Plant Tissue Culture and Hormones Quiz

Plant Cell Biology & Cell Technology

• BIOL3402 course covers techniques in plant cell biology and plant cell cultures.
• Course aims to provide a comprehensive understanding of cell structure, function, and principles/applications of cell culture and instrumentation in biology and biotechnology
• Course content includes: plant cell biology and techniques in plant cell culture (6 lectures + 3 lab hours).
• Assessment includes a 2-hour written exam (50%), quiz (20%), and practical work assessment (30%); lab reports due at the end of each lab session, use Moodle for exam and quiz prep.
• Specific learning outcomes include acquiring fundamental knowledge on plant cell biology and technology, acquiring laboratory techniques in plant cell culture, cooperation with peers, and gaining insight into real-life applications in plant cell biology and technology.

Course Materials

• Refer to Moodle e-notes (choose course from HKU Portal - "My eLearning" tab)
• Consult Find@HKUL for book references and e-reserves using search term "BIOL3402".

Reference Videos

• The living plant cell: An introduction to plant cell biology (AV 581.87 L78 26m) - by Karl J. Oparka , introduction to plant cell dynamics & organisation, especially focusing on dynamic movements of cells.
• Plant Cell Culture: TAFE Publications (AV 571.5382 P71 30m) - covers the fundamentals of plant tissue culture,
• Plant tissue culture Pt. 1, tissue culture & 2, culture technique (AV 581.0724 P71 T6 & AV 581.0724 P71 C) - by Visual Education Productions - discusses various stages of plant tissue culture

Practical Session

• 1-hour lab session starting at 1430
• Isolate and observe mesophyll protoplasts from flowering Chinese cabbage and pericarp of capsicum.
• Examine plant cell samples (starch grain, plasmolysis, guard cells & stomata, xylem, trichomes
• Dress lab coats.
• Submit lab reports by end of session.

Course Assessment Details

• One 2-hour written examination (50% weighting), quiz (20%), and practical work assessment (30%).
• Lab reports should be submitted at the end of each lab session.
• Moodle should be used by students to help prepare for the quiz and written exam, exam consists of multiple choice (40 marks total in section A), and essay questions in Section B (60 marks total across three questions), with each question carrying equal marks.

Microscopy Techniques

• Light microscopy
• Electron microscopy
• Confocal laser scanning microscopy

Plant Cell Biology Lab Session

• Isolating protoplasts
• Using light microscopy in lab, including onion epidermal cells, cytoplasmic streaming, plasmolysis, leaf of African violets, leaf and stem of Arabidopsis (stomata, xylem).
• Comparing different plant shapes and sizes; specialized cells (e.g., xylem)

Plant Cell Structures

• Leaf cells exhibiting: cell wall, vacuole, chloroplasts, and mitochondria.
• Plant cells have different shapes and sizes
• Specialized plant cells (e.g., xylem).
• Trichomes (on African violet leaf surface) protect against pests and reduce evaporation.

Cell Wall : Cellulose Fibrils

• Cell walls consist of cellulose fibrils, providing structural integrity.
• Turgor pressure (osmosis) demonstrated in the movement of water/fluid within cells pressed against cell membranes/walls resulting in wilting if water loss occurs

Plasmodesmata

• Small openings in the cell walls that interconnect cells, facilitating communication for transport and exchange between cells.

Plasma Membrane

• Separates the cytoplasm from the external environment
• Crucial in osmoregulation and transport across membrane

Protoplast

• Plant cells lacking cell walls, containing cytoplasm, nucleus, organelles, and enzymes in cytosol
• Differ in size and content;
• Enzymes (cellulases and macerozyme) used to digest cell walls.
• Some protoplasts are not spherical (due to internal cytoplasmic strands).

Cytoplasmic Streaming

• Directional movement of cytoplasm that converges at the nucleus.
• Organelles within cytoplasm move, observable via light microscopy.

Transvascular Strands

• Act as cytoplasmic bridges in cytoplasmic streaming.
• Increase the exchange surface area for exchange between cytoplasm and vacuole

Vacuole

• Largest organelle in plant cells
• Bound by vacuolar membrane (tonoplast)
• Occupies a large portion of mature plant cells.
• Contributes to adjusting cell size, turgor pressure, and functions in storage/defense.

