tissue culture techniques of banana

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BANANA CULTURE R.Sathes Student Department of Plant Biology and Bio technology Loyola College

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Commercial cultivation of banana using plant tissue culture technique.This provides a good job opportunity and a good business.......

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Page 1: Tissue culture techniques of Banana

BANANA CULTURE

R.Sathes

Student

Department of Plant Biology and Bio technology

Loyola College

Page 2: Tissue culture techniques of Banana

WHAT IS TISSUE CULTURE?

Tissue culture is collection experimental methods of isolation and inoculation of organs, tissues and cell in an artificial medium under in-vitro aseptic condition.

Micro propagation is vegetative propagation of plant using plant tissue culture. This also known as direct differentiation.

Page 3: Tissue culture techniques of Banana

BANANA CULTIVATION IN INDIA Banana is a globally important fruit crop with 97.5 million

tones of production. In India it supports livelihood of million of people. With total annual production of 16.91 million tones from

490.70 thousand ha., with national average of 33.5 T/ha. Maharashtra ranks first in production with 60 T/ha. Banana

contributes 37% to total fruit production in India. Banana occupy 20% area among the total area under crop in

India. Jalgaon is a major Banana growing district in Maharashtra

which occupy 50,000 hectares area under Banana. But most of Banana is grown by planting suckers. The technology development in agriculture isvery fast, it

results in developing Tissue Culture Technique.

Page 4: Tissue culture techniques of Banana

REQUIREMENT FOR BANANA GROWTH

Page 5: Tissue culture techniques of Banana

AGRO CLIMATE

Banana is basically a tropical crop, grows well in temperature range of 13ºC – 38ºC with RH regime of 75-85%.

In India this crop is being cultivated in climate ranging from humid tropical to dry mild subtropics through selection of appropriate varieties like Grandnaine.

The normal growth of the banana begins at 18ºC, reaches optimum at 27ºC, then declines and comes to a halt at 38ºC.

Higher temperature causes sun scorching. High velocity wind which exceeds 80 km phrs

damages the crop.

Page 6: Tissue culture techniques of Banana

SOIL

Soil for banana should have good drainage, adequate fertility and moisture. Deep, rich loamy soil with pH between 6-7.5 are most preferred for banana cultivation.

Ill drained, poorly aerated and nutritionally deficient soils are not suitable for banana.

Saline solid, calcareous soil are not suitable for Banana cultivation.

Avoided soil of low laying areas, very sandy & heavy black cotton with ill drainage.

A soil that is not too acidic & not too alkaline, rich in organic material with high nitrogen content, adequate phosphorus level and plenty of potash are good for banana.

Page 7: Tissue culture techniques of Banana

VARIETIES In India banana is grown under diverse conditions and production

systems. Selection of varieties, therefore is based on a large number of varieties catering to various kinds of needs and situations. Dwarf Cavendish. Robusta. Monthan Poovan Nendran Red banana Nyali Safed Velchi Basarai Ardhapuri Rasthali Karpurvalli Karthali Grandnaine

Grandnaine is gaining popularity and may soon be the most preferred variety due to its tolerance to biotic stresses and good quality bunches.

Page 8: Tissue culture techniques of Banana

GRANDNAINE VARIETY

Page 9: Tissue culture techniques of Banana

PLANTING MATERIAL Sword suckers weighing approximately 500-1000

gm are commonly used as propagating material.

Page 10: Tissue culture techniques of Banana
Page 11: Tissue culture techniques of Banana

WHY TISSUE CULTURE?

Suckers generally may be infected with some pathogens and nematodes.

Similarly due to the variation in age and size of sucker the crop is not uniform, harvesting is prolonged and management becomes difficult.

Therefore, in-vitro colonel propagation i.e. Tissue culture plants are recommended for planting.

They are healthy, disease free, uniform and authentic.

Properly hardened secondary seedlings are only recommended for planting

Page 12: Tissue culture techniques of Banana

ADVANTAGES OF TISSUE CULTURE PLANTING MATERIAL True to the type of mother plant under well

management. Pest and disease free seedlings. Uniform growth, increases yield. Early maturity of crop - maximum land use is possible

in low land holding country like India. Round the year planting possible as seedlings are

made available throughout the year. Two successive ratoons are possible in a short duration

which minimizes cost of cultivation. No staggered harvesting. 95% - 98% plants bear bunches. New varieties can be introduced and multiplied in a

short duration.

Page 13: Tissue culture techniques of Banana
Page 14: Tissue culture techniques of Banana

MICROPROPAGATION

Of Banana

Page 15: Tissue culture techniques of Banana

INITIATION OF SHOOT CULTURES

Shoot cultures of banana start conventionally from any plant part that contains a shoot meristem, i.e. the parental pseudo stem, small suckers, peepers and lateral.

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.

Page 16: Tissue culture techniques of Banana

The optimal size of the explants depends on the purpose.

For rapid multiplication, a relatively larger explants (3-10 mm) is desirable despite its higher susceptibility to blackening and contamination.

When virus or bacteria elimination is needed, meristem-tip culture is the preferred option.

The explants 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.

Page 17: Tissue culture techniques of Banana

The suckers for culture is prepared and taken into the lab.

They are soaked in Bavistin for 18 hours in order to remove the fungus and fungal spores.

In lab first they are washed in running water. Then they are Dipped into detergent water

(teepol) containing for one hour. After that they are taken and washed in tap

water. Washed suckers are taken to LAF chamber

and further sterilization is done.

