Clonal propagation tissueculture

A slide show on clonal propagation in plant tissue culture. A brief look at somatic embryogenesis and mersitamatic cultures

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CLONAL CLONAL
PROPAGATIONPROPAGATION
SYSTEMSSYSTEMS
GROGRO

UP A

UP A
1.SOMATIC EMBRYOGENESIS
2.MERISTEM CULTURES

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Asexual propagation of many new plants Asexual propagation of many new plants
from an individual all with the same from an individual all with the same
genotype.genotype.
In vitro clonal propagation is a type of In vitro clonal propagation is a type of
micro propagation.micro propagation.
Gives rise to genetically identical plants.Gives rise to genetically identical plants.
 Identical also to the parentIdentical also to the parent
WHAT
IS
CLONAL PROPAGATION?

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Clonal propagation Clonal propagation
benefitsbenefits
Variability arising from sexual reproduction and Variability arising from sexual reproduction and
seed formation is omittedseed formation is omitted
Plants with long seed dormancy can be raised Plants with long seed dormancy can be raised
faster than in vivo seed propagationfaster than in vivo seed propagation
Undesirable juvenile phase seen in seed raised Undesirable juvenile phase seen in seed raised
plants does not appear in vegetatively plants does not appear in vegetatively
propagated plants from adult materialpropagated plants from adult material

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Somatic Somatic
EmbryogenesisEmbryogenesis
A plant reproduces naturally through the A plant reproduces naturally through the
development of zygotic embryos.development of zygotic embryos.
Introduction
Division of the
Fertilized eggs
Or zygote
embryos in
Ovule embryo
sac
DifferentiationMaturation New Plantlet

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Somatic Somatic
EmbryogenesisEmbryogenesis
Schematic representation of the major stages in zygotic Schematic representation of the major stages in zygotic
embryo development from pollination to germination.embryo development from pollination to germination.
Introduction

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Somatic Somatic
EmbryogenesisEmbryogenesis
But in somatic embryogenesis, the But in somatic embryogenesis, the
plant is derived from a single somatic plant is derived from a single somatic
cell or group of cellscell or group of cells
It therefore differs from the natural It therefore differs from the natural
pathway of plant reproductionpathway of plant reproduction
Introduction

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Somatic Somatic
EmbryogenesisEmbryogenesis
•Therefore somatic embryogenesis can be defined
as the clonal propagation technique that produces
an unlimited number of genetically identical plants
from a single somatic seed.
•The term “somatic” indicates that the plants are
developed vegetatively.
•The somatic embryo’s development is however
analogous to its zygotic counterpart
•All of the plantlets produced have the same
genetic makeup.

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Somatic Somatic
EmbryogenesisEmbryogenesis
The process has four main The process has four main
phases:phases:
Somatic Embryogenesis Process
Initiation & Proliferation
Maturation
Germination
Greenhouse culture
And Field planting

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Somatic Somatic
EmbryogenesisEmbryogenesis
Explant is placed on a Petri dish containing Explant is placed on a Petri dish containing
Initiation medium Initiation medium (to initiate cell division)(to initiate cell division)
AfterAfter 6 weeks some of the growing tissue 6 weeks some of the growing tissue
converts into embryogenic tissue (white, fluffy converts into embryogenic tissue (white, fluffy
& translucent appearance)& translucent appearance)
Embryogenic tissue continues to proliferate Embryogenic tissue continues to proliferate
as long as its in the initiation mediumas long as its in the initiation medium
Embryogenic tissue can also be frozen & Embryogenic tissue can also be frozen &
stored in liquid nitrogen for future somatic stored in liquid nitrogen for future somatic
embryogenesisembryogenesis..
Initiation & Proliferation of embryogenic tissue

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Somatic Somatic
EmbryogenesisEmbryogenesis
Auxins:Auxins:
2,4-D (2,4-dichlorophenoxy acetic acid)2,4-D (2,4-dichlorophenoxy acetic acid)
2,4,5-T (2,4,5-trichlorophenoxyacetic acid)2,4,5-T (2,4,5-trichlorophenoxyacetic acid)
Inorganic components: potassiumInorganic components: potassium
Organic components: prolineOrganic components: proline
The inorganic and organic components The inorganic and organic components
modulate embryogenesis or callus modulate embryogenesis or callus
responseresponse
Initiation Medium

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Somatic Somatic
EmbryogenesisEmbryogenesis
Stop proliferation and allow tissue to form Stop proliferation and allow tissue to form
mature somatic embyrosmature somatic embyros
Clumps of embryogenic tissus transferred to Clumps of embryogenic tissus transferred to
maturation mediummaturation medium containing plant growth containing plant growth
regulatorregulator
Regulator promotes maturationRegulator promotes maturation
After 6 weeks mature embryos begin to appear After 6 weeks mature embryos begin to appear
on the clumpson the clumps
They resemble embryos found in seeds They resemble embryos found in seeds
Maturation Of Somatic Embryos

