19 march 2025
INTRODUCTION :- Plant tissue culture of turmeric (Curcuma longa) is a technique used to grow turmeric plants in a controlled, sterile environment using small plant parts like rhizome buds or shoot tips. This method helps in rapid multiplication of disease-free and high-quality turmeric plants. It is especially useful for conserving elite varieties and producing planting material year-round, regardless of season.
APPLICATIONS :-
• Mass propagation of elite genotypes.
• Conservation of germplasm.
• Genetic transformation studies.
STAGES OF TURMURIC PLANT :-
- Preparation of Explant
- Initiation
- Shoot Multiplication
- Rooting
- Hardening/Acclimatization
SELECTION OF EXPLANT:-
• Shoots tip
• Rhizome
MATERIALS :-
| Petri Plates | Beaker |
| Test tube | Culture bottle |
| Spatula | Tissue paper |
| Scissors | Distilled water |
| Cotton plug | Sterile distilled water |
| Forecep | Sterile Surgical Blades |
| Scalpel | Autoclave bag |
| Test tube stand | Filter paper |
| Conical flask | Measuring cylinder |
CHEMICALS FOR STERILIZATION :-
| Ethanol |
| Savlon ( Dettol) |
| Liquid soap |
| Sodium hypochlorite |
| Bavistin |
| Antibiotics |
CHEMICALS FOR MS MEDIA :-
| Macronutrients |
| Micronutrients |
| Iron source |
| Vitamins |
| Mesoinositol |
| Sucrose |
| Agar |
| Hormones |
| HCl / NaOH |
PREPARATION OF MACRONUTRIENTS :-
| Macronutrients | Original concentration(mg/l) | Amount of salt after multiplying original concentration by 20X, Solution Volume (250ml) |
| NH4NO3 | 1650 | 8250mg |
| KNO3 | 1900 | 9500mg |
| KH2PO4 | 170 | 850mg |
| MgSO4.7H2O | 370 | 1850mg |
| CaCl2.2H2O | 440 | 2200mg |
PREPARATION OF MICRONUTRIENTS :-
| Micronutrients | Original concentration(mg/l) | Amount of salt after multiplying original concentration by 100X, Solution Volume (250ml) |
| H3BO3 | 6.2 | 155mg |
| MnSO4.4H2O | 22.3 | 557.5mg |
| ZnSO4.7H2O | 8.6 | 215mg |
| Na2MoO4.2H2O | 0.25 | 6.25mg |
| CuSO4.5H2O | 0.025 | 0.625mg |
| CoCl2.6H2O | 0.025 | 0.625mg |
| KI | 0.83 | 20.75mg |
| Vitamins | Original Concentration (mg/l) | Original Concentration multiplying by 1000X, Final solution volume (50ml) |
| Thiamine HCl | 0.1 | 5mg |
| PyridoxinHCl | 0.5 | 25mg |
| Nicotinic | 0.5 | 25mg |
| Glycin | 2.0 | 100mg |
PREPARATION OF IRON SOURCE :-
| Iron source | Original Concentration (mg/l) | Original Concentration multiplying by 400X, Final solution volume (50ml) |
| FeSO4.7H2O | 27.8 | 2780mg |
| Na2EDTA.2H2O | 37.3 | 3730mg |
PREPARATION OF MS MEDIA :-
| Materials | Final Volume ( 250ml ) |
| Distilled water | 100ml |
| Macronutrients | 12.5ml |
| Micronutrients | 0.625ml |
| Iron source | 0.625ml |
| Vitamines | 0.25ml |
| Mesoinositol | 25mg |
| Sucrose | 7.5gm |
| Agar | 2gm |
MESOINOSITOL:-
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 HORMONES:-
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 :-
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. 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 turn8 on UV – ray light for 1 hour.
21 march 2025
METHODOLOGY :-
1. Select mother plants ( Rhizome ) that are 6–12 months old, disease-free, and have high-yield and 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 4% 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 30ml 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.
25 march 2025
RESULT :-
Contamination appeared in both rhizome and shoot tip explants a few days after inoculation.
22 march – 23 march 2025
Cleaned the glassware, prepared the MS media, and autoclaved both the glassware and MS media.
24 march 2025
I gave a hands-on session to Makethon participants on how to prepare the water testing kit.
25 march 2025
During the Makethon. I instructed participating studenty on the preparation of culture MS media and conducted hands-on soil testing sessions with them.
26 march 2025
During the Makethon, I provided hands-on training to the participating students on the inoculation procedure for turmeric explants, some changes the procedure after the discussion of Dixit sir, washing with a brush and soaking in an antibiotic solution for 30 minutes.
27 march 2025
Cleaned the glassware, prepared the MS media, hormones media and autoclaved the glassware, MS media and hormones media.
31 march 2025
Contamination appeared in the rhizome explants a few days after inoculation, while no contamination was observed in the shoot parts.
29 march 2025
Cleaned the glassware, prepared the MS media, and autoclaved both the glassware and MS media.
1 April 2025
Carefully select a healthy turmeric plant and wash the explant ( shoot tip ) 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.
RESULT :-
After 15 days, growth appears in the turmeric explant but contamination is observed in the culture bottle.
4 April – 5 April 2025
Prepare the hormone-containing medium, then sterilize the medium, distilled water, glassware, and other required items using an autoclave.
7 April 2025
Again select a healthy turmeric plant and wash the explant ( shoot tip ) 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.
RESULT :-
growth appears in the turmeric explant but again contamination is observed in the culture bottle.
9 April 2025
Turmeric explants were selected and inoculated following the same procedure as previously used.
15 April 2025
After 15 days of inoculating the turmeric explants, a few contaminations appeared.Then, the contamination of the turmeric plant continued to increase.
The following is an Excel sheet containing plant tissue culture data.
https://1drv.ms/x/c/840e164df4551c42/EXC8xqPVbxpNgi5T4T485zEBE8mvOz70V4I2lsaw145gUA?e=G8XCuh
