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Download Chapters 17 and 18 Tissue Culture and Micropropagation
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Transcript
Tissue Culture Micropropagation • Propagation of Plants Using Tissue Culture Techniques • Accelerated Form of Clonal Propagation Benefits Disadvantages • Makes Mass Propagation Possible • Expensive & Sophisticated • New Cultivars Fast • Trained Personnel • Cultivars With High Market Value • Specialized Techniques • More Genotypes Can Be Propagated • High Labor Costs • Difficult to Propagate Species • High-Volume System • Disease-Free Plant Stock • Contamination • Conservation of Endangered Species • Variability & Off-Type Key Concepts & Terms • Totipotency • Murashige & Skoog Totipotency • Single Cell Has Genetic Program to Grow Into Entire Plant • Developmental Stages of Micropropagation • Explant • in Vivo & in Vitro Environments • Meristem Culture 1 Murashige & Skoog • Skoog – Ratio of Cytokinin to Auxin Controls Growth • Murashige – Developmental Stages Explant • Piece of Plant Used to Initiate Tissue Culture Process • ‘Propagule’ – Medium For Tissue Culture • Ratios of Hormones • Levels of Nutrients in Vivo vs. in Vitro • in Vivo – Within Living Organism • in Vitro – Artificial Environment Outside Living Organism – Growing in Glass (in Test Tube) Developmental Stages of Tissue Culture Meristem Culture • Produce Plants Free of Diseases • No Viruses • Explant – Meristem Dome & Leaf Primordia • Meristems of 0.1 to 0.15 mm Have Been 100% Virus Free – Small Explants More Difficult to Establish Four Stages I. Establishment II. Multiplication III. Root Formation IV. Acclimatization 2 • Explant Source Selection I. Establishment – Avoid Mistakes! – Identify Correct Genotype • Place Tissue Into Culture to Initiate Microshoots – Herbaceous Plants Easier • Important Aspects – Juvenile Stage Easier • True-to-Type – Explant Source Selection – Disinfestation – Culture Medium – Stabilization • Control Pathogens – External Contaminates Are Ubiquitous – Spores Move on Air Currents – Disinfest Explants • Remove Contaminants • Microbe Growth Usually Within Week II. Multiplication • Induce Multiple Shoots • Medium Similar to Stage I • Cytokinin Supports Shoot Initiation – Avoid Greater Than Optimum Cytokinin • Quickly Discard Contaminated Vessels – Disinfest Tools & Working Area – Proper Culture Medium • Subculturing – Harvest at Appropriate Length – Start New Cultures • Divide Into Smaller Pieces • Fresh Medium • 2 to 8 Weeks III. Root Formation • Initiate Roots – in vitro or ex vitro – Don’t Wait Too Long to Subculture • Leaf Yellowing & Necrosis 3 in vitro • Root-Inducing Medium – Different Growth Regulator Ratio • Roots Usually Suboptimal – Poorly-Formed Vascular System ex Vitro • Treat With Auxin • Insert Into Soilless Potting Mix • Place Under Mist • Roots Tend to Be ‘Normal’ – Must Change Morphology • Can Stimulate Rooting of Difficult-to-Root Plants IV. Acclimation • Gradually Move Plants to Open Air • Shift From Heterotroph to Autotroph Leaf Morphology Affected • in vitro Shoots Have Smaller Leaves & Fewer Cell Layers • Leaves Lack Proper Epicuticular Wax – Can Desiccate Quickly – Abnormal Stomata Generating Plantlets Axillary Shoots • Shoot Cultures Are Like Mini Stem Cuttings That Grow From Lateral & Terminal Meristems – Existing Apical Meristems Are Used so Genotype Reliably Reproduced 4 Adventitious Shoots • Initiated Directly on Explant or in Callus Produced From Explant Aseptic Seed Culture • Orchid Seeds Commonly Propagated by Tissue Culture • Higher Multiplication Rates • Increased Numbers of Off-Types Embryo Rescue • Excise Embryo & Germinate in Aseptic Culture • For Embryos That Would Normally Be Aborted Within Seed • Practical Use – Hybrid Crosses – Early-Ripening Cultivars of Many Fruit Trees Micrografting • Unique Uses – – – – – Disease Free Plants Virus Indexing Early Detection of Graft Incompatibility Propagation of Novel Plants Rejuvenation of Scions • Mature Scions on Seedling Understocks – Convenient Way to Send Germplasm Synthetic Seed Production • Development of Embryos From Vegetative Cells & Tissues • ‘Somatic Embryogenesis’ • Enclose Somatic Embryos in Artificial Seed Coats • Manipulation of Agar Components Agrobacterium