1. Gene transfer to plants – vectors and strategies
Bio4600

2. Methods of transformation:
Particle bombardment:
Small particles covered with DNA are introduced into plant cells
Whole plants are regenerated from transformed cells by tissue culture

3. Particle bombardment:
Integration of many and often rearranged copies of foreign DNA in replicating regions

4. Methods of transformation:
Agrobacterium tumefaciens-mediated transformation
Utilization of a natural gene transfer system
Introduction in flowering plants or plant cells
Dicotyledonous plants

5. A. tumefaciens transformation of plant cells
Ti-plasmid with T-DNA
transfer of single-stranded T-DNA
random integration

6. Selection of transformants and generation of transfomed plants

7. General requirements to a T-DNA vector
pVS1
Sm/SpR LB
pnos
nptII
3’nos RB
pKOH110-
(10813bp +)
EcoRI SmaI XbaI SscSJ87I HindIII
Right and Left Borders
E. coli origo
A. tumefaciens origo
Bacterial selectable marker
Plant selectable marker
Cloning sites
Plasmid with vir-genes in A. tumefaciens strain

8. Vector for promoter analysis
SmaI BamHI XbaI SalI PstI HindIII
CCCGGGGATCCTCTAGAGTCGACCTGCAGGCTGCAAGCTT
nos-
ter
gus
nptII LB
StrR, SmR RB
pZP221G
Vector backbone bp
ori
pVS1
pBR322
bom
rep
NheI
ClaI

9. Vector for over-expression and GFP detection
PstI
SmaI
BamHI
NotI
XhoI EcoRI
CmR
ccdB
BglII XbaI
EcoRI
attR1
PstI TL
35S
GAW-A
GFP
ter
attR2
EcoRI XbaI SscSJ87I HindIII RB
pnos
nptII
pKOH35SGAWGFP
3’nos
(ca. 14 kb)
pVS1 LB
Sm/SpR
TL - translasjonsenhancer
ter -terminator

10. Vector for knock-down, RNAi