Organelles

• Mitochondria: respiration and ATP production
• Spherosomes/oleosomes: single-membrane-bound oil droplets
• Cytoskeleton: microtubules and actin filaments
• Nucleus: controls the cell's activities and contains DNA
• Endoplasmic reticulum (ER): synthesis of proteins and lipids
• Chloroplasts: site of photosynthesis, absent in onion epidermis

Origin of Mitochondria and Chloroplasts

• The eukaryotic cells evolved via ancient symbiosis, when an aerobic prokaryote was engulfed in a eukaryotic cell leading to mitochondrial origin and photosynthetic prokaryote engulfed for chloroplast evolution.

Microscopy Techniques

• Confocal microscopy produces optical 'slices' of tissue – useful for examining auto-fluorescent structures in living cells
• For example, examining the movement of vesicles in root hairs.
• Techniques allow visualization of internal structures or functions over time or fluorescence tagging (GFP, RFP, YFP), enabling observation for example of the Golgi apparatus.

Isolation of Protoplasts

• To isolate plant cells
• Observe shape
• Identify cytoplasm, nucleus, organelles and proteins in cytosol;
• Identify variability in size and content

Cytoplasmic Streaming

• Directional movement of cytoplasm
• Movement of structures (organelles) observable via light microscopy

Transvascular Strands

• Bridges enabling cytoplasmic exchange

Possible Exam Questions

• Short notes on the use of confocal laser scanning microscopy in plant cell biology
• Short notes on plasmodesmata and vacuole
• What are differences between three types of microscopy (light, electron & confocal)
• Advantages of plant tissue culture over intact plants
• Discuss secondary metabolite production using plant cell cultures, with examples.
• Important factors in ensuring successful plant cell cultures (composition and method of preparation of the plant tissue culture medium)

Plant Tissue Culture

• Stages (initiation, proliferation, pre-transplant, establishment)
• Advantages over intact plants (e.g., limited space for large-scale propagation, uniform production, exclusion of pests, ease in screening large numbers of plant lines, rapidity in yielding large amounts of identical individuals, ease in plant gene transfer, use as a bioreactor for producing chemicals)
• Applications of plant tissue culture

Different types of In Vitro Plant Culture

• Intact plant culture
• Callus culture
• Embryo culture
• Organ culture
• Suspension culture
• Protoplast culture

Micropropagation

• Plant propagation using small pieces of tissue from a mother plant to grow new plants.
• Techniques widely used in commercial horticulture

Case study: Propagation of Syngonium

• Steps involved: starting with healthy mother plant, removing leaves and cutting into sections, using 10% bleach for 10 minutes, washing, using a sterile rooting medium with cytokinin, incubating at 25ºC (500-1000 foot candles of light), transferring to conventional greenhouse, allowing roots to develop and to be transplanted to high humidity and low-intensity environment

Practical Layout of Plant Tissue Culture Facility

• Preparation area, Transfer room, and Incubation room, with specific instruments, tools and procedures required for each area

Tools: Apparatus

• Culture vessels (petri dishes, plastic containers and flasks)
• Media storage shelves
• Sterile containers and media using autoclave
• Laminar flow cabinets with UV lamps for aseptic transfer
• Sterilized scalpels, blades, and forceps.
• Heat steriliser with dry-heated glass beads for sterilization
• Shakers for suspension cultures
• Plant growth chambers for light and temperature control.

Tools: Environmental Conditions

• Light (controlled light and dark periods)
• Temperature (~25°C, ranging from 17-27°C)
• Microbe-free environment

Techniques for Plant Cell Culture & Cell Technology

• Sterilization and aseptic techniques
• Media preparation
• Explant selection
• Surface sterilization
• In vitro culture establishment
• Scaling-up
• Introducing new genes into plant cells (e.g., by using Agrobacterium tumefaciens, particle bombardment, microinjection, or PEG-mediated Transformation)

Sterilization Techniques

• Wet heat (e.g., 121°C, 103.5kPa (kilopascal), 15-20 min)
• Dry heat (e.g., Oven:160°C 1 h, Flame: 600°C red-hot
• Chemical sterilisation (e.g. using 70% ethanol or 1% Na-hypochlorite)
• Filtration (using pore size - 0.22 µm diameter)
• Irradiation (using UV)

Aseptic Techniques

• Operator's hand washing
• Using HEPA-filtered laminar flowhoods
• Wiping working surfaces with 70% ethanol
• Placing only essential items inside the hood/cabinet

