Micropropagation

Definition, Application,

Advantages/Disadvantages


Definition



Micropropagation refers to a method used for the purposes of propagating or cloning given genotype in vitro. 

In general, there are several methods through which organisms can produce similar copies of themselves. A good example of this is through binary fission where some bacteria are able to divide and give rise to daughter cells similar to the parent.

On the other hand, cuttings (from parents with desired traits) are used in agriculture and botany, etc for the purposes of reproducing plants with similar characteristics to the original plant.

Although these methods result in the production of individuals with similar characteristics (genotype) to the original plant/organism, they differ from micropropagation in that micropropagation specifically involves growing cells/tissue from a given plant/organism in culture under sterility.

While this technique is more sophisticated compared to some of the other techniques used in cloning, it allows for the reproduction of a large number of plants within a short period of time. 

 


Some of the plants that are propagated through micropropagation include:

 

  • Pine
  • Rubber tree
  • Tomatoes
  • Yam
  • Oil palm
  • Jojoba
  • Banana

 



Tissue Culture



In micropropagation, a small piece of tissue from a plant (e.g. from the apical part of the plant, flower or leaves, etc) is used to produce numerous plants.

Following tissue preparation, the first step of micropropagation is known as tissue culture where the small tissue is put in a culture plate (with the appropriate medium). 


Some of the media used here include:

 

  • Linsmaier and Skoog
  • White's Medium
  • Gamborg medium
  • Nitsch and Nitsch

 

Tissue culture is essential for micropropagation because it's used to grow small tissue/cells. Here, the medium provides all the nutrients and other substances (e.g. hormones) that the cells need to proliferate and ultimately produce the shoot and roots. Therefore, tissue culture is one of the most important aspects of micropropagation. 



Steps of Micropropagation



Although micropropagation is generally divided into four main stages, there is a preparative stage that comes before any of the other steps.

For this reason,  the entire process can be divided into five (5) main stages. 



These include:

 


Stage 0: Preparative stage


Also known as the mother plant selection stage, stage 0 is an important step of micropropagation that allows for the ideal plant (with desired characteristics) to be selected and prepared for the next stage. 

This may involve selecting a suitable plant and allowing it to develop in hygienic conditions. Pretreatment also helps reduce the risk of contamination.

Depending on the plant, one of the strategies used here involves growing the plant in a greenhouse under specific conditions required by the plant. As well, some plants like Cordyline spp may be foliated and sterilized (surface sterilization) during this stage.

 

Some of the other strategies used to ensure that the plant remains hygienic for the next stage involve:



· The use of trickle irrigation - Unlike overhead irrigation, this helps minimize chances of spreading infectious organisms (bacteria, fungi, etc)


· Maintaining a temperature of about 25 degrees C and a humidity level of 70 percent - However, this may change depending on the type of plant


· The use of thermotherapy (using heat) in order to eradicate viruses

 

* The primary goal of Stage 0 is to care for the plant and thus obtain starting material that is hygienic and better adapted physiologically.

 


Stage 1: Initiation of Culture


This stage of micropropagation serves to produce a reliable start by establishing culture of the plant selected. 


While it's the second step of five main stages of micropropagation, this stage may also be divided into three (3) main steps that include:

 

a) Cutting a piece of tissue - The first step in this stage involves cutting a small tissue from the plant. While parts of the leaves and flower heads have been used, using the apical part of the plant or an auxiliary bud is often recommended. This is because compared to the other parts of the plant, the apical part and auxiliary buds tend to be less contaminated. Here, cutting a small part of the plant is also suggested to help reduce the risk of contamination. 


b) Disinfecting - The second step involves disinfecting the plant tissue. This is an important step given that the tissue has to be sterilized before it can be grown in culture.

Here, the first step involves washing the plant tissue in water. The tissue is then placed in 95 percent ethanol for a few seconds before being put in about 1 percent mercuric chloride and detergent for about 3 minutes.

A very small amount of the detergent is used- e.g. two drops of the detergent per 100 ml of solution. After 3 minutes, the tissue is rinsed using water (autoclaved water) and placed in sodium hypochlorite and a small amount of detergent for about 10 minutes. Using water, the tissue is again rinsed to remove the detergent. 

 

* If a laminar flow hood is available, then this procedure can be carried out in the hood to improve the final results.


* The disinfection step serves to remove surface contaminants. 

 

c) Growing the tissue in the appropriate medium - The last step of stage two (2) involves placing the tissue in the appropriate medium known as the initiation medium. One of the media commonly used is the Murashige and Skoog medium; However, any nutrient medium used should contain growth hormones as well as the nutrients required for the tissue to actually start growing.

