Methods of Micropropagation

Micro-propagation is the practice of quickly multiplying stock plant material to produce a large number of progeny plants, using contemporary plant tissue culture methods.
Micro propagation is used to multiply dignified plants such as those that have been genetically modified or bred through conventional plant breeding methods. It is also used to provide a sufficient number of plantlets for planting from a stock plant which does not produce seeds, or does not respond well to vegetative reproduction.

Establishment : –
Micro propagation begins with the selection of plant material to be propagated. The plant tissues are removed from an intact plant in a sterile condition. Clean stock materials that are free of viruses and fungi are important in the production of the healthiest plants. Once the plant material is chosen for culture, the collection of explants (s) begins and is dependent on the type of tissue to be used; including stem tips, anthers, petals, pollen and other plant tissues. The explant material is then surface sterilized, usually in multiple courses of bleach and alcohol washes, and finally rinsed in sterilized water. This small portion of plant tissue, sometimes only a single cell, is placed on a growth medium, typically containing sucrose as an energy source and one or more plant growth regulators (plant hormones). Usually the medium is thickened with agar to create a gel which supports the explant during growth. Some plants are easily gr
own on simple media, but others require more complicated media for successful growth; the plant tissue grows and differentiates into new tissues depending on the medium. For example, media containing cytokinin are used to create branched shoots from plant buds.

Multiplication :- 
Multiplication is the taking of tissue samples produced during the first stage and increasing their number. Following the successful introduction and growth of plant tissue, the establishment stage is followed by multiplication. Through repeated cycles of this process, a single explant sample may be increased from one to hundreds and thousands of plants. Depending on the type of tissue grown, multiplication can involve different methods and media. If the plant material grown is callus tissue, it can be placed in a blender and cut into smaller pieces and recultured on the same type of culture medium to grow more callus tissue. If the tissue is grown as small plants called plantlets, hormones are often added that cause the plantlets to produce many small offshoots. After the formation of multiple shoots, these shoots are transferred to rooting medium with a high auxin\ cytokinin ratio. After the development of roots, plantlets can be used for hardening.

Pre-transplant : –
This stage involves treating the plantlets/shoots produced to encourage root growth and “hardening.” It is performed in vitro, or in a sterile “test tube” environment.
“Hardening” refers to the preparation of the plants for a natural growth environment. Until this stage, the plantlets have been grown in “ideal” conditions, designed to encourage rapid growth. Due to the controlled nature of their maturation, the plantlets often do not have fully functional dermal coverings. This causes them to be highly susceptible to disease and inefficient in their use of water and energy. In vitro conditions are high in humidity, and plants grown under these conditions often do not form a working cuticle and stomata that keep the plant from drying out. When taken out of culture, the plantlets need time to adjust to more natural environmental conditions. Hardening typically involves slowly weaning the plantlets from a high-humidity, low light, warm environment to what would be considered a normal growth environment for the species in question.

Transfer from culture :- 
In the final stage of plant micro-propagation, the plantlets are removed from the plant media and transferred to soil or (more commonly) potting compost for continued growth by conventional methods.

This stage is often combined with the “pre-transplant” stage.

Methods of Micro-propagation

Meristem Culture : –
In Meristem culture the Meristem and a few subtending leaf primordial are placed into a suitable growing media. An elongated rooted platelet is produced after some weeks, and is transferred to the soil when it has attained a considerable height. A disease free plant can be produced by this method. Experimental result also suggests that this technique can be successfully utilized for rapid multiplication of various herbaceous Plants.

Callus Culture
A callus is mass of undifferentiated parenchymatous cells. When a living plant tissue is placed in an artificial growing medium with other conditions favorable, callus is formed. The growth of callus varies with the homogenous levels of auxin and Cyotkininn and can be manipulated by endogenous supply of these growth regulators in the culture medium. The callus growth and its organogenesis or embryogenesis can be referred into three different stages.

• Stage I: Rapid production of callus after placing the explants in culture medium
• Stage II: The callus is transferred to other medium containing growth regulators for the induction of adventitious organs.
• Stage III: The new plantlet is then exposed gradually to the environmental condition.

