INTRODUCTION :- Plant tissue culture is a techniques used to maintain or grow plant cells, tissue, or organs in a sterile, nutrient – rich medium under controlled conditions. The principle of tissue culture is “totipotency” (ability of single plant cell to regenerate into whole plant). Gottlieb Haberlandt father of tissue culture, he proposed first idea of growing plant cell in invitro conditions.
ADVANTAGE OF PLANT TISSUE CULTURE :-
- Mass Production – Produce large number of plant in short time, useful for commercial agriculture.
- Disease-free plants – Growing healthy plants stock, eliminate the harmful microorganism like bacteria, fungi, viruses etc.
- Conservation of Rare Species – Tissue culture helps preserve genetics material of rare, or valuable plant species.
- Faster Growth – Fast growing plant tissue culture compare to traditional method like seeds or cuttings.
- Genetics Uniformity – Produces identical plants(clone) with desire traits, maintaining quality and yield.
STAGES OF Tissue Culture :-
1.Initiation – A small pieces of plant tissue (explant) is collected from mother plant. Then sterilized explant to prevent contamination.
2.Establishment – It is placed on a nutrient medium containing essential nutrients, sugars, and hormones.
3.Multiplication – The explant develops into callus or shoots.
4.Differentiation – callus develop into specialized cells, tissues, or organs.
5.Shooting – Shoots develop from differentiation cells.
6.Rooting – Roots develop from shoots.
7.Accilimatization ( Hardening Stage) – Rootes plantlets gradually show external conditions like Light, Temperature, Humidity Then placed greenhouse or nursery.
2 march 2025
The project was initially assigned to Shrutika. After her fellowship ended, I took over and continued the work. In this project, the primary focus is to achieve 100% reduction in contamination in plant tissue culture, and I am continuing this project with the goal of growing 100 banana plants and 50 turmeric plants.
MATERIALS:-
| Petri Plates | Tissue paper |
| Test Tube | Distilled water |
| Test Tube Stand | Sterile Distilled water |
| Beaker | Sterile Surgical Blades |
| Culture Bottle | Autoclave Bag |
| Scissors | Filter paper |
| Spatula | Forecep |
| Scalpel | Conical flask |
| Cotton plug | Measuring Cylinder |
CHEMICALS FOR MS MEDIA:-
| Macronutrients |
| Micronutrients |
| Vitamines |
| Hormones |
| Agar powder |
| Iron source |
| Amino acid |
| NaOH |
| HCl |
CHEMICALS USE FOR STERILIZATION:-
| Ethanol | Bavistin |
| Savlon ( Dettol) | Antibiotic |
| Tween 20 (liquid soap) | Sodium hypochlorite |
PREPRATION OF MACRONUTRIENTS SOLUTION:-
| Macro nutrients | Original Concentration | Amount of salt after multiplying original concentration by 20X, Solution Volume (250ml) |
| NH4NO3 | 1650mg/l | 8250mg |
| KNO3 | 1900mg/l | 9500mg |
| KH2PO4 | 170mg/l | 850mg |
| MgSO4.7H2O | 370mg/l | 1850mg |
| CaCl2.2H2O | 440mg/l | 2200mg |
| CaCl2(fused) | 330mg/l | 1650mg |
PREPARATION OF MICRONUTRIENTS SOLUTION:-
| Micronutrients | Original Concentration | Amount of salt after multiplying original concentration by 100X, Solution Volume (250ml) |
| H3BO3 | 6.2mg/l | 155mg |
| MnSO4.4H2O | 22.3mg/l | 557.5mg |
| ZnSO4.7H2O | 8.6mg/l | 215mg |
| Na2MoO4.2H2O | 0.25mg/l | 6.25mg |
| CuSO4.5H2O | 0.025mg/l | 0.625mg |
| CoCl2.6H2O | 0.025mg/l | 0.625mg |
| Kl | 0.83mg/l | 20.75mg |
PREPARATION OF VITAMINS:-
| Vitamins | Original Concentration | Original Concentration multiplying by 1000X, Final solution volume (50ml) |
| Thiamine HCl | 0.1mg/l | 5mg |
| Pyridoxin HCl | 0.5mg/l | 25mg |
| Niacin | 0.5mg/l | 25mg |
| Glycine | 2.0mg/l | 100mg |
IRON SOURCES:-
| Sources | Original Concentration | Original Concentration multiplying by 400X, Final solution volume (50ml) |
| FeSO4.7H2O | 27.8mg/l | 2780mg |
| Na2EDTA.2H2O | 37.3mg/l | 3730mg |
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:-
| Materials | Final Volume (500ml) | Final Volume (250ml) |
| Distilled water | 250ml | 100ml |
| 20X, Macronutrients | 25ml | 12.5ml |
| 100X, Micronutrients | 1.25ml | 0.625ml |
| 1000X, Vitamins | 1.25ml | 0.625ml |
| 400X, Iron sources | 0.5ml | 0.25ml |
| Mesoinositol | 50mg | 25mg |
| Sucrose | 15gm | 7.5gm |
| Agar powder | 4gm | 2gm |
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:-
- Select mother plant which age is 6-12 olds and do not any disease and high yield desirable traits.
