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Micropropagation – Tissue Culture

Banana
M. Sivaji*, M. Pandiyan, M. Yuvaraj, T. Thilagavathi, R. Sasmitha and M.
Suganyadevi
Agricultural College and Research Institute
Vazhavachanur, Tiruvannamalai, Tamil Nadu (606753), India

Corresponding Author
M. Sivaji
Email: [email protected]
OPEN ACCESS

Keywords

in vitro culture, Micropropagation, Clonal plants and Banana

How to cite this article:

Sivaji, M., Pandiyan, M., Yuvaraj, M., Thilagavathi, T., Sasmitha, R., and Suganyadevi, M. 2020.
Micropropagation – Tissue Culture Banana. Vigyan Varta 1(3): 28-32.

ABSTRACT
Micropropagation (or in vitro propagation) is the most common term used for clonal, true‐to‐
type propagation of plants produced into large number by using a variety of tissue, cell, and
organ culture methods. It implies the aseptic culture of small sections (i.e., explants) of tissues
and organs, in closed vessels with defined culture media and under controlled environmental
conditions. This method has five distinctive stage from explant to whole plant development.
Micropropagation, is one of the most commercially efficient and practically oriented
application of plant biotechnology, resulting in rapid generation of many clonal plants of many
plant species, which are in many cases virus‐ or other pathogen‐free. Besides many useful
applications, in crop improvement this method is useful to generate transgenic plants and
somatically bred plants (Somaclonal variants). Many types of horticulture and agricultural
crops i.e. banana, bamboo, rose, sugarcane etc. are successfully multiplied by this method of
propagation. This method is having lot of advantages over then other method of conventional
propagation method.

INTRODUCTION production of such clones is known as clonal

propagation. Production or development large

G
enerally, plants are propagated either amount of clonal propagation material with
sexually or asexually. Sexual small pieces of plants (explants) referred as
propagation exhibits high micropropagation. This means that only a small
heterogeneity unless the seeds are obtained amount of space is required to maintain or
from inbreeds. Whereas, asexual propagation multiply large number of plants. So
results in genetically identical progenies from micropropagation is defined as “Multiplication
the parent plant, known as clones. Method of

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of genetically identical copies of a parent plant such as rate of contamination and phenolic
under in vitro conditions, usually with an exudation. Plant tissues are commonly
accelerated proliferation of shoots during associated with bacteria and fungus. In tissue
subcultures”. It can also be simply called as “in culture media, presence of sucrose as a carbon
vitro clonal propagation”. source encourages their growth and
consequently kills inoculated tissues.
Stages of Micropropagation Therefore, adequate sterilization methods are
employed to eliminate microorganisms from
Currently, five stages procedure (0 to IV) is explant. Several sterilizing agents or surface
widely accepted and in practice (figure below). disinfectants such as sodium hypochlorite

(2%), calcium hypochlorite and saturated

Stage 0 — Mother Plant Selection: chlorine water are used. Adding some
antioxidants like ascorbic acid into culture
Before initiation of micro-propagation, media and incubating the initial period of
selection of suitable mother plant is crucial in primary culture in citric acid, reduced light or
the whole exercise of propagation. Generally, dark incubation of culture can minimize the
disease free mother explant is selected for the polyphenol compound damage. In addition,
micro-propagation to reduce contamination of liquid media and frequent sub culturing in
cultures. Certain growth parameters of mother liquid media are some of the promising
plant can be improved by pretreatment of methods in controlling phenolics.
mother explant before initiation of cultures (not
shown in figure). Stage II — Multiplication of shoots:

