ueiiia presentation transient transformation of plant cells

8/2/2019 UEIIIA Presentation Transient Transformation of Plant Cells

1/21

Transient Transformation

of Plant Cells


2/21

Content Transient Transformation

Applications Methods

Summary


3/21

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


4/21

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


5/21

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


6/21



7/21



8/21

Agrobacterium tumefacienschanges on Ti plasmid

T-dna cut out

no tumor induction

Transgene between border

sequences

Selection markers


9/21


Binary Vector


10/21

Agroinfiltration Plant into Agrobacterium suspension

Vacuum applied

Bacteria into intercellular spaces

Infection of surrounding cells

in planta


11/21

Agrobacterium tumefaciens in planta

single copy of transgene

rapid, easy

inexpensive

Coinfiltration limited host range


12/21

Viral Vectors Recombinant plant viruses


13/21

Viral Vectors non-replicating

in vivo

Systemic spread

Host restrictions

Examples: TMV, PVX, CMV


14/21

Particle BombardmentHelium gas

Rupture

disk

Macroprojectile

Target cellStops macroprojectile

DNA-coatedmicocarrers


15/21

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


16/21

Particle Bombardment PDS-1000/He, Biolistic

Particle inflow gun

Electrical discharge particle acceleration:

ACCELL technology

Helios gene gun


17/21

PEG Polyethylen Glycol: H(O-CH2-CH2)n-OH

water-soluble polymer

Protoplasts

promotes uptake of naked DNA

mix DNA, protoplasts, PEG/Ca


18/21

Electroporation Protoplasts

electrical discharges

reversible pores in

plasma membrane

Protoplasts easily

killed Efficiency: 40-60%


19/21

Silicon Whiskers Silicon carbide fibers

vortex plant cells, DNA

and whiskers

simple, inexpensive

restricted to cell

suspensions


20/21

SummaryAgrobacterium Particle Bombardment

cheap expensive

single copy multiple copies

tissues, in planta single cells, tissues

limited host range for all species


21/21

Thank you for your Attention!

ueiiia presentation transient transformation of plant cells

Documents