Download Chapters 17 and 18 Tissue Culture and Micropropagation

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
–
–
–
–
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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
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