Micropropagation / Tissue Culture
Micropropagation refers to the production of plants from very small plant parts, tissues, or cells, which are grown asexually in test tubes or containers under controlled nutritional, environmental and aseptic conditions. All biological principles of micropropagation techniques are based on the occurrence or full potential of the cell, which means that a plant cell has the potential to arise as a fully grown plant with various organs.
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Micropropagation vs. Other Propagation Methods:
Micropropagation propagation differs from other vegetative propagation methods:
- A very small plant part (explants) used as starting material
- The explants are kept in small containers with a well-defined culture medium
- Requires extreme aseptic conditions; And
- A large propagation material is produced in a very short time.
- Production of more plants in less time
- production of disease-free plants
- Clonal propagation of parental stock for hybrid seed production
- Year-round nursery production
- Useful in the propagation of dioecious plants
- the reproductive (Breeding) cycle is shortened
- Useful in difficult to propagate plant species
- Useful in germplasm conservation.
- Expensive and sophisticated facilities, trained personnel, and specialized techniques are required
- High cost of production results from expensive facilities and high labour inputs
- Contamination or insect infestation can cause high losses in a short time
- Higher level of somatic variation
- Poor establishment of the plantlets in the field.
Stages of micropropagation
Micropropagation is an integrated process in which cells, tissues, or organs of the selected plants are isolated, surface sterilized, and incubated in a growth-promoting, sterile medium and environment to produce a large number of plants. The different stages are:
Step 0: Mother Plant Selection for Explant Isolation: The mother plant from which the explants are to be isolated should be
- A certified and true representative of the desired species or varieties.
- Healthy and free from pests or diseases
- Must be vigorous enough.
Step 1: Establishment of the explant in culture medium: During this step, the tissue is grown in a suitable culture medium, preferably agar-based media, for activation and multiplication.
Stage 2: Proliferation and Multiplication: In this stage, repeated subcultures are performed to encourage more proliferation, which largely depends on a combination of growth regulators. The duration of this phase is unlimited and largely depends on the choice of the propagator.
Stage 3: Establishment and Rooting of Plants: In this stage, the selected plants are forced to root, which can be achieved by modifying the media and the concentration of growth regulators. The concentrations of cytokinins and sugars are lowered and auxin and light intensity are increased in the laboratory to initiate photosynthesis and other physiological activities simultaneously.
Stage 4. Acclimatization or hardening: Plants grown in culture tubes are adapted to a specific environment with high humidity, low light levels, and constant temperatures. Furthermore, the roots developed in the culture tubes are hairless and therefore fragile, requiring care during the transfer from the culture medium. For better survival rates, plants can be transferred to containers placed in mist chambers where the high relative humidity is maintained. Once new growth appears, these plants can be gradually moved outdoors, increasing their ability to tolerate increased light intensity.
Once the plants are well rooted, they will have to adapt to the greenhouse environment. Rooted plants in culture tubes are removed from the culture vessels and the agar is thoroughly washed to remove the potential source of contamination. Plantlets are transplanted in a more or less conventional manner by a standard pasteurized rooting or in a soil mixture in small pots or cells. Initially, micro plants should be kept in shaded, high-humidity tents or protected from drying out by mist or fog. Several days may be required for new functional roots to form.
Plantlets should be gradually exposed to low relative humidity and high light intensity. Any inactivity or rest conditions that develop may need to be overcome as part of the installation process. These conditions help the plants to adapt to the natural conditions, which also helps them to become easily established in the field.
Various culture techniques such as (i) meristem culture (ii) callus culture (iii) shoot bud regeneration (iv) somatic embryogenesis (v) ovule culture (vi) embryo culture (vii) anther culture and (viii) protoplast culture are employed in micropropagation.
- Meristem culture: Meristem culture involves the culture of both shoot-tip and axillary-bud. Pioneering by Morell in the 1950s, the basis for a technique known as meristem-tip-culturing used short shoot-tips consisting of an apical dome with one or two leaf primordia (0.1–0.5 mm). Meristem tip cultures are now routinely being used mainly in horticultural crops for the eradication of viruses from infected material. Apparently, the virus either does not invade easily or does not multiply rapidly in young meristematic tissue. A simple nutrient medium consisting only of salts, sucrose, and vitamins is used to reduce callus formation. Gibberellic acid is often needed to promote adequate growth and NAA may be needed to encourage root formation.
- Callus culture: A piece of sterile plant tissue of living cells is transferred to the culture medium to induce callus proliferation. Subcultures are then performed on a medium with or without growth regulatory concentrations, eventually resulting in external organs or embryos. In the final stage, the regenerated plants are removed from the in vitro culture and gradually exposed to the external environment so that the plants become fully autotrophic.
- Cell Culture: Cells are maintained in suspension culture to produce free cells and then sub-cultured to reproduce complete plants from single cells. This technique is now useful to induce variability in plant cells and to select desired cell types and reproduce complete plants from these types.
- Embryo Culture: It involves the aseptic excision of the embryo and transferring them to a suitable medium for development under optimal culture conditions. After the embryo develops into a plant in vitro, it is transferred to sterile soil or vermiculite and grown to maturity in a greenhouse. This technique is useful in the production of interspecific and intergeneric hybrids that cannot otherwise be accomplished and is also useful in overcoming embryo abortion.
- Protoplast Culture: From a variety of sources, protoplasts (plants without any rigid cellulose wall but with plasma membrane only allowed to fuse to form a somatic hybrid) in suitable media to regenerate the cell wall are cultured and then cultured in a suitable medium for differentiation and morphogenesis.
- Anther Culture: Anther culture is very important for breeders as it is possible to produce haploid plants that show recessive alleles. These haploid plants can be used to produce homozygous diploids, thus avoiding generations of inbreeding. Additional benefits, such as smaller flowers and longer flowering times, can be ensured by the use of haploid plants as they are generally smaller than their diploid counterparts and will not result in pollination-induced senescence by being sterile. Anther cultures are used to eliminate viruses in Pelargonium species, to produce haploid plants in Lilium species, and to obtain different flower colors in Gerbera.
- Somatic embryogenesis: The greatest potential for clonal multiplication is through somatic embryogenesis, where technically a single isolated cell can first produce an embryo, then a complete plant. Somatic embryogenesis and plantlet regeneration in various species of horticultural plants using mid-rib, leaf, and stem callus on MS-based medium modified with 1.0 – 2.0 mg/l 2,4-D and 0.25-0.50 mg/l BA or kinetin.
Success in the technology and application of tissue culture methods depends on the selection of the appropriate culture medium, which meets the nutritional requirements of cultured cells and tissues. The basic components of all nutrient media are inorganic salts, carbohydrates, vitamins, growth regulators, agar (for solid medium), and water. Other components, including organic nitrogenous compounds, organic acids, and complex substances, may be important but are optional.
1.Chadha, K.L. Handbook of Horticulture (2002) ICAR, NewDelhi
2.Jitendra Singh Basic Horticulture (2011) Kalyani Publications, New Delhi
3.K.V.Peter Basics Horticulture (2009) New India Publishing Agency
4. Jitendra Singh Fundamentals of Horticulture, Kalyani Publications, New Delhi