Download Plant Breeding and Plant Biotechnology

Plant Tissue Culture
Techniques
Huseyin Tombuloglu, Phd.
GBE 304 Spring 2015
Definition
the culture of plant seeds, organs,
tissues, cells, or protoplasts on
nutrient media under sterile conditions.
Tissue culture relies on three fundamental abilities of plant
there are:
Totipotency
Dedifferentiation
Competency
Three Fundamental Abilities of Plants

Totipotency
the potential or inherent capacity of a plant cell to
develop into an entire plant if suitably stimulated.
It implies that all the information necessary for
growth and reproduction of the organism is contained
in the cell

Dedifferentiation
Capacity of mature cells to return to meristematic
condition and development of a new growing point,
follow by redifferentiation which is the ability to
reorganise into new organ

Competency
the endogenous potential of a given cells or tissue to
develop in a particular way
HISTORY
Haberlandt
early 1900’S
• proposed concept of totipotency
• cells cultured under right conditions
• Callus cultured from tree cambium
Gautheret, Nobecourt, Whire
In the 1930s.
• cells kept alive but did not develop
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INORGANIC & ORGANIC SUPLLEMENTS
COUMPOUNDS
Mg/Ml
NH4NO3
1,650.00
KNO3
1,900.00
CaCl2 (anhyd)
332.20
MgSO4 (anhyd)
180.70
KH2PO4
170.00
Na2EDTA
37.25
FeSO4.7H2O
H3BO3
27.80
6.20
MnSO4.H2O
16.90
ZnSO4.H2O
5.37
KI
0.83
Na2Mo4.2H2O
0.25
Sucrose
30,000.00
i-Inositol
100.00
Thiamine.HCl
0.40
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Antibiotics :
Streptomycin, kanamycin
Activated charcoal
Other organic supplements :
Protein, coconut milk,yeast,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.
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Basis for Plant Tissue Culture

Two Hormones Affect Plant Differentiation:
 Auxin:
Stimulates Root Development
 Cytokinin: Stimulates Shoot Development

Generally, the ratio of these two hormones can
determine plant development:

