Plant Biotechnology Exam Notes

Plant Tissue Culture

Plant tissue culture involves growing plant cells, tissues, or organs under sterile conditions. Key techniques include:

  • Aseptic culture for cell multiplication and regeneration.

  • Use of adventitious shoots and roots.

  • Callus formation from differentiated plant cells.

General Terms in Plant Tissue Culture

  • Adventitious: Development from unusual points of origin.

  • Asepsis: Absence of contaminating microorganisms.

  • Callus: Unorganized mass of differentiated cells.

  • Cybrid: Cytoplasmic hybrid from fused cytoplast and a whole cell.

  • Explant: Tissue transferred to artificial medium.

  • Gameto-clone: Plants regenerated from gametes.

  • Meristem culture: Culture of shoot apex.

  • Somaclone: Plants derived from somatic cells.

  • Totipotency: Potential of a cell to form all cell types.

History of Plant Tissue Culture

  • 1902: C. Haberlandt's first attempt to culture isolated plant cells.

  • 1922: W.J. Robbins and W. Kotte cultured isolated roots.

  • 1934: P.R. White demonstrated indefinite culture of tomato roots.

  • 1939: R.J. Gautheret and P. Nobecourt achieved long-term culture of callus.

  • 1941: J. Van Overbeek discovered the nutritional value of coconut endosperm.

  • 1953: W.H. Muir developed single-cell culture techniques.

  • 1955: C.O. Miller and F. Skoog discovered cytokinins.

  • 1957: F. Skoog and C.O. Miller hypothesized regulation of shoot and root initiation by auxins and cytokinins.

  • 1960: E.C. Cocking achieved enzymatic isolation and culture of protoplasts.

  • 1966: S.G. Guha and S.C. Maheshwari cultured anthers and pollen to produce haploid embryos.

  • 1983: M.D. Chilton produced transformed tobacco plants.

Laboratory Requirements for Plant Tissue Culture

  • Aseptic conditions.

  • Appropriate environment for cell division and expression.

  • Essential elements include:

    • Washing and storage facilities.

    • Media preparation and sterilization room.

    • Sterile transfer area.

    • Culture rooms with controlled conditions.

    • Observation area.

    • Transplantation area.

  • Laminar air flow cabinets with HEPA filters are crucial for aseptic manipulations.

Composition of Commonly Used Media

Common media include White's, Murashige and Skoog (MS), Gamborg (B5), Chu (N6), and Nitsch's. They contain macronutrients, micronutrients, and organic supplements like vitamins. MS Medium is widely used.

Totipotency, Dedifferentiation and Competency

  • Totipotency: Potential of a plant cell to develop into an entire plant.

  • Dedifferentiation: Capacity of mature cells to return to meristematic condition.

  • Competency: Endogenous potential of a cell to develop in a particular way.

Cell, Organ, and Tissue Culture

Gottlieb Haberlandt attempted to culture isolated cells in 1898. Successful root cultures were established by White in 1934. Callus cultures were achieved by White, Gautheret, and Nobecourt in 1939. Discovery of cytokinins and coconut milk spurred advancements.

Applied Aspects of Plant Tissue Culture

  • Plant Breeding: Rapid micropropagation, virus-free plants, homozygous lines, somatic hybrids.

  • Industrial Production: Production of natural compounds using cell culture.

  • Germplasm Conservation: In vitro storage of plant parts.

  • Genetic Engineering: Development of genetically modified plants using Agrobacterium-based vectors or direct DNA transfer.

Types of Tissue Culture

  • Seed Culture

  • Embryo Culture

  • Meristem Culture

  • Bud Culture

  • Callus Culture

  • Cell Suspension Culture

  • Anther Culture

  • Protoplast Culture

Culture Environment

  • Light: Measured in PAR (photosynthetically active radiation), typically 60-100 µmol m-2 s-1.

  • Temperature: Usually maintained around 25 pm 2°C.

  • Humidity: High humidity (90-100%) in test tubes can cause stomatal malfunction.

  • Oxygen: Important for root formation.

Plant Cell Culture Media

Media composition depends on the plant species and culture type. Key components are inorganic nutrients, carbon and energy sources, organic supplements, growth regulators, and solidifying agents. The pH is typically 5.0-6.0.

DNA Isolation and Extraction

CTAB technique involves:

  • Lysing cells with CTAB buffer.

  • Extracting with chloroform/iso-amyl alcohol.

  • Precipitating DNA with isopropanol.

  • Washing and resuspending DNA.

Plasmid DNA Isolation

The most common is E. coli XL1-Blue. Methods include phenol-chloroform extraction or using a plasmid isolation kit.

Molecular Biology of Agrobacterium Infection

Agrobacterium tumefaciens facilitates gene transfer. Acetosyringone attracts Agrobacterium. Virulence genes (virA, virB, virC, virD, virE, virG, virH) are essential for T-DNA transfer.

Androgenesis and Gynogenesis

  • Androgenesis: Development of haploid plants from pollen.

  • Gynogenesis: Development of haploid plants from unfertilized female gametes.

Protoplast Culture

Involves enzymatic removal of cell walls to culture protoplasts. Key steps include:

  • Plasmolysis: To stabilize the cells in hypertonic solution.

  • Enzyme treatment with cellulase and pectinase.

  • Culturing protoplasts in suitable media.

Germplasm Conservation and Cryopreservation

  • In-situ conservation: Conserving germplasm in its natural habitat.

  • Ex-situ conservation: Preserving germplasm outside its natural habitat.
    Cryopreservation involves storing germplasm at very low temperatures (e.g., liquid nitrogen at -196°C).

Embryo Culture

Aseptic isolation and growth of an embryo in vitro. Categories include culture of mature, dissected, immature, and inviable embryos.

Secondary Metabolites in Plant Cultures

Cell cultures can produce valuable natural compounds. The process involves:

  • Selection of high-yielding cell lines.

  • Large-scale cultivation.

  • Medium optimization.

  • Elicitation.

  • Biotransformation.

Transgenic Plants Development Methods

  • Direct gene transfer: Includes electroporation, particle bombardment, and microinjection.

  • Vector-mediated gene transfer: Such as Agrobacterium-mediated transformation.

Electroporation involves high-voltage electrical impulses to create pores in cell membranes and for DNA uptake.