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Somatic Somatic
EmbryogenesisEmbryogenesis
Pre-maturation:Pre-maturation:
From globular to torpedoFrom globular to torpedo
Solid BOi2y medium lacking 2,4-DSolid BOi2y medium lacking 2,4-D
Maturation Phase 1:Maturation Phase 1:
Enriched BOi2Y medium; contains a high level of Enriched BOi2Y medium; contains a high level of
sucrose, nitrogen and sulphursucrose, nitrogen and sulphur
Deposition of storage reservesDeposition of storage reserves
Embryos accumulate fresh and dry weight.Embryos accumulate fresh and dry weight.
Maturation Phase 2:Maturation Phase 2:
Modified BOi2Y containing ABA (abscisic acid); Modified BOi2Y containing ABA (abscisic acid);
induces desiccation tolerance.induces desiccation tolerance.
Maturation Of Somatic Embryos

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Somatic Somatic
EmbryogenesisEmbryogenesis
plantlet
A cell
Cell clump
Globular stage
Heart stage
Induced
cells
Torpedo stage
2
nd
medium
1st medium

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Somatic Somatic
EmbryogenesisEmbryogenesis
Maturation Of Somatic Embryos
The morphological stages of somatic embryo development in alfalfa
(Medicago sativa L.)

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Somatic Somatic
EmbryogenesisEmbryogenesis
Individual mature embyos are picked from Individual mature embyos are picked from
clumps.clumps.
Then placed onto germination medium Then placed onto germination medium
Medium contains a mixture of nutrients for Medium contains a mixture of nutrients for
early plant development.(1/2 MS salts + early plant development.(1/2 MS salts +
1%sucrose)1%sucrose)
Mature embryos germinate to form roots and Mature embryos germinate to form roots and
shoots shoots
The plants obtained are referred to as The plants obtained are referred to as
“emblings” / “somatic seedlings” / “somatic “emblings” / “somatic seedlings” / “somatic
derived plantlets”.derived plantlets”.
Germination Of Somatic Embryos

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Somatic Somatic
EmbryogenesisEmbryogenesis
When germinated emblings are large enough When germinated emblings are large enough
they are transplantedthey are transplanted
Into soil for further growth and acclimatization Into soil for further growth and acclimatization
(Greenhouse)(Greenhouse)
After normal greenhouse culture period they After normal greenhouse culture period they
are planted in the fieldare planted in the field
Greenhouse Culture & Field Planting

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Somatic Somatic
EmbryogenesisEmbryogenesis
Provision of cell lines for:Provision of cell lines for:
Genetic engineeringGenetic engineering
Long term gene storageLong term gene storage
Use in researchUse in research
Tree improvement programsTree improvement programs
Pest resistantPest resistant
Fast growingFast growing
More productive (high value plantation More productive (high value plantation
forests; reduce need to harvest existing forests; reduce need to harvest existing
natural forest; conservation)natural forest; conservation)
Applications Of Somatic Embryogenesis

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Meristem Meristem
CulturesCultures
Introduction
What is Meristem culture?
•The cultivation of axillary or apical shoot
meristems, particularly of shoot apical meristem.
•Involves the development of an already existing
shoot meristem and subsequently, the regeneration
of adventitious roots from the developed shoots
•It does not involve the regeneration of a new
shoot meristem

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Its developed from Its developed from
meristamatic cells meristamatic cells
found at the shoot tipfound at the shoot tip
Shoot tip is normally Shoot tip is normally
free of contaminating free of contaminating
organismsorganisms
Produces pathogen Produces pathogen
free clones of plantfree clones of plant
E.g. potato, dahlia, E.g. potato, dahlia,
strawberry etc.strawberry etc.
Meristem CulturesMeristem Cultures
Introduction

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Explants used:Explants used:
The apical meristem and the first 1 or 2 leaf The apical meristem and the first 1 or 2 leaf
primordia below it are excised from the shoot tip primordia below it are excised from the shoot tip
(the meristem itself is not able to grow (the meristem itself is not able to grow
independently unless some leaf primordia are independently unless some leaf primordia are
retained)retained)
Meristem CulturesMeristem Cultures

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Culture Medium used:Culture Medium used:
MS medium has been found satisfactory for most
plant species. But for some species a much lower
salt concentration may be adequate or even
necessary, since the high salt concentration of MS
medium may be toxic.
 E.g. for blueberry, 1/4 MS salts are the best, while
full MS is often toxic. Agar gelled medium is the
most widely used mainly for convenience.
Meristem CulturesMeristem Cultures

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Stages of meristem culture technique:Stages of meristem culture technique:
Meristem CulturesMeristem Cultures
Culture Initiation
Shoot Multiplication
Rooting of Shoots
Transfer of plantlets to
Greenhouse & Field

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Explants:Explants:
Only the meristematic dome and 1 pair of
subtending leaves should be excised.
If larger pieces are taken, it is likely that the virus
will be transmitted.
The size of a meristem plus the subtending leaves
ranges from 0.1-0.5 mm.
The apical dome itself measures from 0.1-0.25
mm depending on the species. There is a balance
in size. The meristem tip must be small enough to
eradicate viruses andother pathogens, yet large
enough to develop into a shoot.
Meristem CulturesMeristem Cultures

