INTRODUCTION :-
Moringa plant tissue culture is a method of growing Moringa (Moringa oleifera) plants under sterile conditions using small plant parts like shoot tips or leaves. This technique enables rapid multiplication of disease-free and genetically uniform plants. It plays a crucial role in conserving germplasm, enhancing propagation rates, and supporting large-scale cultivation for medicinal and nutritional uses.
EXPLANT CAN USE :-
Nodal segments
shoot tips
leaf segments
embryos ( Seeds )
STAGES :-
Preparation of Explant
Initiation
Shoot Multiplication
Rooting
Hardening/Acclimatization
MATERIALS :-
| Petri plate | Test tube |
| Beaker | Test tube stand |
| Conical flask | Scissors |
| Measuring cylinder | Distilled water |
| Culture bottle | Sterile distilled water |
| Forecep | Tissue paper |
| Scalpel | Autoclave bag |
| Spatula | Filter paper |
| Cotton blug | Sterile Surgical bag |
CHEMICALS FOR STERILIZATION :-
| Liquid soap |
| Savlon ( Dettol ) |
| Ethanol |
| Sodium hypochlorite |
| Antibiotics |
CHEMICALS FOR MS MEDIA :-
| Macronutrients |
| Micronutrients |
| Vitamines |
| Iron source |
| 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 |
PREPARATION OF VITAMINS :-
| Vitamins | Original concentration (mg/l) | Original Concentration multiplying by 1000X, Final solution volume (50ml) |
| Thiamine HCl | 0.1 | 5mg |
| Pyridoxin HCl | 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 |
| Vitamines | 0.625ml |
| Iron source | 0.250 |
| Mesoinositol | 25mg |
| Sucrose | 7.5gm |
| Agar powder | 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.
13 April 2025
METHODOLOGY :-
1. Select mother plants ( Nodal part ) that are young shoot tips 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.
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 20 minutes( for 1 jar 10ml sodium hypochlorite and 250ml sterile distilled water).
12. After 20 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 30 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.
21 April 2025
RESULT :-
After one week, no growth was observed in the inoculated moringa explants. And
14 April – 15 April 2025
Cleaned the culture bottles, prepared the MS medium, and autoclaved the MS medium, glassware, and distilled water.
16 April 2025
I again took young nodal segments of moringa and inoculated them using the same process. And some contamination appeared after a few days.
23 April 2025
RESULT :-
No growth was observed in the inoculated moringa explants.
17 April – 18 April 2025
Cleaned the culture bottles, prepared the MS medium, and autoclaved the MS medium, glassware, and distilled water.
25 April 2025
Since taking nodal segments directly from the mother plant did not result in growth, I have now used seeds for plant tissue culture. When nodal segments develop from the seeds, we will take them and inoculate them into fresh medium.
METHODOLOGY:-
1.I take the seeds and soak them for 12 hours, then remove the seed coat.
2.I put the seeds into a cleaned beaker and leave them under running water for 15 minutes.
3.After 15 minutes, I remove the running water, put on gloves, and wash the seeds with liquid soap for 5 minutes ( 10ml liquid soap and 190ml distilled water).
4.Then wash it with distilled water 3 to 5 times.
5.Soak the seeds in Savlon solution for 15 minutes ( 3ml savlon 200 ml double distilled water).
6.Then remove the seeds from the Savlon solution and rinse them 3 to 5 times with double-distilled water.
7.Prepare a 4% sodium hypochlorite solution and soak the seeds in it for 20 minutes; this entire process is carried out under laminar airflow ( 10ml sodium hypochlorite and 190ml double distilled water).
8.Then remove the seeds from the sodium hypochlorite solution and rinse them 3 to 5 times with double-distilled water.
9.Soak the seeds in antibiotic solution for 30 minutes ( 1ml antibiotic and 200ml double distilled water).
10.After 30 minutes, inoculate the seeds into MS medium culture bottles and wrap them with parafilm.
11.Then place the inoculated culture bottles under sterile conditions at 24°C.
30 April 2025
RESULT : –
Contamination appears in the inoculated seeds after 5 to 6 days.
26 April – 27 April 2025
Cleaned the culture bottles, prepared the MS medium, and autoclaved the MS medium, glassware, and distilled water.
30 April 2025
Moringa seeds were taken, soaked, then surface sterilized, and subsequently inoculated into culture media.
6 May 2025
After discussing with Dixit sir, I bought a different type of Moringa seeds, cleaned them properly and subsequently inoculated them into the culture medium.
RESULT :-
Contamination occurs in moringa seeds a few days after inoculation.
9 May 2025
Again, after surface sterilizing the moringa seeds, their inoculation was done in the culture medium.
RESULT :-
After inoculating three culture bottles with moringa seeds, two bottles showed growth after a few days, while one bottle became contaminated.
10 May 2025
Cleaned the culture bottles, prepared the MS medium, and autoclaved the MS medium, glassware, and distilled water.
12 May 2025
Moringa seeds were taken, soaked, then surface sterilized, and subsequently inoculated into culture media.
RESULT :-
Contamination appears in the inoculated seeds after one week.
13 May 2025
Cleaned the culture bottles, prepared the MS medium, and autoclaved the MS medium, glassware, and distilled water.
14 May 2025
Moringa seeds were taken, soaked, then surface sterilized, and subsequently inoculated into culture media.
16 May 2025
Cleaned the culture bottles, prepared the MS medium, and autoclaved the MS medium, glassware, and distilled water.
17 May 2025
Moringa seeds were taken, soaked, then surface sterilized, and subsequently inoculated into culture media.
18 May 2025 – 29 May 2025
Autoclave was not working, so the plant tissue culture work had to be stopped for several days.
30 May 2025
Bought a new variety of moringa seeds from the market and performed plant tissue culture on it.
Moringa seeds are soaked in double distilled water, then remove the moringa seeds from the soaking culture bottle and peel off the outer seed coat. Then, surface sterilize the moringa seeds and inoculate them into the MS media culture bottle.
31 May 2025
Cleaned the culture bottles, prepared the MS medium, and autoclaved the MS medium, glassware, and distilled water.
1 June 2025
I discussed with Dixit sir the problem of Moringa seeds getting contaminated after 15 days of inoculation and the seed coat of Moringa seeds is very hard, which causes the seeds to get damaged while removing it. Dixit sir suggested performing surface sterilization of Moringa seeds before soaking and soaking the seeds in double-distilled water, then placing them in an incubator for 20 hours.
After 20 hours of soaking, remove the Moringa seeds and surface sterilize them. And inoculate them into the MS media culture bottle.
2 June 2025
Take the Moringa seeds, surface-sterilize them, and then soak the seeds in double-distilled water for 20 hours. After 20 hours of soaking, remove the Moringa seeds and surface sterilize them. And inoculate them into the MS media culture bottle.
3 June 2025
Cleaned the culture bottles, prepared the hormone medium, and autoclaved the hormone medium, glassware, and distilled water.
4 June 2025
Take the Moringa seeds, surface-sterilize them, and then soak the seeds in double-distilled water for 20 hours. After 20 hours of soaking, remove the Moringa seeds and surface sterilize them. And inoculate them into the MS media culture bottle.
6 June 2025
Cleaned the culture bottles, prepared the MS medium, and autoclaved the MS medium, glassware, and distilled water.
7 June 2025
After surface sterilizing the moringa seeds, we soak them in double-distilled water for 20 hours. After 20 hours, we remove the seeds, take off their seed coats, and then surface sterilize them again.
8 June 2025
Cleaned the culture bottles, prepared the MS medium, and autoclaved the MS medium, glassware, and distilled water.
9 June 2025
