BY: RAHUL ANDHARIA (MSIWM001)
Plant Tissue culture:
It is an in-vitro aseptic culture method in which plant tissues or cells are grown in an artificial medium under suitable environmental conditions generally to produce clones of plants. The medium used to grow this tissues or cells is known as culture medium. There are various culture medium used based on different requirements.
Principle of Plant Tissue culture and culture medium:
Many plants have the ability to regenerate into a new whole plant which is called as Totipotency. Protoplasts(plant cells without cell wall), pieces of leaves, single cell often is used for generation of new plant using appropriate plant culture medium. Specific environment is created to provide maximum growth and multiplication of the plant in the culture medium. The conditions include providing proper nutrient medium, maintaining temperature and pH and proper liquid and gaseous environment.
Culture medium facilities in-vitro growth and morphogenesis( differentiation of unfolding of undifferentiated cells) in plants. The success of tissue culture lies in the selection of plant culture medium. If an appropriate medium is selected, better results can be obtained. Generally, plants can synthesise their own food material, but the in-vitro plants are primarily heterotrophic, that is they cannot synthesise their own food, so it becomes essential that plant culture medium should consist of the same nutrients as required by the whole plant.
Composition of Culture medium:
Basically there are two factors inconsideration for the composition of plant culture medium:
- Species of the plant- composition of the medium very much depends on the species of the plant used in tissue culture. Each plant specie has different requirements and also requires different environmental conditions for their growth.
- Type of culture material- The composition also depends on the type of material used such as protoplasts, single cells or pieces of leaves. Each material requires different composition.
Thus, composition of culture medium is dependant on the specific requirements of the culture system and it’s formulation. The type of medium used can be solid or liquid medium. The choice of medium completely depends on the culture response and it’s growth.
pH of Medium:
Most optimum pH for tissue culture is in the range of pH 5-6. pH can be adjusted during sterilization, but usually falls after autoclaving. At a higher pH of 7 and lower pH of 4.5 plant cells will no longer grow in culture medium.
General method for medium preparation:
- Prepare stock solutions using high purity chemicals and demineralised water.
- Stock solutions can be stored in glass or plastic containers in freezer and can be used whenever needed.
- As most of the growth regulators are not water soluble dissolve them first in NAOH or alcohol.
Rules for Selection of appropriate culture medium:
Each specie has different culture requirements and therefore selecting an appropriate medium is an integral part of plant tissue culture. De Fosard Et al has described an experiment to select an appropriate culture medium. This is very beneficial especially if the culture system used is not tested.
The steps are as follows:
- Medium is divided into four different categories (a) minerals, (b) auxins, (c) cytokines and (d) organic nutrients like sucrose, amino acids.
- Choose three different concentrations for each of the components that is high, medium and low.
- Try various combinations with the chosen four components. At the end it will lead you to 81 different treatments.
- Denote the four letter code the best among the 81 treatments. For example a medium containing medium salts, low auxin, low cytokinin and high organic nutrients can be represented as MLLH.
- At this stage, test the concentrations of auxins and cytokinins to assess the suitable concentration of the growth regulators.
Types of Culture medium:
There are many number of culture media available. Some of them are listed below:
- MS ( Murashige and Skoog) medium:
As the name suggests this medium was developed by the two scientists, Murashige and Skoog in the year 1962, while working on plant growth regulators. This is the most common culture medium used in labs. Basic formulation includes inorganic salts, nutrients and amino acids.
Purpose: This medium is used to induce callus culture, organogenesis, and micropropogation.
- LS (linsmaier and Skoog) medium:
This medium was developed in the year 1965. Initially, the medium was used to optimise organic nutrient supplements in Tobacco culture. This medium contains Thiamine hypochlorite and inositol. Inositol is primarily involved in primary and secondary metabolism of plants and also is an cofactor in glycolysis and TCA cycle.
Purpose: used in induction of callus culture(growing mass of parenchyma cells is, callus) organogenesis, micropropogation and cell suspension.
- Gamborg (BY) medium:
The medium was particularly used for cell suspension and callus generation of glycine max belonging to plant family, fabaceae by Gamborg in the year 1968. Formulation of medium generally includes inorganic salts, carbohydrates and vitamins. Generally, this medium has high concentration of nitrate and potassium and lower concentrations of ammonia. Soybean root callus formation is being induced by potassium nitrate, while ammonium sulphate helps in plant cell growth.
