Name: SIDRA PERVEEN
Roll No; BBOF16MM048
Subject: CLONAL PROPAGATION THROUGH TISSUE
CULTURE TECHNIQUE
Class: BS Botany 8th
Submitted To: DR. AMIR MUHAMMAD KHAN
Department Of Biological Sciences
Clonal Propagation Through Tissue Culture
Technique:
Definition:
1. Multiplication of genetically identical copies of a cultivar by asexual reproduction
called “Clonal Propagation”.
2. Asexual propagation of many new plants from an individual all with the same
genotype. In vitro clonal propagation is a type of micro propagation. Gives rise to
genetically identical plants. Identical also to the parent.
Introduction:
Due to rapid deforestation and depletion of genetic stocks, concerted efforts must be
made to evolve new methods for mass propagation and production of short duration trees with a
rapid turnover of biomass and induction of genetic variability for the production of novel fruit
and forest trees which are high yielding, resistant to pest and disease associated with increased
photosynthetic efficiency. Tissue culture techniques have already revolutionized the mass scale
propagation of many horticultural crops. The following are some of the areas of tissue culture
which are of prime interest in forestry.
Clonal Propagation in Trees:
The clonal multiplication reproduces clones, which contain all the genetic information of
the parent tree. The term clone is used to mean a genetically uniform plant material derived from
a single individual and propagated exclusively by vegetative means.
Clonal selection and deployment are receiving attention as an intensive forest
management tool for increased wood production. Many wood-based industries in particular, pulp
and paper industries are involved in plantation establishment program using clonal forestry
approaches in the recent past.
Clonal propagation Benefits:
Variability arising from sexual reproduction and seed formation is omitted.
Plants with long seed dormancy can be raised faster than in vivo seed propagation.
Undesirable juvenile phase seen in seed raised plants does not appear in vegetatively
propagated plants from adult material.
Somatic Embryogenesis:
Introduction:
A plant reproduces naturally through the development of zygotic embryos.
Schematic representation of the major stages in zygotic embryo development from
pollination to germination.
But in somatic embryogenesis, the plant is derived from a single somatic cell or group of
cells.
It therefore differs from the natural pathway of plant reproduction.
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..
Somatic Embryogenesis Process:
The process has four main phases:
Initiation & Proliferation
of embryogenic tissue:
Explain is placed on a Petri dish containing Initiation medium (to initiate cell division).
After 6 weeks some of the growing tissue converts into embryogenic tissue (white, fluffy
& translucent appearance).
Embryogenic tissue continues to proliferate as long as its in the initiation medium.
Embryogenic tissue can also be frozen & stored in liquid nitrogen for future somatic
embryogenesis.
Initiation Medium:
Auxin:
2,4-D (2,4-dichlorophenoxy acetic acid)
2,4,5-T (2,4,5-trichlorophenoxyacetic acid)
Inorganic components: potassium.
Organic components: proline.
The inorganic and organic components modulate embryogenesis or callus response.
Maturation Of Somatic Embryos:
Stop proliferation and allow tissue to form mature somatic embryos.
Clumps of embryogenic tissue transferred to maturation medium containing plant growth
regulator.
Regulator promotes maturation.
After 6 weeks mature embryos begin to appear on the clumps.
They resemble embryos found in seeds.
Pre-maturation:
From globular to torpedo
Solid BOi2y medium lacking 2,4-D
Maturation Phase 1:
Enriched BOi2Y medium; contains a high level of sucrose, nitrogen and Sulphur
Deposition of storage reserves
Embryos accumulate fresh and dry weight.
Maturation Phase 2:
Modified BOi2Y containing ABA induces desiccation tolerance.
Maturation Of Somatic Embryos:
Germination of Somatic Embryos:
Individual mature embryos are picked from clumps.
Then placed onto germination medium.
Medium contains a mixture of nutrients for early plant development. (1/2 MS salts +
1%sucrose).
Mature embryos germinate to form roots and shoots.
The plants obtained are referred to as “somatic seedlings” / “somatic derived
plantlets”.
Greenhouse Culture & Field Planting:
When germinated emblings are large enough they are transplanted.
Into soil for further growth and acclimatization (Greenhouse).
After normal greenhouse culture period they are planted in the field.
Applications of Somatic Embryogenesis:
Provision of cell lines for:
Genetic engineering
Long term gene storage
Use in research
Tree improvement programs
Pest resistant
Fast growing
More productive (high value plantation forests; reduce need to harvest existing
natural forest; conservation)
Meristem Cultures
Introduction
The cultivation of 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 meristemthe 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.
Its developed from meristamatic cells found at the shoot tip.
Shoot tip is normally free of contaminating organisms.
Produces pathogen free clones of plant.
E.g. potato, dahlia, strawberry etc.
Explants used:
The apical
meristem
and the first 1 or 2
leaf primordial
below it are
excised from the
shoot tip (the
meristem itself is not able to grow independently unless some leaf primordial are
retained).
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.
Stages of meristem culture technique:
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 and other
pathogens, yet large enough to develop into a shoot.
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 (bacteria/fungi present inside ex plant) a suitable
antibiotic, e.g., fungicide, e.g. Bavistin, may be added to the culture medium.
Shoot Multiplication:
After 2-3 weeks, the cultures are transferred to a shoot multiplication medium designed to
promote auxiliary branching.
The medium generally contains:
A cytokinins (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 cytokinins, 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.
Higher concentrations (>2 mg/l BAP) of cytokinins induce adventitious buds and retard
shoot growth. The latter may necessitate a culture of shoots on basal/low cytokinins/
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 and cost).
Rooting 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.
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.
Transfer 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.
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.
Conclusion:
Meristem tip culture is used successfully to remove viruses, bacteria, and fungi from
plants.
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.
Reference:
https://www.slideshare.net/breal85/clonal-propagation-tissueculture?from_action=save
http://vikaspedia.in/agriculture/forestry/clonal-forestry
https://www.sciencedirect.com/science/article/pii/S0928342096800188
