Tissue culture (TC) is the cultivation of plant cells, tissues, or organs on specially formulated nutrient media. Under the right conditions, an entire plant can be regenerated from a single cell.
Tissue culture technology is important for the production of disease-free, high quality planting material and the rapid production of many uniform plants.
Types of tissue culture
- Seed Culture.
- Embryo Culture.
- Callus Culture.
- Organ Culture.
- Protoplast Culture.
- Anther Culture.
Procedure of Plant tissue culture
The part(s) of the plant used for culturing is known as explants. The explants are cultured in-vitro on a nutrient medium that caters to fulfil its nutritional requirements. The nutrient medium must provide the following:-
- Macronutrients – This includes elements like nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), sulfur (S) which is required for proper growth and morphogenesis.
- Micronutrients – Elements like iron (Fe), manganese (Mn), zinc (Zn) etc., which are also crucial to the growth of tissues.
- Carbon or Energy source – This is one of the most crucial ingredients of the nutrient media. Sucrose is the most widely used carbon source among other carbohydrates that serve to provide C, H, and O.
- Vitamins, amino acids, and other inorganic salts.
DAY – 1(10/10/2021)
- Lab cleaning, lab rearrangement and fumigation.
1st day of training we learnt about lab cleaning and fumigation techniques.
- lab were cleaned by using Dettol and Ethanol
- Rearrange all the instruments which is needed for the tissue culture.
- fumigation were done by using potassium paramagnet and formaldehyde. This method produce a violent chemical reaction that generates considerable heat and releases formaldehyde gas.
- All glassware Bottles, tubes and Petri plates were cleaned and dried using Hot Air Oven and Autoclaved all of them.
2. Distilled water collection and sterilization.
Stored the Distilled water and sterile the water in autoclave for the media preparation.
DAY – 2(30/10/2021)
- Preparation of 1 liter MS stock solution
- stock 1 – Macronutrient (major stock )
| Sr. no. | Chemical | Weight ( mg/lit) | Weight for 20X (mg) for 500ml |
| 1 | NH4NO3 | 1650 | 33000 |
| 2 | KNO3 | 1900 | 38000 |
| 3 | MgSO4.7H2O | 370 | 7400 |
| 4 | KH2PO4 | 170 | 3400 |
| 5 | CaCl2.2H2O | 440 | 8800 |
Dissolve all chemicals should be dissolve one by one in 500 ml D/W.
NOTE:-To make 1 liter MS basal media we have to add 25 ml of macronutrient stock.
- stock 2 – Micronutrient
| Sr.no. | Chemicals | Weight ( mg/lit) | Weight for 200X (mg) for 500ml |
| 1 | H3BO3 | 6.2 | 1640 |
| 2 | MnSO4.4H2O | 22.3 | 4460 |
| 3 | ZnSO4.4H2O | 8.6 | 1720 |
| 4 | KI | 0.83 | 166 |
| 5 | Na2MoO4 | 0.25 | 50 |
| 6 | CoCl2.6H2O | 0.025 | 5 |
| 7 | CuSO4.5H2O | 0.025 | 5 |
NOTE:-To make 1 liter MS basal media we have to add 2.5 ml of micronutrient stock.
- Stock3 -Fe-Na- EDTA
| Sr.no. | Chemical | Weight ( mg/lit) | Weight for 50X (mg) for (200ml) |
| 1 | Na2EDTA.2H2O | 37.3 | 1865 |
| 2 | FeSO4.7H2O | 27.8 | 1390 |
NOTE:-To make 1 liter MS basal media we have to add 1ml of Fe-Na- EDTA stock .
- Stock-4 Vitamin
| sr.no. | Chemicals | mg/lit. | 100x mg |
| 1 | Thiamin HCl | 0.1 | 10 mg |
| 2 | Nicotinic acid | 0.5 | 50 mg |
| 3 | Pyridoxin HCl | 0.5 | 50 mg |
| 4 | Glycine | 2.0 | 200 mg |
- Myo-inositol – 100mg/Lit to be added freshly
- Sucrose 30g/ lit
- pH= 5.8
- Gelrite- 2mg/lit or agar agar -8 gm/lit
Note- for pH calibration Chemical should be used
| 0.1 N NaOH | 100 ml |
| 0.1 N HCl | 100 ml |
- PGR stock – Auxin and cytokine 100, 50 mg/ml
- MS basal media for seed germination (1000 ml)
- MS+ 0.55 mg/liter BAP (500 ml)
- MS + 1 mg/liter BAP ( 500 ml)
DAY – 3(11/11/2021)
Mother plant collection –
- Combretum indicum ( Madhumalati)
- Frerea indica ( Shiv suman )
- Clitoria ternatea ( gokarna)
Explant surface sterilization –
DAY – 4 (18/11/2021)
Inoculation and labelling.
