MICROPROPAGATION OF MEDICINAL & AROMATIC PLANTS / CLONAL PROPAGATION

MICROPROPAGATION
OF MEDICINAL AND
AROMATIC PLANTS
V.K.VIKRAM VARMA
M PHARMACY
(PHARMACOGNOSY)
SECOND SEMESTER
SPER
JAMIA HAMDARD
7 March 2020V.K.VIKRAM VARMA
1

 MICROPROPAGATION
• Micropropagation is the production of whole plant from small
section of plant such as stem tip, node, meristem, embryo or even a
seed
• It is a advanced vegetative propagation technology for producing
large number genetically superior and pathogen free transplants in
a limited time and space
• Multiplication of genetically identical copies of a cultivar by asexual
reproduction is called Clonal propagation.
2

CONTD.
Propagation
Asexual
With the help of
seeds
Sexual
Only by the
vegetative parts.
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 STEPS OF MICROPROPAGATION
• Murashige proposed three (I to III) stages, Debergh and Maene added stage ‘0’.
Currently we have accepted five stages procedure (0 to IV)
oStage 0- Selection & preparation of mother plant (sterilization of the explant
tissues)
oStage I- Initiation of culture (Explant placed into growth media)
oStage II- Multiplication (Explants transferred to shoot media & shoot can be
constantly divided)
oStage III- Rooting (Explant transferred to rooting media)
oStage IV- Hardening (Transferring to soil)
4

CONTD.
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 Selection & preparation of mother plant
• This stage was basically introduced to overcome
the problem of contamination. Stock plants are
grown under more hygienic conditions to reduce
the risk of contamination.
• EXPLANT: Cell, Tissue or Organ of a plant that is
used to start in vitro cultures.
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Initiation of culture
• Explant isolation –any part of the plant can be used
as explant like vegetative parts (Shoot
tip, meristem, leaves, stems, roots) or reproductive
parts (Anthers, pollen, ovules, embryo, seed,
spores). Shoot tip and auxiliary buds are most often
used
• Surface sterilization – Explants are surface sterilized
by treating it with disinfectant solution for a specific
period. Ethyl alcohol, bromine water, mercuric
chloride, silver nitrate, sodium hypochlorite,
calcium hypochlorite etc can be used as
disinfectant.
7

CONTD.
• Washing – Washed with water.
• Establishment of explant on appropriate medium –
There is no one universal culture medium; however
modifications of Murashige and Skoog basal
medium (Murashige and Skoog, 1962) are most
frequently used.
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 Multiplication of shoots
• In this stage, rapid multiplication of the
regenerative system is carried out for obtaining
large number of shoots.
• Stage II mainly involves multiplication of shoots or
rapid embryo formation from the explant. A
growth chamber set at 20–24 °C is used, with a
2000- to 4000-lux light intensity, & a lighting
period of 16 hours or so.
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 Rooting of regenerated shoots or germination of
somatic embryos in vitro
• In this stage, shoots or shoot clusters from
stage II are prepared to transfer to soil.
Shoots are separated manually from
clusters and transferred on a rooting
medium containing an auxin.
10

 Hardening
• Transfer of plantlets to sterilized soil for
hardening under greenhouse
environment.
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 SYSTEM USED TO REGENERATE PLANTLETS BY
MICROPROPAGATION
CELL SUSPENSION
 Produced from friable callus
 Maintained in shaker cultures or bioreactors
PROTOPLAST CULTURE
 Cell culture without cell walls (cellulose added to degrade cell walls)
 Only Plasma membrane remains
 Osmotic pressure is maintained to prevent cell walls from rupturing
(mannitol used)
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 METHODS OF MICROPROPAGATION
AXILLARY BUD PROLIFERATION APPROACH
 Meristem and shoot tip culture
 Bud culture
ORGANOGENESIS
 INDIRECT
 DIRECT
EMBRYOGENESIS
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 AXILLARY BUD PROLIFERATION APPROACH
 Meristem and shoot tip culture
oMorel and Martin (1952) develop the
technique of meristem culture for in vivo
virus of Dahlia from of Dahlia Mosaic
virus(DMV) .
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15
https://dahlia.org/wp-
content/uploads/2018/01/ADS-
DMV_Symptoms_Slides.pdf
Dahlia Mosaic virus(DMV)
CONTD.

oG. Morel (1965) was
developed the technique
shoot tip culture for micro
propagation of orchid
Cymbidium.
oThis method is more
successful in herbaceous
plant.
16CONTD.

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• Buds contain active meristem depending upon the physiological state of
the plant. The various types used in bud culture,
oSingle node culture
oAxillary bud culture
 BUD CULTURE

 ORGANOGENESIS
• Indirect:
oThis pathway includes a callus stage.
oCallus is undifferentiated tissue that develops on or around an injured or
cut plant surface.
• Direct:
oThis pathway is bypasses a callus stage.
oThis method is particularly suitable to herbaceous plants.
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 ORGANOGENESIS FLOW DIAGRAM
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Direct organogenesis
• Auxin/cytokinin 10:1-100:1 induces
roots
• Auxin/cytokinin 1:10-1:100 induces
shoots
Indirect organogenesis
• Intermediate ratios around 1:1 favour
callus growth

 EMBRYOGENESIS
• The process of initiation & development of embryos & embryo like structure
from somatic cells.
• It usually involves a callus intermediate stage which can result in variation
among seedlings.
• Its not a common micropropagation technique but currently being used to
produce superior pine seedlings.
oDirect embryogenesis
oIndirect embryogenesis
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EMBRYOGENESIS
FLOW DIAGRAM

 HOW DOES MICROPROPAGATION WORK?
• Plant cell have the ability to reproduce the whole plant from a single
plant from a single cell. This is called totipotency.
• Totipotency is the ability of single cell to express the full genome in
the cells to which it gives rise by cell division.
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 EXAMPLE: CARROT
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 EXAMPLE:
BANANA

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 EXAMPLE: SUGARCANE

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 EXAMPLE:
POTATO

 ADVANTAGES & DISADVANTAGES
ADVANTAGES
• One to many propagules rapidly.
• Multiplication in controlled lab
conditions.
• Continuous propagation method.
• Potential for disease free plant.
• Inexpensive per plant once established.
• Reduce stock plant space.
DISADVANTAGES
• Specialised equipment requirement.
• More technical expertise required.
• Plant produced may not fit industry
standards.
• Relative expensive to set up.
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 APPLICATIONS
• Soma clonal variation
• Germplasm conservation
• Mutation breeding
• Inducing mutation
• Embryo culture
• Haploid & Dihaploid production
• Molecular farming
• Invitro hybridisation- protoplast
fusion
• Production of disease free plants
• Genetic engineering
• Production of secondary
metabolites
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 REFERENCE
• www.epgpathshala.com
• www.youtube.com
• www.wikipedia.com
• http://theagricos.com/tissue-culture/micropropagation/stages-of-micropropagation/
• http://www.biologydiscussion.com/plant-breeding/micro-propagation/micro-propagation-methods-and-
stages-biotechnology/61317
• https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/micropropagation
• http://www.sakshieducation.com/Story.aspx?nid=73282
• https://microbenotes.com/micropropagation-stages-types-applications-advantages-limitations/
• www.slideshare.com
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MICROPROPAGATION OF MEDICINAL & AROMATIC PLANTS / CLONAL PROPAGATION