Transcript The Agrozobium™ project

Selection-free screening protocol for plant
transformation: an open-source platform
for plant biotechnology
The
GusPlus™
project
A new GUS gene,
Available under BIOSTM licensing,
Pioneering use of the BioForgeTM concept
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GUSPlusTM as a selectable marker
Premise:
Based on our observations, plant tissues could survive and
continue to regenerate after incubation in a low
concentration X-GlcA solution, and potentially in the
presence of the end product of GUS cleavage (indigo).
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Why avoid herbicide/antibiotic selection?
• Lack of freedom to operate (FTO)
• Horizontal gene transfer to weedy relatives or
other biota
• Herbicide/antibiotic may have negative effect
on transformation efficiency
• Cytotoxic treatments in culture may create
unacceptable epigenetic or genetic variability
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Removal of herbicide/antibiotic resistance
genes from GM plants
A number of approaches have been used:
• Separate T-DNA for gene-of-interest and selection marker,
followed by segregation in subsequent generations (Komari T, Hiei Y,
Saito Y, Murai N, Kumashiro T (1996) Plant J 10:165-174 )
• Recombinases, such as Cre/lox recombination system
(Hajdukiewicz, P.T., Gilbertson, L.A. and Staub, J.M. (2001) Plant J. 27: 161–
170.)
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Non-herbicide/antibiotic resistance gene
approaches
• Betaine aldehyde dehydrogenase (BADH)
al., 2001 Curr. Genet. 39: 109-116.)
(Daniell et
• Phosphomannose isomerase (Joersbo et al., 1998 Mol.
Breeding 4:111-117)
• Ac-isopentenyl isomerase (Ebinuma et al., 1997 Proc. Natl.
Acad. Sci. 94: 2117-2121)
• PCR (Popelka et al., 2003 Transgenic Res 12(5):587-96;
De Vetten et al.,
2003 Nat. Biotechnol., 21(4): 439-442)
• GFP
(Jordan 2000 Pl. Cell Rep. 19:1069-1075; Zhang et al., 2001 Mol.
Biotechnol. 17:109-117)
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GusPlusTM approach
• Three model crops: Arabidopsis, rice and
tobacco.
• Mono- and dicotyledonous species
• Three different transformation systems:
• Floral dip
• Leaf disc
• Callus
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GUSPlusTM vectors
Hyg (R)
35S
35S
CAT intron
GUSPlus
pCambia1305.1
Hyg (R)
35S
35S
GRP
CAT intron
GUSPlus
pCambia1305.2
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Tobacco ‘selection’ strategy
• Co-cultivate leaf discs with Agrobacterium
• Transfer to regeneration media containing antibacterial agents but no selection agent
• At various time points incubate tobacco callus or
shoots in X-GlcA (200ug/ml)
• ‘Select’ blue-stained tissues for regeneration
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Selection of transgenic tobacco plants
using GUSPlusTM
Callus
Leaves
Shoot
Leaves
Tobacco callus (upper left) or tobacco shoot (lower left) showing GUS expression (arrow) after incubation
with low concentration X-GlcA. These tissues regenerated into plantlets whose leaves also expressed GUS
(upper and lower right).
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Summary of GUSPlusTM selection for
transgenic tobacco plants
GUSPlus construct
Leaf discs co-cultured
with Agrobacterium
Transgenic
plants
Cambia1305.1
8
14
Cambia1305.2
9
13
Cambia1305.2
25
10
Cambia1305.2
21
7
Total
63
44
Cambia1305.2
(Hyg selection)
63
160
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Rice selection strategy
• Co-cultivate rice calli with Agrobacterium.
• Transfer to callus growth media containing
anti-bacterial agents but no selection agent.
• At various time points incubate rice calli in XGlcA (200ug/ml).
• Select ‘blue’ calli and move to regeneration
media.
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Selection of transgenic rice plants using
GUSPlusTM
Callus
Rice callus expressing GUS after incubation in low concentration X-GlcA.
