BIOL3402 Cell Biology & Cell Technology PDF

BIOL3402 Cell Biology & Cell Technology Techniques in plant cell biology & plant cell cultures Dr. Peng Wang School of Biological Sciences Lab7N/15, Office 7N/16, KBSB [email protected] 1 WHAT A...

BIOL3402 Cell Biology & Cell Technology Techniques in plant cell biology & plant cell cultures Dr. Peng Wang School of Biological Sciences Lab7N/15, Office 7N/16, KBSB [email protected] 1 WHAT ARE YOU EXPECTED TO LEARN? Aim/Course objectives To provide an understanding of the structure and function of cells, and the principles and applications of cell culture and instrumentation in biology and biotechnology HOW CAN THIS BE ACHIEVED? Contents to be covered by Dr. Peng Wang (6 lectures + 3 hrs of lab) Plant cell biology & techniques in plant cell culture HOW IS THE COURSE ASSESSED? One 2-hour written examination (50% weighting), quiz (20%) & assessment of practical work (30%) Hand up lab report by the end of lab session Use your Moodle to prepare for your quiz and written exam SPECIFIC LEARNING OUTCOMES (from these 6 lectures and 1 lab) Students will be able to a. Acquire fundamental knowledge on plant cell biology and cell technology b. Acquire some laboratory techniques on plant cell culture (1 lab session) c. Cooperate and work with other students d. Gain insight into real-life applications in plant cell biology and cell technology To pursue further learning read e-reserves and watch videos to help understand  Content Date Topics Lecture Plant cell biology 1 23/10/24 Wednesday Techniques in plant cell biology 2 Tools for plant cell culture & cell technology 3 29/10/24 [media, equipment, environmental conditions] Thursday Techniques in plant cell culture 4 [aseptic techniques, callus cultures, regeneration] * micropropagation Applications of plant cell culture and technology 5 06/11/24 [*micropropagation, haploid cultures, protoplast cultures, Thursday somaclonal variants, grafting, cryopreservation, secondary metabolites] Plant genetic engineering 6 11/11/24 Laboratory session on plant cell biology & cell technology Practical Monday Isolation of mesophyll and pericarp protoplasts Examine different plant cells 3  Practicals 1 lab session Check grp! Start 1430 Isolate and observe mesophyll protoplasts released from leaves of flowering Chinese cabbage and from pericarp of capsicum Examine samples of different plant cells at display stations Starch grain, plasmolysis, guard cells & stomata, xylem, trichomes Wear your lab coat Hand in your lab report by the end of the session 4 HOW IS THE COURSE ASSESSED? One 2-hour written examination (50% weighting), quiz (20%) & assessment of practical work (30%) Hand up lab report Use your Moodle to help prepare for your quiz & written exam consisting of Multiple choice: Section A: (40 marks) Answer ALL questions Essay Q: Section B: (60 marks) Answer ALL THREE questions. All questions carry equal marks My question(s) could be part of a Q eg 1 (d) of Q1 (a-d)  Course Materials Refer to Moodle e-notes Select your course from “My eLearning” tab after logging in HKU Portal at http://hkuportal.hku.hk Refer to Find@HKUL search “BIOL3402” for Book references Electronic reserves Reference videos (to help you better understand) The living plant cell: an introduction to plant cell biology: Karl J Oparka Introduces the dynamic nature of plant cell organisation, especially dynamic movements within the cell. AV 581.87 L78 26 m Plant Cell Culture: TAFE Publications Covers the fundamentals of plant tissue culture AV 571.5382 P71 30 m Plant tissue culture Pt. 1, tissue culture & 2, culture technique: Visual Education Productions Defines & explains the various stages of plant tissue culture (20+20 m) AV 581.0724 P71 T6 & AV 581.0724 P71 C Plant tissue culture https://www-jove-com.eproxy.lib.hku.hk/science-education/11112/plant-tissue-culture Plant cells and tissues https://www-jove-com.eproxy.lib.hku.hk/science-education/11091/plant-cells-and-tissues 7  L1-2 Plant Cell Biology & Techniques in Plant Cell Biology  Plant cell biology Bark of cork oak tree A similar kind of microscope used by http://www.hagarty-on-wine.com/OnWineBlog/wp-content/uploads/2009/11/IMG_1739.jpg https://www.pinterest.com/pin/561472278521144052/ Robert Hooke 9  Studying about plants informs us about our world Cells were first observed in plants Photograph of cork cells Drawing of cork by Robert Hooke, discoverer of “cells” Photo credit: ©David B. Fankhauser, Ph.D Major microscopic methods used to study plant cell biology 1000x Light microscopy 40x Electron microscopy Confocal laser scanning microscopy Scanning EM 30,000x CLSM Transmission EM 1,000,000 to 2,000,000x 11  Microscopy images Dissecting Light Light mitochondrion TEM SEM CLSM http://princetoninnovation.org/magazine/wp-content/uploads/2015/12/arjuna-cycle-2-image-1.jpg http://www.denniskunkel.com/gallery/plants/58694A.jpg http://themagicschoolbus.blogspot.com/2006/05/cell-structure.htmlA.jpg https://ccrod.cancer.gov/confluence/display/CMCF/Home 12 http://www.iflscience.com/technology/some-spectacular-sem-images-microscopic-world/ http://bloomiq.com/indoorplantstips/african-violets 12  Tour of a plant cell Leaf cell: cell wall, vacuole, chloroplasts, mitochondria 1’44” https://vimeo.com/14797963 13  Main differences between plant and animal cells Plant cells: Green in colour Chloroplast Regular shape Cellulose cell wall Large vacuole Plasma membrane (similar to animal cells) Elodea cells https://orangesdms.wikispaces.com/Plant+Cells+-+BO 14 https://en.wikipedia.org/wiki/Elodea#/media/File:Elodea_canadensis.jpeg  Cell wall: cellulose fibrils Cell wall Provide Glucose structural molecules integrity https://public.ornl.gov/site/gallery/detail.cfm?id=159#credits https://www.google.com.hk/url?sa=i&source=images&cd=&cad=rja&uact=8&ved=2ahUKEwiwt5upuNHgAhVJIIgKHRwBBvsQjRx6BAgBEAU&url=https%3 15 A%2F%2Fwww.miifotos.com%2Fim%25C3%25A1genes%2Fleaf-cell-parts-0b.html&psig=AOvVaw1WeqdQINwBu26vCuK0WfBl&ust=1550996973919670  Demonstration of structural integrity: *turgor pressure (osmosis occurs in video) *Pressure exerted by fluid in cell that presses cell membrane against cell wall Loss of water leads to loss in turgor causing wilting 66” https://www.youtube.com/watch?v=fhsurzE_-J0 16  Cell wall Mainly cellulose β 1-4 linkage Glucose molecules Provides rigidity & strength Opposes turgor pressure Provides a barrier for individual cell How could turgor pressure be possible for the celery if there are barriers among cells? Plasmodesmata control content of osmolytes and water within the cell and turgor pressure can affect the non-targeted movement of molecules starch α 1-4 17 http://www.joostdevree.nl/bouwkunde2/jpgc/cellulose_1_moleculaire_structuur_http_doors-sliding_com.jpg  Plasmodesmata interconnect cells through cell wall openings (pits) https://bio.libretexts.org/@api/deki/files/998/Figure_04_06_02.jpg?revision=1 Persimmon endosperm starch α 1-4 https://commons.wikimedia.org/wiki/File:Fuyu_Persimmon_(Diospyros_Kaki).jpg http://www.carolina.com/images/product/large/304864_ms.jpg 18 http://www.joostdevree.nl/bouwkunde2/jpgc/cellulose_1_moleculaire_structuur_http_doors-sliding_com.jpg Demonstration of cell wall & membrane Light microscopy: onion epidermal