Here’s a detailed, structured overview of plant tissue culture and cloning techniques, including applications, steps, and types of cloning methods used in plants.
1. Plant Tissue Culture Overview
Plant tissue culture (PTC) is the in vitro cultivation of plant cells, tissues, or organs under sterile conditions on a nutrient medium. It allows for rapid multiplication, conservation, and improvement of plants.
Key Principles
- Explants (any plant part: leaf, stem, root, bud, meristem) are cultured aseptically.
- Nutrient medium (Murashige & Skoog [MS] commonly) provides:
- Macronutrients & micronutrients
- Vitamins
- Carbon source (usually sucrose)
- Plant growth regulators (PGRs): auxins and cytokinins
- Controlled light, temperature, and humidity ensure growth.
Advantages
- Rapid clonal propagation of elite plants
- Disease-free plant production (meristem culture)
- Conservation of endangered species
- Production of secondary metabolites
- Genetic transformation and breeding support
2. Cloning Techniques in Plants
Cloning in plants refers to producing genetically identical copies of a plant. Tissue culture provides several cloning techniques:
A. Micropropagation
Definition: In vitro propagation of plants from small explants to produce multiple identical plantlets.
Stages
- Initiation: Selection and sterilization of explants.
- Multiplication:
- Explants produce multiple shoots or callus.
- Media enriched with cytokinins for shoot proliferation.
- Rooting:
- Shoots transferred to auxin-rich media to induce roots.
- Acclimatization: Gradual adaptation to soil/greenhouse conditions.
Applications
- Commercial propagation of ornamental, medicinal, and rare plants.
- Production of uniform planting material (e.g., Aquilaria spp., bananas, orchids).
B. Organogenesis
Definition: Formation of organs (shoots or roots) from explants or callus.
- Direct Organogenesis: Organs form directly from explant tissue without callus formation.
- Indirect Organogenesis: Organs develop from callus tissue.
Key PGRs:
- Cytokinins → shoot induction
- Auxins → root induction
Applications: Mass propagation of woody plants and elite genotypes.
C. Somatic Embryogenesis
Definition: Formation of embryos from somatic (non-reproductive) cells, which then develop into complete plants.
- Direct: Embryos arise directly from explants.
- Indirect: Embryos arise via callus formation.
Applications:
- Synthetic seed production
- Cloning of endangered species
- Large-scale propagation in bioreactors
D. Protoplast Culture and Fusion
Definition: Culturing plant cells without cell walls (protoplasts).
- Protoplast fusion allows hybridization between different species that cannot cross sexually.
Applications:
- Somatic hybrid production
- Genetic improvement and breeding
E. Meristem and Shoot Tip Culture
Definition: Culture of actively dividing apical meristems to produce virus-free plants.
Applications:
- Virus elimination in crops (potatoes, sugarcane)
- Rapid multiplication
F. Synthetic Seeds
Definition: Somatic embryos encapsulated in a protective gel matrix for storage, transport, and planting.
Applications:
- Conservation of endangered plants
- Large-scale propagation
3. General Steps in Plant Tissue Culture
- Explant Selection & Sterilization
- Culture Initiation on sterile media
- Induction of Callus or Shoots using PGRs
- Multiplication of shoots or embryos
- Rooting / Embryo Germination
- Acclimatization in greenhouse conditions
4. Commonly Used Plant Growth Regulators (PGRs)
| PGR Type | Examples | Role |
|---|---|---|
| Cytokinins | BAP, Kinetin, TDZ | Promote shoot proliferation |
| Auxins | IAA, IBA, NAA, 2,4-D | Promote root induction, callus formation |
| Gibberellins | GA3 | Stimulate elongation, seed germination |
| ABA | Abscisic Acid | Somatic embryo maturation, stress response |
5. Applications of Plant Tissue Culture and Cloning
- Mass propagation of elite and endangered plants
- Production of disease-free planting material
- Secondary metabolite production in bioreactors
- Genetic engineering and transformation studies
- Conservation of germplasm and rare species