Generally, most of the media used have been shown to consist of half ( ½) strength LePoivre basal salt mixture, auxin hormone, 3 percent sucrose, 5mg/L benzylaminopurine, as well as 0.8 percent agar with a pH of 5.7.

 

* Part of the plant that is cut for culture (e.g. apical part or axillary bud) is known as the explant.


* Cells of the apical are constantly dividing which is one of the reasons the apical tissue is suitable for culture.


* In culture, nutrients allow the cells to divide and produce a mass of cells known as a callus.

 

Given that high cell proliferation during stage I results in the production of a callus, this presents an opportunity to increase the number of plantlets.

Depending on the number of plantlets required, the callus can be further cut into small pieces (making sure not to cause any contamination) which are then further grown in culture.

Because several parts are obtained from the same callus, then all the plantlets will all have the same characteristics. 

 


Stage II - Multiplication stage


The third stage of micropropagation is characterized by growth which results in the development of the vegetative shoot. To induce growth and elongation of the vegetative shoot (from the callus/mass of cells), the appropriate mixture of auxins and cytokinins is used.

In particular, cytokinin (e.g. kinetin), which is a regulator of plant growth promotes the development of the shoot as well as its elongation from the mass of cells in culture. 

 


Stage III: Shoot elongation and root development

 

In this stage of micropropagation, the shoot continues to elongate in culture. In addition, however, it's also the stage in which the roots are induced to develop. By this stage, the plant tissues have given rise to a small shoot. However, some of the roots have not yet developed. 

For this reason, auxins, are used to induce the development of roots which prepares the plant to be planted - Auxins are a type of plant growth regulator that promotes the development of the plant including the development of parts of the plant like the roots. 

Here, however, it's worth noting that for some of the plants (e.g. herbaceous species), roots are quickly produced in the culture and therefore using the hormone (auxin) is not necessary. 

 


Stage IV: Acclimatization (transplantation)


Following the rooting stage, the plant is ready to be planted in soil so that it can continue growing. This, however, generally involves growing the plant in greenhouse conditions to allow it to adjust (acclimatization ). Here, a greenhouse provides the plant with the conditions it needs to continue growing.

Transfer of plantlets from tissue culture to soil exposes the plant to new conditions. For instance, unlike the conditions in a culture plate/container, there is a higher risk of infection when the plantlet is planted in soil. 

Given that most of the plantlets that grow in culture do not produce cuticles, growing them in greenhouse conditions is also beneficial because it prepares them for other new conditions like changing light and temperature intensities that would otherwise desiccate them. 

Following the acclimatization period in greenhouse conditions, the plantlets can then be transferred into the farms or gardens so that they can continue growing and developing. 

 


Applications



For Agronomic Crops


Agronomic crops are a wide variety of plants that are primarily produced for food. However, some of these plants are also used to produce fuel or for land restoration purposes.

Because of their many uses, these crops occupy vast amounts of land in different parts of the world. Some examples of agronomic crops include beans, cotton, sugar cane, sugar beets corn, and wheat among many others. 

Although a number of other methods and techniques such as those used to produce transgenic plants are currently being used to improve the overall quality of plants/crops, micropropagation is also being used for the commercial production of a few plants/crops like sugarcane. 

Generally, one of the reasons this method has not been exclusively used to propagate many horticultural plants/crops is because they are mostly propagated using seeds. However, it's still widely used for producing hybrids with the majority of these crops. 

For this reason, micropropagation still plays an important role in the production of high-quality agronomic crops. In doing so, researchers have been able to reproduce the desired characteristics of different types of plants/crops. 

 

* In agriculture, micropropagation is also used to produce animal feeds/fodder. The method has also proven effective in producing hybrids that provide the nutritional requirements of different types of domestic animals



In Forestry


One of the other applications of micropropagation is in forestry. The main reason for using this technique in forestry is that it allows researchers to produce trees with characteristics similar to the trees that already grow in certain regions. Therefore, using this technique, it becomes possible to produce trees that are suited to a specific area or region. 

For instance, in India, teak is one of the most important trees. This is because it is durable and has been shown to be insect resistant. For this reason, it's an important source of timber in the country.

In India and the neighboring countries where this type of tree grows, a single bud is capable of producing as many as 500 trees a year. 

This makes micropropagation an important technique that can be used for reforestation as well as to ensure that the people will have timber and other products they need for their day to day lives. 

Apart from India, the application of micropropagation in forestry has become popular in a number of other countries including New Zealand,  France, and other parts of Europe.

