Biotechnology: Class 12 — Chapter 7: Plant Tissue Culture

• Theodor Schwann & Matthias Schleiden (19th Century): Drew attention to the cell as the basic unit of life with the capacity to divide and grow.
• Gottlieb Haberlandt (1902): Regarded as the Father of Plant Tissue Culture. He pioneered PTC by attempting to culture fully differentiated plant cells on simple nutrient media and proposed that plant cells are capable of resuming uninterrupted growth and regenerating embryos from vegetative cells.
• Vitamins & Auxins (1930s): Discovery of their necessity for plant tissue growth on synthetic media gave a significant boost to PTC.
• Natural Supplements (1940s-1970s): Coconut water, corn milk, and orange juice were introduced to stimulate embryo development and callus culture.
• Skoog & Miller (1950s): Established that the morphogenic fate of cultured cells is influenced by the exogenous balance of auxins and kinetin. High auxin-to-kinetin ratio causes rooting; low ratio causes shooting; intermediate levels cause callus proliferation.
• Murashige & Skoog (MS) Media (1962): Developed a highly successful artificial culture medium with high salt concentrations, which remains the most widely used nutrient media in PTC.

• Organogenesis: Inducing the formation of vegetative organs (shoots or roots) from cells or tissues. It involves:
  1. Dedifferentiation: Specialised cells of explants revert to an undifferentiated state and divide to form a mass of cells called callus.
  2. Redifferentiation: Formation of organ primordia (shoot or root) from the callus, controlled by relative concentrations of auxins and cytokinins.
• Somatic Embryogenesis: The process of forming an embryo from somatic cells. The resulting somatic embryos follow embryogenic pathways similar to zygotic embryos but originate from vegetative tissues and are typically larger.

1. Washing Area: Dedicated space to wash and store glassware, plasticware, and labware.
2. Media Preparation Facility: Space and components to prepare nutrient media.
3. Sterilisation Facility: Autoclaves or micropore filters to sterilise media, instruments, and vessels.
4. Controlled Environment Culture Room: Facilities to maintain cultures under regulated light, temperature, and humidity.

1. Media Selection & Sterilisation: Preparing suitable nutrient media and sterilising it via autoclaving or filtration.
2. Explant Selection: Choosing healthy starting material (e.g., root/shoot apical meristems, leaves, cotyledons).
3. Surface Sterilisation: Treating explants with disinfectants like sodium hypochlorite, followed by sterile distilled water washes. (Explants from aseptically grown seedlings do not require surface sterilisation).
4. Inoculation: Transferring the sterile explant onto the nutrient media under aseptic conditions.
5. Incubation & Culture Growth: Growing cultures in a controlled room to regenerate callus or organs.
6. Shoot Regeneration & Elongation: Transferring cultures to shoot-induction media.
7. Rooting: Excising regenerated shoots and transferring them to a rooting medium.
8. Hardening & Acclimatisation: Transferring plantlets to sterilised soil in pots within a greenhouse before field transfer.

• Macronutrients: Required in millimolar (mM) concentrations. Includes Carbon (C), Calcium (Ca), Hydrogen (H), Potassium (K), Magnesium (Mg), Nitrogen (N), Oxygen (O), Phosphorus (P), and Sulphur (S).
• Micronutrients: Required in micromolar (μM) concentrations. Includes Boron (B), Cobalt (Co), Copper (Cu), Iron (Fe), Manganese (Mn), Molybdenum (Mo), and Zinc (Zn).
• Organic Supplements: Includes amino acids (arginine, asparagine, glycine, proline) as nitrogen sources, and vitamins (thiamine/B1, nicotinic acid/B3, pyridoxine/B6).
• Carbon Source: Sucrose (at 2-5% concentration) is the most preferred carbon source. Glucose, fructose, and mannose are also used.
• Plant Growth Hormones: Required in minute quantities. The five main classes are Auxins, Cytokinins, Gibberellins, Abscisic acid, and Ethylene. Auxins and cytokinins are primary for organogenesis.
• Gelling Agent: Required for solid media. Agar is the most commonly used gelling agent.
• pH Regulation: Usually adjusted to 5.8-6.0. High pH increases medium hardness; low pH leads to poor solidification and affects nutrient uptake and salt solubility.
• Antibiotics/Antifungals: Used selectively to suppress bacterial and mould infections.

A tissue culture technique used to rapidly multiply plants without sexual reproduction, producing genetically identical offspring (clones).

• Advantages: Rapid multiplication in small spaces, season-independent, useful for multiplying non-fertile, rare, or endangered plants.
• Commercial Success: Widely used in agriculture and forestry (e.g., potato, banana, carnation, chrysanthemum). Banana tissue culture is highly profitable in India, producing over 400 million plantlets annually.

Produced by encapsulating somatic embryos within a protective, nutrient-rich matrix.

Structure of an Artificial Seed: Consists of a somatic embryo enclosed in a gelling agent (like calcium alginate) containing nutrient elements, growth regulators, an artificial seed coat, and antibiotic/antifungal agents to mimic conventional seeds.

• Applications: Long-term storage, rapid propagation of elite species and hybrid varieties. Successfully applied in carrot, grapes, and sandalwood.

Haploid plants contain a single set of chromosomes ($n$). They are produced via anther, pollen, or ovary culture.

• Double Haploids: Treating haploids with colchicine doubles their chromosome number, creating completely homozygous diploid plants. This allows recessive traits to express immediately, accelerating breeding programs. Examples include brassica, broccoli, rice, sorghum, and tobacco.

Somatic hybridisation bypasses sexual incompatibility barriers by fusing protoplasts from distantly related species.

• Methodology: Cell walls are digested using cellulases and pectinases. Isolated protoplasts are fused using polyethylene glycol (PEG).
• Cybridisation: The process of combining the cytoplasmic genome (mitochondria/chloroplasts) of one parent with the nuclear genome of another parent, producing cybrids. Useful for transferring cytoplasmic traits like male sterility.

Viruses cause severe yield losses, especially in vegetatively propagated crops. Because apical and axillary meristems are generally free of virus particles, culturing tiny meristem explants (less than 1 mm long) allows the regeneration of completely virus-free plants. Successfully used in sugarcane, banana, and potato.

Genetic variations that accumulate in somatic cells and are expressed in plants regenerated from tissue culture.

• Boon: Source of novel agronomic traits (e.g., disease-resistant sugarcane, banana, tomato, and high-yielding wheat).
• Bane: Can introduce unwanted traits, compromising clonal uniformity in commercial micropropagation.

Plants produce secondary metabolites (alkaloids, flavonoids, tannins, steroids) for environmental interactions and defense. These are highly valuable in medicine and industry.

• In Vitro Production: Cell and tissue cultures (especially hairy root systems) provide a controlled, season-independent source of these compounds, preventing the overharvesting of rare plants.