MICROPROPAGATION

Generally plants propagate by sexual and asexual methods.
Sexual method: here fertilization of ovary taken lace pollen grains and the resulting plants show high degree of heterogenecity.
Asexual Methods: here the cells undergo mitosis and the resulting plants will be genetically identical to parents.
Multiplication of genetically identical copies of plants is known as clonal propagation.
Clonal propagation of plants through tissue culture is known as Micropropagation.
Explants (starting material used to initiate tissue culture
The explants widely used for tissue culture include
a) Meristem
b) Shoot-tip
c) Auxiliary buds
a) Meristem
This is the terminal portion of shoot tip containing a group of actively dividing cells.
b) Shoot-tip
Shoot-tip/ shoot apex also contains a group of actively dividing cells with one-three leaf premordia.
c) Auxiliary buds
These are actively dividing cells present in the axile portion of the node.
Murashige in 1978 recognized four stages (stage I, II, III, IV) for Micropropagation. Stage I, II, III is performed in-vitro. Later in 1981, Debergh and Maene introduced stage 0 for Micropropagation.
The stages include:
a) Stage 0:- selection of stock plant
b) Stage I: - Establishment of aseptic culture.
c) Stage II: - multiplication of explant on defined medium.
d) Stage III:- Rooting
e) Stage IV: - hardening
Stage 0
Stock plants having derived charades are selected.
Maintained in controlled environment conditions for 3 months
They are grown in low humidity, irrigation and without systematic microbial infection.
Stage I
The selected explants (derived from stock plants) are prepared for inoculation.
The explants are surface sterilized by using chemicals such as 0.1% Hgcl2 or 5% sodium hypochlorite or 70% alcohol or a combination of all these chemicals.
These surface sterilized explants are then inoculated onto MS medium supplemented with vitamins, sucrose and growth regulators.
These cultures are incubated at 3000-10000Lux light intensity with 16hrs photoperiod.
Note: Auxin stimulates callus formation.
Cytokinin (1 – 3 mg/l BAP) is good for Micropropagation.
Stage II
This is the longest period.
Single shoots develop from apical shoots.
These are excised into nodal explants.
The nodal explants are further inoculated on cytokinin medium to proliferate multiple shoots.
Note: Multiple shoots can also be obtained directly from explants by organogenesis or somatic embryogenesis)
In most plants, explants are known to produce 1-3 shoots in 4-5 weeks. This would give upto 510-612 plants in one year if all plants survive.
Stage III
Individually produced shoots in stage II are inoculated in fresh medium (with auxins) for rooting.
Or in some cases rooting is induced directly in the soil in high moisture condition.
In case of somatic embryos, they are allowed to germinate in the medium and then transferred to soil.
The plantlets obtained are slowly transferred to soil for hardening.
Stage IV
The plantlets are first prepared for soil conditions by keeping them in medium containing peat /vermiculite/fearlite which holds more moisture.
This makes plants to become resistant to moisture, stress and disease making plants completely autotrophic from their heterotrophic.
The plantlets are protected from direct sunlight.
Humidity is gradually decreased.
During this period, plants will form well developed roots and the aerial tissues will for cuticular wax.
Thus, the plants acclimatize themselves and become suitable for transfer into the field.
Note:
Some species grow in vitro (in tab) from brittle, glassy and water soaked shoots and this is known as vitrification.
Vitrification is due to poorly developed vascular bundles, abnormal functioning of stomata etc.
This can be overcome by addition of high concentration of agar (1%), bottom cooling of culture tubes etc.
Applications of Micropropagation
1. Alternative method of vegetative propagation.
2. A small amount of plant tissue is sufficient to produce millions of clones in a year
3. Requires less space for large number of plants.
4. Plants with high yield and vigour can be obtained.
5. Disease free plants are produced from this method.
6. Helps in germplasm storage and saving of endangered species.
7. Provides speedy international exchange of plant material.

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