We had inoculated seed part, nodule section and leaf part of plant. all of them have different procedure to surface sterilization and inoculation
- Combretum indium ( Madhumalati)
- Tap water washing with tween 20 by using 1% concentration for 30 min.
- Washing with distilled water inside laminar.
- Sterile distilled water, glasswares,culture water and miscellaneous and UV treatment 20-30 min.
- Wash explant with distilled water twice.
- Deep explant in 0.1% HgCl2 and wait for 2-3 min
- 3-5 times wash with D/W
- Deep explant in 70% alcohol leaf or nodule section wait 30 sec-1 min
- 3-5 times wash with D/W
- Drain out sterilized explant for inoculation.
- cut down edges of leaf by using sterile surgical blade and then cut leaf into 4 parts.
- by using sterile forceps place the leaf explant in media bottle and tubes.
- place the explant at four corner on equal distance from each other and make sure that don’t place explant very close to edge of bottle.
- Seed Culture – Frerea indica ( Shiv suman )
- we had pods of shiv suman. That pods were collected by Lokhande sir.
- Tap water washing with tween 20 by using 1% concentration for 30 min.
- Washing with distilled water inside laminar.
- Sterile distilled water, glasswares,culture water and miscellaneous and UV treatment 20-30 min.
- Wash explant with distilled water twice.
- Deep explant in 0.1% HgCl2 and wait for 2-3 min
- 3-5 times wash with D/W
- Deep explant in 70% alcohol and wait 2-3 min
- 3-5 times wash with D/W
- Drain out sterilized explant for inoculation.
- after surface sterilization that pods were collected in sterile dry petri plate.
- by using Sterile surgical blade and forceps make a cut superficial to take out the seeds from that pod. don’t give deep cut, it will be damage to explant seeds.
- pull out the seeds from pod and cut the lower part of it.
- inoculate in culture bottles and tubes as we done for Madhumalati
Seed Culture
- Clitoria ternatea ( gokarna)
- we had pods of gokarna. That pods were collected from Vigyan Ashram campus
- Tap water washing with tween 20 by using 1% concentration for 30 min.
- Washing with distilled water inside laminar.
- Sterile distilled water, glasswares,culture water and miscellaneous and UV treatment 20-30 min.
- Wash explant with distilled water twice.
- Deep explant in 0.1% HgCl2 and wait for 2-3 min
- 3-5 times wash with D/W
- Deep explant in 70% alcohol and wait 2-3 min
- 3-5 times wash with D/W
- Drain out sterilized explant for inoculation.
- after surface sterilization that pods were collected in sterile dry petri plate.
- by using Sterile surgical blade and forceps make a cut superficial to take out the seeds from that pod. don’t give deep cut, it will be damage to explant seeds.
- pull out the seeds from pod and cut the lower part of it.
- inoculate in culture bottles and tubes as we done for shiv suman.
Tobacco
Incubation and Observation.
Conditions- Temp- 25-30 degree celius
Humidity above 60%
Light- 16 hr- Daylight and 8 hr dark.
31/12/2021
Prepared 38 bottle of 1.5 lit MS basal media with three BAP concentrations 0.25mg/ml, 0.5 mg/ml, and 1 mg/ml, respectively.
Jan 2022
Sub culturing of in vitro grown seedlings sesuvium(Feria, Tobbaco, gokarn)
15 jan2022
Medium- 0.25 and 0.5 BA,MSB
Prepared 23 bottle for sub culturing of tobacco and Feria
3 time media prepation were wrong due to improper pH.
We have total 23 bottle of tobbaco and 7 bottle of Feria
Feb 2022
Sub culturing of tobacco plant- MS media prepared varying PGR concentration 0.5 and 1 mg per ml.
Prepared 28 bottle for sub culturing 4 bottle of fully growth plant at 27th feb 2022.
March 2022
Sub culturing of tobacco plant- MS media prepared varying PGR concentration 0.5 and 1 mg per ml.
Prepared sub culturing 28 bottle
April 2022
May 2022
Prepared 18bottle for sub culturing 4 bottle of fully growth plant at May 2022
June 2022
Prepared 58 bottle for sub culturing 4 bottle of fully growth plant at June 2022
July 2022
Prepared 25 bottle and 14 test tube for sub culturing 4 bottle of fully growth plant at July 2022
August 2022
3/08/2022
Taking all type of photos all plant( Ferari indicum and turmeric plant
Nov 2022