Page 18: Tissue culture techniques of Banana

In LAF chamber the suckers are sterilized using mercury chloride

Two different concentrations are used for sterilization purpose.

First the sucker sterilized using 0.12% of HgCl2. The sucker put into the bottle containing HgCl2 Shake well for 2 min.

After that HgCl2 is removed and the sucker washed using distilled water. At first the distilled water added and the bottle shaked for 1 min. then the water removed and fresh distilled water added shaked for another one min. The water removed.

This step repeated under following timings 2min,3min,5min and 12 min.

Page 19: Tissue culture techniques of Banana

After 1st sterilization a layer of the sucker removed carefully.

The suckers are again sterilized using 0.1% HgCl2 for 5 min.

After that they are washed thoroughly with distilled water in following timings 1min,1min,2min,3min,5min,12min.

Finishing the above process another layer of sucker is removed.

The suckers are ready for inoculation.

Page 20: Tissue culture techniques of Banana

SUCKERS

Layer removal

1st Layer2nd LayerInner Layer

1st sterilization 2nd sterilization

Removal of a layer Removal of a layer

Page 21: Tissue culture techniques of Banana

STERILIZATION PROCESS

Page 22: Tissue culture techniques of Banana

MEDIUM USED FOR BANANA CULTIVATION For banana micro propagation, MS-based media

are widely adopted. Generally, they are supplemented with sucrose as a carbon source at a concentration of 30-40 g/l. Media are poured in a glass bottle where suckers are propagated.

Usually two types of growth regulators used, a cytokinin and an auxin, are added to the banana growth medium.

Their concentration and ratio determines the growth and morphogenesis of the banana tissue

In most banana micro propagation systems, semi-solid media are used. As a gelling agent agar (5-8 g/l) is frequently added to the culture medium.

Page 23: Tissue culture techniques of Banana

PROBLEM OF BANANA PROPAGATION 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.

Page 24: Tissue culture techniques of Banana

TRANSFER TO GROWTH ROOM

Banana shoot-tip cultures are incubated at An optimal growth temperature of 28 ± 2°C In a light cycle of 12-16 h with a photosynthetic

photon flux (PPF) of about 60 µE/m2s1. Air condition will be working all the time to

provide needed temperature. Which also provide clean dust free environment.

Page 25: Tissue culture techniques of Banana

GROWTH ROOM

Page 26: Tissue culture techniques of Banana

INOCULATE SUCKER & SUCKER AFTER 2 WEEKS

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ARISE OF SHOOTS FROM SUCKERS

Page 28: Tissue culture techniques of Banana

SUB CULTURING OF BANANA

After 2 weeks the suckers will become greenish in colour.

2 weeks after that multiple shoot will arise from the base of the suckers.

The shoots are cut at the base separated and placed in a fresh medium.

In each bottle three shoots are inoculated. After a week multiple shoots arise from the

inoculated shoot. Again they are separated and placed in a new fresh medium.

Page 29: Tissue culture techniques of Banana

SUB CULTURED BANANA

Page 30: Tissue culture techniques of Banana

SUB CULTURING AND INOCULATED CULTURES

Page 31: Tissue culture techniques of Banana

The sub culturing is done until the require amount of plant needed.

The shoots are every day checked for contamination and the contaminated shoots are transferred to a fresh medium.

Meanwhile a set of well grown healthy shoots are taken for rooting.

Page 32: Tissue culture techniques of Banana

WELL GROWN HEALTHY SHOOTS

Page 33: Tissue culture techniques of Banana

ROOTING OF BANANA SHOOTS

Rooting of banana shoot is done in bottle containing charcoal medium.

For rooting IAA Used as growth regulator (for commercial purpose).

Medium without hormone gives good results. It will take 2 weeks for rooting and fresh

roots are arise at the base of the shoot.

Page 34: Tissue culture techniques of Banana

ROOTED PLANTLET

Page 35: Tissue culture techniques of Banana

DIFFERENT STAGES OF BANANA CULTURE

Page 36: Tissue culture techniques of Banana

ACCLIMATIZATION OF BANANA PLANTLET

Rooted plantlet are kept in basal medium for certain time.

Then there are taken for acclimatization process where they gradually trained to the in-vivo temperature and light.

There are two stages in hardening process Primary Hardening Secondary Hardening

The hardened plantlets are carefully maintained in green house.

Page 37: Tissue culture techniques of Banana

PLATES USED FOR PRIMARY HARDENING

Page 38: Tissue culture techniques of Banana

SECONDARY HARDENING

Page 39: Tissue culture techniques of Banana

TRANSPORTING THE PLANTLETS.

The plantlets after acclamatization should be transported to the required place. Normal transportation is done where the plants are placed and grown in Plastic Bags.

Hence the well grown plants removed to provide the space in green house for the next cycle of plants and also to lower the cost of storage.

Page 40: Tissue culture techniques of Banana

SCOPE OF BANANA CULTURE

Banana have lot of useful properties which built the pathway for banana growers.

Normal banana suckers which obtained directly from the plantation grows in 12 to 15 month where PTC plantlet will grow in 10 months.

The suckers are collected for PTC are healthy and high yielding so the daughter plantlets have the same character of the mother plant.

The banana is a short term crop so it provides immense opportunity to produce PTC plants through out the year.

Care full measures should be taken in growing PTC banana which can give a good job opportunity and income for the producers.

Page 41: Tissue culture techniques of Banana

Entrepreneurship is living a few years of your life

like most people won’t, so that you can spend the

rest of your life like most people can’t.

YOU CAN WIN

Page 42: Tissue culture techniques of Banana

Thank You