Transient Transformation of Plant Cells

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Transient Transformationof Plant Cells
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Content

• Transient Transformation
• Applications
• Methods
• Summary

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Transformation

• Definition The introduction of exogenous genes
  into plant cells, tissues or organs
• Indirect biological vector
• Agrobacterium tumefaciens
• Viral vector
• Direct physical chemical processes
• Particle Bombardment
• Polyethylene Glycol (PEG)
• Electroporation
• Silicon Whiskers

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Stable vs Transient

• Stable
• inherited by Progeny (Segregation)
• Transformation Integration Regeneration
• time consuming
• not very efficient
• Transient not inherited
• might be integrated
• fast, easy
• no regeneration necessary

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Fields of Application

• rapid results
• verify transformation constructs
• to study gene expression and regulation
• function of promoters, transcription factors
• Expression of transgenes and their function
• in different tissues /on cellular level
• under different conditions

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Agrobacterium tumefaciens
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Agrobacterium tumefaciens
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Agrobacterium tumefaciens

• changes on Ti plasmid

• Transgene between border sequences
• Selection markers

• T-dna cut out
• no tumor induction

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Agrobacterium tumefaciens

• Binary Vector

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Agroinfiltration

• Plant into Agrobacterium suspension
• Vacuum applied
• Bacteria into intercellular spaces
• Infection of surrounding cells
• in planta

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Agrobacterium tumefaciens

• in planta
• single copy of transgene
• rapid, easy
• inexpensive
• Coinfiltration
• limited host range

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Viral Vectors

• Recombinant plant viruses

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Viral Vectors

• non-replicating
• in vivo
• Systemic spread
• Host restrictions
• Examples TMV, PVX, CMV

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Particle Bombardment
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Particle Bombardment

• for all species, tissues and cell cultures
• no pre-treatment of tissue necessary
• more than 1 bombardment possible
• direct gene transfer
• Efficiency 10000 cells/shot
• DNA often gets fragmented, multiple copies

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Particle Bombardment

• PDS-1000/He, Biolistic
• Particle inflow gun
• Electrical discharge particle acceleration
  ACCELL technology
• Helios gene gun

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PEG

• Polyethylen Glycol H(O-CH2-CH2)n-OH
• water-soluble polymer
• Protoplasts
• promotes uptake of naked DNA
• mix DNA, protoplasts, PEG/Ca

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Electroporation

• Protoplasts
• electrical discharges
• reversible pores in plasma membrane
• Protoplasts easily killed
• Efficiency 40-60

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Silicon Whiskers

• Silicon carbide fibers
• vortex plant cells, DNA and whiskers
• simple, inexpensive
• restricted to cell suspensions

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Summary
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Thank you for your Attention!