8/2/2021 Banana Expert System

Planting Material
SUCKERS
SEED
 
MICRO-PROPAGATED PLANTS
MACRO-PROPAGED PLANTS

Suckers
In India, use of conventional suckers cannot be completely avoided because of some regional special varieties
tissue culture technique is yet to be perfected. further for resource poor small scale growers the cost of tissue
culture plants stills a hurdle in adoption. there are three types of planting material used for propagation namely,
Suckers, Peepers,Rhizome.

Natural regeneration of cultivated bananas through suckers is very slow due to hormone-mediated apical
dominance of the mother plant.
A plant produces only 5-20 suckers during its life time of 12-14 months. For accelerating the propagation
rate, suckers with growing buds or cut rhizomes called ‘bits’ and  peepers’ are used.
Several good bits, each with a centrally placed germinating eye can be cut from an unbunched rhizome
after trimming the roots.
Selection of appropriate mother plant for raising new propagules either through in vivo or in vitro methods
is important.
About one kilogram uniformly sized rhizomes or bits, well-trimmed around the growing sprout are the best
starting material.
In some parts of India rhizomes are sun dried for 2-3 days after paring and pralinage treatment (trimmed of
all roots, dipped in mud slurry and sprinkled with nematicide) and stored in shade for a week before
planting.

Selection of mother plants

Mother plant should be  healthy, true to type and free from diseases and pests,especially virus diseases.
 The male flowers buds should be retained to check the presence of virusdiseases (male flower buds
exhibit symptoms of late infection of viruses like BBTV and BBrMV).
Mother plants should be raised under roofless insect proof shade net withsufficient height.
Mother nursery must be located away from other banana plantations with an isolation distance of 500 m to
maintain purity and to avoid spread of virus diseases.
Mother plants should be grown under very good management conditions so as to facilitate the true
expression of traits.
Individual plants should be tagged with a master code number so that the plantlets developed could be
traced back to the mother plant.
Pedigree record and source of each mother plant should be maintained and catalogued.
Once indexed, the mother suckers can be maintained in field or concrete rings with frequent decapitation
to facilitate production of more auxiliary buds. They also serve as explants for culture initiation.

Suckers

a) Sword Sucker

A sucker next to but only superficially attach to the mother rhizome

with broad leaves at an early stage. Water suckers produce inferior
fruit (not healthy banana clump) and are therefore not recommended.

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b) Water sucker

A sucker next to but only superficially attach to the mother rhizome with
broad leaves at an early stage. Water suckers produce inferior fruit (not
healthy banana clump) and are therefore not recommended.

Peepers
Peepers (very young suckers) produce late and poor crop. Four month
old suckers and split rhizomes(each having about 2.0 Kg) produced
heavier bunches compared with those obtained from peepers.

Rhizomes
Whole or split rhizomes can also be used when suckers are not
available. Bits of rhizome of 2.0 Kg or more may be planted in the
nursery for sprouting or directly sown in the main field. for quick
multiplication of a variety rhizome bits may be used. Though the plants
will require little longer time to fruit.

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Seed

Wild banana fruit Seed

Seed propagation is common in wild species which are diploid and undergo normal meiosis, fertilization
and seed set.
The extent of seed set, germinability and dormancy depends on the species.
In  Ensete, the only other genus of Musaceae, seed propagation is the only means of perpetuation since
sucker production is absent.
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Seeds are generally brown to black in colour, 2-6 cm in diameter, round or triangular in shape, and mostly
compressed in appearance.
Fruits of wild species are inedible, being full of seeds that are enveloped in thin mucilaginous pulp
In India, this propagation system is not followed because all cultivated commercial bananas are
parthenocarpic.   

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Micro-Propagated Plants
Introduction
Micropropogation is the practice of rapidly multiplying stock plant material to produce a large number of progeny
plants under aseptic conditions, using modern plant tissue culture methods.
Steps involved in the micro propagation of banana :

1. Selection of pedigree mother plant.

2. .Establishment and maintenance of mother block nursery.

