Samacheer Kalvi 12th Biology Chapter 7 Exercise Solutions

Totipotency is the ability of a single plant cell to divide and regenerate into a complete organism. Every living plant cell contains the full complement of genetic information (genome) needed to produce an entire plant. This property is the fundamental basis of plant tissue culture — it allows a small piece of plant tissue (explant) to be cultured in vitro and grown into a whole plant under appropriate hormonal and nutritional conditions.

Callus is an unorganised, undifferentiated mass of cells that forms when an explant is cultured on a nutrient medium containing growth hormones. The cells of the explant undergo dedifferentiation — they lose their specialised structure and revert to a meristematic (dividing) state. This is stimulated by a balanced ratio of auxin and cytokinin in the medium. The callus can later be induced to redifferentiate into shoots, roots, or embryos by changing the hormone balance.

Auxin and cytokinin are plant growth regulators that control organogenesis in tissue culture. A high auxin to cytokinin ratio promotes root formation (rhizogenesis). A high cytokinin to auxin ratio promotes shoot formation (caulogenesis). A balanced ratio of both hormones maintains callus growth without differentiation. This hormonal control was first demonstrated by Skoog and Miller and is called the auxin-cytokinin ratio concept.

Somaclonal variation is the genetic variation that arises in plants regenerated from tissue culture. It occurs due to mutations, chromosomal changes, or epigenetic alterations during the tissue culture process. It can be undesirable when the goal is to produce genetically identical clones (micropropagation). However, it can be desirable in plant breeding as a source of novel traits, such as disease resistance or improved yield, which can be selected and propagated.

Somatic hybridisation is the process of fusing protoplasts (cells without cell walls) from two different plant species to create a hybrid. The cell walls are removed using cellulase and pectinase enzymes, and the protoplasts are fused using polyethylene glycol (PEG) or electrofusion. The fused cell divides and regenerates into a hybrid plant. Example: Pomato — a somatic hybrid of potato and tomato created by fusing their protoplasts.

Anther culture involves culturing anthers (containing haploid microspores) on nutrient medium to produce haploid plants. Haploid plants have only one set of chromosomes. These haploids can be treated with colchicine to double their chromosome number, producing homozygous diploids (dihaploids). This is important in plant breeding because it shortens the time needed to develop pure lines from several generations to just one step.

Steps of micropropagation:

1. Selection of explant: A small piece of healthy plant tissue (shoot tip, node, leaf segment) is selected as the explant.

2. Surface sterilisation: The explant is sterilised using disinfectants (sodium hypochlorite, mercuric chloride, or ethanol) to remove all microorganisms. Aseptic conditions are essential.

3. Culture initiation: The sterilised explant is placed on a sterile nutrient medium (MS medium) containing macronutrients, micronutrients, vitamins, sucrose, agar, and growth hormones.

4. Shoot induction: Cytokinin (e.g., BAP) in the medium promotes multiple shoot formation from the explant. This is the multiplication stage.

5. Subculture: Growing shoots are transferred to fresh medium to encourage further multiplication. Multiple subcultures can produce thousands of shoots.

6. Rooting: Individual shoots are transferred to a rooting medium containing high auxin (e.g., IAA, IBA, NAA) to induce root formation.

7. Acclimatisation (hardening): Plantlets are gradually exposed to external conditions (reduced humidity, natural light) in a greenhouse before transplanting to soil. This step is critical because in vitro plantlets have thin cuticles and stomata that are not fully functional.

Diagram description: Draw a flow chart — Explant → Sterilisation → Culture on MS medium (cytokinin) → Multiple shoots → Subculture → Rooting medium (auxin) → Rooted plantlets → Hardening → Soil (field transfer).

