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BANANA TISSUE CULTURE

INTRODUCTION

Plant Tissue Culture is culturing of any part of the plant in a specially defined growth
media under aseptic laboratory condition in Petri dishes, test tubes or in any other
suitable glass containers. The plant nutrient media consists of macro and micro salts,
vitamins and desired levels of plant growth hormones. Depending upon the plant
species genetic nature and with help of above supportive media various forms of
callus/embryos/shoots/ roots or direct plantlets can be induced. Obtaining plants
through the above techniques is called as regeneration. With the recent technology
brake through it is now possible to obtain complete plants of many species through
single cell culture.
Banana plants produced from Tissue Culture are free from diseases at the time of
supply and they give high yields since they are made from selected high yielding
mother plants. If proper care is taken, as per instructions, they grow into strong
healthy plants and give high yields of good quality fruits. Since they are produced
under controlled laboratory conditions using selected nutrients, they usually give
yields one or two month earlier than conventionally propagated plants.

1. MICROPROPOGATION

Production of large number of plant lets within a short time, with less labor costs
through culture media in 2 3 different stages under laboratory conditions. Large
scale and commercially viable technologies were developed and being successfully
adopted in Europe for rising cut flowers (Floriculture), Ornamental and other plants.

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2. HORTICULTURE

Raising stocks of disease and virus free selected elite clones (varieties) can be supplied
to the farmers as plant material. Uniform, true to type, high yielding and early
harvesting was reported in case of Banana, Papaya, Pineapple. Few others are being
followed now in various parts of the world.

3. PROTECTION OF INTELLECTUAL PROPERTY RIGHTS

Developing suitable DNA fragments as probes by using restricted fragment length
polymorphism (RFLP) mapping techniques to protect seed companies developed
varieties and to avail exclusive marketing rights with scientific authentication and
proof.

The production of Banana in India is estimated at 5.8 million tonnes from an area of
0.33 million Ha. as per an estimate. India occupies the third place in annual Banana
Production. However, in spite of large potentialities, there is no appreciable presence
in the export trade. This is due to several factors, chief among them is Poor Yield.
Several local varieties are grown in preference to the Cavendish group. It is amply
clear that wherever clones from Cavendish group are grown, the yields are high and
consistent. One of the major impediments to extending the area under cultivation is
non-availability of disease diagnosed planting material. Tissue Culture or micro
propagation alone can solve this problem.




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Banana (Musa spp.) are large herbaceous plants with their center of diversity in
Southeast Asia (Simmonds, 1976). The main method of propagation is by means of
daughter suckers formed at the base of the pseudostem. Traditionally, sword suckers
with narrow leaves are the preferred planting material for vegetative propagation.

The major constraint for conventionally propagating Banana is the lack of ready
availability of large quantities of sword suckers at any given time. The problem is felt
more acutely in non-availability of sword suckers consistently.

4. ADVANTAGES

Initiation and establishment of rapidly multiplying aseptic shoot cultures can
eliminate the problem of low sucker multiplication rates effectively and economically.
Tissue Culture micro propagation is attractive in a number of situations like:

a) Large number of uniform propagules can be generated in a relatively short
period of time.
b) Variability encountered in size and propagules density especially in clones
suckering erratically can be minimized.
c) It could allow for rapid bulking of novel clones when used in concert with
breeding programms.
d) It would facilitate transcontinental exchange of disease diagnosed planting
material. With refinement in preservation techniques, in-vitro culture of
Bananas can be immense value in germplasm conservation.



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5. BREIF HISTORY OF TISSUE CULTURE IN BANANA

Realizing the importance of shoot meristem culture in getting rid of the plant disease
causing pathogens and its subsequent mass propagation. Berg and Bustamante (1974)
attempted technique. However, this procedure is not completed effectively in the
absence of suitable diagnosis. Soon after reports of Banana plantlets produced by in-
vitro shoot tip culture came from Taiwan (Ma and Shill, 1974).

6. TISSUE CULTURE TECHNOLOGY

Successful micro-propagation strategies are as a result of interplay of plant material,
the culture milieu and the micro-environment.

7. LABORATORY FACILITIES

Ideally a plant Tissue Culture facility must consist of separate rooms for media
preparation, aseptic transfer, culture incubation and illuminated rack systems.
Aseptic conditions constitute the central theme in such exercise.

8. MEDIA PREPARATION AREA

Media preparation area consists of a large table to keep media, cooking utensils like
jacketed stainless steel vessels along with a tap to dispense media in glass containers.

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In addition to this various other equipment like de-mineral water unit, pH meter,
sensitive balance, trays and trolleys, water distillation unit and autoclave etc., which
are called critical equipments.





