Plant Tissue Culture Notes

Asexual Reproduction

• Naturally occurs via vegetative propagation, examples include:
  • Tubers
  • Stolons
  • Reproductive leaves

Artificial Asexual Reproduction

• Achieved through methods like:
  • Cutting
  • Grafting (Scion and Stock)
  • Layering
  • Micropropagation

Unique Features of Plant Cells

• High phenotypic plasticity, allowing adaptation to environmental changes.
• Totipotency: The ability of a single cell to divide and differentiate into an entire organism.

Culture Media Preparation

• Macronutrients (e.g., nitrogen and potassium) in millimolar (mM) concentrations.
• Micronutrients (e.g., boron and iron) in micromolar (μM) concentrations.
• Vitamins: Myo-inositol (B8), thiamine (B1).
• Carbohydrates: Sucrose or glucose.
• Organic supplements: Amino acids, casein hydrolase, coconut milk.

Plant Hormones

• Auxins (e.g., IAA - Indole-3-acetic acid).
• Cytokinins (e.g., kinetin).
• Gibberellins.
• Abscisic acid.
• Effects of hormone concentrations on growth:
  • No growth: Nutrient agar medium with no hormones.
  • Callus formation: Low IAA (2 mg/liter), Low Kinetin (0.2 mg/liter).
  • Root formation: High IAA (2 mg/liter), Low Kinetin (0.02 mg/liter).
  • Shoot formation: Low IAA (0.02 mg/liter), High Kinetin (1 mg/liter).

Supports

• Agar.
• Filter paper bridges.
• Preparation:
  • Prepare stock solution (10× or more concentrated).
  • Sterilize media (by heat or filtration).
  • Grow in vitro under aseptic and controlled conditions.
  • Media can be liquid, semisolid, or solid.

Culture Types

• Callus culture.
• Cell suspension culture.
• Protoplast culture.
• Seed culture.
• Embryo culture.
• Endosperm culture.
• Anther culture.
• Meristem culture.
• Hairy root culture.

Callus Culture

• Initiated from surface-sterilized explants, typically young tissue.
• Dedifferentiation occurs in the dark.
• Most calluses cannot photosynthesize.
• Callus can be compact or friable.
  • Friability can be increased by altering media components, using liquid or semi-solid media, or by subculturing.

Cell Suspension Culture

• Derived from friable callus of suitable size.
• Agitation in liquid medium is required.
• Subculture regularly.
• Growth is determined by:
  • Cell number.
  • Fresh or dry weight.
  • Packed cell volume.

Plant Transformation

• Process: Cell suspension $\rightarrow$ Cell transformation $\rightarrow$ Transformed callus $\rightarrow$ Transgenic callus $\rightarrow$ Transgenic plants.
• Alternative process: Cell suspension culture $\rightarrow$ Cell culture in bioreactor $\rightarrow$ Protein products $\rightarrow$ Downstream processing.

Transformation Methods

• Introduction of exogenous DNA into a cell.
  • Agrobacterium-mediated transformation using Agrobacterium tumefaciens with a tumor-inducing (Ti) plasmid.
  • Biolistic transformation using a particle gun with biological ballistics.

Bioreactor

• Special vessel for culture growth in controlled conditions.
  • Types: Stirred, bubble column, airlift, wave-mixed.
  • Agitation can be mechanical or pneumatic.

Production Advantages

• Lower cost.
• Shorter time.
• Greater scale.
• Better quality.
• Less contamination.
• Easier purification.

Protoplast Culture

• Directly from plant, preferably leaf mesophyll tissue, or indirectly from cell suspension.
• Isolation methods:
  • Plasmolysis and dissection.
  • Pectinase and cellulase.
• Visualization: Calcofluor white stains cellulose, fluorescein diacetate (FDA) stains viable protoplasts.

Factors Affecting Protoplast Technology

• Protoplast isolation:
  • Physiological state of tissue and cell material.
  • Pre-treatment.
  • Enzymes.
  • Osmoticum.
  • pH.
  • Time.
  • Temperature.
  • Purification techniques.
• Protoplast culture and regeneration:
  • Plating density.
  • Culture media composition.
  • Culture media supplementation.
  • Light.
  • Temperature.
  • Donor material.

Low-Density Protoplast Culture Needs

• Preconditioned media.
• Feeder layer.
• Co-culturing.

Protoplast Culture Applications

• Protoplast-mediated transformation:
  • By electroporation or chemicals (e.g., Ca^{2+} + polyethylene glycol).
  • Typically for transient expression.

Liposome-Mediated Transformation

• Carry DNA by phospholipid vesicles with aqueous core to increase stability.
• Mechanisms:
  • Liposome-protoplast fusion.
  • Chemical endocytosis.
  • Leakage of liposomal contents after contacting with the cell plasma membrane.

Somatic Hybridization

• Fusion of 2 protoplasts to form a hybrid cell & subsequent development of hybrid plant after selection.
• Results in tetraploid plants (4n).
• Selection of ideal hybrid cells based on visual markers or growth requirements.

Cybridization

• Fusion of a nucleated and an enucleated protoplast to form a cytoplasmic hybrid.

Protoplast Fusion Advantages

• Cytoplasm transfer.
• Genetic recombination between sexually incompatible species.
• Genetic recombination in asexual or sterile plants.