INTRODUCTION

By definition, members of single clone have a same genotype. Further, different

varieties of vegetatively propagated plants are distinct clones. Conventionally,
vegetative reproduction is achieved by cutting, budding, grafting e.t.c. Tissue
culture is also enable rapid clonal propagation of plants; this is usually called
micropropagation.

Musa sp. consists of two gropes i.e. Banana group for fresh fruit and plantain
group for cooked food. The first group is most popular for the farmer. Banana is
characterized morphologically by the pseudostem arises from an underground
corm. This corm forms large number of small corms, which has potential to form
sucker. Traditionally this sucker is used as seedling, however, the ability of corm
to form sucker has become a constrain in large-scale cultivation and can not insure
to produce bacterial viral free seedling.

In vitro culture has a great advantage for mass propagation of various vegetatively
propagated crops. Commercial application of banana by In vitro culture usually
uses shoot multiplication. This technique can increase the rate of seedling
production and improve the seedling quality, such as uniform and true to parent
type. The average rate of shoot formation produced by this technique is 4-5 shoot
per month.

Naturally banana has large potential to produce large number of corms. Thus the
in vitro technique for inducing cormlet initiation followed by its development to
form micro sucker is prospective method to improve the commercialized of
banana in vitro technique.

The biggest advantage of micro-propagation is uniformity, the produced plants are

true to its parent type. They mature at a same time and decrease the cost of crop.
The micro-propagated plantlets are free from bacterial as well as viral diseases.
There are so many banana plants are developed which free from bunchy top of
banana. Micro-propagated plants can be transported easily with in a limited period
of time. The chief advantage of micropropagation is the extremely high
multiplication rates, 106 plants/ year. Very small plants can be used as
micropropagation, which is impossible with conventional techniques. Plants can
be maintained in vitro in a pathogen free state. Such plants are easy to export since
there is no quarantine problem, and there packing is easier due to its smaller size.
Micropropagation can be carried out through out year independent of season.

The main problem with micropropagation is high production cost and limited
application of this technique to more valuable to ornamental crops and some fruit
trees.
 MATERIALS AND METHODS

Material required
Young banana pseudostem, knife, water, sodium hypoclorite solution (1%),
autoclave, betadin, Murashinge and skoog (MS) medium, 6-benzylamino purine
(BAP), geberlalic acid (GA3 ), Indole acetic acid (IAA), Naphthalene acetic acid
(NAA), e.t.c.

Explant sterilization
Young banana of G-9 were selected from field and uprooted. The remaining
portion of pseudostem is washed to remove all traces of soil. Young sucker as well
as the shoot tips of pseudostem is used as the sources of apical meristem slice.
After washing the pseudostem and suckers are stripped and placed in a sodium
hypoclorite solution (1%) plus two drops of tween 100. All subsequent treatment
was carried out under aseptic condition.

The apical meristem were washed 4 times with sterile distilled water, and trimmed
into square block ranging from 7-10 mm in thickness, depend upon the size of the
apical meristem and then deepen into the betadin for 30 min, and then washed 5
times with sterile water. The apical meristem was then cut into slice of 3-mm
thickness. A middle slice containing the leaf bases. All slices containing part of
the corms were pushed 1 to 2 mm into the cultural; media. 10 slices were used for
each treatment and all treatment were repeated five times.

Cultural Media
Modified Murashige and Skoog (MS) medium supplemented with 100 mg/l Myo-
Inositol, 0.4 mg/l Thiamine HCl, 150mg/l citric acid, 02 mg/l L-cystein was used
for all stages. The experiment consist of three stages, i.e 1st stage of cormlet
initiation,2nd stage of cormlet development and 3rd stage is plantlet rooting. The pH
was adjusted to 5.7 with KOH and HCl. 20 ml vol. of was dispensed into vials and
autoclaved at 21 psi for 30 minutes. The liquid media with paper brig was used on
the 1st stage and solid media with the addition of 0.7% Difto Bacto Agar was used
on the 2nd and 3rd stages.
Composition of MS medium

Stock
Compound Amount per lit.
Solution(mg/100ml)
Macronutrient
NH4NO3 1650 mg
KNO3 1900 mg
MgSO4.7H2O 370 mg
KH2PO4. 170 mg
KI 83 1 ml
CaCl2.2H2O 440 mg

Micronutrient
H3BO3 620
MnSO4.4H2O 2230
ZnSO4.7H2O 860 1 ml
Na2MoO4.2H2O 25
CuSO4.5H2O 2.5
CoCl2.6H2O 2.5
Na2EDTA.2H2O 37.3 mg
FeSO4.7H2O 27.8 mg

Vitamins
Nicotinic Acid 5
Thiamine HCl 1 10 ml
Pyridoxine HCl 5
m-Inositol 1000

Growth Regulator
2,4-D 100 1 ml or as req.
Kinetin 10 1 ml or as req.

Others
Glycine 2 mg
Sucrose 30 g
pH 5.5
Agar 6g
Treatment

Stage I: Cormlet initiation

Treatments in this stage were arranged by combining (1) between each of 0, 5, 10,
15, and 20 mg /l of 6- benzylamino purine (BAP) and each of 0, 10, 20, 30 and
40%of sucrose, (2) between each of 0, 5, 10, 15, and 20 mg/l of BAP and each of
0, 5, 10, 15, and 20 mg/l of ancymidol and (3) between each of 0,5,10,15, and
20mg/l BAP and each of 0, 5, 10, 15, and 20mg/l of paclobutrazol.