cells before and after plasmolysis (brought about by soaking in sugar solution) normal cells Cell wall flaccid cells (water loss Red onion Plasma membrane no turgor pressure) https://organicfoodsandcafe.com/wp-content/uploads/2017/05/8935-pg.png 19 http://www.microscopy-uk.org.uk/mag/art97/maysnp2.html http://www.microscopy-uk.org.uk/mag/imgfeb11/027-hm.jpg Plasmolysis followed by osmosis… Plasmolysis (water loss from cell) occurs when cell in hypertonic solution eg 20% sucrose Addition of water causes osmosis and cells become normal again Light microscopy of Elodea cells 38” 20 https://www.youtube.com/watch?v=zVvHn6Sj9PQ  Plasma membrane = Plasmalemma Functions: Separates cytoplasm from external environment Transport across membrane Role in osmoregulation 21 http://cache.diomedia.com/170h/01/AP/AP/01AP-APXY.jpg Protoplast: plant cells lack cell wall Observe shape cytoplasm with nucleus, organelles, proteins, enzymes in cytosol Heterogeneous in size & content Use of enzymes (cellulases & macerozyme) Tobacco protoplasts to digest cell wall 20 µm https://www.researchgate.net/profile/Yaroslav_Blume/publication/2 95830728/figure/fig2/AS:342364313931789@1458637421881/Fres hly-isolated-tobacco-protoplasts-Scale-bar-indicates-20mm.png 22 Some protoplasts may not be spherical because of the presence of internal cytoplasmic strands 23 extracted from https://www.youtube.com/watch?v=CDCbhDOsZ84 Cytoplasmic streaming = directional movement of cytoplasm converging at nucleus organelles in cytoplasm in motion: watch movie (light microscopy) 1’18” 24 extract from The living plant cell : an introduction to plant cell biology: Karl J. Oparka  Transvascular strands act as cytoplasmic bridges in cytoplasmic streaming increase surface area between cytoplasm and vacuole for exchange between them transvascular strands nucleus vacuole 50 µm Confocal microscopy: Transient expression of GFP fusion proteins in onion Rodriguez-Llorente et al. 2003 (Eur. J. Biochem. 270) epidermal cell 25  More on plasma membrane, protoplast, cytoplasmic strands … Video: Plant Cell Motility and Laser Microsurgery of Cytoplasmic Strands https://www.youtube.com/watch?v=CDCbhDOsZ84 Plant cells shaped by rigid cell walls. Osmotic forces press plasma membrane tightly against walls. Walls can be removed with enzymes (YOU WILL DO THIS IN CLASS PRACTICAL). Remaining structures (protoplasts*) bordered by the plasma membrane (usually spherical due to non-directional osmotic forces.) During cytoplasmic streaming, cytoplasmic strands change length, branching and attachment therefore protoplast shape may change. Laser microsurgery provides proof that inner tension built by cytoplasmic network is moving the cell. If strand is cut, network collapses without destroying cell integrity. As a result osmotic forces can take over and round the protoplast but cytoplasmic streaming still functions in intracellular transport. Vacuole biggest organelle Transmission electron microscopy Schematic 27 https://plantcellbiology.masters.grkraj.org/html/Plant_Cellular_Structures7-Plant_Cell_Vacuoles_files/image003.jpg  Vacuole bound by vacuolar membrane (tonoplast) occupies greater portion of mature cell ~30% but can fill up to 90% of intracellular space large fluid-filled vesicles allows cells to adjust size and turgor pressure contains inorganic ions, sugars & enzymes functions in storage (e.g. proteins in seed vacuoles) & defense The Vacuole Song 4 min https://www.youtube.com/watch?v=evW93DtSoZY 28  Organelles Mitochondria Function in respiration Obtaining energy from sugar metabolism for ATP Spherosomes/ oleosomes Oil droplets up to 1 mm in diameter Bound by a single membrane Cytoskeleton Microtubules, actin filaments Nucleus Control centre, DNA, nucleolus Endoplasmic reticulum (ER) No streaming Chloroplasts 29 Photosynthesis, absent in onion epidermis http://i2.wp.com/blog.gymlion.com/wp-content/uploads/2015/02/Purple-heart-plant.jpg Spherosomes Spiderwort 20” Spherosomes moving through the stamen hair cells of Tradescantia plant (400X) Demonstrate the presence of cytoplasmic strands in light microscopy https://www.youtube.com/watch?v=pY-sBlJPevE 30 Origin of mitochondria and chloroplasts Originated from ancient symbiosis A eukaryotic cell engulfed an aerobic prokaryote and established an endosymbiotic relationship – mitochondrion Eukaryotic cells containing mitochondria then engulfed photosynthetic prokaryotes - chloroplast Photosynthetic prokaryote Chloroplast Mitochondrion Aerobic prokaryote 31 https://www.nature.com/scitable/content/ne0000/ne0000/ne0000/ne0000/14747702/U1CP2-3_MitoChhloroOrigins_revised.jpg  Techniques in plant cell biology: Light microscopy Chloroplasts Guard cells Cell division How useful is it? http://www.microscopy-uk.org.uk/mag/imgmay11/13mayo11wd-2.jpg https://qph.fs.quoracdn.net/main-qimg-62810fd85bf364a50d862ac130d12414-c 32 https://dccdn.de/pictures.doccheck.com/images/c2d/ab9/c2dab9e053e6f74ef3b8ccca2823faef/89963/l_1469540196.jpg  Methods used to study plant cell biology: Light microscopy Fixed specimen placed and oriented in embedding mould Ribbon of sections being cut from paraffin block using a rotary microtome Sections are 4 µm thick and show little distortion or disruption Block of sample ready for microtomy (stable storage) https://drp8p5tqcb2p5.cloudfront.net/fileadmin/_processed_/csm_Embedding_028_sized_e0018decbe.jpg https://drp8p5tqcb2p5.cloudfront.net/fileadmin/_processed_/csm_Sci_Lab_Histo_Lead0008_03_152b838287.jpg 33 https://drp8p5tqcb2p5.cloudfront.net/fileadmin/_processed_/csm_Sci_Lab_Histo_Lead0009_03_e5b2e7490b.jpg  Methods used to study plant cell biology: Light microscopy Standard staining or immuno- histochemical labelling method can be applied to paraffin sections Paraffin section mounted on microscope Series of sections lifted from the slide after floated out on microtome for further processing warm water to flatten it http://drp8p5tqcb2p5.cloudfront.net/fileadmin/_processed_/csm_Sci_Lab_Histo_Lead0009_03_e4502ff4f6.jpg https://drp8p5tqcb2p5.cloudfront.net/fileadmin/_processed_/csm_Sci_Lab_Histo_Lead0010_03_ce6abed86c.jpg 34  Microtomy 55” Preparing paraffin sections using a microtome 35 https://www.youtube.com/watch?v=TKve7Jq3TBs  Immuno-histochemical staining 1’32” https://www.youtube.com/watch?v=ir8QzfADa4w 36  Immuno-stained sample Coloured or fluorescent signal released Specific antibody (mouse) from reacting with substrate Anti-mouse antibody (goat) Anti-mouse antibody conjugated with alkaline phosphatase/ Horse Radish Peroxidase Visualization of specific protein Fixed cell sample  Methods used to study plant cell biology: Light microscopy Seed sections stained with specific antibodies which recognize a protein expressed in embryos (to localize its site of expression) Properties: - sectioning & immuno-staining - cell sacrificed in immunostains - labour & time consuming 38  Methods used to study plant cell biology: Electron microscopy Electrons replace light as primary radiation source, increasing resolution Subcellular organelles seen cw Can be immunostained cw Gold particle Antibody g Disadvantages: - more time in fixing er - cell sacrificed v, vacuole; cw, cell wall; g, Golgi apparatus; er, endoplasmic reticulum - dynamic motion