormones, enzymes etc.
 Production of genetically modified organisms (GMOs) by recombinant
DNA Technology
 Tissue culture
 Production of somatic hybrids, cybrids
 Diagnosis of infectious diseases
 Gene therapy to treat inborn diseases
 Gene banks, DNA clone banks.

5. LANDMARKS IN BIOTECHNOLOGY
Before 6000 BC : Yeasts were used to make wine and beer
About 4000 BC : Yeasts were used to make bread
1876 : Louis Pasteur established germ theory of fermentation
1897 : Edward Buchner extracted enzymes from yeast
1902 : Haberlandt predicted the concept of tissue culture
1904 : Hanning’s attempt of embryo culture
1912 -1914 : Large scale production of acetone, butanol and glycerol using bacteria
1919 : The word “Biotechnology “ was first used by Karl Ereky
1925 : Use of embryo culture technique in interspecific crosses by Laibach
1941 : The term “genetic engineering’ was first used by a Danish microbiologist, A.
Jost.

6. 1944 : Large Scale Production of Penicillin
1954 : Cell culturing techniques were developed
1957 : Regulation of organogenesis by changing auxin kinetin ratio by Skoog and Miller
1958 : Purification of DNA Polymerase
1959 : Regeneration of embryos from callus and cell suspensions of Carrot Daucus carota by Reinert
and Steward
1960 : Enzymatic method of cell wall degradation by Cocking
1962 : Mining of Uranium with the aid of microbes, Acidithiobacillus ferrooxidans (Canada).
1964 : Anther culture in Datura by Guha and Maheswari
1969 : First in vitro synthesis of an enzyme, Ribonuclease by Ralph F. Hirschmann
1970 : In vitro synthesis of gene by Har Gobind Khorana
Identification of Specific Restriction nucleases by Hamilton O. Smith, Thomas Kelly and Kent
Wilcox.
1973 : First successful protoplast fusion by Power
First successful genetic engineering experiments by Stanley Cohen and Herbert Boyer.
1975 : Use of plasmid vectors for gene cloning
Establishment of rDNA technology by Boyer and Cohen
1976 : Expression of yeast genes in E.coli
1977 : Use of genetically engineered bacteria to synthesize human growth protein ; Methods for rapid
DNA Sequencing.

7. 1978
: Production of human insulin by genetically engineered E.coli
Human Genomic library constructed
1980
: Marketing of human food of fungal origin (U.K) – Fusarium venenatum marketed as Quorn.
1983
: Approval for the use of insulin produced by genetically engineered microbes
The Polymerase Chain Reaction (PCR) was developed by Kary Mullis
First genetic transformation of plant cells by Ti Plasmid – European researchers and Monsanto.
1985
: DNA finger printing technique was developed by Dr. Alec Jeffreys
1988
: The first successful production of a genetically modified crop (soy beans) – Round up Ready
resistant to glyphosate.

8. 1990 : Official launching of Human Genome Project
Development of Random Amplified Polymorphic DNA (RAPD) technique by William & Welsh
1991 : First test of gene therapy on human cancer patients –Copies of ADA gene -Adenosine deaminase
enzyme.
1994 : The Flavr Savr tomato introduced , the first genetically engineered whole food approved for sale.
1998 : First cloned mammal (the sheep Dolly)
2000 : First Plant genome was sequenced (Arabidopsis thaliana )
A draft of the human genome is completed by Celera Genomics and the Human Genome Project
Production of Golden rice
2001 : The sequence of human genome published
2002 : First crop plant genome (Rice – Oryza sativa ) was sequenced
2003 : The Human Genome Project is completed, providing information on the locations and sequence of
human genes on all 46 chromosomes

9. Plant Tissue Culture: Principles and
Techniques
Cellular Totipotency
 Ability of a single plant cell to regenerate a whole plant
 Exploited in Plant tissue culture
 German botanist Gottlieb Haberlandt (1902) - Father of plant
tissue culture - predicted the concept of tissue culture under
in vitro conditions by using an artificial medium. He reported
the culture of isolated single palisade cells from leaves in
Knop’s salt solution enriched with sucrose.
 Steward et.al., demonstrated the development of embryoids
from the secondary phloem of Carrot.