3. Selection of superior planting material for in vitro initiation.

4. .Rouging at various stages of proliferation.

5. Rooting, primary hardening and rouging.

6. Secondary hardening and rouging off-types .

7. Virus indexing and genetic fidelity testing at various stages of micro propagation.

Tissue culture protocols

Selection of superior initial planting material

The sucker should be healthy and not less than 60-80 days of age.Growing meristem should be of 1.0
cubic cm in size.
Micropropatation of Musa involves establishment of aseptic culture of shoot tips. This is achieved by
disinfection,excision and incubation of explants.
NaCl is the common disinfectant and the laboratory grade is normally used at concentrations ranging from
0.5-1.0%.
If the explant is disinfected after excision a shorter treatment time (5 minutes).Sucrose is the most
preferred carbon source used at a concentration of 2-4%(W/V).Vitamins, thiamine, Nicotinic acid and
pyridoxine are frequently used.
Amino acid glycine is being used as an imediate source of nitrogen cultured tissues.Commonly used
reducing agents are ascorbic acid – citric acid (1.0 and 1.5% W/V respectively) which are used in various
stages like disinfecting solution itself or after disinfection.Auxins and cytokinons are commonly used for
rooting and shooting respectively.

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The widely used auxins are IAA, NAA and IBA. BAP is the cyotkinin of choice for in vitro shoot bud
proliferation.Ph is usually maintained at 5.8, which is prone to changes over culture duration.
The optimum incubation temperature should be between 24-26°C.Generally the light intensity maintained
ranges from 1,500-3,000 lux.Higher levels of 3,000-10,000 lux during the later stages improve the survival
rate of plant lets up on transfer to soil.

Culture initiation

Shoot cultures of banana start conventionally from any plant part that contains a shoot meristem, i.e. the
parental pseudostem, small suckers, peepers and lateral buds.
The apex of the inflorescence and axillary flower buds are also suitable explants for tissue culture initiation.
Overall, it is important to select explant material from preferably mature individuals.
For rapid in vitro multiplication of banana, shoot tips from young suckers of 40-100 cm height are most
commonly used as explants.
 From the selected sucker a cube of tissue of about 1-2 cm³ containing the apical meristem is excised.
This block of tissue is dipped in 70% ethanol, surface sterilized in a 2% sodium hypochlorite solution, and
after 20 min rinsed three times for 10 min in sterile water.
Subsequently a shoot tip of about 3 × 5 mm, consisting of the apical dome covered with several leaf
primordia and a thin layer of corm tissue, is aseptically dissected.
Larger explants have the merit of consisting of a shoot apex bearing more lateral buds which rapidly
develop into shoots.
The explant is then further reduced in size (0.5-1 mm length), leaving a meristematic dome with one or two
leaf initials.
Meristem cultures have the disadvantage that they may have a higher mortality rate and an initial slower
growth.
The explant is placed directly on a multiplication-inducing culture medium. For banana micropropagation,
MS-based media are widely adopted.
Generally, they are supplemented with sucrose as a carbon source at a concentration of 30-40 g/l.
Banana tissue cultures often suffer from excessive blackening caused by oxidation of polyphenolic
compounds released from wounded tissues. These undesirable exudates form a barrier round the tissue,
preventing nutrient uptake and hindering growth. Therefore, during the first 4-6 weeks, fresh shoot-tips are
transferred to new medium every 1-2 weeks.
Alternatively, freshly initiated cultures can be kept in complete darkness for one week.
Antioxidants, such as ascorbic acid or citric acid in concentrations ranging from 10-150 mg/l, are added to
the growth medium to reduce blackening, or the explants are dipped in antioxidant solution (cysteine 50
mg/l) prior to their transfer to culture medium.
Usually two types of growth regulators, a cytokinin and an auxin, are added to the banana growth medium.

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In most banana micropropagation systems, semi-solid media are used. As a gelling agent agar (5-8 g/l) is
frequently added to the culture medium. Liquid media are superior for shoot multiplication, but for
maximum plant production and survival ex vitro, one culture cycle on semi-solid medium is also needed.