9. TISSUE CULTURE TRANFER AREA

The most preferred arrangement for aseptic transfer is a separate air conditioned dust
free room equipped with the requisite number of laminar airflow clean benches fitted
with ultraviolet lamps. A high level of cleanliness ensures a reduced risk of
contamination. All working surface is thoroughly cleaned and disinfected prior to
sterile transfer. It is desirable that the transfer room and the laminar flow hood be
used exclusively for aseptic manipulations. Other equipment needed for sterile
manipulations include scalpels with removable blades, forceps if varying lengths, an
ethanol dip, an alcohol lamp/Bunsen/burner/incinerator/hot bead sterilizer etc. A
stereoscopic zoom microscope with a cold light source is optional and required only
for micro dissections.

10. PLANT GRWOTH ROOM

A clean culture incubation room with provision for temperature, light and humidity
control is preferred. Good results can be obtained with a RH of 40%. Cultures are
normally kept in racks, stacked on shelves illuminated by cool, white fluorescent
lamps (40 Watts.). The light intensity thus obtained is in the order of 30 to 50 m.e.m-2

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sec-1. Separate incubation culture room and light rack arrangements enclosed in the
drawing and designs separately.


11. EXPLANT PREPARATION AND DISINFECTION

Sword suckers are carefully removed from field grown fruiting Banana plants and
washed thoroughly in tap water and a solution of the diluted detergent teepol. All
traces of teepol and the extraneous rhizome tissue are carefully chopped with a
stainless steel knife. Trimmed suckers are now soaked in a solution of Bavistin (0.5%)
a fungicide and streptocycline antibiotic for six to eight hours. Shoot tips containing
rhizome tissue and measuring 2.5 to 3.5cm in length are isolated. Surface sterilized
with chlorine-saturated distilled water for 15 to 20 mins. Further operations are all
carried out under a laminar flow chamber. All traces of chlorine are removed by
washing several times with autoclaved, sterile distilled water.

The sterilized shoot tip, explants are handled using sterilized stainless steel scalpels.
Cut surfaces of the rhizomatous tissue and leaf bases are further trimmed so that
shoot tips finally contain at least six to eight overlapping leaf bases enclosing auxiliary
buds. The explants are now ready for inoculation and measures 1 to 2. cm. It is then
immersed into the sterile solid medium present in the culture vessel.


12. INDUCTION OF GROWTH

Cultures should be incubated in the basal nutrient media supplemented with plant
growth regulators. Thereupon the healthy, contamination free explants should be
taken for next multiplication stage.

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13. MASS MULTIPLICATION

Contamination free explants are further cultured on multiplication media
supplemented with plant growth hormones which help in proliferation of axillary buds
(cytokinins) into multiple shoots. These shoots are divided and multiplied to bulk up
the multi culture stock. The multiplication cycles are restricted to 8 because Banana is
genetically highly unstable.

14. SHOOTING

Multi cultures are further divided and transferred to shooting media which is
composed of auxins (PGR) to get the elongation. In this stage leaves will develop and
the whole plant will grow upto 4 to 5 cm.

15. ROOTING

Plantlets from shooting media are separated and single plantlets are transferred to
media containing charcoal and auxins. In this stage roots will develop and plants will
be ready for dispatch from laboratory.

16. AGAR WEENING OF PLANTS

Well developed single plantlets need to be removed from the culture incubation room
and exposed to ambient conditions in the culture vessel for four to five days. The
plantlets are then carefully removed and the roots washed in running tap water.




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17. PRIMARY HARDENING


A quick dip in 0.5% Bavistin solution follows and finally in-vitro plants are transferred
to trays containing sterilized coco peat. These trays are kept under tunnels made of
transparent PP Plastic sheets to maintain the humidity above 80%. These tunnels
should be under 50% to 75% shade nets. Primary hardening will take atleast 4 weeks
depending upon the climatic conditions. In final week these trays are gradually
exposed to 50% shade by removing plastic sheets. These plantlets are sprayed with
fungicides, bactericide, and water soluble fertilizers as per schedule.

18. SECONDARY HARDENING


Primary hardened plants after 4 to 5 weeks are transferred to Poly bags (Nursery
Bags) of suitable size. Soil mixture is prepared by mixing sand, soil and farm yard
manual into 1:2:1 ratio. The plants are kept in these Poly bags for 6 to 8 weeks under
50% shade. Humidity is maintained around 60% to 70% and regular foliar sprays of
plant protection chemicals and water soluble fertilizers are given regularly. Any
possible variation if observed is discarded at this stage. The plant ready for sale will
be having 5 to 6 opened leaves and almost 1 feet in height







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PHOTOGRAPHS SHOWING DIFFERENT

STAGES OF

TISSUE CULTURE BANANA PLANT






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B BA AN NA AN NA A M MU UL LT TI IP PL LI IC CA AT TI IO ON N




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B BA AN NA AN NA A R RO OO OT TI IN NG G
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B BA AN NA AN NA A P PL LA AN NT TS S R RE EA AD DY Y F FO OR R
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