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

pCambia1305.1Hyg (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 14Cambia1305.2 9 13Cambia1305.2 25 10Cambia1305.2 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 8pCambia1305.1 60 1 pCambia1305.2 420 12pCambia1305.2 160 5pCambia1305.2 225 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 1pCambia1305.2 236 5pCambia1305.2 334 3pCambia1305.2 150 2pCambia1305.2 90 0

pCambia1305.2 225 2Total 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 plantsBrian Weir, Heidi Mitchell, Tuan Nguyen, Richard Jefferson

BIOSTM licensingDraft License: Mat Berman (UC) Mike Rabson, Marie Connett Porceddu,

Richard Jefferson; Commentable website: Steve Irwin, Nick dos Remedios;

BioForgeTM distributive collaboration websiteCollabnet® and CAMBIA’s BIOS Initiative

Funded by the Rockefeller Foundation, Monticello Research Foundation and Horticulture Australia