Ppt GM Plants

8/12/2019 Ppt GM Plants

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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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