Plant Tissue Culture

Plant tissue culture is as technique for economic propagation of plants. This training is aimed for the under graduate students so that they become familiar with this upcoming and very important aspect of bio technology. The plants chosen are Sarpagandha, Kalmeg, Aloe vera, Brahmi and Poplar
Plant Tissue Culture
Introduction:
Plant Tissue Culture and genetic engineering are the two most widely used methods for crop improvement in plant breeding. Plant tissue culture is an emerging tool for plant biotechnology and mainly implied for the propagation of some economically important cops of agriculture, horticulture, forestry, endangered, rare and threatened plants.

Definition:
"Plant Tissue Culture" is the technique of in vitro maintaining and growing plant cells, tissues or organs aseptically on artificial medium in suitable containers under controlled environmental conditions. It also known as "micro propagation."
 
History
Tissue culture technique is based on "totipotent" nature of plant cell or phenomenon of totipotency i.e each and every plant cell has inherent capacity to develop into a complete plant.
  1. The concept of totipotency was given by Haberlandt (1902) also known as "Father of Plant Tissue Culture".
  2. Practical application of totipotency was shown by Steward (1932) when he developed a complete carrot plant from a single cell obtained from root of wild carrot.
  3. F. Skoog and Miller (1975) demonstrated the importance of growth hormones in the plant tissue culture.
 
Technique of Plant Tissue Culture
The technique of tissue culture can be carried out into the following 3 phases
(a) Pre - experiment phase
(b) Experimental phase
(c) Post - experiment phase
(a) Pre - experimental phase
In includes the following steps:

1. Sterilization of Room / culture Room
For this, fumigation method can be applied or mopping with formaline solution can be done. This will help in removing micro-organisms from the culture room.
(Yuvak Quami Ekta Committee and Chitransh A D P G College Bhopal)

2. Washing of glassware
Proper washing protocol should be fallowed as it is the prime step of the experiment. If washing is not proper, it may bring contamination.

3. Choice of media
Plant cells and tissues require a proper nutrient medium for three growth and development. Therefore the proper choice of medium is as important as other steps. Depending upon the morphology and nutrient requirements of plant, medium is selected. In plant tissue culture, there are different medias eg:- M.S. medium (Murashig & Skoog medium), White Medium, Gambog etc.
The medium must contain the following components:-
  • Nutrients
  • Carbohydrate Source - Sucrose
  • Solidifying agent - Agar
  • Water
  • Growth Regulators
  • Special Additives.

 
Basic nutrition
  • Macronutrients - N, K, Ca, P, Mg, S.  
  • Micronutrients - Mn, Fe, Zn, B, Cu, Mo, Co, I,
  • Vitamin's - Thiamine, Pyridoxine, Nicotinic acid, glycine, Riboflavin, calciumpant othinate  etc.
  • Additives - Coconut milk, casein hydrolysate, yeast  extract, malt extract, adenine sulphate, glutamine, activated charcoal.
 
Growth Regulators
Phyto-hormones are the most important ingredient of the medium. The concentration and selection of growth regulator is the deciding step in any experiment of plant tissue culture.
The concentrations of phytohormones in plant tissue culture medium are usually present in milligrams (mg), parts per million (ppm) or micromoles. Every phytohormone has their specific function.
(i) Auxins:- used to induce - Embryogenesis, Rooting Callusing, Maturation. Natural auxins- IAA = 3 - Indole acetic acid
Synthetic auxins
(1) 2,4 - D = 2,4, Dichlorophenoxy acetic acid.
(2) IBA = 3 - Indolebutyric acid
(3) NAA = 1- Napthylacetic acid
(Yuvak Quami Ekta Committee and Chitransh A D P G College Bhopal)
 
(ii) Cytokinin:- Used to induce - Embryogenesis, shootinitiation, shoot buds flowering egs
(a) BAP - Benzyl aminopurine
(b) KN - Kinetin
(c) Zeatin - 4 hydroxyl - 3-methyl -Trans -2butenylanunopurine
(d) 2ip - N6 - (2 - isopentyl) - adenine
 
(III) Others:-
  • Gibberellins: Influence growth and development in a variety of ways eg :- by increasing stem length, promoting flowering and fruiting.
  • Absciseic acid: Maturation of embryos.
  • Ethylene
 
(iv) Preparation of Stock solutions:-
The stock solution of different compositions of medium are prepared according to the protocols and kept in a refrigerator.
In our Biotech lab, we have prepared MS medium. Its two components MA & MB has been prepared and kept as a stock.
Stock solutions of phytohormones have also been prepared.
 
(b) Experimental Phase
It includes following stops:
(1) Media preparation:

All the ingredients are mixed carefully. The Ph of medium (MS) should be 5.5 to 5.8.
(2) Sterilization of Nutrient Medium:
Proper autoclaving for 20-30 minutes of medium should be done.
(3) Collection of Ex-plants:
In plant tissue culture technique, a single cell or a group of cells is taken for culture, which is known as ex-plant. (Ex-plant can be any part of the plant being it a leaf, shoot, stem, leaves).
- Explants can be obtained either from fields or from controlled cultured plants.
- The regeneration of plants in in-vitro conditions is largely dependent upon the mature of explants.
(Best results are shown by those explants, which have obtained from controlled cultured plants.)
In our lab we have selected the following:
(a) Rauwolfia Serpentine ( Sarpagandha)
(b) Bacopa monnieri (Brahmi)
(c) Aloe vera (Ghrita kumari)
(d) Poplar
(e) Kalmeg (Andrographis penniculata)
These are all medicinal plants and in Indian herbal industry which is at blooming stage, with very high demand.
(4) Sterilization of Explants:
Ex-plants being it apical buds, axillary buds, leaves are sterilized properly. The process of killing microbial contaminants on the surface of explants materials is known as surface - sterilization.
Collected explants were thoroughly washed with 2-4 drops of surfactant (Tween 20 or any soft liquid swap) for 10 min under running tap water. Surfactants seduce the surface tension of material being cleaned there by making the disinfectant solution more effective.
After washing with tap water properly the explants were subjected to chemical sterilization (HgCl2 or NaOCl) in the laminar airflow. It is then washed 3-4 times with sterilized double distilled water. Now the explants are ready for inoculation on required medium.
(5) Inoculation of Explants:
The inoculation of sterilized explants is carried out in laminar airflow cabinet. Laminar airflow is sterilized with UV for 30 minutes followed by 15 minutes flow of motor before inoculation. The forceps are further sterilized by flame with repeated dipping in ethanol. Finally the explants is inoculated in the medium. The mouth of the culture vial is immediately re-plugged.
(6) Incubation:
Incubation of culture vials is carried out in incubation room under proper light conditions of 16 hours photoperiod and 8 hours dark period at a temperature of 25+30C and relative humidity of 50 to 60 percent.
(7) Observations:
The cultures are observed after one week and after wards with proper growth sub cultured.
 
(c) Post-Experimental Phase
Transfer to greenhouse
After complete plant developments, cultured plants are acclimatized/ hardened in the greenhouse. The healthy and tolerating plants are finally transferred to fields.