10.  Skoog et.al., reported the role of cytokinins in
inducing callus.
 Skoog and Tsui (1948) reported the role of auxin in
induction of callus.
 Skoog and Miller (1957) suggested that organogenesis
could be induced in cultured callus tissues by varying
the ratio of auxin and cytokinin.
 A higher kinetin (cytokinin) to auxin ratio promotes
only shoot development – Caulogenesis
 A low kinetin to auxin ratio promotes only root
development - Rhizogenesis

11. Terminology
 Plant Tissue Culture – The process where by small
pieces of living tissue (explant ) are isolated from a
plant and grown aseptically for indefinite periods on or
in a semi – defined or defined nutrient medium.
 Explant - Part of the plant used for culture – may be
taken from a root (carrot), stem or leaf (Tobacco),
basal plate (Onion), grains (Paddy, Wheat) excised
endosperm (maize), embryo (rye).

12. Differentiation – Developmental change of a cell which
enable it to do specialized functions.
Dedifferentiation – Reversion of mature cells to meristems –
involves renewed and enhanced RNA and protein synthesis
leading to the formation of new cellular components
needed for meristematic activity.
Callus – Unorganized / undifferentiated mass of cells
produced by the growth and division of cells of explant.
Redifferntiation – Ability of a dedifferentiated cell to form a
whole plant or plant organ.

13. Callus

14.  Somatic Embryogenesis (Somatic Embryogeny)
Formation of embryo – like structures called somatic
embryos (embryoids or asexual embryos) on the
cultured callus tissue.
 Somatic embryo generally originate from single cells
which divide to form a group of meristematic cells –
becomes isolated by breaking cytoplasmic connections
with the other cells around it and subsequently by
cutinization of the outer walls of this differentiating
cell mass. The cells of meristematic mass continue to
divide and give rise to globular, heart – shaped,
torpedo and cotyledonary stage.

15. Image:https://biocyclopedia.com/

16. Image:https://www.biotecharticles.com/

17. Direct Somatic embryogenesis – Somatic embryos are raised
directly from a single cell or a group of cells or from an entire
explant tissue.
 No callus phase during the development
 Eg. Explants of Ranunculus spiteratus , Citrus species
Indirect somatic embryogenesis – The explant tissues are induced
to produce callus which will develop into proembryos, which
undergo regeneration (organogenesis – formation of root, shoot
etc.) and develop into plant lets in the medium – organogenetic
embryogeny. The plantlets are formed by caulogenesis
(development of adventitious shoots) or rhizogenesis (development
of adventitious roots) directly from the callus tissue. Plant lets thus
produced are unipolar in nature – only shoot or root directly arises
from the callus, the other portion arise away from the site of the
origin of shoot or root. There is no direct physical contact between
the root and shoot of the plant let – connected by callus tissue.

18. Tissue Culture Medium
 Nutrient preparation on or in which a culture is grown
 Nutritional requirements vary with different parts/
species
Basal Medium – A medium containing essential
nutrients such as carbon source, inorganic salts and
vitamins.
Synthetic Medium – A medium composed of
chemically defined components.

19. Basic Components / Chemical Composition of
a Tissue Culture Medium
 Inorganic nutrients
– Macronutrients C, H2, O2, N, P, K, Ca, S, Mg
- Micronutrients Fe, Zn, Mn, Cu, Mo
 Different tissue culture media provide different
concentrations of the inorganic nutrients
 Carbon Source – Sucrose, 20 -50 g/l

 Vitamins – Thiamine (B1 ), Inositol (B8 ), Pyredoxine
(B6), Nicotinic acid (Niacin, Vitamin B3)

20.  Growth Regulators – Auxins - IAA, IBA, NAA, 2, 4 – D
 Supports cell division
 Callus growth
 Somatic embryo induction
 Rooting

21. Cytokinins – Kinetin (Furfurylamino purine ),
BAP (Benzyl Amino Purine)
 Promote cell division
 Regeneration of shoot
 Somatic embryo induction
 Enhance proliferation and growth of axillary buds

22. Gibberellins – GA3 (Gibberellic acid)
 Promotes shoot elongation
 Somatic embryo germination
 Used in apical meristem culture.

23. Abscisic Acid (ABA) – Promotes formation of
somatic embryo and shoot bud regeneration.
Organic Supplements – Coconut milk, yeast
extract, corn milk, malt extract, tomato juice,
Potato extract, Casein hydrolysate.

