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Selection-free screening protocol for plant transformation: an open-source platform for plant biotechnology The GusPlus ™ project PowerPoint PPT Presentation


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Selection-free screening protocol for plant transformation: an open-source platform for plant biotechnology The GusPlus ™ project. A new GUS gene, Available under BIOS TM licensing, Pioneering use of the BioForge TM concept. TM. GUSPlus TM as a selectable marker. Premise:

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Selection-free screening protocol for plant transformation: an open-source platform for plant biotechnology The GusPlus ™ project

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Selection-free screening protocol for plant transformation: an open-source platform for plant biotechnologyThe GusPlus™ project

A new GUS gene,

Available under BIOSTM licensing,

Pioneering use of the BioForgeTM concept

TM


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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

  • 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 approaches

  • 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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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 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 using GUSPlusTM

Callus

Leaves

Leaves

Shoot

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


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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 X-GlcA (200ug/ml).

  • Select ‘blue’ calli and move to regeneration media.


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Selection of transgenic rice plants using GUSPlusTM

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 plantlets

expressed GUS in leaf material.


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Summary of GUSPlusTM selection for transgenic rice plants


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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

Leaf

Seedling

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


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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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GUSPlusTM will be available for use under the conditions of a BIOSTM 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 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

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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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


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