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.
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 | Iron Source |
| Micronutrients | HCl |
| Agar Powder | Sucrose |
| Vitamines | Amino acid |
| Hormones | NaoH |
CHEMICALS USE FOR STERILIZATION:-
| Ethanol | Bavistin |
| Savlon ( Dettol) | Liquid Soap |
| Tween 20 | Sodium Chloride |
PREPRATION OF MACRONUTRIENTS SOLUTION:-
| Macro nutrients | Concentration | Solution Valume (250ml) |
| NH4NO3 | 1650 | 8.25 |
| KNO3 | 1900 | 9.5 |
| KH2PO4 | 170 | 0.85 |
| MgSO4.7H2O | 370 | 1.85 |
| CaCl2.2H2O | 440 | 2.2 |
| CaCl2 | 440 | 1.65 |
PREPARATION OF MICRONUTRIENTS SOLUTION:-
| Micronutrients | Concentration | Solution Valume (250ml) |
| H3BO3 | 6.2mg/l | 620mg |
| MnSO4.4H2O | 22.3mg/l | 2230mg |
| ZnSO4.7H2O | 8.6mg/l | 860mg |
| Na2MoO4.2H2O | 0.25mg/l | 25mg |
| CuSO4.5H2O | 0.025mg/l | 2.5mg |
| CoCl2.6H2O | 0.025mg/l | 2.5mg |
| Kl | 0.83mg/l | 83mg |
PREPARATION OF VITAMINS:-
| Vitamins | Concentration | Solution Valume (50ml) |
| Thiamine HCl | 0.1mg/l | 0.005g |
| Pyridoxin HCl | 0.5mg/l | 0.025g |
| Niacin | 0.5mg/l | 0.025g |
| Glycine | 2.0mg/l | 0.1g |
IRON SOURCE:-
| Source | Concentration | Solution Valume 50ml |
| FeSO4.7H2O | 27.8mg/l | 2.78g |
| Na2EDTA.2H2O | 37.3mg/l | 3.73g |
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 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 like test tube, Petri Plates, conical flask, beaker, measuring cylinder, forecep, spatula , scalpel, surgical blades etc.then dried in hot air oven for 1 to 2 hours at 180°C it so no water droplets remain in glassware.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 inoculate explant in culture bottle growing slowly.
