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Making transgenic plants
contribution by Ann DepickerVIB, Ghent University, Belgium
Cost Exploratory Workshop:
What role of GM technology in futurecompetitiveness of European agri-food sector?
5 November 2008 Ljublijana, Slovenia
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the teams of Jef Schell and Marc Van Montaguunderstanding crown gall induction
1978 This people are what we called theThis people are what we called theGhent Crown gall groupGhent Crown gall group
GM technology: Where do we come from?
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Three bacterial elements were foundThree bacterial elements were found
to be required for Tto be required for T--DNA transfer to plants:DNA transfer to plants:
therefore, the whole interior part of
the T-DNA could be deletedand substituted with any other DNA sequence.
Chromosomal genes
Virulence genesT-DNA border sequences
Genes in the T-DNA are required
for tumor growth and opine synthesisbut not for T-DNA transfer:
UNIVERSITEIT
GENT
1982
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cggcaggatatattcaattgtaaa
tggcaggatatataccgttgtaat
accgtcctatatatggcaacatta
gccgtcctatataagttaacattt
T-DNA
virregion
ori
LB
RB
Left terminal repeat = LB
Right terminal repeat = RB
UNIVERSITEIT
GENT
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1983
thethe
firstfirsttransgenictransgenic
plantsplants
the first selectable
markers:
kanamycin and
chloramphenicol
resistance
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() ()
,
LB RB
T-DNA
80
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mid 90s: first GM crops
focus on
insect resistance: BT variants
herbicide resistance
UNIVERSITEIT
GENT
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2000 till now:
world wide spread and increasing use of GM crops exceptin Europe
Not to grow GM crops in Europe except in Spain is a
political decision:
Import of approved GM crops is allowed in Europe butcommercial production of approved GM crops is not
allowed
The consumer has a choice: labeling of GM crop derivedfood/feed is obligatory. However separation of the chainswill become more and more costly
UNIVERSITEIT
GENT
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.
Pest tolerance (viruses, fungi, nematodes..)
A biotic stress tolerance (drought, salt, high light..) Yield stability
Improved nitrogen uptake efficiency
Growth in alkaline or Fe-restricted soils Food quality cfr Cathie Martin
Bio-energy production
Molecular farming cfr Eva Stoger oils, pharmaceuticals, high value proteins..
Phytoremediation
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What do we have to make
transgenic plants?
a series of vector systems
various transgene elements
a selection system
a target plant
The use of the transgenic plant determines which
criteria are used to screen for a best GM
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* * * *
* *
*
( )
()
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exon1 exon2intron1 intron2 exon3
mRNA AUG5UTR
UAAUAGUGA AAUAAA poly A
coding sequenceor cDNA
3UTR
promoter and terminator: transcriptional control
2. 5UTR and 3UTR: where the transcriptional fusions aremade3. introns are present if a genomic sequence is used4. coding sequence: especially the codon usage:
optimizing the gene sequence can increase proteinexpression levels several fold
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What are the transformation frequencies?What are the transformation frequencies?
This is relevant for the question whether selection for aThis is relevant for the question whether selection for a
transformed plant is needed or whether a transformedtransformed plant is needed or whether a transformed
cell could be screened for?cell could be screened for?
If transformation frequencies are far below 1 %,a selection marker is needed. This is no
problem for research but there is a lot ofopposition to the use of resistance markers intransgenic crops.
The main reason is the fear for spread ofantibiotic resistance genes.