Suspension Culture :- 

A cell suspension culture refers to cells or groups of cells dispersed and growing in an aerated liquid culture medium (Street, 1997, Thorpe1981) is placed in a liquid medium and shaken vigorously or balanced dose of hormones. Suezawa et al. (1988) reported Cyotkininn induced adventitious buds in kiwi fruit in a suspension culture Sub- culture for about a week.

Embryo Culture : –

In embryo culture, the embryo is excised and placed into a culture medium with proper nutrient in aseptic condition. To obtain a quick and optimum growth into plantlets, it is transferred to soil. It is particularly important for the production of interspecific and intergeneric hybrids and to overcome the embryo abortion.

Protoplast Culture : –

In protoplast culture, the plant cell can be isolated with the help of wall degrading enzymes and growth in a suitable culture medium in a controlled condition for regeneration of plantlets. Under suitable conditions the protoplast develops a cell wall followed by an increase in cell division and differentiation to grows into a new plant. The protoplast are first cultured in liquid medium at 25 c to 28 c with a light intensity of 100 to 500 lux or in dark and after undergoing substantial cell division, they are transferred into solid medium congenial or morphogenesis in many horticultural crops response well to protoplast culture.

Advantages : –

Micro-propagation has a number of advantages over traditional plant propagation techniques:

• The main advantage of micro-propagation is the production of many plants that are clones of each other.
• Micro-propagation can be used to produce disease-free plants.
• It can have an extraordinarily high fecundity rate, producing thousands of propagules while conventional techniques might only produce a fraction of this number.
• It is the only viable method of regenerating genetically modified cells or cells after protoplast fusion.
• It is useful in multiplying plants which produce seeds in uneconomical amounts, or when plants are sterile and do not produce viable seeds or when seed cannot be stored (see recalcitrant seeds).
• Micro-propagation often produces more robust plants, leading to accelerated growth compared to similar plants produced by conventional methods – like seeds or cuttings.
• Some plants with very small seeds, including most orchids, are most reliably grown from seed in sterile culture.
• A greater number of plants can be produced per square meter and the propagules can be stored longer and in a smaller area.

Disadvantages:- 

Micro propagation is not always the perfect means of multiplying plants. Conditions that limit its use include:

• It is very expensive, and can have a labour cost of more than 70%.[fusion_builder_container hundred_percent=”yes” overflow=”visible”][fusion_builder_row][fusion_builder_column type=”1_1″ background_position=”left top” background_color=”” border_size=”” border_color=”” border_style=”solid” spacing=”yes” background_image=”” background_repeat=”no-repeat” padding=”” margin_top=”0px” margin_bottom=”0px” class=”” id=”” animation_type=”” animation_speed=”0.3″ animation_direction=”left” hide_on_mobile=”no” center_content=”no” min_height=”none”][clarification needed]
• A monoculture is produced after micro propagation, leading to a lack of overall disease resilience, as all progeny plants may be vulnerable to the same infections.
• An infected plant sample can produce infected progeny. This is uncommon as the stock plants are carefully screened and vetted to prevent culturing plants infected with virus or fungus.
• Not all plants can be successfully tissue cultured, often because the proper medium for growth is not known or the plants produce secondary metabolic chemicals that stunt or kill the explant.
• Sometimes plants or cultivars do not come true to type after being tissue cultured. This is often dependent on the type of explant material utilized during the initiation phase or the result of the age of the cell or propagule line.
• Some plants are very difficult to disinfect of fungal organisms.

The major limitation in the use of micro propagation for many plants is the cost of production; for many plants the use of seeds, which are normally disease free and produced in good numbers, readily produce plants (see orthodox seed) in good numbers at a lower cost. For this reason, many plant breeders do not utilize micro propagation because the cost is prohibitive. Other breeders use it to produce stock plants that are then used for seed multiplication.
Mechanization of the process could reduce labour costs, but has proven difficult to achieve, despite active attempts to develop technological solutions.

By:- Abhishek Bahuguna
Department of Horticulture.
Uttaranchal (P.G.) College Of Bio-Medical Sciences & Hospital

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