24. Types of Media Based on Consistency
 Liquid Medium
 Semisolid Medium (0.5% Agar)
 Solid Medium (1 % Agar)

25. Different Culture Media
M S Medium (Murashige and Skoog Medium, 1962)
 Most widely used high salt medium
 Good for both monocots and dicots
 Whites medium (1963)
 L.S. Medium (Linsmaier and Skoog, 1965)
 B5 Medium ( Gamborg et.al.1968)- Soybean callus
culture, Taxus, Linum etc.
 N6 Medium (Nitsch and Nitsch,1969) – Anther culture
 SH Medium (Schenk and Hilderbrandt, 1972) – Culture of
monocots

26. Composition of M S Medium for Plant tissue
culture
Constituents Concentration –mg/ l
Macronutrients
NH4NO3 1650
KNO3 1900
CaCl2 2H2O 440
MgSO4. 7H2O 370
KH2PO4 (Potassium dihydrogen
phosphate)
170

27. Constituents Concentration –mg/ l
Micronutrients
KI 0.83
H3BO3 (Boric acid) 6.2
MnSO4 .4H2O 22.3
ZnSO4. 7H2O 8.6
Na2MO4. 2H2O (Sodium molybdate) 0.25
CuSO4.5H2O 0.025
Co Cl2.6H2O 0.025
FeSO4. 7H2O 27.8
Na2EDTA. 2H2O (EDTA – Ethylene diamine –
tetra acetic acid)
37.3

28. Constituents Concentration –mg/ l
Vitamins and Hormones
Inositol 100
Nicotinic acid or Niacin (Vitamin B3) 0.5
Pyridoxin – HCl 0.5
Thiamine – HCl 0.1
IAA 1 -30
Kinetin 0.04 – 10
Carbon Source
Sucrose 30000
PH 5.8

29. Preparation of MS Medium
 Requirements
 Constituents of M S Medium
 Conical flasks (100 ml, 250ml,500ml,1000 ml)
 Measuring Cylinders (100, 1000ml)
 Pipettes (1ml, 5ml, 10ml)
 Distilled water
 IN NaOH, IN HCl
 Autoclave

30. Procedure
 Prepare macronutrients solution in 100ml distilled water
 Prepare micronutrients solution in 100 ml distilled water.
 Add macronutrient and micronutrient solutions to 700 ml
distilled water taken in a 1000ml conical flask.
 Add sucrose, vitamins and hormones (Vitamins and
hormones can be added after autoclave for better results )
 Make the final volume of the medium by addition of more
distilled water.
 Adjust the pH to 5.8

31.  Agar is added if a solid medium is required
 Pour the medium to culture vessels
 Plug with non – absorbent cotton wool wrapped in
cheese- cloth
 Autoclave for 20 minutes
 Cooled medium can be used for inoculation or stored
at 40
C.

32. Simple method of Preparation of Culture
Medium
 Required quantity of commercially available
powdered medium is dissolved in distilled water.
 Add sucrose & other constituents
 Add agar , if a solid medium is required.
 Adjust the pH
 Sterilize the medium by autoclaving

33. Aseptic Techniques in Tissue Culture
 Sterilization
 Process of destroying or physically removing all forms
of microbial life including vegetative cells, spores and
viruses from a surface, a medium or an article.
 Methods employed depend on
 Purpose for which sterilization is carried out
 Material which has to be sterilized
 Nature of the microorganisms that are to be removed or
destroyed .

34. METHODS OF STERILIZATION BY DRY HEAT
 Flaming
 Instruments like forceps, scalpels, needles etc. are flame
sterilized by dipping in 95% alcohol. Mouth of the culture
vessels are also flame sterilized.
 Hot Air Oven
 Poor penetration capacity for dry heat - long periods of
exposure is necessary to destroy bacterial endospores. The
holding time and temperature is 1600 C for 1 - 2 hrs.
 Dry glass wares are wrapped in paper
 Oven should not be over loaded.
 Materials should be arranged in a manner that allows free
circulation of hot air in between the objects.
 Allow the oven to cool before the door is opened. Otherwise
the glasswares may get cracked by sudden or uneven
cooling.

35. Hot Air Oven

36. Sterilization by Moist heat
 Moist heat has more rapid penetration power than dry
heat because water molecules conduct heat better
than air.
 Moist heat kills microorganisms by coagulating their
proteins.
 More effective than dry heat.
 Lower temperatures and less time of exposure is
needed than dry heat sterilization
 Instruments used for moist heat sterilization –
 Autoclave (Chamberland,1884) / Pressure Cooker

37. Autoclave
 A double jacketed steam chamber equipped
with devices which permit the chamber to
be filled with saturated steam and
maintained at a designated temperature
and pressure for any periods of time.
 Articles are placed in the sterilizing
chamber.
 Steam is maintained in the steam jacket. As
steam flows from the steam jacket into the
sterilizing chamber cool air is forced out
and a special valve increase the pressure to
15 psi above normal atmospheric pressure.

