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Questions and Answers
Which component of plant cells is responsible for photosynthesis?
Which component of plant cells is responsible for photosynthesis?
What is the significance of auxin and cytokinins in plant cell culture?
What is the significance of auxin and cytokinins in plant cell culture?
Which technique would most likely be used to prevent contamination in plant cell cultures?
Which technique would most likely be used to prevent contamination in plant cell cultures?
Who is known as the 'Father of plant tissue culture'?
Who is known as the 'Father of plant tissue culture'?
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What was one of the significant contributions of Skoog and Miller in 1957?
What was one of the significant contributions of Skoog and Miller in 1957?
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Which of the following is NOT a type of microscopy used to study plant cells?
Which of the following is NOT a type of microscopy used to study plant cells?
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What essential function do plasmodesmata serve in plant cells?
What essential function do plasmodesmata serve in plant cells?
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Which historical figure first introduced the concept of cell theory?
Which historical figure first introduced the concept of cell theory?
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What happens to plant growth when cytokinin concentration is high and auxin concentration is low?
What happens to plant growth when cytokinin concentration is high and auxin concentration is low?
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Which plant hormone is primarily responsible for root development?
Which plant hormone is primarily responsible for root development?
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What is the role of indole-3-acetic acid (IAA) in plant growth?
What is the role of indole-3-acetic acid (IAA) in plant growth?
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What is the significance of the balance of cytokinin and auxin concentrations in plants?
What is the significance of the balance of cytokinin and auxin concentrations in plants?
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What condition promotes minimal cell proliferation in plant tissue culture?
What condition promotes minimal cell proliferation in plant tissue culture?
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What does increasing auxin concentration lead to in the context of plant growth?
What does increasing auxin concentration lead to in the context of plant growth?
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Which of the following statements about plant cells is true?
Which of the following statements about plant cells is true?
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When cytokinin concentration is increased, what is the expected effect on root growth?
When cytokinin concentration is increased, what is the expected effect on root growth?
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What is the typical production scale for plant cell cultures?
What is the typical production scale for plant cell cultures?
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Which of the following is a common problem associated with plant cell cultures?
Which of the following is a common problem associated with plant cell cultures?
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Which equipment is used for scaling up plant cell cultures?
Which equipment is used for scaling up plant cell cultures?
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What process is used to introduce new genes into plant cells?
What process is used to introduce new genes into plant cells?
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What needs to happen after introducing new nuclei into protoplasts?
What needs to happen after introducing new nuclei into protoplasts?
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What is one characteristic that complicates the bioengineering of plant cells?
What is one characteristic that complicates the bioengineering of plant cells?
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What role do plant growth regulators play in tissue culture?
What role do plant growth regulators play in tissue culture?
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What is one significant feature of plant cell cultures that impacts their growth?
What is one significant feature of plant cell cultures that impacts their growth?
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What effect do haploids have on varietal development?
What effect do haploids have on varietal development?
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Which stage involves the production of haploids?
Which stage involves the production of haploids?
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What is the final step in the process described?
What is the final step in the process described?
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At which stage are trials conducted to verify performance?
At which stage are trials conducted to verify performance?
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What role does inbreeding play in the described process?
What role does inbreeding play in the described process?
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What is the typical timeline for commercialization following initial trials?
What is the typical timeline for commercialization following initial trials?
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What is the primary purpose of using haploid cultures in plant breeding programs?
What is the primary purpose of using haploid cultures in plant breeding programs?
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What generation does the selfing or backcrossing process occur in?
What generation does the selfing or backcrossing process occur in?
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What is indicated at the 3rd year of the varietal development process?
What is indicated at the 3rd year of the varietal development process?
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During the propagation of Syngonium, what is the temperature at which the plantlets should be incubated?
During the propagation of Syngonium, what is the temperature at which the plantlets should be incubated?
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What happens to monoploid plants derived from haploid cultures?
What happens to monoploid plants derived from haploid cultures?
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What treatment is necessary to achieve homozygous dihaploid plants from haploid cultures?
What treatment is necessary to achieve homozygous dihaploid plants from haploid cultures?
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What initial structure develops from dissected anthers in anther culture?
What initial structure develops from dissected anthers in anther culture?
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In which stage of plant development are haploid plantlets generated from microspore-derived embryos?
In which stage of plant development are haploid plantlets generated from microspore-derived embryos?
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What is the light condition recommended for establishing plantlets in a greenhouse after rooting?
What is the light condition recommended for establishing plantlets in a greenhouse after rooting?
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Why is colchicine used in the production of dihaploid plants from haploid cultures?
Why is colchicine used in the production of dihaploid plants from haploid cultures?
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What is the primary use of cultured multipotent cells (CMCs) as described?
What is the primary use of cultured multipotent cells (CMCs) as described?
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Which of the following plants is NOT mentioned in relation to CMCs?
Which of the following plants is NOT mentioned in relation to CMCs?
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What is the critical ecological issue associated with harvesting taxol from the Pacific yew?
What is the critical ecological issue associated with harvesting taxol from the Pacific yew?
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What cancers is taxol specifically noted to be effective against?
What cancers is taxol specifically noted to be effective against?
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How much pure taxol is required annually in the USA?
How much pure taxol is required annually in the USA?
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What do de-differentiated cambial meristematic cells produce?
What do de-differentiated cambial meristematic cells produce?
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What process do cultured multipotent cells (CMCs) undergo to function effectively in production?