arrested 39 Xiao et al., 2008 Plant Molecular Biology 68, 571-583 Arrowheads: gold particles (immuno-gold labeling)  Confocal microscopy 2’40” https://www.youtube.com/watch?v=BYwHLhgP1qI 40  Confocal Laser Scanning Microscopy Use laser on specimen to produce optical ‘slices’ of tissue Can use live cells that are auto-fluorescent 29” Click video to watch 41 https://d2v9y0dukr6mq2.cloudfront.net/video/preview/4Ku9x8aIg/videoblocks-slicing-through-a-healthy-green-apple-to-reveal-its-juicy-insides-filmed-in-stop-motion_btzpngvlz__SB_PM.mp4 https://png.pngtree.com/element_origin_min_pic/16/11/16/95f8999cf5edcb9700d6942b17e7a672.jpg  Confocal Laser Scanning Microscopy - No actual slicing - Sample not sacrificed - 3D images - Can provide a time series of event - Requires computer processing Arrowheads, nuclei Confocal images showing localization of auto-fluorescence GFP-tagged fusion proteins transiently expressed in onion epidermal cells 42 Contrast confocal with light microscopy shown here: time lapse of root growth of Cichorium intybus Production of root hairs is obvious but no details recorded in light microscopy Only limited growth structure could be observed with a light microscope 7” More info by Photos were taken every 5 minutes with Nikon D5100 and AF-S VR Micro-Nikkor 105mm f/2.8G using confocal microscopy… https://www.youtube.com/watch?v=36VNPsONv8g 43 https://en.wikipedia.org/wiki/Chicory Use of Confocal Laser Scanning Microscopy Time lapse of dividing cells that form a new root tip Red dots are the cell nuclei Cell wall proteins in green Different fluorescent- tagged probes can be used at same time 50’ https://www.youtube.com/watch?v=IjGgt8rOg_0 44  Confocal microscopy & GFP tagging shows movement of vesicles through a root hair Fastest growing plant cells: root hair Requires new membrane & cell wall materials Requires compounds synthesized in ER, modified in Golgi apparatus & packed in vesicles Vesicles delivered to plasma membrane by motor proteins along cytoskeleton How do vesicles find target membranes? Specific protein-protein interaction Interactions between small proteins on vesicle surface and proteins on target membrane One group of these, RabGTPases are visibly because they are tagged: RabGTPase-GFP fusion 45  Fantastic vesicle traffic GFP-tagged protein (RabGTPase) in action https://www.youtube.com/watch?v=7sRZy9PgPvg Germination of Arabidopsis seeds, zooming on root hairs, which display movement of fluorescent vesicles through the cytoplasm, and finally one can see an animation of myosin VI motor proteins dragging the fluorescently- marked vesicles 2’24” along actin filaments This animation is made possible from results achieved by CLSM Author: Daniel von Wangenheim 46 http://www.chlorofilms.org/index.php/crpVideo/display/videoid/17  Confocal laser scanning/electron microscopy & fluorescence tagging (GFP, RFP, YFP) to examine function and organisation of the Golgi apparatus "Sweet Home Apparatus" - the ultimate Golgi music video, Author: Anne Osterrieder https://youtu.be/cnK7RT1q0bA  Plant Cell Biology Lab session 1. Isolating protoplasts 2. Use of light microscopy in laboratory a) Inner epidermis of onion epidermal cell cell walls, vacuoles, plasma membrane cytoplasmic streaming plasmolysis in high salt or high sugar solution b) Leaf of African violet trichomes c) Leaf and stem of Arabidopsis stomata, xylem (functions in water conduction when cells are dead) 3. From comparison of 2a, b, c: Plant cells come in different shapes and sizes. Some plant cells are specialized cells (e.g. xylem)  African violet leaf feel fuzzy due to presence of trichomes / hairs Functions: protect against pests, reduce evaporation 2 types present on upper leaf surface: non-glandular trichomes (hairs) small glandular trichomes (small) Right SEM: top-non-glandular trichomes bottom-small glandular trichomes Trichomes from leaves of the model plant Arabidopsis in light microscopy Trichome  Representative scanning electron microscopy images of trichomes on plants A. Erect glandular trichome on the stem of M. sativa. B. Nonglandular trichome on a rosette leaf of Arabidopsis. C. Procumbent trichome on the petiole of M. truncatula. D. Glandular trichomes on a female bract of Cannabis sativa. E. Glandular trichomes on a bract of H. lupulus. F. Nonglandular trichome on a leaf of M. truncatula. G. Types VI (small arrow) and I (large arrow) trichomes on a leaf of S. lycopersicum. Bars = 100 µm. Dai, X., et al. Plant Physiol. 2010;152:44-54 Copyright ©2010 American Society of Plant Biologists Guard Cells (Leaf) in Light Microscopy Plant cells come in different shapes and sizes -variation Different shapes of plant cells… Some plant cells are specialized cells xylem vessel element potato tuber cell (thickened walls) (starch grains) EM : Ultrastructureof the vascular cambium (A) cross-section (B) radial longitudinal section CZ, cambial zone P, phloem V, vessel element X, xylem. Scale bars equal (A) 5 μm (B) 20 μm. Chaffey Phys. Plantarum114: 594-600 J.M. Agiulera et al. Food Res. Int. 34: 939-947 For more, see http://www.olympusconfocal.com/gallery/plants/index.html  Possible exam questions Write short notes on the use of confocal laser scanning microscopy in studying plant cell biology. Write short notes on “plasmodesmata” and “vacuole”. What are the differences between the three types of microscopy used in studying plant cell biology? 54  Plant Cell Biology The living plant cell [videorecording] : an introduction to plant cell biology Karl J. Oparka Scottish Crop Research Institute 26 min AV 581.87 L78  BIOL3402 Cell Biology & Cell Technology Tools & Techniques in plant cell biology & plant cell cultures Dr. Peng Wang School of Biological Sciences Lab7N/15, KBSB [email protected] 1  Recap from L1-L2 Plant cells: Chloroplast Cell wall Vacuole Cell-Cell communication: plasmodesmata Instruments to study plant cells: Light Microscopy Electron Microscopy Confocal Laser Scanning Microscopy 2  L3-4 TOOLS & TECHNIQUES FOR PLANT CELL CULTURE & CELL TECHNOLOGY TOOLS: media, equipment, environmental conditions TECHNIQUES: aseptic techniques, callus cultures, regeneration micropropagation  Historical Perspective on Plant Cell Culture 17th Century first observed cells and gave that name Robert Hooke 1838 Schwann cell theory and Schleiden cell = smallest biological unit that could be considered totipotent a single cell can develop into a complete plant (realized by plant tissue culture) 1. All living organisms are composed of one or more cells 2. The cell is the most basic unit of life 3. All cells arise only from pre-existing cells Matthias Jakob Schleiden Theodor Schwann (1804–1881) (1810–1882) https://en.wikipedia.org/wiki/Matthias_Jakob_Schleiden https://en.wikipedia.org/wiki/Theodor_Schwann Historical Perspective on Plant Cell Culture 1902 Haberlandt first to consider culturing cells aseptically in a nutrient solution. Father of plant tissue culture. 