Tissue Culture, Micropropagation Method: Somaclones, Somatic Hybridisation, Practice Problems and FAQs

You all know that with the increasing population, the demand for food is increasing at a high pace. Although the traditional methods of breeding of crops were practised at full efficiency by the farmers, they were not able to fulfil the huge crop demands of the nations. Now the question is, what really is missing in the traditional crop breeding system? Now that we are getting ample amounts of food resources around us, what made this possible?

Fig: Traditional plant breeding

During the 1950's, the scientists came up with a new method of plant breeding called tissue culture. This new technique enticed everyone with its speed and efficiency. It is surprising to know how this method works. It uses just a part of the plant and generates a completely new plant. Isn’t it interesting? Well, there is m

Fig: Tissue culture

With the use of this technique, thousands of plants can be grown in a short time. Do you know what this technique is called? Do you know a hybrid called pomato has been produced by fusing potato and tomato. Ever thought how is this made possible? Let's find out the answers to all these questions in this article. Let’s also understand how these new advancements in the agricultural field have changed the face of plant breeding around the world.

Table of contents

• Tissue culture
• Steps of plant tissue culture
• Micropropagation
• Application of plant of tissue culture
• Somatic hybridisation
• Advantages and disadvantages of plant tissue culture
• Practice Problems
• FAQs

Tissue culture

It is the technique of maintaining and growing plant cells, tissues or organs in an artificial medium in suitable containers under controlled environmental conditions. This technique takes place in vitro in sterile conditions. Different approaches are used to make the lab environment free of contamination, i.e. to kill all microorganisms. Sterilisation is the process of killing microorganisms. Physical and chemical procedures, such as autoclaving and fumigation, are used to accomplish this.

In the year 1902, Gottlieb Haberlandt gave the hypothesis that the culture of plant tissues is possible. This idea remained unfolded until the 1950s when the scientists gave the hypothesis that the plant cells have the capacity to generate a whole new plant. As Gottlieb gave the first idea of culture of plant tissues, he is known as the father of plant tissue culture.

Fig: Gottlieb Haberlandt

Totipotency

The ability of a plant cell to divide, grow and generate an entire plant is called totipotency. Plant tissue culture takes advantage of this potential of plant cells.

Fig: Totipotency of plant cells

Steps of plant tissue culture

The steps of plant tissue culture are as follows:

Collection of explant

Explant refers to any plant portion (cell, tissue, or organ) that is used to continue plant tissue culture. It could be a stem tip, a root, a leaf, nodes, or any other component of the plant.

Fig: Parts of the plant used as explant

Preparation of nutrient media

Nutrient media is a synthetic medium that contains nutrients required for plant growth. The components used to prepare the nutrient medium are as follows:

Organic components

Organic salts are the first component of the nutritional medium. Carbon sources such as carbohydrates are commonly included. Sucrose is the most commonly used carbohydrate source. Vitamins are another organic component. For example, thiamine is commonly employed in the nutrient medium.

Inorganic components

Inorganic salts are divided into two categories. 

Micronutrients 

They are required in smaller amounts, hence the name micronutrients is used. Manganese (Mn), Iron (Fe), Molybdenum (Mo), Zinc (Zn), Copper (Cu), and Boron are common examples (B). 

Macronutrients 

They are another form of inorganic salts found in plant tissue culture medium. They are required in greater quantities for plant growth than micronutrients, hence the name macronutrients is ued. Nitrogen (N), sulphur (S), phosphorus (P), potassium (K), magnesium (Mg), and calcium (Ca) are the six primary macronutrients that are added to the nutrient medium.

Solidifying agent

Agar is a solidification agent that is melted and added in the nutrient media. The nutrient media is then autoclaved before the solution containing the components solidifies, to eliminate germs. It involves heating the items to 121° C for at least 30 minutes. This helps to get rid of bacteria and other common contaminants.

Growth hormones

Growth hormones are proteins and break down when heated, they are introduced to the feeding medium after autoclaving. These plant growth hormones can help, hinder, or improve plant development. As a result, these hormones are utilised in various combinations to help the plants flourish. The combination of auxin and cytokinin is mostly utilised. Auxin helps in root formation while cytokinin promotes the formation of axillary shoots.

Fig: Components of nutrient medium

Sterilisation

The explants, culture vessels, media and the instruments used for plant tissue culture must be free from contamination. The explants are treated with specific anti-microbial chemicals like sodium hypochlorite and then washed with sterile distilled water. This process is commonly called surface sterilisation. The vessels, media and instruments are treated with steam, dry heat or alcohol. Media can be subjected to filtration also. These steps are called sterilisation. Autoclaves are commonly used for sterilisation. 

Inoculation

Explant is transferred to a nutrient medium in aseptic circumstances with the use of forceps during inoculation.This is normally performed in a laminar air flow. 