Culture proliferation

First subculture is done after 4 weeks of inoculation.By this time, explants enlarge to about double the
original size.The blackened surface is scraped off and corm base is reduced to 0.5cm thickness.
Reducing the thickness of basal corm tissue decreases blackening,eventually chances of proliferation of
non-meristamatic tissue outgrowing the meristamatic tissue.Any growing apical shoots, is cut to activate
the axillary buds.By the end of first subculture shoots may be seen with 1-3 side buds. The blackened
surface is scraped off, and vertical cuts given in subculture-I are extended till the corm base, so that four
quardrants are obtained.By the end of second subculture cycle is repeated at 4 weeks interval to increase
the proliferation rate. After 5-6 subculture cycles, the proliferated buds are put in a regeneration medium
containing 1/10cycles, the proliferated buds are put in a regeneration medium containing 1/10th BAP.The
shoots develops are then rooted in 1/2MS medium with IBA and activated charcoal.After a month, the
rooted plants are ready for hardening.

Hardening

Micropropagated plants are delicate in nature as they are grown under artificial conditions of High humidity
and optimum light intensity.Rooted shoots of 6-10cm tall with well ramified roots are washed free off agar
medium and taken to the micropots containing soilrite and then shifted to growth tunnels for further
establishment.After an appropriate period of hardening, the plants are taken out of the micorpots roots are
dipped in a fungicidal solution to reduce the risk of damage by fungal diseses.Then repotted in a mixture
containing 1:1:1:1 of soil:sand:soilrite and FYM.
Once after repotting they are shifted under 75% shade nets where they are exposed to 60-70% RH slight
intensity of 40-45 mol/m2/sec for about 10-12 days.Next stage , they are maintained at 50-60%  RH and
light intensity of 200 mol/m2/sec for about 10-15 days and at an intensity of 600 mol/m2/sec for another
month.These plantlets are shifted to polybags containing 1:1:1 of sand :soil:FYM and maintained in green
house (light intensity of 600-700 mol/m2/sec)until field planting.During primary and secondary hardening
plants should be roughed for somaclonal variations. This could be for vegetative deformities,foliar
deformities like variegation, rosette foliage others like dwarfism etc.Important points to be taken care
during secondary hardening ofTC plants are as follows:

1. The rooting media should be 100 per cent free from pathogen.

2. Water used for irrigating the plants should be free from pathogens and nematodes.

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3. Strict rouging by trained personnel should be done to remove the types once in 7 days

4. Sample plants from each batch should be randomly virus indexed (atleast 10 plants from each
batch/explants).
5. While shifting of primary hardened plants, two longitudinal cuts of net pots should be given to facilitate
further corm growth.

Manuring and plant protection in nursery

Plantlets should be 2-3 weeks old before any fertilizer application is taken up.100ml water containing 05.kg
Urea, 2g Super phosphate and 1g Muriate of potash can be applied per plant.The manuring is repeated by
doubling the dosage after three weeks.
Spraying of commercially available micronutrient mixtures during sixth week would help in better
establishment both in nursery and field.Sanitary measures are strictly adopted in the nursery to avoid the
risk of damage by pests and diseases.
Pest and diseases encountered during the process of hardening are to be given due attention at
appropriate times especially foe soil borne nematodes and aphids which transmit BBTV.The maximum
number of subculture cycles should be adhered to 7 or less to check off types to the minimum.

Ideal tissue culture plant

1. A well hardened plant should be minimum of 30 cm in height and should have a pseudostem
circumference of 5.0-6.0 cm after 45-60 days of hardening.
2. .The plant should have 5 photosynthetically active leaves and inter foliar space must be not less than 5.0
cm.
3. The plant should have approximately 25-30 active roots at the end of secondary hardening stage.

4. The length of active roots should be more than 15 cm with a good number of secondary roots.

5. The poly bag should be size (20.0cm in length and 16 cm in diameter)with potting media filled to ¾ full of
the bag.
6. The media/potting mixture approximately should weight about 750-800g.On dry weight basis.

