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 )
root segments
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.
METHODOLOGY :-
1. Select mother plants ( Nodal part )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 20ml 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 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.