Purpose: used mainly in protoplasts culturing.
- NN ( Nitsch and Nitsch )medium:
This medium was primarily first used by Nitsch, to develop anther culture in Nicotiana, which belongs to family solanaceae. This medium is rich in thiamine, folic acid and biotin which supports anther growth.
Purpose: Particularly used in in-vitro anther culture.
- White’s medium:
The medium was developed for root culture of Tomato by P.R white in the year 1963. This was the first medium to be developed for root culturing. The medium has lower concentrations of salts and has higher concentration of Mgso4.
Purpose: widely used for shoot and callus culture. It is more appropriate for the culture of Musa and Daucus species.
Constituents of culture medium:
Elements play a vital role in plant growth and regulation. Elements differ in their physiological functions.
It consists of micro and macro nutrients. Concentration of macronutrients is greater than 0.5mmol/1-, while for micronutrients it’s less than 0.5mmol/1-.
- Macronutrient elements:
It consists of six different types of elements mainly nitrogen, phosphorus, potassium, calcium and sulphur. Ideal concentration for calcium, phosphorus, sulphur and magnesium is 1-3mm mol, while for potassium and nitrogen it is 25mm mol.
They are required in smaller amounts by tissues and cells. Commonly iron, manganese, copper, zinc, molybdenum and boron are the micronutrients. Iron requirement in the medium is very vital. Iron and copper in chelated form are used in the medium.
- Carbon and energy sources: Tissues and plant cells in the culture are usually heterotrophic, so they require external source of energy and carbon for their growth. Sucrose is the most commonly used form of energy source. During autoclaving process, sucrose will hydrolyse into glucose and fructose. Plants uses more amount of glucose than fructose and glucose is considered as efficient as sucrose.
- Organic supplements: They include vitamins, amino acids, organic extracts, activated charcoal, organic acids and antibiotics.
- Vitamins– plant cells produce vitamins but in less amounts. So it is necessary to add vitamins to the medium. Vitamins like thiamine, riboflavin, niacin, biotin, folic acid, pantothenic acid and ascorbic acid are added.
- Amino acids: organic nitrogen in form of amino acids like L-arginine, L-glutamine, L-cysteine and L-arginine are more easily taken up by plants than inorganic nitrogen.
- Organic extracts: Yeast Caseinhydrolysate, orange juice, tomato juice, coconut milk are used.
- Activated charcoal: activated charcoal can stimulate growth of certain plant cells like carrot, tomatoes and orchids. Also it can remove certain toxic components from the medium which can otherwise hinder in cell growth.
- Organic acids: succinate, citrate, fumarate and malate helps in plant growth.
- Antibiotics: antibiotics bare generally added to prevent growth of microbes. Low amounts of kanamycin and streptomycin are added.
- Growth Regulators: Phytohormones promote plant growth, differentiation and development.
- Auxins: it induces cell elongation, cell division and formation of callus in cell cultures. At lower concentrations, auxins can promote root formation and high concentration it helps in callus formation.
- Cytokinins: cytokinins are derivatives of adenine. They are involved in shoot differentiation, cell elongation and in somatic embryo formation. It also helps in promoting RNA synthesis and thereby stimulating enzyme and protein activity.
- Gibberellins: Ga3 is the most commonly used gibberellin among 20 different types of gibberellins known. Ga3 helps in growth of cultured cells, enhances growth of the callus and makes dwarf plantlets to elongate. They are generally known to promote or inhibit tissue cultures depending on the species used.
- Abscisic acid: plays major role in induction of embryogenesis in the culture. Also helps in callus formation.
- Solidifying agents: solidifying agents are used in preparation of solid medium or semi solid medium. Most commonly used agents are:
- Agar: It is a polysaccharide extracted from seaweeds. Main advantages of using agar is that it cannot be digested by plant enzymes, remains stable at cultural temperature and doesn’t react with the media constituents. 0.5 – 1% concentration of agar can form gels.
- Gelatine: it is used at higher concentration(10%) but has limited success, as it melts at lower temperature so has limited use.
Considering all the above factors, culture medium is the most important part of tissue culture. Without proper culture medium, tissue culture cannot be performed.