7. Plantlets should be free from any visual symptoms of leaf spot, pseudostem rot and physical deformations.

8. Plantlets should be free from the presence of root pathogens like Erwinia rot symptoms, nematode lesions
and root knots.Random checking of roots is very essential at the time of procurement.
9. Those exhibiting abnormal growth must be discarded.

Management of pest and diseases during secondary hardening

Erwinia head rot or Tip over disease

The disease is widespread in banana growing areas of the world.The casual agent is Erwinia carotovora
(Jones).This disease is also noticed in the secondary hardening stage of banana tissue culture production.

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Rotting of collar region is the commonest symptom this disease.The leaves of affected plant show epinasty
and dry out suddenly.If the affected plant are pulled out, the plant topple from the collar region leaving the
corms with their roots where as plants affected with nematodes cab be pulled out along with corm and
roots.This is a distinguishable feature for soft rot disease.
In early stage of infection dark brown or yellow, water soaked areas are visible in the cortex
area.Drenching of tissue culture plants kept in polybags with 0.1% Emisson or 2% bleaching powder can
control the disease.

Parasitic nematodes infestation

Banana tissue culture plants are highly susceptible to parasitic nematodes such as root knot nematode
(Meloidogyne incognita), lesion nematode (Paratylenchus coffeae), burrowing nematode (Radopholus
similes) and spiral nematode (Helicotylenchus multicinctus).These nematodes enter the plant roots
through contaminated water or through the pot mixture used for hardening of tissue culture plants.
The tender toots of affected tissue culture plant become weak and cannot absorb nutrients as the roots are
damaged.In case of root-knot nematodes are predominantly present in the primary and secondary
hardening stage of tissue culture plants.
In case burrowing or lesion nematode infection, the roots exhibit extensive reddish brown lesions in the
cortex can be seen when cut longitudinally.Use water, soil and other pot mixture material free of parasitic
nematodes.
Fumigated pot mixture can be used for secondary hardening.Application of 2-3gm of carbofuran per plant
in the secondary hardening stage can protect against invading nematodes.Appliction of VAM or neem cake
also would ensure vigour and health of the plants.

Factors to select off-type production

1. Off-type can arise at any time in culture.

2. They may arise from stimulation of adventitious buds.

3. Growth regulators don’t directly induce mutagenesis (their effect is indirect).

4. Nursery screening procedures are available for rouging off-types.

5. Cavendish types can be identified by morphological characters.

6. Molecular markers can be used.

Tissue culture Unit

Infrastructure
Lab Facilities

1. Washing room.

2. Media preparation room.

3. Inoculation(room).

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4. Growth(room)

Hardening Facilities

1. Transfer area.

2. Green house / shade area.

3. Nursery

Quality Control

1. Selection of clones.

2. .Explant.

3. Virus indexing.

4. Number of multiplication cycles.

5. Overall quality of the plants.

Technical Supervision and monitoring

1. Monitoring of the production process and the staff involved there in.

2. Technical competence of the production supervisory staff.

3. Operators.

Merits
1. Rapidity in propagation and abundant availability.

2. Uniformity of micro propagated plants for both genotypic and phenotypic  characters.

3. The plants produce higher yield, because the mother plants are selected from high yielding clones, hence
the progenies tend to produce heavy and uniform bunches with uniform maturity.
4. The TC plants are healthy in quality because they are free pests and pathogens.

5. Easy to transport for longer distances with cheaper cost than the conventional suckers .

6. Unlike suckers, the tissue culture plants can be produced any time in the year for planting in any agro
ecological zones.

Demerits
1. Inability to lower high frequency of off-types in certain cultivars (Somaclonal Variants).

2. Inability to deal with endogenous bacteria or bacteria-like contaminants contributing to significant losses in
and immediately after, culture.

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Macro-Propagated Plants
Importance of Macro propagation

It can rapidly multiply plantlets to distribute a new variety or replace plants in disease-affected fields.
It gives relatively healthy plants if source suckers are from healthy mother plants and contamination is
minimized during the process.
It can be done locally at low cost and with little training: a private person or a farmers’ organization can
launch this activity.
It can teach awareness of principles of plant health.