Fig: Inoculation

Incubation

In a sterile area called a growth room, the inoculated plant tissues are cultured for 3 - 4 weeks. This is known as incubation. It is kept at 24° C in 50 - 60% humidity with an illuminating source. 

Fig: Incubation

The growth of the plant is influenced by factors such as light, humidity, and temperature in the growth room. Explants divide to generate callus during incubation (dedifferentiation). 

Callus and plantlet formation

A callus is a mass of dividing undifferentiated cells. Callus splits to form distinct parts of the plant, eventually forming a plantlet by redifferentiation.

Fig: Incubation - Formation of plantlet

Hardening

The plantlets are removed from the nutrient medium and transferred to greenhouses. They grow in sterile soil. This process is called hardening. If the plant grows, it indicates the plantlet will survive and eventually form a whole new plant from a tissue. During this process they are kept in reduced light and high humidity for a suitable period of time. This process makes the plantlets capable of tolerating the relatively harsher environments outside the culture vessels. 

Fig: Plantlet in greenhouse

Establishment in the field

After hardening the plantlets are transferred to the fields or greenhouses. 

Micropropagation

Micropropagation is the artificial generation of a large number of plants utilising the technique of plant tissue culture. Plantlets grown using the plant tissue culture technique are identical copies of the parent plant. Somaclones are genetically identical plants created utilising the somatic tissue or cells of plants in plant tissue culture. For example, plants of tomatoes, apples etc. can be produced for commercial purposes by this method. 

Fig: Micropropagation

Application of plant tissue culture

There are four main applications of plant tissue culture. 

• Production of disease free plants
• Somatic hybridisation
• Production of transgenic plants
• Production of mutant plants

Production of disease free plants

The specific areas in the plants which take part in the formation of new cells are called the meristems. In virus infected plants, the meristems are virus free as these cells are actively dividing and viruses cannot replicate themselves in these cells. When meristems are used in plant tissue culture, the plantlets obtained are disease free.

Fig: Production of disease free plants

Somatic hybridisation

It is the process of generation of a hybrid plant by the fusion of somatic cells of two species or varieties of plants. The steps in the process of somatic hybridisation are as follows:

Isolation of protoplast

Protoplast is the part of the living cell without a cell wall. First the somatic cells of the two plants are isolated i.e., tomato and potato. The cell wall of the somatic cells are broken down to obtain the protoplast. The enzymes like cellulase and pectinase are used to break down the cell wall to expose the protoplast of the cells.

Fig: Isolation of protoplasts

Fusion of protoplasts

The isolated protoplasts obtained are fused by physical methods like electrofusion (using brief high voltage electric current) or chemical methods using PEG or Polyethylene glycol or sodium nitrate. Both the cytoplasm and nuclei are fused. 

Fig: Fusion of protoplasts

Cell wall formation

The cell wall gradually emerges over the protoplasts that have fused. This is now a new cell with both potato and tomato traits, as the cell wall emerges. The fused protoplasts are allowed to grow in the culture medium. 

Fig: Formation of hybrid cell

Tissue culture is now being used to create the entire new plant, that is a somatic hybrid of potato and tomato. The somatic hybrid obtained from tomato and potato is pomato.

Fig: Somatic hybrid of tomato and potato - Pomato

Although the generation of pomato was a success, it didn't meet the expectations for the desired characteristics. Hence, the commercial production of pomato never took place. Somatic hybrids are commonly used for gene transfer, transfer of cytoplasm and production of useful allopolyploids. 

Production of transgenic plants

The gene transferred to an organism by genetic engineering is called transgene. The newly created organism by this method is called a transgenic organism. Plants with desirable traits are produced by this method. 

Production of mutant plants

Mutations are induced by adding mutagens to the single cell liquid cultures normally. The cells from this culture are washed and transferred to solid culture for raising plants. 

Advantages of plant tissue culture

The advantages of plant tissue culture are many as follows:

• The plants can be cultivated at any time of the year, regardless of the season.
• Plantlets are produced in a short period of time from a small piece of plant tissue.
• The newly created plants are disease-free.
• Tissue culture does not need a vast room or field to produce plants.
• It produces exact copies of parent plants with good traits like fruits, flowers etc.
• This method is used for mass propagation of plants for commercial purposes.
• This method is used to grow ornamental plants like chrysanthemums, dahlias, orchids etc.

Disadvantages of plant tissue culture

Plant tissue culture has many advantages. But there are some disadvantages also they show up.

• Setting up a laboratory facility is expensive.
• Equipments and reagents are costly
• If a single plant is susceptible to disease in the culture, the entire batch will be susceptible to the same disease.
• A lot of highly trained people are required to perform the procedure with utmost care.
• If precautions are not taken care of during the procedure, the whole stock may get infected.