Induced Growth • Agrobacterium Can Transfer T-DNA Into Chromosomes of Plant Cells • Genes Code for Auxin & Cytokinin • Infected Tissues Make Their Own Hormones • A. rhizogenes Induces Infected Plants to Make Roots on Above-Ground Parts • Cultures Used to Produce Materials Such as Pharmaceutical Compounds 5 Tissue Culture Environment Temperature • 68-81°F • Avoid High Temps • Bulblet Formation – Alternate Day/Night Temp Cycles • Cool Storage – Suspend or Slow Growth When Demand For Plantlets Is Down • Duration/Photoperiod Light – 12 to 16 Hours – Can Trigger Photoperiodic Responses in vitro • Intensity – High Light Can Cause Loss of Chlorophyll & Leaf Necrosis • Quality – Different Wavelengths to Acclimate Plants – Light Inhibits Root Growth Gases Special Problems • All Closures & Caps Somewhat Permeable to Gases • High CO2 Levels Have Improved Growth of Some Species in Research Projects 6 Hyperhydricity • Excess Water Uptake – Translucent, Water-Soaked Succulent Appearance • Change Agar Internal Pathogens • Do Not Assume Absence • Source Material • Contamination During Culturing – Humans – Environment Excessive Exudation • Usually Phenolic Compounds Cause Brown Material Shoot-Tip Necrosis • Ca Deficiency • Refine Medium • Tend to Inhibit Development • Treat With Antioxidant • Transfer Often Habituation Variation • Automatic Growth in Cultures That Previously Required Auxin or Cytokinin for Growth 7 Genetic or Chimeral Effects • Not Always Undesirable • Chimeral Breakdown Possible • Callus Can Produce Adventitious Shoots • Control Genetic Variation Transient Phenotype Variation • Different Growth Patterns in Postculture • Usually Disappear – Promote Axillary Shoot Proliferation – Limit Number of Subcultures – Lower Growth Regulator Levels • Vigor Often Enhanced – Plants May Have Been Rejuvenated to More Juvenile Phase • Branching often Enhanced – Useful For Such Species as Hosta, Begonia, Aster, Mum, Rose, Syngonium, Ferns & Other Foliage Plants Lab Facilities & Procedures – Carryover Effect of Cytokinin in Multiplication Medium • Separate Facility Facilities & Equipment • Restricted Entry • Clean, Clean! 1.Research Labs • Separate Prep, Transfer & Growing Areas 2.Large Commercial Facilities • Service Areas, Offices, Cold Storage 3.Individual Nurseries, Hobbyists 8 • Equipment Preparation Area – Refrigerator – Scales or Balances – Autoclave • Kitchen – Clean Glassware – pH Meter – Media Prepared – Heating Plate • Clean Surfaces – Stirrer & Mixing Device – Filters to Sterilize Non-Autoclavable Ingredients Transfer Area – Water Purification Equipment – Equipment to Decontaminate Explants – Media Dispenser – Storage For Flasks & Bottles • Explants Are Inserted Into Culture • Transfers or Subcultures • Laminar Airflow Hood – Filtered Air Passes Outward – Prefilter to Remove Dust & Microbial Spores • UV Germicidal Lamps Growing Area • Grow Cultures in Separate, Lighted Facility • Can Control Lighting & Temp • Helpful to Have Several Rooms Containers For Cultures • Pyrex or Less-Expensive Glass • Test Tubes, Flasks • Petri Dishes • Nonabsorbent Cotton Plugs • Metal or Plastic Covers • Second Cover to Hold Moisture & Reduce Infection 9 Media – Organic Compounds • Carbohydrates • Vitamins • Ratio of Ingredients Varies • Hormones & Growth Regulators • Some Formulations Common • Ingredients – Inorganic Salts • Macro- & Micro Elements • Stock Solutions (100x) & Stored in Refrigerator – Gelling Agents • Agar – Obtained From Red Algae Aseptic Procedures – Powder Available – Characteristics » Melts When Heated • Disinfest » Change to Semisolid Gel at Room Temp – Remove Contaminants From Explant » Biologically Inert – Methods • Liquid – Nutrient Solution Sometimes – Can Exchange Solution Without Reculturing – Support to Keep Explants & Cultures From Sinking • Microbes – Spores on Surfaces – Settle Out of Air or Are Blown on Air Currents • Aseptic Procedures Done in Transfer Hood or Box • Wash • Cut Material Into Small Pieces • Wash in Running Tap Water • Quick Dip in Alcohol • Place in Disinfesting Solution in Hood • Wear Gloves & Clean Lab Coat • Sterilize All Tools & Containers – Alcohol or Other Sterilant – Bacti-Incinerator • Cool Before Use to Prevent Damage to Tender Plant Tissue – Sides & Surfaces of Chamber – Hands & Arms 10