Stage I — Establishment of Aseptic Culture: The main objective of the stage II is the
multiplication of organs like shoot and
Micro-propagation begins with successful increasing their numbers considerably and
establishment of cultures of selected plant shoot elongation. They can give rise to new
material. The initial steps of micro-propagation individual plant. Multiplication of shoots
are generally associated with several hurdles
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through tissue culture involves four routinely shoot meristem is probably responsible for
used methods like: apical dominance. However, apical dominance
can be reversed by synthesis of cytokinin in
a) Callus mediated multiplication axillary buds or entry of cytokinin into the buds.
b) Adventitious shoots mediated In micro-propagation strategy mass
multiplication multiplication of plants can be accomplished by
c) By apical or axillary shoots enhanced axillary branching, which involves
placing nodal explant containing 1 to 2 axillary
Callus Mediated Shoot Multiplication: buds on the media in horizontal position (in
vitro layering) for enhanced branching.
Innumerable number of plants can be
Synthetic cytokinin like BAP and kinetin play a
accomplished by callus culture. In general
prominent role in releasing unsprouted axillary
auxins particularly 2,4, D favors callus
buds and its further proliferation. The shoot
induction in cultures. Ratio of auxins to
multiplication cycle can be repeated for several
cytokinins could play a decisive role in re-
times until millions of shoots are produced.
differentiation of shoots from the callus. The
callus system can be maintained for several Stage III — In Vitro Rooting:
weeks by sub-culturing every 4 weeks. Callus
mediated shoot multiplication creates Shoots or plantlets obtained during stage II are
variations among clones, this process called as exceedingly small and do not contain roots
Soma clonal variation. enable to grow in soil, even fails to utilize soil
nutrients. Therefore, adequate steps are taken in
In vitro Multiplication of Adventitious stage III to grow individual plantlets that can
Shoots: carry out photosynthesis and survive without
external supply of carbohydrate. Therefore, in
In this method, adventitious shoots arise
vitro grown shoots must be transferred to a
directly from the tissues of the explant and does
rooting media contain more Auxin and less
not involve callus mediated regeneration. The
cytokinin. In addition to the favourable role of
regeneration by direct organogenesis via
auxins in root induction, strength of media is
adventitious shoot initiation refers to initiation
especially important. Media containing half or
of shoots directly from stem, tuber, bulb, leaf
quarter strength of the salts can be useful in the
tissues other than leaf axils. Most of the
induction of roots in vitro. Supplying riboflavin
commercial practical propagation involves
and other adjuvants enhances root induction
adventitious bud formation. The main
process in stage III.
advantage of micro-propagation by direct
adventitious shoot regeneration produce Stage IV — Transplantation or Hardening:
numerous small shoots rapidly from each
explant, so propagation ratio is extremely high, Once in vitro rooting process completes,
which is advantageous for commercial plantlets are ready to be transferred from the
ornamental industry to produce large number of aseptic tissue culture container into the soil.
plantlets. Tissue culture plants generally lack protective
cuticle layer and hairy roots, therefore are
Axillary Shoot Proliferation: subjected for light and temperature shock. High
rate of water loss due to lack of protective
Axillary shoots developed from axillary buds
cuticle layer and root hairs, therefore every
present in the axils of each leaf. In leaf axils
chances of exposure of seedlings to light and
unsprouted status of axillary buds is due to
temperature shock. Plantlets are not developed
apical dominance exhibited by growing shoot
on their own. The photosynthetic minimum
tip region at the top. Synthesis of auxin in apical
period (7 days) is required for them to be
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capable of producing their own source of • Poor Acclimatization to the field is a