1904 Hannig developed embryo culture by isolating immature embryos in vitro and obtaining viable plantlets 1957 Skoog and differentiation dependent on auxin and cytokinins Miller (plant growth regulators) 1965 Vasil and regenerated tobacco plant from single cell derived from Hildebrandt suspension culture 1985 Horsch et al. infection and transformation of tobacco leaf discs by Agrobacterium tumefaciens and regeneration of transformed plant 5  Lab Exhibits Arabidopsis seedlings germinating in solid agar plate Arabidopsis Arabidopsis plantlets potted plants in liquid medium in soil  What is plant tissue culture? = in vitro* plant culture A technique to Aseptically culture plant cells, tissues, and organs in liquid or on solid (agar) medium supplemented with suitable nutrients Culture propagates as a mass of undifferentiated cells for an extended period of time, or they can be regenerated into whole plants  Workflow of plant tissue culture Initiation Explant Callus Proliferation shoot & root Pre-transplant Plantlet Establishment Plant 8 Chrispeels, M.J. and D.E. Sadava. 1994 Plants, genes, and agriculture Why is plant tissue culture important? Essential technique in study of plant biology- basic and applied traditional plant sciences plant molecular biology plant biochemistry plant development plant genetic engineering Commercial production of disease-free valuable horticulture crops by micropropagation (practice of rapidly multiplying stock plant material to produce progeny plants, using modern plant tissue culture methods) 9  Advantages of tissue cultures over intact plants 1. Ability to grow plant cells in liquid culture on a large-scale for the commercial production of secondary metabolites or recombinant protein in bioreactor no variation in productivity no endangering wild plants 2. Shortens time in obtaining homozygous lines in breeding programmes by the production of ‘dihaploid’ plants from haploid cultures in micropropagation of crop plants for food, fuel etc 3. Isolation of protoplasts followed by somatic fusion of related species creates opportunities in transfer of desirable traits in crops, i.e. cross distantly-related species by protoplast fusion to regenerate novel hybrid---overcome reproductive isolation 10  Advantages of tissue cultures over intact plants 4. Feasibility in screening large numbers of plant lines in limited space e.g. to achieve salt/herbicide-resistant variants screening programmes of cell (rather than plants) for advantageous characters 5. Uniform production of horticultural species from limited material in short period of time by micropropagation e.g. using meristem and shoot cultures to yield large numbers of identical individuals from limited material or absence of seeds 6. Exclusion of pests (fungi, bacteria, viruses, insects, nematodes) e.g. removal of viruses using propagation from meristem 11 … applications of plant tissue culture include: Tool of research Plant propagation Disease elimination Plant improvement Production of secondary metabolites Conservation of rare species 12  Tools & Techniques for Plant Cell Culture & Cell Technology What are tools for plant cell culture and cell technology? 13 Tools: Equipment - culture vessels (petri dish, plastic containers, flasks), - shelves for storage of containers and media - autoclave to sterilise containers and media - laminar flow cabinet (fitted with UV lamps) for aseptic transfer - scalpels, blades, forceps sterilised by flaming or by using dry-heated glass beads in a heat steriliser - shakers for suspension cultures - plant growth chamber with light and temperature control http://img.medicalexpo.com/images_me/photo-g/75366-5097151.jpg Tools: Environmental conditions - light (controlled light and dark periods) - temperature ~ 25 o C (17-27 o C) - microbe-free environment  Techniques for Plant Cell Culture & Cell Technology 1. Sterilisation and aseptic techniques 2. Preparation of media 3. Selection of explant 4. Surface sterilisation 5. Establishing in vitro culture 6. Scaling–up 7. Introducing new genes to plant cells Extracted from Plant Cell Culture. TAFE Publications. AV 571.5382 P71 16  1. Sterilisation 1. Wet heat: 121 o C 103.5 kPa (kilopascal) 15-20 min 2. Dry heat: Oven:160 °C 1 h Flame: 600 °C red-hot 3. Chemicals: 70% ethanol; 1% Na-hypochlorite 4. Filtration: pore size - 0.22 µm diameter 5. Irradiation: UV 17  Aseptic techniques Operator’s hands must be washed Laminar flow hood has HEPA-filtered air Working surfaces of laminar flow cabinet must be swiped with 70% ethanol Place only essential items in hood HEPA = High Efficiency Particulate Air removes at least 99.97% of airborne particles 0.3 µm in diameter https://beta-static.fishersci.com/images/F30069~wl.jpg https://www.cdc.gov/handhygiene/images/Animated-Logo-Clean-Hands-Count.gif 18 https://upload.wikimedia.org/wikipedia/commons/thumb/b/b1/HEPA_Filter_diagram_en.svg/607px-HEPA_Filter_diagram_en.svg.png  Lamina air flow scheme HEPA filter Clean filtered air Room air Contaminated air 19 http://www.lamsys.com/files/img/boxes/air-282-eng.jpg http://img.medicalexpo.com/images_me/photo-g/75366-5097151.jpg  Importance of a Biological Safety Cabinet in protection & air flow 1’22” Extracted from https://www.youtube.com/watch?v=HDKqOMybf4A 20  Use of laminar flow hood (wiping with ethanol, creating sterile environment in hood) 4.5 min https://www.youtube.com/watch?v=ORT01wl8vlY 21 http://img.medicalexpo.com/images_me/photo-g/75366-5097151.jpg  2. Preparation of media Most commonly used is MS Medium Murashige and Skoog Medium Must be prepared and sterilised; Some components are heat-labile* *added after autoclaving by filter-sterilisation pH of media 5.0-6.5; best 5.7; must < 7.0 村重 http://ecx.images-amazon.com/images/I/41DkB73mUNL.jpg http://ik.imagekit.io/bfrs/tr:w-200,h-160,pr-true,cm-pad_resize,bg-FFFFFF/image_titanbiotech/data/TP003.jpg http://www.sigmaaldrich.com/content/dam/sigma-aldrich/product3/167/z364754.tif/_jcr_content/renditions/z364754-large.jpg 22 http://www.emdmillipore.com/INTERSHOP/static/WFS/Merck-Site/-/Merck/en_US/Freestyle/BI-Bioscience/Cell-Culture/cell-culture-images/prepare_millex250.jpg  Media composition Macronutrients Micronutrients Vitamins Carbon source (sucrose) Organic N Agar (solidifying agent or support system) Plant growth regulators (critical for successful callus growth differentiation) Undefined mixtures sometimes added (coconut milk, yeast extract) 23  Macronutrients (major inorganic nutrients in mM amounts) For nucleic acid () and protein (□) synthesis □ Nitrogen as nitrate NO3- and ammonium NH4+ Phosphorus as phosphate PO43- □ Sulphur as sulphate SO42- Potassium – cell growth Calcium – cell wall synthesis Magnesium – enzyme co-factors, membrane integrity 24  Micronutrients (in µM quantities) Trace elements: Iron (Fe) (enzyme co-factor; in chelated form) Manganese (Mn) Zinc (Zn) Boron (B) Copper (Cu) Cobalt (Co) Molybdenum (Mo) 25  Plant growth regulators Hormones synthesized by plants that affect growth & development Synthetic derivatives are available Explants which produce enough auxin do not require more (similar with cytokinins) Stock solutions may have to be kept in the dark e.g. Indole-3-acetic acid (IAA) is light-sensitive 26  Cytokinin high auxin low Cytokinin to shoot Cytokinin high Send out auxin high shoots Increasing cytokinin concentration Callus Importance of plant growth regulators Plant cells are totipotent (can regenerate) cytokinins to shoot, auxins to root Cytokinin low auxin high Send out The ratio is roots important Cytokinin low Minimal cell Auxin to root auxin low proliferation Increasing auxin concentration 27 Chrispeels, M.J. and D.E. Sadava. 