Decapitation

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Macropropagation is an excellent option for producing low cost quality planting material.This is a simple
method because of the ease of multiplication, saves cost of producing planting material and has the
potential of producing 50-60 shoots per sucker in 4-5 months.
Macropropagation is achieved by two methods and could be adopted either in the field conditions (in situ)
or in the nursery (ex situ). It involves, decapitation, decortication and hardening.4-6 month old plant is
headed back, the pseudostem is cut down and cross cuts/ incisions are made on the growing meristem so
as to stimulate the production of lateral buds.This method results in the production of 9-15 uniform shoots
per plant in a short span of time and is highly suitable for small and marginal farmers whose requirements
of planting material are relatively small.
Suckers of choice varieties can be maintained in a nursery either in sawdust bed or in a big, bottomless
concrete pot.The initial planting material should preferably be certified as virus free and multiplied at farm
level under an insect proof net house.

Decortications

The pseudostem of the mother corm or sword sucker is cut transversely 2 cm above the collar region and then
the apical meristem is removed leaving a cavity of 2 cm diameter and 4 cm depth.Decapitation and decortication
activate the lateral buds giving rise to more side shoots.Generally, the corms that have already flowered give
better results than corms that have not yet flowered.Hence, healthy corms left in the field after harvesting are also
a potential explant both for in situ and ex situ mass multiplication.
Ex situ mass multiplication
Sword suckers are pared partially (trimmed of all roots and the outer surface scraped) and the growing points are
excised out with a sharp knife. These corms are surface sterilized by dipping in 0.3 % bavistin for 15 min, allowed
to dry for a day and then planted in the initiation medium, usually comprising rice husk or sawdust, though the
latter is much preferred.The initiation medium should essentially provide anchorage, moisture supply and proper
aeration to the roots.Before use, the medium is moistened and decomposed for a period of 2-3 weeks to allow
dissipation of the build up heat during decomposition.The medium could be enriched with sterilized
vermicompost, biofertilizers and rooting hormones like IBA (2,500 ppm). 200 g of VAM/Azospirillum is mixed with
10 kg of vermicompost and 500 g of the foresaid mixture is applied per corm.This treatment induces the
production of lateral buds through an enhancement in the population of Pseudomonas and Aspergillus species,
while IBA induces rooting in the developing multiple shoots.
Secondary decortications

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Development of
multiple shoots
After 30-35 days of first decortication, 3-4 buds emerge from the mother corm, depending on the variety.When
the side shoots attain a height of 15-20 cm with 3-4 leaves, secondary phase decortication is done by heading
back with a sharp knife followed by 3-4 transverse cuts.This encourages production of multiple shoots.Third
phase decortication is again attempted after 20-25 days but with greater care as the shoot buds are located very
close.Thus by the end of 140-150 days, a total of 50-60 shoots are produced from a single sucker.
Hardening

High humidity
Multiple Plantlets Detached plantlets
chamber
 
The lateral sprouts of 8-10 cm length are shifted to pro-trays containing equal parts of cocopeat and vermiculite
and after sufficient watering left in a shade net (70 % shade) at 80-90 % humidity.High humidity is achieved by
intermittent misting.Sprouts are usually maintained in the pro-trays for a period of 15-20 days and then shifted to
polythene bags of size 6’x 4’ and thickness of 120 gauge for secondary hardening.At this stage, the plants are
maintained at 50 % shade and 40-50 % humidity.Watering is done on alternate days and the plants are ready for
field planting in 30-45 days.
In situ mass multiplication
In situ production of suckers is induced chemically by pouring 4 ml of 40 ppm BAP into the decorticated cavity
and covering the individual mats with a mixture containing equal parts of sandy loam and poultry manure to 5 cm
above the ground level.Such chemical induction of lateral buds could be done on the first generation suckers and
continued up to third generation suckers.This method leads to the production of 45-50 shoots in a short span
months.The suckers are separated from the mother corm and subsequently rooted in sterile soil medium under
intermittent misting.

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