What process do cultured multipotent cells (CMCs) undergo to function effectively in production?
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What is one consequence of the harvesting method used for taxol from the Pacific yew?
What is one consequence of the harvesting method used for taxol from the Pacific yew?
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Which microscopy technique provides the highest magnification for studying plant cells?
Which microscopy technique provides the highest magnification for studying plant cells?
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What is the primary component of the plant cell wall that provides structural integrity?
What is the primary component of the plant cell wall that provides structural integrity?
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Which feature distinguishes plant cells from animal cells in terms of shape?
Which feature distinguishes plant cells from animal cells in terms of shape?
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Which of the following accurately describes turgor pressure in plant cells?
Which of the following accurately describes turgor pressure in plant cells?
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What effect does loss of water have on plant cells in terms of turgor pressure?
What effect does loss of water have on plant cells in terms of turgor pressure?
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Which component of the plant cell wall is involved in opposing turgor pressure?
Which component of the plant cell wall is involved in opposing turgor pressure?
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What happens to celery cells when they lose turgor pressure?
What happens to celery cells when they lose turgor pressure?
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What is the effect of a high cytokinin and low auxin concentration on plant growth?
What is the effect of a high cytokinin and low auxin concentration on plant growth?
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Which statement accurately describes the behavior of indole-3-acetic acid (IAA) in plant cells?
Which statement accurately describes the behavior of indole-3-acetic acid (IAA) in plant cells?
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What happens when both cytokinin and auxin concentrations are low?
What happens when both cytokinin and auxin concentrations are low?
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What role do cytokinins play in plant tissue culture?
What role do cytokinins play in plant tissue culture?
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How does increasing auxin concentration affect callus formation?
How does increasing auxin concentration affect callus formation?
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How do plasmodesmata contribute to the movement of substances within plant cells?
How do plasmodesmata contribute to the movement of substances within plant cells?
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What effect does a hypertonic solution have on plant cells as illustrated by plasmolysis?
What effect does a hypertonic solution have on plant cells as illustrated by plasmolysis?
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In plant cells, cytoplasmic streaming primarily assists in which function?
In plant cells, cytoplasmic streaming primarily assists in which function?
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What is the primary reason for observing the shape of protoplasts?
What is the primary reason for observing the shape of protoplasts?
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What is the relationship between turgor pressure and the osmotic state of plant cells?
What is the relationship between turgor pressure and the osmotic state of plant cells?
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What role does the plasma membrane play within plant cells?
What role does the plasma membrane play within plant cells?
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How does plasmolysis affect plant cell functionality?
How does plasmolysis affect plant cell functionality?
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Which statement about cytoplasmic streaming is true?
Which statement about cytoplasmic streaming is true?
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What is the significance of protoplasts in studying plant cells?
What is the significance of protoplasts in studying plant cells?
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What is the primary function of RabGTPases in vesicle trafficking?
What is the primary function of RabGTPases in vesicle trafficking?
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What type of microscopy is particularly highlighted for studying the Golgi apparatus?
What type of microscopy is particularly highlighted for studying the Golgi apparatus?
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Which component of the vesicle transport system is associated with myosin VI motor proteins?
Which component of the vesicle transport system is associated with myosin VI motor proteins?
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What is one role of trichomes found on the leaves of plants like the African violet?
What is one role of trichomes found on the leaves of plants like the African violet?
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What is the primary function of mitochondria in plant cells?
What is the primary function of mitochondria in plant cells?
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Which statement accurately describes the origin of mitochondria and chloroplasts?
Which statement accurately describes the origin of mitochondria and chloroplasts?
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Which of the following types of trichomes are NOT present on the upper leaf surface?
Which of the following types of trichomes are NOT present on the upper leaf surface?
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In which type of microscopy can cytoplasmic strands in plant cells be observed?
In which type of microscopy can cytoplasmic strands in plant cells be observed?
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What type of plant cell is characterized by its role in water conduction?
What type of plant cell is characterized by its role in water conduction?
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What is the primary visualization technique used to examine cytoplasmic streaming and plasmolysis?
What is the primary visualization technique used to examine cytoplasmic streaming and plasmolysis?
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What distinctive feature do spherosomes possess?
What distinctive feature do spherosomes possess?
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Which protein interaction is key for the targeting of vesicles to their final destination?
Which protein interaction is key for the targeting of vesicles to their final destination?
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Why are chloroplasts absent in onion epidermis cells?
Why are chloroplasts absent in onion epidermis cells?
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What visual marker is often used in microscopy to study protein interactions and vesicle movement?
What visual marker is often used in microscopy to study protein interactions and vesicle movement?
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What is a primary characteristic of guard cells in plant leaves?
What is a primary characteristic of guard cells in plant leaves?
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What is a key role of the cytoskeleton in plant cells?
What is a key role of the cytoskeleton in plant cells?
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What is the significance of using fluorescent tagging in microscopy studies?
What is the significance of using fluorescent tagging in microscopy studies?
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What is the primary role of the endoplasmic reticulum (ER) in plant cells?
What is the primary role of the endoplasmic reticulum (ER) in plant cells?
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Which cellular component acts as the control center in plant cells?
Which cellular component acts as the control center in plant cells?
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How do chloroplasts contribute to the energy needs of plant cells?
How do chloroplasts contribute to the energy needs of plant cells?
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Study Notes
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).
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Description
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.