1994 Plants, genes, and agriculture  Plant growth regulators Examples of auxins include: IAA (indoleacetic acid which is natural-occurring) for callus induction at 10-30 μM IBA (indolebutyric acid) for rooting shoots at 1-50 μM NAA (naphthaleneacetic acid) synthetic analogue of IAA (0.2-2 μM root induction, 2-20 μM for callus induction) 2,4-D (dichlorophenoxyacetic acid) synthetic auxin for callus induction and maintenance (1-50 μM) Examples of cytokinins include: BAP (6-benzylaminopurine) for callus induction (0.5-5 μM) and for inducing shoots (5-50 μM)  3. Selection of explant Any part of a plant Root, stem, petiole, leaf, flower … Healthy growing plant Actively dividing specimen Young tissues (e.g. leaf with stalk or petiole) More responsive to initiation Disease free Differs between species 29  4. Surface sterilisation Vital in removing microbial contamination Surface-sterilization 1-10% bleach or 70% ethanol 1% sodium hypochlorite: Seed – 10 min; Leaves – 1-2 min follow by rinses in sterile water 30 Extract from Plant tissue culture Pt. 2, culture technique: Visual Education Productions AV 581.0724 P71 C  4. Surface sterilisation 1’30” 31 Extract from Plant tissue culture Pt. 2, culture technique: Visual Education Productions AV 581.0724 P71 C 5. Types of in vitro plant cultures: From experimental to industrial scale I. Intact plant culture II. Callus culture III. Embryo culture IV. Organ culture V. Suspension culture VI. Protoplast culture 32  I. Intact plant culture Orchid Hoffmannseggella cinnabarina Germinated seeds Fruit Flower 1 cm 1 mm Non-germinated seeds Sowing of orchid seed in vitro to form seedling and then whole plant 1 cm 33 Asymbiotic seed germination and in vitro seedling development of the threatened orchid Hoffmannseggella cinnabarina (2012) In Vitro Cell.Dev.Biol.- Plant 48:500–511  II. Callus culture Colourless mass of cells dedifferentiated from differentiated tissue On solid medium (agar) callus cells can occur in lumps In liquid medium plant cells can occur as individual cells or in small clusters to form suspension cultures Occur on cut surfaces Gradually cover and seal the damaged area Can be maintained indefinitely if necessary nutrients are given and free from contamination Must be sub-cultured regularly into fresh growth medium Can be induced to re-differentiate into whole plants by changing the growth media 34 Techniques in plant tissue culture To initate the culture, explant is aseptically incubated on/in medium which will nourish the plant cells Concentration of the growth regulators (auxin and cytokinin) is significant in initiation and development of culture e.g. callus…. to generate multiple embryos ….shoots (micropropagation) Conditions for micropropagation may differ for various species e.g. Tobacco is easy to regenerate…. calli (plural), callus (singular) In Lab Session, start culture from tobacco leaf discs (differentiated tissue) to get callus tissue (undifferentiated) tobacco leaf discs tobacco calli  Example: Calli from kenaf - Hibiscus cannabinus stem 47” Optimal conditions have to be determined empirically 37 https://www.youtube.com/watch?v=uzuZF2pnXbs&list=PLFBzKJdqylfJsn4DybOcVlEFe9q7Tv5h5  How does one initiate a callus culture? 1. Selection of explant (source of culture) Physiological state dependent Young healthy tissue is best! 2. By trial and error and LUCK Differs between species Appropriate nutrients & growth regulators 38 Techniques in in vitro plant cell culture i) aseptic techniques ii) initiation of callus cultures iii) regeneration of tissue cultures 39  III. Embryo culture 3 6 15 Mature embryos at 3, 6 and 15 d cultures; Bar: 1 cm isolate wheat embryos for callus induction 40 Front. Plant Sci., 30 August 2016 | https://doi.org/10.3389/fpls.2016.01302  IV. Organ culture isolate organ e.g. meristem, shoot-tip, root, anther, ovule for explant culture 41 Crop Protection 30 (2011) 1425e1429  V. Suspension culture Incubate at 27°C 100 rpm Discard dark brown culture Subculture at end of log growth phase Free from microbial contamination 2’7” Suspension culture derived from a tissue or callus Usually aggregates of 20-100 cells Suspension cultures grow faster than callus culture 42 Extracted from Plant cell culture. TAFE Publications AV 571.5382 P71  VI. Protoplast culture 20” Isolation of protoplasts from organ by enzymatic digestion of cell wall and maintain the propagation in liquid medium 43 https://www.youtube.com/watch?v=5-xm1EoLrW4 Four stages of tissue culture process Initiation Cut, disinfect, sterilize media and preliminary growth Proliferation Growth of explant to plantlets Cut and multiplication Constant divide and transfer Pre-transplant Growth of plantlets, initiate root & shoot development Favourable media and condition for growth Establishment Transplant to conventional growth condition (soil) 44  Regeneration of tissue cultures Initiation Plant cells are totipotent Pre-transplant Establishment Bottom view Explant Root & Proliferation regeneration Shoot Regenerated tobacco plant in soil Callus formation 45  Lab Exhibits Plant cells are totipotent African violet: African violet potted in soil stages in regeneration Tobacco calli on plate 46  Tissue culture Initiation 1 Proliferation 2 Pre-transplant Establishment 4 3 47 Extracted from Plant Tissue Culture Techniques Vocational Education Production AV 581.0724 P71 C  Micropropagation: use of tissue culture for propagation of ornamental plants Can start with limited mother stock Require less space Require much less time in producing similar number of offspring Offsprings are identical to mother plant; clones Offsprings are disease free 48  Case study: Propagation of Syngonium Healthy mother Remove all leaves Section with 10% bleach 10’ plant and cut into leaf bud Rinses in water sections selected After 4-6 Allow to proliferate, Incubate at 25 °C Place explant in weeks, divide and subculture 100-500 foot liquid medium with plantlets sections in medium candles of light micro- and macro- with cytokinin nutrients, sucrose, Extracted from Plant Tissue Culture Techniques Vocational Education Production AV 581.0724 P71 C a.a. and at pH 5.049 https://keyserver.lucidcentral.org/weeds/data/media/Images/syngonium_podophyllum/syngonium_podophyllum1.jpg  Case study: Propagation of Syngonium Multiplication of plantlets in Incubate at 25 °C, 500-1000 foot Allow the root agar medium & initiate roots candles of light for 3 weeks system to form After rooting, transfer to Transplant the plantlets for conventional green house establishment at high humidity & for further development low light intensity in green house 50 Extracted from Plant Tissue Culture Techniques Vocational Education Production AV 581.0724 P71 C Practical layout of plant tissue culture facility Preparation area Tissue culture Incubation room Transfer room Isolation and Maintenance Making of media disinfection of of explant & Sterilization of explant callus apparatus Transfer of Glass/Plastic explant & Plantlets ware callus Balance, pH meter etc. Temperature, Disinfectant, light & laminar flow humidity control 51  Preparation area Making