Auxin ↓Cytokinin = Root Development
  Cytokinin ↓Auxin = Shoot Development
 Auxin = Cytokinin = Callus Development
EQUIPMENT & APPARATUS
 VESSELS & GLASS WARE :
• All the glassware should be of Pyrex.
• Large test tubes,flasks,graduated pipettes etc.. are used.
 EQUIPMENT :
•
•
•
•
•
•
•
•
Scissors,scapels,foreceps are used for explants preparation.
A spirit burner for flame sterilization.
Hot air oven.
A PH meter.
A BOD incubator.
Laminar air flow chamber.
A balance to weigh nutrients.
Data collection and recording room.
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Knife
Laminar air flow chamber
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 ESTABLISHMENT OF PLANT TISSUE CULTURE
In vitro culturing of plant tissue culture involves the following
steps.
 Collecting & sterilization of glassware tools/vessels.
 Preparation of explant.
 Surface sterilization of Explant.
 Production of callus from explant.
 Proliferation of culture.
 Sub culturing of callus.
 Suspension culture
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 EXPLANT PREPARATION
EXPLANT : It is defined as a portion of plant body, which has
been taken from the plant to establish a culture
• Explant may be taken from any part of the plant like
root,stem,leaf,or meristematic tissue like cambium, floral parts
like anthers, stamens etc..
•Age of the explant.
• Homozygous plants are preferred.
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flower
leaf
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 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
sodium hypochlorite
sterile water
STEM : running water
sodium hypochlorite
sterile water
LEAF : surface clean
Hgcl2
sterile water
dried
explant
 PRODUCTION OF CALLUS FROM EXPLANT
•
•
•
•
Sterilized explant is transferred aseptically onto defined
medium.
Transfer to incubator.
Temperature (25 ± 2 ͦ) and light is necessary for callus
production.
Callus produced with in 3-8 days.
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 PROLIFERATION OF CULTURE
• if callus is well developed, it should cut into small pieces &
transferred to another fresh medium containing hormones, which
supports growth.
•The medium used for production of more amount of callus is
called proliferation medium.
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15 DAYS
30 DAYS
 CALLUS GROWTH
50 DAYS
80 DAYS
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 SUBCULTURING OF CALLUS
•After sufficient growth of callus it should be periodically
transferred to fresh medium to maintain viability of cells.
•This subculture will be done at the interval of 4-6 weeks.
•After a maximum growth transfer into a pottling soil under
required condition.
 SUSPENSION CULTURE
•It contains a uniform suspension of separate cells in a liquid
medium
callus
liquid medium
agitated continuously
finally cells separated
sub-culture the cells
•This can be achieved by rotary shaker attached within the
incubator at a rate of 50-150 rpm.
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Protoplasts
Landmark:
1960: E. C. Cocking (Univ Nottingham) isolated
protoplasts by treating explants with
concentrated cellulase isolated from a fungus.
[Commercial cellulase and macerozyme were
not available till 1968].
Tobacco protoplasts
Protoplast fusion
1. Somatic hybrids
2. Cybrids A cytoplasmic hybrid (or cybrid a portmanteau of the two words
Inter-specific fusions
Datura innoxia X D. stramonium = D. straubii
Tomato X Potato
=
Tomoffel
4n Synkaryon
2n x 2n
(O. Schieder)
(G. Melchers)
Somatic hybrids
2n Heterokaryon
Fusion of haploid protoplasts (derived from anther cultures)
n + n= 2n
Cybrid Technology
Mixing two cytoplasm without hybrid formation.
TomTato plant
tomoffel
Haploid Culture
Haploid plant (n) = recessive mutations displayed
n+n= double haploid
Occur spontaneously in inter-specific cross or induced by
irradiating pollen prior to pollination. Extremely poor efficiency.
Later: Microspore cultures.
Protoplast fusion: gametic hybridization
Haploid cells
nXn
Protoplast (n)
2n (synkaryon)
Anther culture techniques/ fusion has been
extensively used in rice breeding program
Applications of tissue culture to plant breeding
1. Haploid production (rice, wheat and barley)
2. Triploid production (fruits and poplar)
3. Embryo Rescue/ Wide hybridization (numerous examples)
4. Somatic hybridization (scientific examples, few commercial products)
5. Somaclonal Variations (
Tomato with altered color,
taste and texture by Fresh World Farms;
Imidazolinone resistant maize,
American Cyanimid;
Bermuda grass (Brazos R-3) with increased resistance to fall armyworm
etc.)
1. Production of disease free plants.
2. Clonal propagation
3. Secondary metabolite production (eg. Taxol production from cell
cultures derived from the bark cuttings of pacific yew tree)
4. Germplasm conservation (cryopreservation)
 TYPES OF CULTURE
Callus culture
Suspension culture
Root tip culture
Leaf or leaf primordial culture
Shoot tip culture
Complete flower culture
Anther & pollen culture
Ovule & embryo culture
Protoplast culture
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Callus
culture
Ovule culture
Protoplast culture
Suspension
culture
Root tip
culture
Leaf primordial
culture
Pollen culture
Shoot tip culture
Flower culture
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 ADVANTAGES :
Availability of raw materials.
Fluctuation in supplies & quantity.
Easy purification of the compound.
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Modifications of chemical structure.
Disease free & desired propagule.
Crop improvement.
Bio-synthetic pathway.
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Seedless watermelon triploid that produces excellent fruit with a dark
green skin, consistent round shape, sweet taste (with an average Brix
sugar content level of 11 degrees), deep red color, density and texture.
In addition, it has intermediate resistance* to both Anthracnose and
Fusarium wilt race 1.
The available genetic resources for citrus fruit are predominantly
diploid. Nonetheless, polyploidy can give citrus fruit interesting
characteristics. Thus, the genetic breeding programmes aim to
create triploid varieties, which are sterile and produce seedless
fruit.