What is Micropropagation?
Micropropagation is a method of plant propagation using extremely small pieces of plant tissue taken from a carefully chosen and prepared mother plant, and growing these under laboratory conditions to produce new plants. It is widely used in commercial horticulture.
Plant tissue culture refers to the practice of growing plant material in laboratories in all forms, including micropropagation, and also other techniques which although not always of immediate practical use in horticulture are very important in other fields such as plant science research and plant breeding.
Methods of Micropropagation
Meristem Culture
In this method of micropropagation, subtending leaf primordial and a meristem is placed into their respective growing media culture and allowed to grow. After some weeks, an elongated rooted plantlet is produced.
Once after they reach a considerable height, these plantlets are transferred into the soil. In this method, a disease-free plant can be produced and can be successfully used for rapid multiplication of various herbaceous plants.
Callus Culture
In this method, selected plant tissue is placed in an artificial growing medium culture until the callus is formed. After the production of call us, they are transferred into a culture medium containing plant growth regulators for the induction of adventitious organs. After a few weeks, a new plantlet is exposed gradually to the environmental condition.
Suspension Culture
In this method of micropropagation, cells or groups of cells are dispersed and allowed to grow in an aerated and sterile liquid culture medium.
Embryo Culture
In the method of embryo culture, the embryo is extracted and placed into a culture medium with proper nutrient in aseptic condition.
Protoplast Culture
In this method, the plant cell is isolated and cultured in an appropriate medium to reform the cell wall and callus. Later, under suitable conditions, the cell develops a cell wall followed by an increase in cell division and cellular differentiation and grows into a new plant.
Steps of Micropropagation
Pre-Stage 1: Selection of Stock Plants
This step includes selecting the source plant based on the desirable characters. The stock plants chosen for culturing purposes are grown for their multiplication on a large scale. After selection, these plants are transferred and maintained in a favorable growth environment.
The condition generally maintained for the growth of explants for about three months include low humidity, irrigation, and the absence of any systemic microbial infection. The plants are checked and observed regularly for their health conditions.
Stage 1: Establishment of Aseptic Culture
In this stage, the selected plants are prepared for culturing purposes. The preparation includes cutting the selected source of explant to a suitable size that should fulfill the goal of culturing.
The explants are pretreated and sterilized with certain chemicals before they are inoculated in the growth medium.
The chemicals used to surface sterilize the shoot tip, axillary buds, and meristem are 5% sodium hypochlorite, 0.1% mercuric chloride, or 70% alcohol. (You can also use PPM for the surface sterilization step).
The sterilization time varies depending on the procedure and the explant to be cultured. Sometimes the sterilization process is done in combination with other methods depending on the level of surface contamination.
After surface sterilizing the explants, inoculate them on a growth culture medium containing components based on species to be grown and method to be performed.
The explants are generally inoculated in basic Murashige Skoog (MS) medium (or any other suitable media) supplemented with growth regulators (like auxin, cytokinin), vitamins, and sucrose.
The inoculated explants are incubated at a 16-h photoperiod at 3000–10,000 flux light intensity.
Stage 2: Multiplication of Explants
In this stage, the cultured explants are provided with suitable culture media and growth conditions for the multiplication of shoots and embryo development. This stage requires a considerable amount of time for shoot regeneration from explants.
Then, the regenerated shoots are excised into multiple explants for the multiplication purpose. In some cases, many nodal explants are obtained excising the single shoot (as in the case where only a single shoot is obtained from the apical shoot).
For the multiplication, cytokinin rich media is used to inoculate the nodal explants. In a period of 4 to 5 weeks, 5-6 explants are produced from a single shoot. For the maximum survival of the plants, provide suitable media supplemented with growth regulators and keep them in a suitable environment.
Stage 3: Rooting of Regenerated Shoots or Somatic Embryo Germination
This stage can be called the rooting stage. In this stage, the grown shoots are transferred to a fresh medium supplemented with auxins to induce rooting in the regenerated shoots. The rooting is induced by individually separating each shoot and then transferring it into the rooting medium.
Sometimes, rooting is directly induced in the soil having high moisture. In some other cases, the first hardening of the plant is done and then they are slowly transferred to soil.
For example, in somatic embryogenesis, the explants are allowed to form plantlets and then are transferred to soil for their growth and acclimatization. The hardening medium should be either pearlite, peat, or vermiculite.
These mediums should hold considerable moisture and be maintained at high humidity. Only the properly grown plantlets with roots should be transferred to the soil.
Stage 4: Acclimatization or Transferring of Plantlets to Soil
The plants grown in vitro conditions are very sensitive to diseases and pathogen attacks. The transfer of plants to the soil should be carefully performed.
When the in vitro plants are exposed to the natural environment, they have to undergo several variables of culture conditions.
To protect them from sudden shock, hardening of the plantlets are performed to avoid stress, moisture, and several diseases.
Also, during the acclimatization, the roots of the plants should be well developed and cuticular layers should be formed which makes them suitable for the transfer to open fields.
In some cases, vitrification of the cultures is observed in which the plantlets look brittle, water-soaked, and glossy. It can be due to abnormal functioning of stomata, poorly developed vascular bundles, and abnormal wax. This condition can lead to the loss of many plantlets.
Advantages of Micropropagation
Micropropagation has a number of advantages over traditional plant propagation techniques:
- The main advantage of micropropagation is the production of many plants that are clones of each other.
- Micropropagation 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).
- Micropropagation 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 of Micropropagation
Micropropagation is not always the perfect means of multiplying plants. Conditions that limit its use include:
- Labour may make up 50–69% of operating costs.
- All plants produced via micropropagation are genetically identical clones, 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 micropropagation 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 micropropagation because the cost is prohibitive. Other breeders use it to produce stock plants that are then used for seed multiplication.
Mechanisation of the process could reduce labour costs, but has proven difficult to achieve, despite active attempts to develop technological solutions.
Applications of Micropropagation
The various applications of micropropagation are:
- Plant tissue in small amounts is sufficient for the production of millions of clones in a year using micropropagation. It would take a great deal of time to produce an equal number of plants using conventional methods.
- The technique of micropropagation provides a good alternative for those plant species that show resistance to practices of conventional bulk propagation.
- An alternative method of vegetative propagation for mass propagation is offered through micropropagation. Plants in large numbers can be produced in a short period. Any particular variety may be produced in large quantities and the time to develop new varieties is reduced by 50%.
- Large amounts of plants can be maintained in small spaces. This helps to save endangered species and the storage of germplasm.
- The micropropagation method produces plants free of diseases. Hence, disease-free varieties are obtained through this technique by using meristem tip culture.
- Proliferation of in vitro stocks can be done at any time of the year. Also, a nursery can produce fruit, ornamental, and tree species throughout the year.
- Increased yield of plants and increased vigor in floriculture species are achieved.
- Fast international exchange of plant material without the risk of disease introduction is provided. The time required for quarantine is lessened by this method.
- The micropropagation technique is also useful for seed production in certain crops as the requirement of genetic conservation to a high degree is important for seed production.
- Through somatic embryogenesis production of synthetic artificial seeds is becoming popular nowadays.