of sterile media and reagents Balance, pH meter, stirrer heater, autoclave, dishwasher http://www.scalesgalore.com/global/images/product_2/alt/alt_7451.jpg http://store.clarksonlab.com/images/products/detail/SP131015Q.gif http://www.sperdirect.com/images_products/benchtop-ph-mv-meter-248lar.jpg http://www.autoclavesale.com/blog/wp-content/uploads/2015/07/Vertical_Autoclave_VA-SD.jpg http://oconchemicals.ie/media/catalog/category/LABORATORY_BOTTLES_1.jpg http://www.mikro-polo.com/files/mpwww2/userfiles/menuimages/Sigma-Aldrich.jpg https://images-na.ssl-images-amazon.com/images/I/31kEa6dBoXL._SX342_.jpg https://www.sks-science.com/images/14080-01.jpg 52 https://www.spectrumchemical.com/OA_MEDIA/H_301974.gif https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcQ7wPa1ycslh6a9hq7RBukqJMzTyAGXq55EFXUIJXXJMIKYPsLl2A  Transfer room Disinfection of plant surface Use of laminar flow hood to prevent contamination Use of dissecting microscope https://ae01.alicdn.com/kf/HTB1Ogr_NVXXXXbUXVXXq6xXFXXX2/227055173/HTB1Ogr_NVXXXXbUXVXXq6xXFXXX2.jpg https://carlschrammel.files.wordpress.com/2012/07/0011.jpg?w=199&h=300 https://cdn.shopify.com/s/files/1/2593/8322/products/[email protected]?v=1514526624 https://assets.fishersci.com/TFS-Assets/CCG/product-images/F11509-01~p.eps-650.jpg https://thumbs.dreamstime.com/z/scalpel-white-background-clipping-path-34541775.jpg https://www.vetpak.co.nz/files/8d7c012478470ca19a60e836166fd4d7n/54/Group%2B-%2BAlcohol%2BSpray.JPG https://i5.walmartimages.ca/images/Large/230/079/230079.jpg 53 http://www.tka.it/Img_sito/HOLAF.png https://i.ebayimg.com/images/g/PawAAOSw2tFaWfL9/s-l300.jpg https://images-na.ssl-images-amazon.com/images/I/31xA0RzLLVL.jpg http://img.frbiz.com/nimg/9e/7c/3decda581ff888dc6eed16bd34cf-98x98-1/trinocular_zoom_stereo_microscope_with_greenough_optical_system.jpg  Growth room Shelves for holding clones Controlled light and dark periods Controlled temperature (17-27°C) Greenhouse https://cs4.pikabu.ru/post_img/2016/06/30/12/1467320168191840147.jpg http://www.gemination.com/Images/TissueCultureGroup.jpg 54 http://www.stuart-equipment.com/adminimages/SSL2(1).jpg https://upload.wikimedia.org/wikipedia/commons/thumb/1/18/Callus1.jpg/1200px-Callus1.jpg http://www.aralab.pt/wp-content/uploads/2016/07/PROJ9-Aralab-Tissue-Culture-Rooms-2.jpg http://www.conviron.com/sites/default/files/images/plant-growth-chamber-pgc-flex.png 6. Scaling up plant cell cultures Lab scale: 5 L Production scale: 10-30+L Chrispeels, M.J. and D.E. Sadava. Plants, genes, and agriculture.  6. Scaling up Lab scale: 5 litres Production scale: 10-30+ litres Use of bioreactors Air-lift fermenter Stir tank fermenter Bioengineering problems Plant cells are large Cells tend to clump together Cells easily damaged by shearing Only grows at high density Produce foams Relatively slow growth rate Specific requirement for oxygen https://www.intechopen.com/source/html/51512/media/fig4.png 56 https://upload.wikimedia.org/wikipedia/commons/b/bd/Real_life_bioreactor.png  7. Introducing new genes to plant cells Make protoplasts from cell suspensions Prepare free nuclei Introduce the nuclei into the protoplasts (of different plant) to form hybrids Promote nuclei uptake (and fusion) with polyethylene glycol (PEG 4000) Regeneration of protoplasts to callus then plantlets New strain with valuable characteristics 57  Possible exam questions Write short notes on the preparation of plant explant. Describe the essential equipment used in plant cell culture. List the advantages in using plant tissue culture. What is the role of plant growth regulators in tissue culture? 58 Video on tools and techniques Plant cell culture Ann Lawrie, Tim Rolfe 30 min AV 571.5382 P71 Sources of explant, callus induction, liquid cultures (protoplast suspension cultures, somatic hybridisation) 1. Sterilisation and aseptic techniques 2. Preparation of media 3. Surface sterilisation 4. Using explant & meristem in micropropagation 5. Establishing callus 6. Establishing cell suspension culture 7. Scaling –up (bioreactors-airlifts & stir-tanks) 8. Introducing new genes (protoplasts) Video further describes: media – composition, preparation equipment environmental conditions aseptic techniques initiation of callus cultures regeneration of tissue cultures  BIOL3402 Cell Biology & Cell Technology Applications of plant cell culture and technology Plant genetic engineering Dr. Peng Wang School of Biological Sciences Lab7N/15, KBSB [email protected]  Content Date Topics Lecture Plant cell biology 1 23/10/24 Wednesday Techniques in plant cell biology 2 Tools for plant cell culture & cell technology 3 29/10/24 [media, equipment, environmental conditions] Thursday Techniques in plant cell culture 4 [aseptic techniques, callus cultures, regeneration] * micropropagation Applications of plant cell culture and technology 5 06/11/24 [*micropropagation, haploid cultures, protoplast cultures, Thursday somaclonal variants, grafting, cryopreservation, secondary metabolites] Plant genetic engineering 6 11/11/24 Laboratory session on plant cell biology & cell technology Practical Monday Isolation of mesophyll and pericarp protoplasts Examine different plant cells 2  Practicals 1 lab session Check grp! Start 1330 Nov 11 Isolate and observe mesophyll protoplasts released from leaves of flowering Chinese cabbage and from pericarp of capsicum Examine samples of different plant cells at display stations Starch grain, plasmolysis, guard cells & stomata, xylem, trichomes Wear your lab coat Write UID on the lab report and submit your lab report to your demonstrators by the end of the session 3  Recap from Last Lecture Plant Tissue Culture: Stages: initiation->proliferation->pre-transplant->establishment Advantages of tissue culture over intact plants Applications of tissue culture and why it is an important technique Plant Culture Lab Design: Equipment Environmental conditions Essentials of tissue culture Lab area layout  L5-6 APPLICATIONS OF PLANT CELL CULTURE & TECHNOLOGY  Applications of plant cell culture & technology i. Micropropagation ii. Haploid cultures iii. Protoplast cultures, new varieties through protoplast fusion iv. New varieties through somaclonal variants & grafting v. Preservation and cryopreservation of germplasm vi. Secondary metabolites via plant cultures vii. Plant genetic engineering i) Micropropogation q Micropropagation is a method of plant propagation using extremely small pieces of plant tissue taken from a carefully chosen and prepared mother plant, and growing these under laboratory conditions to produce new plants. q It is widely used in commercial horticulture. From Wikipedia  Case study: Propagation of Syngonium Multiplication of plantlets in Incubate at 25 °C, 500-1000 foot Allow the root agar medium & initiate roots candles of light for 3 weeks system to form After rooting, transfer to Transplant the plantlets for conventional green house establishment at high humidity & for further development low light intensity in green house Extracted from Plant Tissue Culture Techniques Vocational Education Production AV 581.0724 P71 C ii) Haploid cultures is an in vitro culture method where you use haploid cells (pollens or ovules) to develop a whole plant Current Biology 