Importance of Biological Safety Cabinet

• Protection of human from contamination
• Proper air flow in the cabinet

Preparation of Media

• MS Medium and Murashige and Skoog Medium is frequently used
• Sterilizing media
• Heat-labile components are added after autoclaving by filter sterilization
• Media pH at 5.0-6.5, best 5.7; and must be <7.0

Media Composition

• Macronutrients (nitrogen (as nitrate or ammonium), phosphorus (as phosphate), and sulphur (as sulphate), potassium, calcium, magnesium)
• Micronutrients (iron, manganese, zinc, boron, copper, cobalt, molybdenum)
• Vitamins
• Carbon source (sucrose)
• Organic N
• Agar (solidifying agent)
• Plant growth regulators (like auxin and cytokinin)
• undefined mixtures

Plant Growth Regulators

• Hormones affecting plant growth and development
• Synthetic derivatives are available.
• Explants may produce enough auxin so do not need further addition (similarly with cytokinins).
• Stock solutions may need to be kept in the dark (e.g., Indole-3-acetic acid (IAA) is light-sensitive).

Selection of Explant

• Any part of a plant (root, stem, petiole, leaf, flower)
• Healthy growing plant
• Actively dividing specimen
• Young tissues
• Disease free
• Varies based on species

Surface Sterilization

• Vital for removing microbial contamination
• Using 1-10% bleach or 70% ethanol, along with sodium hypochlorite (1%) solution
• Rinsing in sterile water follows.

Types of In Vitro Plant Cultures

• Intact plant culture
• Callus culture
• Embryo culture
• Organ culture
• Suspension culture
• Protoplast culture

Introducing new genes into plant cells

• Using protoplasts from cell cultures
• Preparing free nuclei to form new hybrids with polyethylene glycol (PEG 4000)
• Regenerate protoplasts to callus then to plantlets, generating new strains with valuable characteristics

Plant Biotechnology

• Large-scale plant cell culture for secondary metabolites progress rapidly, but few commercialized.
• Product R&D requires substantial investment (10 years).
• Cheap production is essential by introducing genes (encoding enzymes into cell lines/crops).
• Using plant cells as a bioreactor for converting easily obtained complex metabolites into high-value products.
• This is only possible in instances where the required synthesis involves one or two crucial enzymes (e.g., in producing pyrethrins, using a single enzyme).

Agrobacterium tumefaciens

• Natural plant pathogen that contains Ti plasmid causing crown galls.
• Used for plant genome modification in lab (e.g., introducing a gene into many plant species through Ti plasmid to synthesize and accumulate specific chemicals)
• Foreign DNA introduction via Agrobacterium transfer, particle bombardment, microinjection, or PEG-mediated transformation

Methods to introduce recombinant genes

• Agrobacterium-mediated transformation
• Particle bombardment
• Microinjection
• Polyethylene glycol (PEG)- mediated transformation of protoplasts

History of plant transformation

• Timeline of successful plant transformation

Examples of genetically engineered crops

• Cultivar, Trait, and agronomically traits for crops developed by recombining DNA (e.g. herbicide tolerance, virus resistance, insect resistance, modification of seed oils, insect resistance, modified seed protein storage)

Other examples of plant genetic engineering

• Engineering of Rose Flavonoid Biosynthetic Pathway to successfully generate blue-hued flowers accumulating delphinidin
• Production of taxol in tissue cultures from Pacific yew to increase and identify best producers (24-fold difference observed)

Preservation and Cryopreservation of Germplasm

• Storing germplasm for future use (e.g., using cryo-preservation techniques)

Secondary metabolites from plant cultures

• Unusual and complex chemicals

• Pharmaceuticals

• Fragrances

• Flavor compounds

• Dyes

• Insecticides

• Plant cells grown in bioreactors for valuable chemicals (e.g., using eastern hemlock, and mint).

Valuable secondary metabolites

• Primary metabolites (sugars, amino acids, nucleotides, chlorophyll).
• Secondary metabolites (natural products with no direct role in plant growth; protection against predation, herbivory, pathogens)
• Examples: opium poppy, codeine, Catharanthus roseus (Madagascar periwinkle), ajmalicine, coptis japonica, berberine, Rosmarinic acid.
• Estimated world market for selected plant products (quantified in US$ million).

Test your knowledge on plant tissue culture, the roles of plant hormones like auxins and cytokinins, and foundational concepts in plant biology. Learn about the techniques used to prevent contamination in cultures and the contributions of significant figures in the field. This quiz covers essential aspects of plant cells and their functions.