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CocultivationCocultivation withwith AgrobacteriumAgrobacterium
and nonselective regenerationand nonselective regeneration
Conclusion:After cocultivation with Agrobacterium, selection is required to obtainArabidopsis root explant transformants, but no selection is required toobtain tobacco protoplasts transformants
=>different tissues or types of cells have a differentcompetence for transformation
transformation of root explants of 0 transformantsArabidopsis thaliana /172 plants
transformation of protoplasts of 26 transformantsNicotiana tabacum /140 plants(18%)
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Regenerated plants Number
Isolated 84With a transiently expressed C T-DNA 4/84 (5%)With an integrated C T-DNA (transformation) 0/84 (
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Cotransformation frequencies are much higher
than expected from the transformationfrequencies: this means that especially theintegration is limiting the transformationfrequencies
Co-transformed T-DNAs often co-integrate at thesame site and this results in Inverted and
tandem repests of integrated T-DNAs
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Transformation frequencies are
determined by:
accessibility of the plant cell to be
transformed
Agrobacteriumattachment efficiency andT-DNA transfer
competence of the plant cell for T-DNAintegration
division of the transformed cell
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Plant transformation is, in many cases, a very-low
frequency event; therefore, selection is needed.
In most cases, selection is based on the inclusion into the
culture medium of a substance that is toxic to plants.The classical selectable markers confer resistance to
antibiotics and herbicides.The recent alternatives are metabolic selection markers (eg.
pmiand dao) and easily screenable visual markers (eg.dsred, gfp).
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, , ,
The P35S-dao1 gene (from yeast Rhodotorula gracilis) catalyzes theoxidative deamination of a range of D-amino acids=> provides positive and negative selection
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A gene useful as reporter for transformation:the Green Fluorescent protein (GFP)
WT
Advantage of GFP: the assay is non-destructive
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Also seed specific Dsred expression allows to pick
immediately the transformed Arabidopsis seedswithout any other selection
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Removal of the selectable marker?
Different ways: cotransformation and
subsequent segregation, removal via sitespecific recombination, or screening via PCRbased methods for transformants
Why remove the selectable marker? Primarily to avoid problems with horizontal gene
transfer to pathogenic bacteria. However, this doesnot seem to happen.
Also to allow subsequent transformation withadditional transgenes: can be circumvented withcrossing and PCR screening.
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TT--DNA integrationDNA integration
D
T-DNA integration occurs through illegitimaterecombination:
First, the T-strand is made double strandedin the plant cell
Then the ds DNA is integrated at randompositions
Parallels are seen with double strand breakrepair (DSB) and non-homologous end joining(NHEJ) via single strand annealing mechanism
=> T-DNA integration makes use of the plant DNA doublestrand break repair system
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plant DNA preinsertion site
T-DNA
Target site deletion
69 T-DNA plant DNA recombination sites
were sequenced and subdivided in 3 classes:- 10 % end to end ligations
- 50 % junctions with microhomology
- 40 % junctions with filler DNA
RB junctionLB junction
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T-DNA integration: integration site can not (yet) becontrolled
T-DNA: random integration: in genes and between genes- no homology between the T-DNA ends and the plant DNA target
- preference for open chromatin regions
plant target DNA shows a deletion of approximately 10 to100 basepairs
the ends of the T-DNA are often processed (truncated) upto about 100 basepairs
=> sequence the integration site and subsequent T-DNAplant junctions to enrich for clean events in between genes
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T-DNA integration
Some transformants contain a single T-DNA copy;however most transformants contain many T-DNA copiesat one locus or at 2 or 3 loci
Truncated copies may be present and also non-T-DNAor vector DNA may occur (skipping of the bordersequences)
Many transformants contain unlinked point mutations;translocations occur in 10 to 20 % of the transformants
and also aneuploidy is found more often then expected.