27, R1089–R1107, October 23, 2017 ii) Haploid cultures Use of haploid cultures Monohaploid plant has only 1 set of chromosomes generally in which the original number (in diploid) has been halved Monoploids are sterile Production of dihaploid plants from haploid cultures helps in breeding programmes; homozygous lines quickly achieved  Example: How to produce haploid plant from anther culture Anthers dissected Callus develop on Haploid plantlets Haploid plant and transferred to surface of cultured generated from transferred to soil agar anther; microspore-derived Deeply stained tissue = embryo initiation embryos microspores or haploid pro-embryo 1. Calli develop to haploid plants 2. Homozygous dihaploid can be achieved by colchicine* treatment *mitotic inhibitor; blocks mitosis at metaphase and prevents microtubule formation, Morrison R & DA. Evans Nature Biotechnology 6, 684 - 690 (1988) hence chromosomes do not pull apart; inducing polyploidy  Impact of haploids on varietal development (shortens time) Years HAPLOIDS INBREEDING 0 F2 Production of □ haploids □ 1 □ Selfing or □ □ backcrossing 2 Initial field trials □ □ to achieve 3 Trials to verify □ stability □ performance □ 4 □ Commercialization F8 5 Initial field trials 6 Trials to verify 7 performance 8 9 Commercialization Morrison R & DA. Evans Nature Biotechnology 6, 684 - 690 (1988)  iii) Protoplast cultures ØProtoplasts are isolated plant cells without cell wall Generally enzymatic degradation of cell wall using 1-2% cellulase ØIdeal for gene transfer: one transformed protoplast regenerates to one plant (not a chimera) ØSource for protoplasts Problem lies in ability to sustain division in protoplasts and regenerate from protoplasts Protoplasts derived from embryos may be better than from leaf in terms of regeneration potential ØAlso useful for somatic hybridization (protoplast fusion) https://www.researchgate.net/profile/Yaroslav_Blume/publication/295830728/figure/fig2/AS:342364313931789@1458637421881/Freshly-isolated-tobacco-protoplasts- Scale-bar-indicates-20mm.png Sources of protoplast… embryo, leaf… Maize protoplasts a) embryogenic-derived protoplast (top) b) mesophyll-derived protoplast (bottom) Lusardi et al. 1994. Plant Journal 5: 571-582  New varieties through somatic hybridization of protoplasts (protoplast fusion) 4 stages in somatic hybridisation: 1. Isolation of protoplasts: species A & B 2. Fusion of protoplasts A & B (AB) Fusion aided with polyethylene glycol 3. Regeneration from selected tissues (AB) 4. Analysis of regenerated plants (AB)  New varieties through somatic hybridization of protoplasts Virtually any combination of protoplasts can be induced to undergo fusion Allows generation of hybrids between sexually incompatible species Facilitates genetic modification of sterile or sub-fertile species and of species with long life cycles Following fusion, heterokaryons are formed consisting of nuclei from two genera/ species/ varieties à select stable lines  iv) New varieties through somaclonal variation & grafting Disadvantage of vegetative propagation is the chance of somaclonal variation Somaclonal variation = genetic variation due to genetic instability during vegetative propagation Variation seen in plants produced by plant tissue culture Types of variation: changes in chromosome number & structure, DNA sequence Factors that affect mutation frequency 1. Method of vegetative propagation mutation increases with use of adventitious shoots induced by regulators use of single-node or axillary-bud method for propagation reduces mutation 2. Number of subcultures increases mutation 3. Starting material decrease mutation with use of undifferentiated tissue e.g. pericycle, procambium, cambium 4. Type of regulator used e.g. 2,4-D and naphthalene acetic acid (NAA, synthetic auxin), cytokinin increase mutation  Selection of disease-resistant variant Somaclonal variation can occur after repeated subculture (even if started from a single cell culture) Disadvantage: genetic instability Advantage: feasibility of novel variation e.g. Carrot somaclonal variants selected and tested for resistance to the leaf spot pathogen, Alternaria dauci (fungus that causes total necrosis of mature leaves) https://ecommons.cornell.edu/bitstream/handle/1813/43265/carrot-leaf-blight-FS-NYSIPM.pdf?sequence=1&isAllowed=y  New plant species through grafting Grafting = fusion of adjacent tissues Whole nuclear genomes transferred through grafting to create a new polyploid species Many crops (wheat, cotton, canola, Arabica coffee, leek, oat and peanut) are allopolyploids* * allopolyploids are species that contain >2 chromosome sets derived from different species by sexual hybridization Now… allopolyploids and chromosome addition lines (i.e. transfer of single chromosomes) can be generated asexually by grafting which allows new strategies for crop improvement P. Hare (Nat Biotech 32: 887 (2014)  New plant species through grafting Stem-to-stem grafting of transgenic hygromycin-resistant cigarette tobacco (Nicotiana tabacum; 48 chromosomes) to transgenic kanamycin-resistant tree tobacco (Nicotiana glauca; 24 chromosomes) After fusion, graft site is cut and cultured on regenerative media containing both antibiotics Plants resistant to both antibiotics had 72 chromosomes (sum of 48+24) N. tabacum N. glauca P. Hare (Nat Biotech 32: 887 (2014) https://i.ytimg.com/vi/9-szCqJeynw/maxresdefault.jpg https://upload.wikimedia.org/wikipedia/commons/thumb/3/3e/Nicotiana-glauca-20080330.JPG/1200px-Nicotiana-glauca-20080330.JPG  New plant species through grafting New species named Nicotiana tabauca grows better and has intermediate traits N. tabacum N. glauca N. tabauca N. tabacum N. glauca N. tabauca P. Hare (Nat Biotech 32: 887 (2014)  v) Preservation and cryopreservation of germplasm Germplasm can be stored and micropropagated when needed Plant embryos can be encapsulated Carrot embryo encapsulated in a hydrated gel. Courtesy of S. Satoh Orchid embryos & germination Chrispeels, and Sadava. 1994. Plants, genes, and agriculture. https://www.ias.ac.in/article/fulltext/reso/006/05/0039-0047 http://www.discoverbiotech.com/image/image_gallery?uuid=9032cf54-4838-403c-940c-534f6da6b5ea&groupId=11406&t=1333348742484  Cryopreservation Seeds are usually stored at cold temperatures for long periods For cell cultures (e.g. suspension cells, somatic and zygotic embryos) without subculture, during which somaclonal variation and contamination may occur use -196°C (liquid nitrogen) ALL cellular activity is inhibited Need to thaw & regrow when needed 2X Cryoprotectant solution (1M dimethylsulfoxide (DMSO), 1M glycerol, 2M sucrose) added to 1 volume of cell suspension vi) Secondary metabolites from plant cultures Plants can produce an amazing assortment of chemicals CO2 vitamin A vanillin vitamin C caffeine morphine  Secondary metabolites for plant cultures Unusual and complex chemicals Cinnamon, star anise & Pharmaceuticals nutmeg Lavender Fragrances Flavor compounds Dyes Insecticides Plant cells can be grown in bioreactors (cells suspended by aeration) to establish cultures that yield these valuable chemicals Eastern hemlock Mint Colours from plants http://media-cache- ec0.pinimg.com/600x/21/82/2c/21822c318b9 44cd8ae22ff0061eff6ac.jpg