1. Banana Culture R.Sathes Student
Department of Plant Biology and Bio technology
Loyola College

2. 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.

3. 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 million tones from 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.

4. Requirement for Banana Growth

5. 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.

6. Soil
Soil for banana should have good drainage, adequate fertility and moisture. Deep, rich loamy soil with pH between 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.

7. 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.

8. Grandnaine variety

9. Planting Material
Sword suckers weighing approximately gm are commonly used as propagating material.

11. 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

12. 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.

14. Micropropagation
Of Banana

15. 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 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.

16. 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 ( 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.

17. 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.

18. 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.

19. 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.

20. Suckers Layer removal 1st Layer 2nd Layer Inner Layer
1st sterilization
2nd sterilization
Removal of a layer
Removal of a layer

21. Sterilization process

22. 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 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.

23. 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 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.

24. Transfer to Growth room
Banana shoot-tip cultures are incubated at
An optimal growth temperature of 28 ± 2°C
In a light cycle of 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.

25. Growth Room

26. Inoculate sucker & Sucker after 2 weeks

27. Arise of shoots from suckers

28. 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.

29. Sub Cultured Banana

30. Sub culturing and inoculated cultures

31. 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.

32. Well grown healthy shoots

33. 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.

34. Rooted Plantlet

35. Different Stages Of Banana Culture

36. 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.

37. Plates used for primary Hardening

38. Secondary Hardening

39. 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.

40. 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.

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42. Thank You