 

* In forestry, the shoot multiplication cycle ranges from 2 to 6 weeks. This is a significantly short period of time that allows for many trees (plantlets) to be produced within a year.

 


In floriculture


Floriculture is a branch of horticulture that is primarily focused on the cultivation of flowers as well as other ornamental plants. This is a large industry that has been reported to grow at a rate of 15 percent annually.

Because of the high demand for different types of flowers and other ornamental plants from different parts of the world, tissue culture in micropropagation has proved very effective for producing different varieties. 

While the technique is particularly effective for producing plants with similar characteristics to the original plant, experts in the field have developed methods of using micropropagation for hybridization in order to produce an array of unique flowers with different characteristics. 

One of the biggest benefits of using this method is the fact that it allows farmers to meet the high demand for flowers within a short period of time. 

 


Advantages of Micropropagation


In the different sectors and industries where micropropagation is applied, the technique has a number of advantages that include:

 

Short growth time - One of the biggest advantages of micropropagation is the fact that it takes a short period of time to produce many plantlets. Once a small tissue is grown in a medium, the callus that is produced can be cut into smaller pieces and again grown in culture so that it can continue to grow. Here, these tissues grow within a short period of time before they can be transferred for continued growth. 

 

Initial small tissue - In micropropagation, only a small initial tissue is required. Here, the tissue obtained from the plant is first grown in culture so that the cells can divide to form a callus (a mass of cells).

This callus can then be cut into smaller pieces and again grown in culture to produce many plantlets. Therefore, even a small tissue can be used to produce numerous plants within a short period of time.  

 

Disease-free plantlets - During the preparation of the tissue (explant) to be used for micropropagation, steps are taken to ensure that the tissue is sterile before it can be grown in culture. In addition, all the equipment are sterilized before culture.

For this reason, most of the plantlets are likely to be free of diseases and pathogens that can these diseases. If proper care is taken when growing the plantlets in greenhouse conditions or in the farm/garden, then these plants would be free of diseases and pathogens.

 

Space - Given that the initial growth involves using small tissues, then space is not a big issue in micropropagation. If steps are taken to minimize chances of contamination, then a small space can be used to initiate growth of the tissue to produce plantlets that can then be grown in a greenhouse setting.

Here, however, the space is also dependent on the number of plantlets being produced. 



Disadvantages of Micropropagation


While micropropagation has many advantages, there are also various disadvantages associated with this method.


These include:

 

Labor intensive and high cost - Micropropagation is a complex method that consumes a lot of time. As mentioned, tissues obtained from a plant have to be properly sterilized in order to minimize the risk of contamination. In addition, conditions required for growth have to be monitored to ensure that ideal conditions are maintained.

Because of the complex nature of this process and the time taken to ensure that all protocols are followed, the cost may be too high for some people. 

 

Poor resilience - Essentially, micropropagation involves using a small tissue from the plant of interest in order to produce plants with similar characteristics. This may prove to be an issue in cases where the plant is less resilient to diseases and given environmental conditions.

More resources may be required to ensure that the plantlets are protected. As a result, this may further increase the cost of producing quality plants. 

 

Diseases/pathogens - One of the biggest advantages of micropropagation is that only a small tissue is required to produce many plantlets that can then be grown on a farm or garden.

In cases where the tissue was not properly sterilized, then any diseases or pathogens that it carries would be passed down to all the plantlets.

Moreover, in a situation where these plantlets are transferred to a farm or garden, then the disease/pathogens may be spread further to other crops and plants. 

More plant biology related articles:


Leaf Structure under the Microscope

Photosynthesis

Page on Plastids

Learn more about Chloroplasts

Mesophyll Cells

Meristem Cells

Budding

Sporulation

Transgenic Plants


Return to Tissue Culture

Return to Cell Culture 

Return to Binary Fission 

Return from Micropropagation to MicroscopeMaster home








References



P. Debergh and R.H. Zimmerman. (1991). Micropropagation Technology and Application. 

 

Richard A Dixon, R. A. Dixon, and Robert A. Gonzales. (1995). Plant Cell Culture: A Practical Approach.

 

Saurabh Bhatia and Kiran Sharma. (2015). Micropropagation: Modern Applications of Plant Biotechnology in Pharmaceutical Sciences. 

 

Subodh Kumar Datta Datta, Jour Pl, and Sci Res. (2019). Need based Tissue Culture in Floriculture : A Success story. 

 

 

Links 

 

https://www.researchgate.net/figure/Advantages-and-disadvantages-of-tissue-culture-based-Agrobacterium-mediated_tbl3_319252279


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