=> screen for transformants with a single T-DNA copy andand inbreed this event for several generations
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PCR analysis
T-DNA
a b
- Allows to screen transgenic plants for integration of T-DNAs that do notcontain a selection marker
- Allows to screen transgenic plants for the presence of silenced T-DNAs
- PCR reaction for internal T-DNA fragments does not allow thedetermination of copy number of the integrated T-DNAs
- Different transformants with the same T-DNA can not be distinguished
- Allows to screen mixture of plants/crops or food/feed for the presenceof GM plants
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T-DNA / plant junctions
T-DNA
probe
T1T2T3T4T5T6
The T-DNA plantjunctions are differentin every transformant;
the number offragments indicatesthe number of T-DNAcopies
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PCR ANALYSIS
CLEAN TRANSGENE
1 SCREENING AND DETECTION
2 CONSTRUCT SPECIFIC DETECTION
3 EVENT SPECIFIC DETECTION
12
3
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Transgene expression variation
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10000
1000
100
10
1
0.1
0.01
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MQM18.05
F12B173.3
F5A1826.30
F12P1924.25
F14J2218.05
T18A2019.80
F14J1620.6
F2D107.2
T4K2210.75
FK24
F2K3
CK2L102 CK2L72
CK2L36
CK2L111 CK2L6
CK2L129
F2I90.22 F14B2
18.1
F13A1019.25
F2K16
F2Hsb21
F5E62.05
F21A1414.15
F4F1519.6
CK2L107
CK2L70
CK2L94
F14M1912.35
L23H314.90
F8B414.85
FH33
F2Hsb20
F2Hsb31
F2Hsb22 CK2L7
T22J188.25
CK2L129
CK2L148
Characterization of the T-DNA integration position in19 single-copy transformants
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21 single copy T-DNA transformants, selected on the expression ofan antibiotic resistance marker, were identified and characterized
In 19/21 single copy lines, gusexpression was similar and not
silenced; in 2/21 lines transgene expression was more than 20-foldlower - In one of those lines, methylation of the transgene was clearlydemonstrated
Integration into an intergenic or genic region, into an exon or anintron, in sense or antisense orientation, did not result indifferential transgene expression
The presence of binary vector sequences in 2 single copy lines didnot have a negative influence on transgene expression
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Single-copy transformants were not the highest expressers
This implies that multicopy loci are not always inducing transgene
silencing. What is triggering the silencing in multicopy loci is notknown
Only very few transformants have no expression of the GUS
reporter gene: this means that complete silencing of a transgene inthe first generation is rather rare.
The silencing degree varies in different transformants and is in
leaves typically between 20 and 200 fold.
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New generation of GM plants: fi drought tolerance
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f
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Plate-based screen of > 1,500 overexpressedtranscription factors
Drought assay (soil grown)
~ 40 different transcription factors regulate droughttolerance
NF-YB (Nelson et al., 2007)
Improved drought physiologySurvival assay
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Nelson et al., 2007 Mendel Biotechnology & MonsantoMolecular phenotyping indicatesNew mode of action
Field efficacy trials
Healthier transgenics:
Less leaf rollingHigher chlorophyll index
Higher photosynthesis rateCooler leaf temperature
Higher stomatal conductance
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www.mendelbio.com
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Increased ABA sensitivityNo effect on photosynthetic yield
Molecular phenotyping: > 80 at least 2.1 fold upregulated.Enrichment for Osmotic Adjustment genes
Functional equivalence in cyanobacteria
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Functional equivalence in cyanobacteria
and diatoms between ferredoxinand
flavodoxinunder iron deficiency
Other Fd-dependent reactions
Fld
Photosynthetic microorganisms compensate Fd decline by inducing Flavodoxins.
Flds: ~19kDa with 1 noncovalently bound flavin mononucleotide as prosthetic group
Not sensitive to oxidative conditions Efficient replacement of Fd in NADP+ reduction, nitrogen fixation, sulfite reduction,. Flds are restricted to prokaryotes and some eukaryotic algae. Lost in evolution to vascular plants (~Fe abundance in coastal regions).
Plants expressing a cyanobacterial flavodoxin in chloroplasts develop
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p g y p pincreased tolerance to various sources of environmental stress
pfld4-2pfld12-4
18 h at 500 mol quanta m-2 s-1 and 40oC
20 days at 500 mol quanta m-2 s-1 and 9oC
3-day water deprivation regime
18 h to a focused light beam of 2,000 mol quanta m-2 s-1
20 min to UV-C radiation
UV-AB radiation for 24 h
1,200 mol quanta m-2 s-1 for 24 h
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Future of GM plants in Europe? will depend on the sound and flexible re-evaluation of the
legal framework
this will determine whether there is a market for GM cropsin Europe
Anyway,
the technology is available to introduce a variety of traits
the perspectives promise a future which plantbiotechnology optimists have dreamed of since many years
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