Practice Problems

Q 1. What is callus?

a. A mass of undifferentiated cells
b. An embryonic tissue growing in the culture
c. A totipotent cell used for plant tissue culture
d. A growth hormone used in nutrient media

Answer: The term ‘callus’ refers to a mass of proliferating and undifferentiated cells formed in tissue culture. It is a mass of cells that have not yet differentiated into specialised plant structures. Callus separates into separate plant components, finally creating a plantlet (redifferentiation). Hence, option a is correct.

Q 2. In which of the following procedures polyethylene glycol is used?

a. Transfer of explant to nutrient medium
b. Solidifying culture media
c. Fusion of isolated protoplasts
d. Micropropagation of plantlets

Answer: Protoplast fusion can be done by an electric field or chemically by adding 20 - 40 percent polyethylene glycol to the protoplasts, which produces protoplast aggregation and then protoplast fusion. After the protoplasts have fused, the somatic hybrid must be chosen. Hence, option c is correct.

Q 3. Assertion (A): After inoculation, the cultured plant tissues are incubated in a growth room.

Reason (R): It provides well-controlled sufficient light, temperature, humidity, and air quality for the tissue culture to grow.

a. Both (A) and (R) are true, but (R) is not the correct explanation of (A)
b. Both (A) and (R) are true, and (R) is the correct explanation of (A)
c. (A) is true, but (R) is false.
d. Both (A) and (R) are false.

Answer: Explant is inoculated with forceps and transferred to a nutrient medium under aseptic conditions. The cultured plant tissues are transported to a sterile growth environment for incubation after inoculation. The room is kept at the right temperature, with enough light, humidity, and air quality for the plant tissue to grow. Explants divide to form callus throughout the incubation period (dedifferentiation). A callus is a mass of undifferentiated cells that are dividing. Callus separates into separate plant components, finally creating a plantlet (redifferentiation). Hence option b is correct.

Q 4. Match the following.

a. A - II, B - III, C - I, D - IV, E - V
b. A - II, B - I, C - III, D - V, E - IV
c. A - IV, B - III, C - II, D - I, E - V
d. A - IV, B - V, C - I, D - III, E - II

Answer: Any plant part (cell, tissue, or organ) that is used to continue plant tissue culture is referred to as an explant. It can be a stem tip, a root, a leaf, node, or any other plant component. Micropropagation is the technique of artificially producing a huge number of plants using plant tissue culture. Plantlets created by the plant tissue culture process are exact replicas of the parent plant. Somaclones are genetically identical plants grown in plant tissue culture using somatic tissue or cells. Totipotency refers to a plant cell's ability to divide, expand, and form a whole plant. Somatic hybridisation is the process of fusing protoplasts of somatic cells obtained from different kinds and or species of plants on an appropriate nutritional medium to produce somatic hybrids. For example, a somatic hybrid created by fusing the protoplasts of tomato and potato is called pomato. Hence, option c is correct.

Q 5. Define somatic hybridisation. Describe the steps involved in this process?

Answer: Somatic hybridization is the process of fusing protoplasts of somatic cells obtained from different kinds and possibly species of plants on an appropriate nutritional medium to produce somatic hybrids. For example, The somatic hybrid generated by fusion of the protoplasts of tomato and potato is known as pomato.

The steps involved in the process of somatic hybridisation are as follows:

• Single cells are isolated from the plants that are selected.
• Using enzymes like pectinase and cellulase, the cell wall is removed. These enzymes expose the naked protoplast by digesting the cell wall.
• Under aseptic circumstances, the separated protoplasts of chosen cells are united to produce hybrid protoplasts on a specific nutritive medium. 
• The use of polyethylene glycol (PEG) or a short high voltage electric current (electrofusion) might cause this fusion.
• The hybrid protoplast is cultivated on an appropriate nutritional medium, where it regenerates its cell wall and begins to divide into new plantlets. 
• These plantlets are known as somatic hybrids.

FAQs

Q 1. What is the use of cytokinin and auxin in plant tissue culture?
Answer: In tissue culture, cytokinins can accelerate cell division and induce the production of axillary shoots. They generally function as auxin antagonists. Cell division and root differentiation are aided by auxins. In cultures, auxins cause cell division, cell elongation, and callus development.

Q 2. What are the uses of micropropagation in plant tissue culture?
Answer: The micropropagation is utilised for the fast vegetative replication of ornamental plants and fruit trees. This tissue culture approach yields multiple plants. Each of these plants will have the same genetic makeup as the original plant from which it was developed and are called somaclones.

Q 3. Name the person who started tissue culture in India?
Answer: Shipra Guha-Mukherjee is an Indian botanist. She started tissue culture in India. She worked on plant tissue culture, plant molecular biology, biotechnology and molecular biology.

Q 4. What is the most important application of plant tissue culture?
Answer: Micropropagation using meristem and shoot culture to produce large numbers of identical individuals.

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