Micropropagation – Stages, Methods and Applications

Micropropagation: Have you heard of Callus? It is an undifferentiated mass of cells, and this is formed In vitro from the artificial vegetative propagation method called Micropropagation or Tissue Culture. Micropropagation is an advanced method of artificial propagation of plants and is extensively used in agriculture, horticulture, the development of virus-plants, hybrid plants like pomato, etc.

What is Micropropagation?

Micropropagation, which is also known as tissue culture, is the method of artificial propagation of plants. It is the rapid artificial vegetative propagation of plants under In vitro conditions of high light intensity, controlled temperature, and a defined nutrient medium.

Fig: Micropropagation

Micropropagation Diagram

The steps or the stages of the Micropropagation is shown through a diagram given below:

Fig: Stages of Micropropagation

Stages of Micropropagation

Micropropagation includes five different stages and are as follows:

Stage 0: Selection of a Stock Plant
This is the first and the preliminary step in micropropagation that involves the selection and growth of stock plants for about three months under controlled conditions before using them for culture initiation.

Stage I: Culture Initiation and Establishment In this second step, the initiation and establishment of an explant in a suitable culture medium are achieved. 
i. Explant: The selected plant material used for In vitro culture is called explants. 
ii. The explants should be washed first in the running water for about \(20\) minutes. In order to remove the dirt and dust. 
iii. These explants should be trimmed, and surface sterilization should be done with \({\rm{1 – 2\% }}\) sodium hypochlorite solution or \({\rm{0}}{\rm{.1 \% }}\) mercuric chloride solution for \({\rm{10 – 15}}\) minutes. 
iv. Then these explants should be rinsed with double distilled water to remove the surface sterilant.
v. The most commonly used explants are organs, axillary buds and shoot tips. 
vi. Inoculation: Inside the inoculation chamber, the explants are then introduced into the culture tubes containing nutrient medium with sterilized forceps. This is called inoculation.
vii. Callogenesis: Within \({\rm{8 – 10}}\) days, the cut end of explants starts to divide to form a mass of undifferentiated cell called Callus, and the process is known as Callogenesis.

Stage II: Shoot Formation
Stage II mainly involves the development of the embryo and then the shoots from the given explant. 
i. Since these embryoids are developed from the vegetative cells of the explants, they are called Somatic embryoids. 
ii. The embryoids are cut and separated from the Callus and introduced into the separate culture tubes containing nutrient mediums having a higher concentration of cytokinin and low concentration of auxins which helps in the shoot development. A growth chamber set at \(20 – {24^ \circ }{\rm{C}}\) is used and with a \(2000\)- to \(4000\)-lux light intensity for a period of around \(16\) hours.
iii. Inside the culture tube, each embryo or shoot bud develops into plantlets or microshoot.

Stage III: Rooting of the Shoots
This stage involves the transfer of shoots to a medium containing plant hormones where the concentration of auxins is higher than the cytokinins, which helps in the rapid development of roots. In vitro, rooting of shoots is generally preferred.

Stage IV: Transfer of Plantlets in the Greenhouse Environment
This stage involves the establishment of plantlets in the soil, and this process is known as acclimatization. 
i. The plantlets or the microshoots are transferred to the plastic bags containing sterilized soil from the laboratories. 
ii. These bags are placed in a greenhouse, and the plantlets grow and get adjusted to the natural environmental conditions. This is called hardening. 
iii. After several weeks, the hardened plants are transferred to the pots or garden soil.

What is the Process of Micropropagation?

Plant tissue culture is a novel and innovative technique to grow high quality, disease-plants easily, quickly, and in a large quantity by culturing various plant parts. The methods of micropropagation are as follows:
1. Meristem Culture
It is the method of cultivation or production of the axillary or apical shoot meristem. It involves the development of an already existing shoot meristem and subsequently the regeneration of adventitious roots from the developed shoot. The process of meristem culture is shown below through a flow chart:

Fig: Meristem Culture Flow chart

2. Anther Culture
This culture method is the culture of anther. It helps in the production of haploid plants used for hybridization experiments. 

3. Cell Culture or Suspension Culture
In this method of micropropagation, the cells or groups of cells are dispersed and allowed to grow in an aerated and sterile liquid culture medium.

4. Embryo Culture
In this method, the embryos are removed from the developing seeds and are placed on a suitable medium to obtain seedlings. It is the culture of mature or immature embryos. It is used to produce disease-plants and used to overcome the abortion of embryo at early stages.
Embryo culture can be applied for:
i. Recovery of interspecific hybrids.
ii. Propagation of orchids.
iii. Overcoming dormancy.

Examples of Plants Developed through Micropropagation

Some of the examples of plants that are developed through micropropagation are plants such as palm, plantain, banana, tomato, pine, date, eggplant, jojoba, pineapple, rubber tree, cassava, yam, sweet potato, etc.

Applications of Micropropagation

Micropropagation has become a suitable alternative to conventional methods of vegetative propagation of plants, and some of the applications or advantages are as follows:
i. A large number of plants can be grown from a piece of plant tissue (explant) within a short period.
ii. The micropropagation technique can be carried out throughout the year, irrespective of the seasonal variations.
iii. Meristem tip cultures are generally employed to develop pathogen-plants.
iv. Micropropagation requires minimum growing space. Hence, a large number of plant species can be maintained inside culture vials in a small room in a nursery.
v. This method is the only viable method of regenerating genetically modified cells after protoplast fusion.
vi. The anther culture or pollen culture helps to produce haploid plants, which can be used to establish pure line breeds after restoring ploidy. 
vii. The cell culture and callus culture are done for the production of secondary metabolites such as flavours, fragrance, essential oils, pigments, alkaloids, etc.
viii. It involves the fusion of protoplasts of two plant cells of different species. This fusion of protoplasts of unrelated species results in the development of hybrids called somatic hybrids. Example: Pomato is a somatic hybrid of potato and tomato. 

Fig: Application of Micropropagation

Limitations of Micropropagation

Some of the limitations or disadvantages of micropropagation are as follows:
i. Micropropagation techniques require intensive labour, and this often limits their commercial application. 
ii. Automation can reduce the labour required.
iii. The plants grown from this method find a problem in acclimatizing to the new natural environment.
iv. During the micropropagation, several slow-growing microorganisms contaminate and grow in cultures. 

Summary

Micropropagation is an advanced artificial vegetative propagation technique. This is very useful and has become a frequently used technique in the plant’s vegetative propagation process. This method helps in getting disease-plants in very little time. This method also does not require more space and can produce a large number of plantlets using only tissue, organs or any part of the plant. 

Frequently Asked Questions (FAQs) on Micropropagation

The answers to the most commonly asked questions about Micropropagation are provided here:

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