Tissue culture is a biotechnological technique that involves the in vitro cultivation of plant cells, tissues, or organs under sterile and controlled conditions. It enables researchers to grow plant material independently of the parent organism, making it useful for genetic modifications, conservation, and large-scale plant propagation.
Tissue culture is widely used in agriculture, horticulture, and medicine due to its ability to produce genetically identical clones, conserve rare plant species, and generate disease-free planting material. It also plays a crucial role in biotechnology for secondary metabolite production and plant genetic transformation.
Principles of Tissue Culture
Tissue culture is based on two fundamental biological concepts:
1. Totipotency
This principle states that every plant cell has the inherent ability to regenerate into a complete plant under the right conditions. By supplying specific nutrients and plant growth regulators (PGRs), a single cell can develop into an entire plant, making tissue culture an effective method for plant propagation and genetic engineering.
2. Cell Differentiation and Dedifferentiation
- Differentiation refers to the process where cells specialize into distinct tissues such as roots, stems, and leaves. Plant hormones like auxins and cytokinins regulate this process in tissue culture.
- Dedifferentiation is the ability of specialized cells to revert to an undifferentiated state, forming callus (a mass of unorganized cells). This callus can later be induced to redifferentiate into roots and shoots, facilitating plant regeneration.
Factors affecting tissue culture success:
- Aseptic conditions – Cultures must be free from microbial contamination.
- Nutrient medium – The medium must contain essential macronutrients, micronutrients, vitamins, and PGRs.
- Environmental factors – Optimal temperature (22–27°C), humidity, and light intensity influence growth.
Techniques of Plant Tissue Culture
Several techniques are used in plant tissue culture, each serving a specific purpose. These techniques allow for plant propagation, genetic improvement, and conservation.
1. Sterilization of Plant Material
- Before culturing, plant tissues (explants) must be surface-sterilized using chemical agents such as sodium hypochlorite or ethanol to remove microbial contaminants.
- Sterile conditions are maintained using laminar airflow cabinets to prevent infections.
2. Preparation of Nutrient Medium
The culture medium provides essential nutrients for plant growth. The most commonly used medium is Murashige and Skoog (MS) medium, which contains:- Macronutrients (e.g., nitrogen, phosphorus, potassium).
- Micronutrients (e.g., iron, zinc, manganese).
- Plant growth regulators (e.g., auxins, cytokinins).
- Sucrose as an energy source.
- A gelling agent (agar) to solidify the medium.
3. Inoculation of Explants
- A small piece of plant tissue (explant) is placed onto the prepared nutrient medium under sterile conditions.
- The explant is incubated under controlled environmental conditions (light, temperature, and humidity).
4. Callus Induction and Proliferation
- If the explant is treated with high auxin concentrations, a callus (an undifferentiated mass of cells) forms.
- The callus is subcultured onto fresh media to promote growth.
5. Organogenesis (Shoot and Root Formation)
- By adjusting auxin and cytokinin levels, callus tissue can be induced to form shoots and roots.
- High cytokinin-to-auxin ratios promote shoot formation, while high auxin levels encourage root development.
6. Hardening and Acclimatization
- After in vitro plantlets are formed, they are gradually exposed to natural environmental conditions in greenhouses before being transferred to soil.
- This process ensures their successful adaptation to external conditions.
Types of Plant Tissue Culture
Callus Culture
A callus is a cluster of undifferentiated plant cells formed in response to wounding. When an explant is placed on a nutrient-rich medium supplemented with auxins and cytokinins, it stimulates callus formation. Callus cultures serve as a foundation for regenerating whole plants through organogenesis or somatic embryogenesis, depending on the applied growth regulators.
Cell Suspension Culture
When friable callus fragments are transferred to a liquid medium and subjected to constant agitation, a cell suspension culture is established. This technique enhances nutrient absorption and accelerates cell proliferation. Suspension cultures facilitate large-scale production of plant metabolites, single-cell cloning, and protoplast isolation for genetic modification.
Anther/Microspore Culture
In this method, anthers or isolated microspores are cultured to induce haploid plant development. The process either follows a callus-mediated route or directly mimics zygotic embryogenesis. Microspore culture drastically reduces the time required to generate homozygous plants, making it a valuable tool for plant breeding, hybrid production, and genetic research.
Protoplast Culture
Protoplasts, which are plant cells devoid of cell walls, can regenerate into whole plants or be fused to create somatic hybrids. Enzymatic digestion or mechanical disruption is used to isolate protoplasts, which are then cultured in liquid or solid media. This technique is widely employed in genetic engineering and somatic hybridization but requires precise handling due to the fragile nature of protoplasts.
Embryo Culture
Embryo culture involves growing isolated embryos in vitro, bypassing seed dormancy or incompatibility barriers. This approach is particularly useful for rescuing hybrids from interspecific crosses or cultivating species with naturally dormant or sterile seeds. It plays a crucial role in plant breeding programs and conservation efforts.
Meristem Culture
By culturing the apical meristem, plants can be regenerated without viral infections. This technique, also known as meristem tip culture or shoot tip culture, is widely used for the propagation of disease-free plant varieties. It is especially effective in eliminating systemic viral infections, ensuring the production of healthy and genetically stable plant stock.
Applications of Tissue Culture
Micropropagation
- Large-scale production of identical plants.
- Conservation of rare and endangered species.
- Transformation of plants with desirable genes (e.g., pest resistance).
- Production of genetically modified crops.
Secondary Metabolite Production
- Production of valuable compounds such as alkaloids, flavonoids, and essential oils in pharmaceutical industries.
Disease-Free Plant Production
- Elimination of viruses and pathogens through meristem culture.
Somatic Hybridization
- Fusion of protoplasts from different species to create new hybrids.
Germplasm Conservation
- Long-term storage of genetic material through cryopreservation.
Post a Comment