Introduction

1. Introduction • Tissue culture is method of isolation and inoculation of tissues in artificial medium under in vitro condition. • Banana is major fruit crop in India and Maharashtra stands first in banana production and productivity in India. • Conventionally banana is grown by planting suckers. However, Tissue cultured plants are becoming popular in farmers.

2. Shoot tip or meristem tip culture • Meristem Tip Culture is a separation of undifferentiated plant cells from shoot tips. • Banana are propagated by Meristem Tip Culture as this technique produces virus free plant stocks. • Steps involved in meristem tip culture are: • Selection, preparation and sterilization of explant • Preparation and sterilization of culture media • Inoculation and incubation of Explant • Sub culturing of explants on media • Hardening of seedlings

3. Steps in Banana shoot tip culture Steps in Banana shoot tip culture

4. Selection and Preparation of Explant Selection • Shoot tips of young suckers of 40 to 100 cm height are used as a explants for rapid in vitro multiplication of banana. • Tissue of about 1 to 2 cubic cmcontaining the apical meristem is separated from the banana suckers. Preparation • Selected explant from the banana sucker is reduced to small- small pieces (0.5 to1 mm length) leaving the meristem dome with one or two leaf.

5. Explants used for banana shoot tip culture Young sucker for micro-propagation Sword (left) and water sucker (right) View of mother plant and sucker Suckers separated from mother plant

6. Sterilization of Explant • Explant soaked in Bavistin for 18 hrs and then washed under running water. • It is then dipped in laboratory detergent like Teepol or Dettol for one hour and then washed with tap water. Sterilization of explants in chamber • Explants separated from Suckers further processed in a surface sterilized Laminar Flow chamber. • Explant treated with mercuric chloride for 2 min and then washed 3 times with distilled water.

7. Removal of subsequent layers from the suckers and sterilization for banana shoot tip culture

8. Preparation of Tissue Culture Medium • MS media is used for banana micro-propagation. Culture medium is sterilized by autoclaving at 121oC for 30 minutes. • Medium is added with sucrose sugar @ 30 to 40 gm/litre as carbon source and gelling agent Agar agar @ 5 to 8 gm/litre for giving semi-solid nature to media. • Cytokinins and Auxins are added in to medium which decides growth and morphogenesis of the tissue explanted on culture medium.

9. Incubation at Growth Room • After successful inoculation of sterilized explant on aseptic culture medium it is transferred to growth room. • Culture bottles are incubated at 28 (+/-2)oC and exposed to light for 12 to16 hours. • Light intensity adjusted to • Aseptic conditions are maintained inside the growth room. Initiation of banana shoot after one week of inoculation

10. Sub Culturing • Transfer of explant to fresh culture medium is called as sub culturing. • Banana shoots become greenish in colored in 2 weeks, the shoot is cut from base and placed on fresh culture medium added with appropriate growth regulators. • Sub culturing is done under aseptic conditions. Shoots are kept in growth room conditions for 2 weeks • Multiple shoot initiation starts in one week, the shoot is again separated and kept in fresh medium for multiplication.

11. A. Sub culturing under aseptic conditions B. Sub cultured explants in growth room Well grown banana shoots after two weeks of sub culturing on MS medium

12. Rooting of Regenerated Shoots • Rooting of regenerated shoots is done on the MS medium added with charcoal power. Indole Acetic Acid (IAA) is used as growth regulator for rooting. • MS medium without any growth hormone also gives good results. • For better establishment of fresh roots, keep explant on rooting medium for 2 weeks under aseptic condition. Root development in banana shoots after transfer to rooting medium

13. Advantages of Banana Tissue Culture • Tissue cultured plants are true to type, pest and disease free, with uniform growth, healthy, vigorous and early maturing. • Banana plantation is possible all round the year. More than 95% plants bears bunches • New and rare varieties of banana can be introduce and multiplied by tissue culture in short duration.