- Cut the mother plant in desirable shape and placed dry tray.
- 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 :-
2nd Stage :
After 30 days we prepared hormones media ( ms media + BAP + IAA ).
| Materials | Final Volume (250 ml) |
| Distilled water | 100ml |
| Macronutrients | 12.5ml |
| Micronutrients | 0.625ml |
| Iron source | 0.625ml |
| Vitamines | 0.25ml |
| Mesoinositol | 25mg |
| Sucrose | 7.5gm |
| Agar | 2gm |
| BAP | 0.750ml(1mg//) |
| IAA | 0.125ml( 1mg/l) |
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.
Some banana plants dried up in the second stage, 20 days after inoculation.
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11 march – 12 march 2025
The glassware was cleaned, the glassware and distilled water were sterilized, and the MS medium was prepared.
14 march 20
I established and carried out protocols for the plant tissue culture of banana explants under sterile conditions (Yogesh). And inoculated the explants into culture bottle media.
15 march 16 march 2025
I prepared hormone-containing media for subculturing the banana plants that are 28 to 30 days old.
17 march 2025
After 28 to 30 days, several banana explants were subcultured onto fresh media containing hormones, and each plant was divided into two parts for further propagation.
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31 March 2025 :-
Third Stage :
I cut some banana plants into two parts from the 2nd stage to the 3rd stage. And then inoculated them onto a fresh hormones medium.
Four Stage :
I cut banana plants into two parts from the 3rd stage to the 4th stage. And then inoculated them onto a fresh hormones medium.
Five Stage :
I cut banana plants into two parts from the 4th stage to the 5th stage. And then inoculated them onto a fresh hormones medium.
25 April 2025
Some banana plants are growing very slowly, while others are showing symptoms like yellowing leaves, and one plant has developed fungal contamination.
The contaminated plant is then cut into two parts, soaked in an antibiotic solution for 30 minutes, and subsequently inoculated onto a fresh hormone medium. But after one week, the banana plants showed signs of fungal infection again.
5 May 2025
I cut banana plants into two parts from the 4th stage to the 5th stage. The plant grew without showing any contamination.
26 May 2025 – 27 May 2025
Then I discussed the problem with Dixit sir, and he suggested that after sterilizing the brown paper, it should be placed in the laminar airflow , with 3 to 4 sheets in a glass beaker, and after putting on the gloves, they should be cleaned with ethanol before proceeding with the next steps. After that, I cut the banana plants at the 3rd stage into two parts and inoculated them into fresh hormone medium, and they are now growing.
2 June 2025
I cut the 5th stage banana plants into two parts. And then inoculated them onto a fresh hormones medium.
7 June 2025
I cut the 5th stage banana plants into two parts. And then inoculated them onto a fresh hormones medium.
11 June 2025
I cut the 3rd stage banana plants into two parts. And then inoculated them onto a fresh hormones medium.
14 June 2025
I cut the 4th and 3rd stage banana plants into two parts. And then inoculated them onto a fresh hormones medium.
16 july 2025
l cut the 5th stage of Banana plants into two parts. And then inoculated them onto a fresh hormones medium.
23 July 2025
After discussing with Dixit Sir, I prepared charcoal medium for root induction in banana plants that are at the 6th stage.
CHARCOAL MEDIUM :-
In banana plant tissue culture, charcoal medium is mainly used during the rooting stage. Activated charcoal helps by removing harmful substances and extra plant hormones from the medium, which prevents tissue browning and promotes healthy root growth. It also improves the quality of the plantlets, making them stronger and better for transfer to soil.
PREPARATION OF CHARCOAL MEDIUM :-
| Materials | Final Volume ( 250ml ) |
| Macronutrients | 12.5ml |
| Micronutrients | 2.5ml |
| Vitamins | 2.5ml |
| Iron Source | 2ml |
| Mesoinositol | 25mg |
| Sucrose | 7.5gm |
| Agar | 2gm |
| BAP | 1ml |
| IAA | 0.125ml |
| Charcoal | 1gm |
| Distilled Water | 100ml |
13 August 2025
Some banana plants at the 7th stage were transferred to charcoal medium.
29 August 2025
The 6th stage banana plants were transferred into charcoal media.
3 September 2025
Again 6th stage banana plants were transferred into charcoal media.
21 September 2025
After one month of inoculation, the charcoal media plants were transferred to the polyhouse. For that, I autoclaved the cocopeat and took the banana plants out from the bottles, then soaked them in the fungicide (Bavistin) for 10 minutes. After that, I planted them in the cocopeat.
8 October 2025
Transferred 5 bottle plants to the 7th stage charcoal media.
10 October 2025
Some banana plants at the 7th stage were transferred to charcoal medium.
October 2025