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Meristem CulturesMeristem Cultures

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Culture Initiation:Culture Initiation:
surface sterilization of explants and establishing
them in vitro.
detection and elimination of contamination
Generally, a Growth Regulator-free basal medium
is used.
In cases of heavy contamination or endophytic
contamination (bacteria/fungi present inside
explant) a suitable antibiotic, e.g., trimithoprim,
and/or fungicide, e.g. Bavistin, may be added to
the culture medium.
Meristem CulturesMeristem Cultures

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Shoot MultiplicationShoot Multiplication::
After 2-3 weeks, the cultures are transferred to a
shoot multiplication medium designed to promote
axillary branching.
The medium generally contains:
a cytokinin (usually 1-2 mg/l, but up to 30 mg/l has been
used) either alone or in combination (BAP is the most
commonly used cytokinin, but with some species, e.g.,
blueberry, garlic, rhododendrons etc., 2- ip is much more
effective. )
with an auxin (commonly 0.1-1 mg/l), chiefly depending
on the plant species.
 NAA, IBA and IAA are generally employed.
2,4-D is not used as it promotes callusing.
Meristem CulturesMeristem Cultures

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Shoot MultiplicationShoot Multiplication::
Higher concentrations (>2 mg/l BAP) of cytokinin
induce adventitious buds and retard shoot growth.
The latter may necessitate a culture of shoots on
basal/low cytokinin/ GA3 medium for shoot
elongation before they can be rooted.
Therefore, a GR combination should be
determined to obtain optimum shoot multiplication
rates with the minimum risk of adventitious shoot
buds and, if possible, without the need of shoot
elongation step (to save time, labour and cost).
Meristem CulturesMeristem Cultures

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Rooting of ShootsRooting of Shoots::
the rooting medium has:
low salt, e.g., 1/2 or even 1/4 salts of the MS medium,
 reduced sugar levels (usually1g/l)
reduced salts being essential for rooting in some
species like Narcissus.
In some species, e.g., Narcissus, strawberry etc
rooting occurs on Growth Regulator-free medium.
But in most species, 0.1-1 mg/l NAA or IBA is
required for rooting.
In plants like Citrus, however, a pulse treatment
with an auxin (10 min with 100 mg/l NAA or IBA)
gives optimum rooting.
Meristem CulturesMeristem Cultures

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Rooting of ShootsRooting of Shoots::
Shoots are usually rooted in an agar medium mix
The cut ends of shoots are treated with a suitable
auxin solution or powder mix, transplanted in pots
and kept under high relative
humidity and low light intensity.
Rooting takes about 10-15 days, depending
mainly on species.
Plantlets with 0.5 to 1 cm roots are usually
transplanted into pots since longer roots tend to
get damaged.
Meristem CulturesMeristem Cultures

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Transfer to SoilTransfer to Soil::
Rooted shoots are removed from the medium
Agar sticking to their roots is washed with tap
water
Then transplanted into plastic cups containing a
suitable potting mix.
Plants are kept in a high (90%) humidity and,
initially low light intensities.
High humidity can be attained by:
 fog (water drops 10um or less)
 mist, or
 a clear plastic to cover individual (plastic bags) or
groups(plastic sheets) of plants.
Meristem CulturesMeristem Cultures

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Transfer to SoilTransfer to Soil::
The potting mix should not be too wet and water
not form on the plantlets.
Therefore fog is preferred over mist.
The humidity is gradually decreased to the
ambient level after about 7- 15 days, and the light
intensity is increased. The plants are finally
exposed to greenhouse conditions.
On a laboratory scale, individual plants may be
covered with clear plastic bags and irrigated daily
with 2-3 drops of water or ¼ MS salts. After 7-10
days, the bags may be removed gradually.
Meristem CulturesMeristem Cultures

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Meristem tip culture is used successfully to
remove viruses, bacteria, and fungi from
plants.
Why virus eradication works:Why virus eradication works:
Virus distribution is uneven in a plant and is
much less in a meristem.
Viruses cannot travel quickly enough through
plasmodesmata to keep up with actively
growing tip.
Meristem CulturesMeristem Cultures
Conclusion:

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http://hugroup.cems.umn.edu/Archive/Research/plant/http://hugroup.cems.umn.edu/Archive/Research/plant/
plant.htmlplant.html
http://www.plant.uoguelph.ca/research/embryo/synseehttp://www.plant.uoguelph.ca/research/embryo/synsee
ds.htmds.htm
http://www.ias.ac.in/currsci/jul25/articles18.htmhttp://www.ias.ac.in/currsci/jul25/articles18.htm
http://trc.ucdavis.edu/egsutter/plb171/lecturespdf4/14-http://trc.ucdavis.edu/egsutter/plb171/lecturespdf4/14-
Virus%20elimination.pdfVirus%20elimination.pdf
http://www.rocw.raifoundation.org/biotechnology/MScBhttp://www.rocw.raifoundation.org/biotechnology/MScB
ioinformatics/planttissueculture/lecture-ioinformatics/planttissueculture/lecture-
notes/lecture-08.pdfnotes/lecture-08.pdf
REFERENCE: