Selection-free screening protocol for Selection-free screening protocol for plant transformation: an open-source plant transformation: an open-source
platform for plant biotechnologyplatform for plant biotechnology
The The GusPlusGusPlus™ project project
A new GUS gene, Available under BIOSTM licensing,
Pioneering use of the BioForgeTM concept
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GUSPlusGUSPlusTMTM as a selectable marker 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?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 Removal of herbicide/antibiotic resistance genes from GM plantsgenes from GM plants
• 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.)
A number of approaches have been used:
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Non-herbicide/antibiotic resistance gene Non-herbicide/antibiotic resistance gene approachesapproaches
• Betaine aldehyde dehydrogenase (BADH) (Daniell et al., 2001 Curr. Genet. 39: 109-116.)
• 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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GusPlusGusPlusTMTM approach 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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GUSPlusGUSPlusTM TM vectorsvectors
35S 35S GRP CAT intron GUSPlus
pCambia1305.2
Hyg (R)
35S 35S CAT intron GUSPlus
pCambia1305.1
Hyg (R)
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Tobacco ‘selection’ strategyTobacco ‘selection’ strategy
• Co-cultivate leaf discs with Agrobacterium
• Transfer to regeneration media containing anti-bacterial 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 Selection of transgenic tobacco plants using GUSPlususing GUSPlusTMTM
Callus
Shoot
Leaves
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 GUSPlusSummary of GUSPlusTMTM selection for selection for transgenic tobacco plantstransgenic 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 strategyRice 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 X-GlcA (200ug/ml).
• Select ‘blue’ calli and move to regeneration media.
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Selection of transgenic rice plants Selection of transgenic rice plants using GUSPlususing GUSPlusTMTM
Callus Leaf Tips
Rice callus expressing GUS after incubation in low concentration X-GlcA.GUS-expressing callus was cultured on regeneration media and some of the developing plantletsexpressed GUS in leaf material.
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Summary of GUSPlusSummary of GUSPlusTMTM selection for selection for transgenic rice plantstransgenic 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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ArabidopsisArabidopsis selection strategy 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 Selection of transgenic Arabidopsis Arabidopsis plants using GUSPlusplants using GUSPlusTMTM
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 GUSPlusSummary of GUSPlusTMTM selection for selection for transgenic transgenic ArabidopsisArabidopsis plants 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
pCambia1305.2 225 2
Total 1159 13
Cambia1305.2(Hyg selection) 979 19
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SummarySummary• 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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GUSPlusGUSPlusTM will be available for use will be available for use under the conditions of a BIOSunder the conditions of a BIOSTM license license
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
BIOSTM-compliant IP licenses will instead contain covenants under which the licensee must agree to:• 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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The intent of the improvement-sharing The intent of the improvement-sharing and non-assertion requirements and non-assertion requirements is that no one licensee can hijack the is that no one licensee can hijack the technology, and it can be used technology, and it can be used - for humanitarian purposes - for humanitarian purposes or or - to make a profit - to make a profit
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
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GusPlusGusPlusTM in the BioForge in the BioForgeTM Project 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.
The The GUSPlusGUSPlus™ project project
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
Funded by the Rockefeller Foundation, Monticello Research Foundation and Horticulture Australia