Advantages of micropropagation:

• Rapid multiplication — thousands of plants from a single explant in a short time
• Genetically identical plants (somaclones) — preserves desirable traits
• Virus-free plants — shoot tip culture eliminates pathogens
• Year-round production — not dependent on seasons
• Conservation of endangered species through mass propagation

The nutrient medium (commonly Murashige and Skoog medium / MS medium) is the foundation of tissue culture. Its composition:

1. Macronutrients (required in large amounts):

• Nitrogen (N), Phosphorus (P), Potassium (K), Calcium (Ca), Magnesium (Mg), Sulphur (S)
• These are essential for protein synthesis, energy transfer, cell division, and membrane function

2. Micronutrients (required in trace amounts):

• Iron (Fe), Manganese (Mn), Zinc (Zn), Boron (B), Copper (Cu), Molybdenum (Mo)
• Iron is provided as Fe-EDTA for better absorption; micronutrients act as enzyme cofactors

3. Vitamins:

• Thiamine (B1), Nicotinic acid, Pyridoxine (B6), Myo-inositol
• Thiamine is essential; others promote growth

4. Carbon source:

• Sucrose (2–4%) — provides energy because tissue culture cells are heterotrophic (cannot photosynthesise)

5. Growth hormones (phytohormones):

• Auxins: IAA, IBA, NAA, 2,4-D — promote root formation and callus growth
• Cytokinins: BAP, Kinetin — promote shoot formation and cell division
• The auxin:cytokinin ratio determines organogenesis

6. Solidifying agent:

• Agar (0.6–1%) — provides a semi-solid support for the explant

7. pH: 5.6–5.8 (slightly acidic, optimal for nutrient uptake)

8. Other additives: Amino acids (glycine), organic acids, activated charcoal (to absorb toxins)

Importance: The medium provides all essential nutrients, energy, and hormonal signals required for cell division, differentiation, and plant regeneration. Aseptic conditions and correct hormone balance are critical for successful tissue culture.

Somatic embryogenesis is the process of forming embryos from somatic (non-zygotic) cells in tissue culture. Unlike zygotic embryos formed by sexual reproduction, somatic embryos arise from somatic cells without fertilisation.

Types of somatic embryogenesis:

• Direct: Embryos form directly from the explant without an intervening callus stage
• Indirect: Explant first forms callus, then embryos develop from the callus

Steps of indirect somatic embryogenesis: 1. Explant cultured on medium with high auxin (2,4-D) → callus formation 2. Callus transferred to medium with reduced or no auxin → embryoids develop 3. Embryoids mature on medium with ABA (abscisic acid) 4. Mature embryoids germinate into plantlets on hormone-free medium

Difference between somatic embryogenesis and organogenesis:

Production of synthetic seeds:

• Somatic embryos are encapsulated in a protective gel (sodium alginate matrix)
• The gel coating provides mechanical protection and contains nutrients
• Encapsulated embryos can be stored, transported, and planted like natural seeds
• Advantages: Easy handling, storage, and transport; clonal propagation of elite plants; preservation of rare species

Somatic hybridisation is the fusion of protoplasts from two different plant species to create a hybrid that cannot be produced by conventional sexual crossing.

Steps of somatic hybridisation: 1. Protoplast isolation: Cell walls of two plant species are removed using enzymes — cellulase (digests cellulose) and pectinase (digests pectin) — to release protoplasts (naked cells without cell walls)

2. Protoplast fusion: Protoplasts from two species are brought together and fused using:

• PEG (Polyethylene Glycol) method — chemical fusogen
• Electrofusion — brief electric pulse fuses membranes

3. Selection of hybrid cells: Fused protoplasts (heterokaryons) are identified and selected using genetic markers or fluorescence. Unfused and self-fused protoplasts are eliminated.

4. Cell wall regeneration: Hybrid protoplasts regenerate their cell walls in the culture medium

5. Division and callus formation: Hybrid cells divide to form callus

6. Plant regeneration: Callus is induced to form shoots and roots through organogenesis, producing a complete hybrid plant

Applications:

• Creation of novel hybrids that are impossible through sexual crossing (e.g., Pomato — potato + tomato)
• Transfer of disease resistance and stress tolerance traits between species
• Production of cytoplasmic male sterile lines for hybrid seed production
• Combination of nuclear and cytoplasmic genomes from different species

Limitations:

• Many somatic hybrids are genetically unstable
• Regenerated plants often show abnormal morphology
• Protoplast isolation and fusion are technically demanding
• Not all protoplast fusions result in viable hybrids
• Chromosome incompatibility between widely unrelated species may prevent hybrid survival