https://upload.wikimedia.org/wikipedia/commons/b/b0/Mint-leaves-2007.jpg https://upload.wikimedia.org/wikipedia/commons/thumb/0/0f/Photobioreactor_PBR_500_P_IGV_Biotech.jpg/220px-Photobioreactor_PBR_500_P_IGV_Biotech.jpg https://upload.wikimedia.org/wikipedia/commons/b/b1/Bioreaktor_quer2.jpg http://images.freehdw.com/510/3d-abstract_other_alpine-purple_28983.jpg https://florafinder.org/LargePhotos/DF/Tsuga_canadensis-9189065F2F.jpg http://www.vosci.com/templates/images/79505_cimet-2.jpg  Valuable secondary metabolites Primary metabolites are sugars, amino acids, nucleotides, chlorophyll etc. Secondary metabolites are natural products They have no direct role in growth Coumarins Qu s ino nnin ne s Ta & development of the plant Carbohydrate Fla metabolism vo Glycosinolates n oi d Protect against s Photo- synthesis des Predation by insects, mammals o li d Ni abo me sm tab aci keti tro lis t me atty ge m Poly n F Pathogens Cy lyco an s i d g o g es s ne en rpe ic Te > 50,000 chemically-different types Alkaloids Restricted distribution: some made by one family or species only https://upload.wikimedia.org/wikipedia/commons/thumb/3/3b/Opium_pod_cut_to_demonstrate_fluid_extraction1.jpg/2 https://upload.wikimedia.org/wikipedia/commons/thumb/5/5c/Codein_- 20px-Opium_pod_cut_to_demonstrate_fluid_extraction1.jpg https://i.dailymail.co.uk/i/pix/2014/06/24/article-2667253-1F12B2FB00000578-521_634x390.jpg _Codeine.svg/220px-Codein_-_Codeine.svg.png https://upload.wikimedia.org/wikipedia/commons/thumb/c/c2/Catharanthus_roseus_white_CC-BY-SA.jpg/250px-Catharanthus_roseus_white_CC-BY-SA.jpg http://westlandgreenhouses.com/wp- https://upload.wikimedia.org/wikipedia/commons/thumb/7/7e/Ajmalicine.png/220px-Ajmalicine.png https://cf.ltkcdn.net/herbs/images/std/180010-425x315-Huang- content/uploads/2014/04/Colius-190x160.jpg https://upload.wikimedia.org/wikipedia/commons/thumb/0/0f/Berberin.svg/220px-Berberin.svg.png Lian-Coptis-Rhizome.jpg https://upload.wikimedia.org/wikipedia/commons/thumb/3/3a/Rosmarinic_acid Medicinal plants.png/220px-Rosmarinic_acid.png https://farm6.static.flickr.com/ 5491/10407989365_9abb2f6f8 b_m.jpg Opium poppy Pain killer, cough medicine & for control of diarrhea Codeine found in sap (latex) of pod Coleus blumei Rosmarinic acid reduces inflammation Ajmalicine Anti-hypertensive drug for treatment Coptis japonica Catharanthus roseus of high blood Madagascar periwinkle pressure Huang lian 黃連 Roots contain berberine anti-inflammatory, anti-diabetic and lower cholesterol Coptis japonica  Plants produce many valuable products Estimated world market for selected plant products Compound Estimated Retail market value Medicinal (US$ M) Caranthus roseus roots Ajmalicine: lowers blood pressure 5 Alkaloid in Codeine: opiate 90-100 opium poppy Diosgenin -wild Corticosteroids: skin disease, eye drops 300 Mexican yam Ephedra (Ma Hung) Ephedrine, pseudoephedrine: decongestion 100 Bark of Chinchona tree Quinine: anti-malarial 50 Caranthus roseus Vinblastine, vincristine: antineoplastic (some cancer) 50-75 Flavor and fragrance Cardamon 25 Cinnamon 4-5 Spearmint 85-90 Agrichemical Chrysanthemum Pyrethrins: natural insecticides 20 Chrispeels, and Sadava. Plants, genes, and agriculture.  Plant products from cultures Examples of secondary metabolites produced in cell culture & root culture Compounds Plant species Remark Type of Yield culture Berberine Coptis japonica Lowers cholesterol Cell 0.8 g/L Rosmarinic acid Coleus blumei Anti-microbial Cell 3.6 g/L Oxidant inflammatory Shikonin Lithospermum Suppress HIV Cell 1.5-4.0 g/L erythrorhizon Atropine Atropa Root 3.7 g/kg dry wt belladonna Scopolamine Atropa painkiller Root 0.24 g/kg dry wt belladonna Scopolamine Duboisia painkiller Root 11.6 g/kg dry wt leichhardtii Chrispeels, and Sadava. Plants, genes, and agriculture.  How can tissue culture technology help? Use clonal selection from single cells or protoplasts to select cells that overproduce the desired metabolite A good assay for the metabolite is available Coloured metabolites can be selected visually Different sources (such as varieties or cultivars) of the same plant species must be tested The clone must be checked for stability since subcultures will be expected in tissue culture Conditions for cell culture must be optimised Variables exist for different species  Metabolites from root cultures Roots of plants naturally produce secondary metabolites, perhaps due to interaction with pathogens and predators e.g. nicotine in tobacco leaves produced in roots and transported Alkaloid scopolamine is produced abundantly in roots, but not in clonally selected cultured cells, hence one has to use root cultures for production Agrobacterium rhizogenes, which causes hairy root disease, can induce root formation  Agrobacterium rhizogenes Causes hairy root disease Infection of A. rhizogenes leads to production of large root mass Contains root-inducing (Ri) plasmid Oncogenic genes on A. rhizogenes T-DNA are not well s ne ge T-DNA understood vir pRi Genes in the T-DNA will be incorporated into the plant genome Mulberry infected with A. rhizogenes Image credits: William M. Brown Jr., Bugwood.org; Reprinted from Dhakulkar, S., Ganapathi, T.R., Bhargava, S. and Bapat, V.A. (2005). Induction of hairy roots in Gmelina arborea Roxb. and production of verbascoside in hairy roots. Plant Sci. 169: 812-818 with permission from Elsevier.  Agrobacterium rhizogenes is useful Wounded plant for natural product Emerging roots at wound site synthesis Scale -up Reprinted from Guillon, S., Trémouillaux-Guiller, J., Pati, P.K., Rideau, M. and Gantet, P. (2006). Hairy root research: recent scenario and exciting prospects. Curr. Opin. Plant Biol. 9: 341-346 with permission from Elsevier.  Use of cultured multipotent cells to obtain Cambial Roberts and Lolewe (2010) Plant natural products from cultured multipotent cells. Nature Biotech 28: 1175-1176 Meristematic Cells  Use of cultured multipotent cells Suspension cultures of CMCs provide an attractive way to produce plant natural products DeDifferentiated Cambial Cells Meristematic Cells CMCs applicable to Panax ginseng Taxol cuspidata Roberts and Lolewe (2010) Plant natural products from cultured multipotent cells. Nature Biotech 28: 1175-1176 Ginkgo biloba  Production of taxol Taxol from the Pacific yew is an anticancer drug Effective against ovarian cancer, breast cancer, melanoma, and colon cancer by inhibiting cell division and prevents cell migration USA require about 250 kg of the pure drug each year, i.e. bark of 360,000 mature (60- to 75-year-old) trees each year Stripping the bark kills the trees! Taxol also abundant in the needles of the Pacific yew Needles are a renewable resource but approval for use in treatment has not been approved Price of taxol ~$200,000 to $300,000 per kg https://upload.wikimedia.org/wikipedia/commons/thumb/5/59/Taxol.svg/430px-Taxol.svg.png  Production of taxol Tissue culture initiated by screening bark tissues of Pacific yew to identify best producers 24-fold difference in taxol abundance observed Now 1 L of tissue culture medium contains about 1 to 3 mg taxol, comparable to extraction from 25 g of bark  Large-scale plant cell culture is limited Rapid progress (past 10 years) in l