Stage 2nd: - Cormlet development

Treatments in this stage included Geberalic acid (GA3), Indole acetic acid (IAA)
and BAP at 0, 0.5, 1, 1.5 and 2mg/l.

Stage 3rd: plantlet development

Treatments in this stage included Indole butyric acid (IBA) naphthalene acetic
acid

Cultural maintenance
All culture were maintain at temperature of 25-28°C and 16h photoperiod. The
light intensity (cool white fluorescent lamps; 40 cm above vials), that supported
maximum growth at the various stage of development, maintained at 2000 lux.

Observation
Cormlet initiation
The earliest sign of cormlet development occurred on apical meristem slices at 10-
14days after culture, which indicated by swelling of the explants. Cormlet initials
first observed on the tope and/ or sides of explants. Within 10-15 days after
formation, cormlet contained 3-4 leaf bases.
The range of initial formation depends upon the plant growth regulator, plant
growth retarded and sucrose present in the initiation media. BAP, sucrose and
ancymidol affected cormlet induction. No cormlet was formed when the induction
media was devoid either BAP or sucrose or ancymidol. Paclobutrazol has no
positive effect on corm formation. When the concentration was below 10mg/l
apical meristem slices explants remained green. Inclusion of 20mg/l
paclobutrrazol caused plantlet to turn brown and dies within 30days.
Effect of ancymidol on cormlet formation was directly related to the concentration
of BAP. Ancymidol addition at 15 mg/l increases cormlet induction and the
number of cormlet per explant. Simultaneous incorporation of 20 mg/l BAP
decreases cormlet induction. High concentration of ancymidol decreases the
cormlet induction.
Appeared in this experiment, that cytokinin (BAP), growth retardant (ancymidol)
and energy sources promote cormlet formation. This phenomenon suggested the
medium components are essential for corm formation on banana.

Cormlet development
After formation of cormlet, it was transferred to cormlet development media. By
visual observation showed that the initialized cormlet developed better if the
original explant was transferred intact to the cormlet development media. The
thicker the original apical meristems slice more initialized cormlet produced,
inferred the thicker slice contained grater number of pre-existing meristem.
Transferring of original explant for developed of the initialized propagules was
reported by Teo, which shoot of rattan can be developed from globular bodies.

During passage on cormlet development media, initialized cormlet produced

additional new initialized cormlet produced additional new initialized cormlets. In
sequent transfer, production of new initialized cormlets and growth occurred
simultaneously. These results suggest that initialized cormlets still contained the
residue of BAP or ancymidol, which used in initiation media.

Micro suckers developed from cormlet of banana reported here has a characteristic
like the micro sucker produced directly from shoot tip. Development of initial
cormlet of banana to form micro sucker depends on the presence both of GA3 and
IAA in media. GA3 at 0.5-1 mg/l was optimum concentration needed to promote
maximum cormlet development. At 2 mg/l or 0 mg/l GA3, cormlet failed to
develop. These results showed that concentration of GA3 needed on in vitro
culture depends on the plant cultured.

Plantlet developed
The rooting of micro sucker occurred within 10-15 days after transferring into
rooting media. The type and level concentration of auxin significantly affected the
rooting ability. When auxin were omitted from rooting medium the rooting
process did not occur. Low concentration of NAA and IBA (0.5 mg/l) favors root
development in the study. IBA and NAA at 0.25 mg/l, produced small number of
root, and high concentration of this auxin inhibited root development.

Using IAA, root formation occurred over wide range of concentration (0.25 mg/l),
but IAA at 2 mg/l also inhibited the root development. In concentration range of
0.25 mg/l –1mg/l, there was no apparent correlation between IAA concentrations
with root number. High concentration of auxin inhabits the rooting synthesis.
 TABLE: Effect of auxin on rooting of micro sucker derived from cormlet.

Auxin Root number

Indole butyric acid(mg/l)
0 0
0.25 0
0.5 8
0.75 2
1 0
2 0
Indole acetic acid (mg/l)
0 0
0.25 3
0.5 6
0.75 6
1 4
2 0
Naphthalene acetic acid(mg/l)
0 0
0.25 3
0.5 8
0.75 1
1 0
2 0

Subculturing
After a period of time, it becomes necessary to transfer organs and tissue to fresh
media. This is particularly true of tissue and cell cultures where a portion of tissue
is used to inoculate new culture tubes or flasks. This is known as subculturing. In
general, callus cultures are subcultured at every 4-6 weeks, while suspension
culture need to be subcultured every 3-14 days. Plant cell and tissue culture may
be maintained indefinitely by serial subculturing.

Transferring into the soil

Plant obtained from this experiment were successfully acclimatized and
transferred into the polybag. The method developed here could increases the rate
of plantlet production from 4 on shoot tip culture, which commercially applied to
12 micro sucker / explant after 7 week culture.

Reference
(1) Biotechnology by B.D. SINGH
(2) Plant breeding principles and method by B.D. SINGH
(3)