carbon. In addition to these problems, in vitro common problem (hyperhydricity)
grown roots are highly tender and their vascular • Risk of genetic changes if 'de novo'
systems are poorly developed. In order to regeneration is used
compound these problems regenerated plants • Mass propagation cannot be done with all
are subjected to hardening and is carried out by crops to date. In cereals much less success
removing test tube grown plantlets and washed is achieved.
under distilled water to remove media sticking • Regeneration is often not possible,
to the basal portion of the shoots, plantlets are especially with adult woody plant material.
then placed in sterilized soil or humus soil plus
sand (1:1) or vermiculate soil. These are Tissue culture Banana
completely covered by plastic sheet or directly
placed in green house to provide 90 to 100 Banana is an important fruit crop, widely grown
percent humidity for upto 15 days. Humidity is in developing countries. Bananas are generally
gradually reduced to 90% then to 80% and propagated by vegetative means, which has
finally to 70%. The acclimatized plants are then many disadvantages attributed to low quality of
transferred to the field. planting material (pest and disease infested),
fidelity and easy availability. Micropropagation
Advantages of Micropropagation: in banana is feasible with advantages of high
multiplication rates and production of high-
• Production of large number of plantlets in quality planting materials with high genetic
short duration, thus helping in rapid fidelity.
introduction and dissemination of new
varieties. Banana shoot tip culture
• A high degree of genotypic and phenotypic
uniformity of the progeny plants can be A. Initiation of shoot tip culture:
maintained.
• Shoot tips can be extracted from the
• Season independent production of planting
pseudostem, suckers, peepers or lateral
material can be achieved.
buds which contain a shoot meristem and
• Micropropagated plants exhibit uniform
they are taken from healthy disease-free
growth and maturity.
mother plants for shoot tip culture.
• Production of disease-free planting
• The suckers are carefully cut to expose the
material can be achieved.
shoot tip of 10 cm3 and cut further to about
• Micropropagation enables growers to
3 cm diameter and 5 cm length.
increase the production of plants that
• To prevent browning of shoot tips, they are
normally propagate very slowly such as
stored in antioxidant solution (100 mg
Narcissus and other bulbous crops.
Ascorbic acid + 150 mg Citric acid per litre
• It would be possible to rapidly multiply
of sterile water), till they are moved to
endangered species and those that are
laminar airflow chamber.
difficult to propagate vegetatively through
• Afterwards, the shoot tips are disinfected
conventional methods.
with 70% ethanol for 30 seconds and later
Disadvantages of Micropropagation with 0.1 % mercuric chloride for 10
minutes. Surface sterilized shoot tips are
• Expensive laboratory equipment and washed three times using sterile water. The
service outer surface of explant exposed to
• No possibility of using mechanization sterilizing agent is removed and the
• Plants are not autotrophic explants trimmed using surgical blade to

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bring the final size to about 3-4 cm length C. Rooting:

and 1-2 cm diameter.
• Decapitation and wounding of shoot tips • After 5-6 subculture cycles, the
are carried out to overcome apical proliferated buds are transferred to rooting
dominance and to encourage axillary bud medium containing auxin and activated
proliferation. The explants are inoculated charcoal. After a month, the rooted
under sterile conditions inside Laminar plantlets are ready for hardening.
Airflow Chamber.
D. Hardening:
• The optimum incubation temperature
should be in the range of 24-26 °C.
• Once the plantlets are ready for shifting
Generally, the light intensity is maintained
outside the laboratory, they are carefully
at 1,500-3,000 lux. Initially, the cultures
acclimatized to adapt to the green house
are maintained at 16 h light/8 h dark cycle.
and later to least protected field conditions.
B. Shoot multiplication: During hardening, the plantlets undergo
physiological adaptation to changing
• First subculture is done after 20-25 days of external factors like water, temperature,
initiation when the explants turn green in relative humidity, and nutrient supply.
colour. For sub-culturing, the outer dead • The hardened plantlets are carefully
tissue from the base of explant is removed maintained in green house until
and one or two leaf bases are peeled till the transportation.
fresh meristematic tip gets exposed.
REFERENCES
• During the second subculture, the central
meristem produces clusters of
George, E.F. and Sherrington, P.D., 1984. Plant
proliferating buds and 1-3 axillary buds get propagation by tissue culture: handbook
regenerated from the basal parts of and directory of commercial
explants around the central apical laboratories, Part 1. Exegetics, Westbury,
meristem. UK.
• In further sub-culturing, the explant is cut
into three to four pieces and each slice with Maene, L. and Debergh, P., 1985. Liquid
two to three proliferating clusters is medium additions to established tissue
cultures to improve elongation and
inoculated to individual culture bottles.
rooting in vivo. Plant cell, tissue and
• During fourth and fifth subcultures, a
organ culture. Vol.5, No.1, pp:23-33.
single clump contains about 15-25
proliferating shoots. http://www.biologydiscussion.com/biotechnol
• To minimize somatic variation, the sub- ogy/clonal-propagation/micro-
culturing is restricted to a maximum of propagation-technique-factors-
seven cycles when each bottle contains 25- applications-and-disadvantages/10732
30 plantlets with well-developed shoots
and roots.

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