PLANT TISSUE CULTURE(BANANA)

CHEMICALS USE FOR STERILIZATION:-

PREPRATION OF MACRONUTRIENTS SOLUTION:-

PREPARATION OF MICRONUTRIENTS SOLUTION:-

PREPARATION OF VITAMINS:-

IRON SOURCES:-

MESOINSITOL:-

When we prepare 1 litre ms media then we will take 100mg mesoinositol.

SUCROSE (3%):-

When we prepare 1 litre ms media then we will take 30gm sucrose.

Preparation of ms (Murashige and Skoog ) media:-

PREPARATION OF HORMONES:-

(Cytokinin & Auxin Solution Preparation)

BAP Stock:-100mg BAP in conical flask (250ml) dissolve with ethanol or 1M NaOH solution and BAP powder dissolve completely in solution and make final volume 100ml solution with Distilled water.

IBA Stock:- 100mg of IBA in conical flask (250) dissolve with ethanol or 1M NaOH solution and IBA powder dissolve completely in solution and make final volume to 100ml with Distilled water.

pH CHECK:- Prepare solution media pH range is 5.8 – 5.9 by using HCl or 1M NaOH.

Agar:- Take 8 gm agar powder and add in media ,mix with glass rod then in microwave the media for 14 minutes and agar dissolve completely in media.

STERILIZATION PROCESS :-

• First, we have cleaned glasswares such as test tube, Petri plates, conical flask, beaker, measuring cylinder, forecep, spatula , scalpel, surgical blades etc.Then, the cleaned items were dried in a hot air oven at180°C for 10 to 12 hours to ensure no water droplets remained. Then we have wrapped glassware in autoclave bag then put in autoclave with distilled water and culture media.

• Autoclave work at high pressure and temperature ( 15 psi, 121°C) for 15 to 20 minutes and kill the microorganisms. After sterilization removed glassware from sterilizer. And placed in laminar air flow then when we use it, we will turn on UV – ray light for 1 hour.

METHODOLOGY:-

1. Select mother plant which age is 6-12 olds and do not any disease and high yield desirable traits.
2. Cut the mother plant in desirable shape and placed dry tray.
3. Wash the cut explant in running water for 15 minutes.

4.Handle the plant material with sterile glovs .

5. Then prepare total volume 300ml soap solution ( 30ml soap liquid and 270ml distilled water).

6. Wear glove hand and wash the plant material for 5 minutes.

7.Then remove the soap solution and placed in glass beaker.

8. Cut the material desirable size with the help of knife and remove the upper cover of material.

9. Then soak the material in savlon solution ( 3ml savlon and 300 ml distilled water) for 15 minutes.

10. Then wash the plant material from sterile distilled water 3 to 5 time in bottle beaker.

11. Prepare 1% sodium hypochlorite solution and taking sodium hypochlorite solution into a laminar air flow and soak the plant material in sodium hypochlorite solution for 15 minutes( for 1 jar 50ml sodium hypochlorite and 250ml sterile distilled water).

12. After 15 minutes then remove plant material from sodium hypochlorite solution and wash the plant material 3 to 5 times in sterile distilled water.

13. Then cut the plant material required culture bottle size with the help of knife on sterilized brown paper.

14. Prepare antibiotic solution using Cefotaxime antibiotic( 200ul antibiotic and 200ml sterile distilled water) and soak the plant material in this solution for 20 minutes. Using only fresh antibiotic solution.

15. Then inoculate the plant material in culture medium( bottle beaker) and remove laminar air flow placed inoculate culture bottle at 24°C with photo periods of 6 to 7 hours light and 8 hours darkness for tissue culture sterile environment.

OBSERVATION:-

After one week inoculated explant in culture bottle growing slowly.

Result :- A few days after the plant gets contaminated, signs start to show on the banana plant.

10 march 2025

Then, remove the contaminated plant from the culture bottle, soak it in an antibiotic solution for 30 minutes, cut it into two parts, and inoculate it into fresh media.

17 march 2025

After subculturing the banana plant, contamination reappears within a few days. Once a plant is contaminated, it tends to remain contaminated.

3 march – 4 march 2025

• Autoclave the used media to sterilize it before disposal.

• Discard the used media from the culture bottle.

• Soak the empty culture bottle in chromic acid (for cleaning or decontamination) for 24 hours.

• After 24 hours, remove the culture bottle from the chromic acid and wash it with liquid soap.

• Then dry it in a hot air oven at 180°C for 12 hours.

• Preparation of ms media.

5 march 2025

Carefully select a healthy banana plant and wash the explant thoroughly. Sterilize it using a liquid soap solution, followed by soaking in an antibiotic solution for 30 minutes. After sterilization, inoculate the explant into a culture bottle under aseptic conditions.

After a few days, growth appears in the banana explant and no contamination is observed in the culture bottle.

7 march 2025

Prepare the hormone-containing medium, then sterilize the medium, distilled water, glassware, and other required items using an autoclave.

8 march 2025

SUBCULTURING :-

After 30 days we prepared hormones media ( ms media + BAP + IAA ).

After preparing the hormone-supplemented medium, the explant is taken from the culture bottle and cut into two parts on sterile paper. Then, each part is inoculate into fresh culture bottles containing the same hormone medium.