GUS-expressing callus was cultured on regeneration media and some of the developing plantlets
expressed GUS in leaf material.
Leaf Tips
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Summary of GUSPlusTM selection for
transgenic rice plants
GUSPlusTM
construct
Calli co-cultured
with Agrobacterium
Transgenic
plants
pCambia1305.1
90
8
pCambia1305.1
60
1
pCambia1305.2
420
12
pCambia1305.2
160
5
pCambia1305.2
225
2
Total
955
28
pCambia1305.2
(Hyg selection)
955
190
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Arabidopsis selection strategy
• Floral dip of Arabidopsis with Agrobacterium
• Allow plant to grow and set seed.
• Germinate seed then incubate seedlings in X-GlcA
(200ug/ml).
• Transfer ‘blue’ seedlings to soil and assay mature
plants for GUSPlusTM expression.
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Selection of transgenic Arabidopsis
plants using GUSPlusTM
Seedling
Leaf
Arabidopsis seedling screened using low concentration X-GlcA showing GUS expression
in the roots (arrows)
Leaf from same plant after 2 weeks growth in soil, stained with X-glcA to show GUS
expression.
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Summary of GUSPlusTM selection for
transgenic Arabidopsis plants
GUSPlusTM
construct
Seedlings screened
Transgenic
plants
pCambia1305.2
124
1
pCambia1305.2
236
5
pCambia1305.2
334
3
pCambia1305.2
150
2
pCambia1305.2
90
0
225
2
Total
1159
13
Cambia1305.2
(Hyg selection)
979
19
pCambia1305.2
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Summary
• Transgenic plants obtained using GUSPlusTM as the only
selectable marker
• Selection system appears to work for mono- and
dicotyledonous plants and for different transformation
systems
• Use of GUSPlusTM gene avoids perceived negative aspects of
herbicide or antibiotic selection
• Use of GUSPlusTM gene overcomes FTO issues
• Unlike PCR, GUSPlusTM allows routine monitoring of
transgenic material.
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GUSPlus will be available for use
under the conditions of a BIOS license
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Traditional intellectual property licenses contain covenants
under which the licensee must agree to:
•
•
•
•
Royalties and/or milestone payments
Exclusive or non-exclusive, with various restrictions on field of use
(often) Grantback of improvements to licensor
(often) Assistance to licensor in maintaining patent monopoly
BIOS -compliant IP licenses will instead contain
covenants under which the licensee must agree to:
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• No royalties, only costs of maintaining protected commons
• Non-exclusive only
• Sharing of improvements and technology data for regulatory
purposes
• No assertion of improvement patent rights against other
licensees
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BIOS licenses will be granted to entities that
agree to the covenants:
• Universities
• Public good research institutions
• Private companies, small, medium or large, wanting to use
and improve the technology to make products
The intent of the improvement-sharing
and non-assertion requirements
is that no one licensee can hijack the
technology, and it can be used
- for humanitarian purposes
or
- to make a profit
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GusPlusTM in the BioForgeTM Project
• www.BioForge.net is a distributive cooperation website
modeled on SourceForge, used by the global software
development community to bring together project needs,
ideas and usage data from people in diverse locations and
time zones.
• BioForgeTM will use GUSPlus as one of the model co-operative
projects for creating a protected commons of shared
methodology.
• We hope this project will serve as an example of restoring
public-good norms and trust in agricultural biotechnology.
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The
GUSPlus™
project
Funded by the Rockefeller Foundation, Monticello Research
Foundation and Horticulture Australia
A new screening protocol for transgenic plants
Brian Weir, Heidi Mitchell, Tuan Nguyen, Richard Jefferson
BIOSTM licensing
Draft License: Mat Berman (UC) Mike Rabson, Marie Connett Porceddu,
Richard Jefferson; Commentable website: Steve Irwin, Nick dos Remedios;
BioForgeTM distributive collaboration website
Collabnet® and CAMBIA’s BIOS Initiative