38.  The temperature rises to 1210 c
 The super heated steam coagulates the proteins of
microorganisms resulting in complete destruction of
all forms of microbial life, including bacterial
endospores.
 Autoclave is used to sterilize culture media,
solutions, glass wares, plastic wares etc.

39. Sterilization by Filtration
 Removal of microorganisms from liquids or
gases using filters
 Membrane filter is held in a suitable assembly
and is sterilized by autoclaving before use.
 Thermolabile (heat – sensitive) materials are
sterilized by filtration – GA3 (Gibberellic acid),
Zeatin, ABA (Abscisic acid), Vitamins, enzymes
etc.
 The solution is passed through a membrane filter
of 0.45 µm or lower pore size and then the
microorganisms are trapped in the pores of the
filters. The solution that drips through the filter
is collected in a previously sterilized container.

40. Sterilization using Laminar Air Flow
Cabinet
 Used to create an aseptic working space by blowing filter
sterilized air through an enclosed (on all sides except one)
space.
 Types –
 Vertical flow unit – Air is directed downward
 Horizontal flow unit – air is directed outward
 A Laminar air flow hood has
 A Fluorescent tube
 UV tube
 Air blower
 A coarse filter
 HEPA filter (0.3m) Image: http://cepclab.org.in/

41. Working Principle of Laminar Air Flow
 Swab the surface of laminar air flow cabinet with alcohol
to remove dust.
 Switch on UV light for 30 minutes to kill germs.
 Open the front cover sheet to keep the desired materials.
 Switch on the air blower – produces air flow with uniform
velocity along parallel flow lines.
 A small motor blows air into the unit through a coarse filter
– removes large dust particles.
 Air then passes through a 0.3m HEPA (High Efficiency
Particulate Air filter ) – air coming out will be ultra clean.
 Wipe the working space with alcohol to reduce
contamination.

42. Sterilization by wiping with 70% alcohol
 Platform of Laminar Air flow cabinet
 Hands of the operator

43. Steps in Plant Tissue Culture Technique
 1. Collection and sterilization of explant
 Explant - Part of the plant used for culture
 Root (Carrot)
 Stem or leaf (Tobacco)
 Basal Plate (Onion)
 Grains (Paddy, Wheat)
 Excised endosperm (Maize)
 Embryo (Rye)
 Young tissues are more suitable than mature tissues.
 Explants obtained from field should be thoroughly washed under
running tap water – remove dirt and epiphytotic microbes.

44.  Treat with – 1% Cetavlon solution (10 -15 minutes) –
reduces bacterial contamination
 Surface sterilization – The plant materials used for
culture are treated with an appropriate sterilizing
agent to inactivate the microbes present on their
surface.
 Procedure depends on the source and type of tissue of
the explant.

45.  Submerge the materials in a dilute solution of surface
sterilizing agents –
 Calcium hypochlorite – 9 -10%
 Sodium hypochlorite – 2%
 Mercuric chloride – 0.1 – 1 %
 Silver nitrate – 1%
 Bromine water – 1 -2%
 Hydrogen peroxide 10 – 12%
 Antibiotics 4 -50mg/l
 After treatment, the explant is rinsed with water to remove the
sterilizing agents.

46. 2. Callus Induction
 Inoculate the surface – sterilized pieces of explant into
a suitable culture medium in a culture vessel in an area
near the flame of a spirit lamp.
 Press the explant in the medium to bring good contact
between the explant and the medium.
 Mouth of the culture bottles are plugged.
 Maintain the inoculated explants in an incubator
(25  20 C)
 The room must have a small amount of illumination.

47.  During incubation , cells of explants divide – callus
(3 -8 days of after incubation)
 Actively growing , unorganized, soft, brittle mass of cells.
3. Proliferation of Callus tissues
Production of more callus tissues (callus proliferation) in a
medium containing altered composition of hormones
(proliferation medium)
Callus is cut into small pieces and inoculated into
proliferation medium – callus tissues multiply more rapidly
by fast cell division and growth.

48. 4. Sub – Culture of Callus (Secondary
Culture)
Transfer of callus tissues to a fresh medium
at regular intervals (4 weeks) to maintain
the cells in a viable condition.
5. Regeneration of plant lets
Callus undergo redifferentiation and form
plant lets either through somatic
embryogeny or organogenetic embryogeny.
Plant lets are transferred to pots and kept
in a green house for proper acclimatization
(hardening). After hardening, the plant
lets are transferred to the main field for
planting.

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