slide1
Download
Skip this Video
Download Presentation
Selection-free screening protocol for plant transformation: an open-source platform for plant biotechnology The GusPlus ™ project

Loading in 2 Seconds...

play fullscreen
1 / 21

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


  • 155 Views
  • Uploaded on

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:

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Selection-free screening protocol for plant transformation: an open-source platform for plant biotechnology The GusPlus' - rachel


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
slide1

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

gusplus tm as a selectable marker
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).

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
removal of herbicide antibiotic resistance genes from gm plants
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:

non herbicide antibiotic resistance gene approaches
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)
gusplus tm approach
GusPlusTM approach
  • Three model crops: Arabidopsis, rice and tobacco.
  • Mono- and dicotyledonous species
  • Three different transformation systems:
    • Floral dip
    • Leaf disc
    • Callus
gusplus tm vectors
GUSPlusTM vectors

Hyg (R)

35S

35S

CAT intron

GUSPlus

pCambia1305.1

Hyg (R)

35S

35S

GRP

CAT intron

GUSPlus

pCambia1305.2

tobacco selection strategy
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
selection of transgenic tobacco plants using gusplus tm
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).

rice selection strategy
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.
selection of transgenic rice plants using gusplus tm
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.

arabidopsis selection strategy
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.
selection of transgenic arabidopsis plants using gusplus tm
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.

summary
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.
gusplus tm will be available for use under the conditions of a bios tm license
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
slide19

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
gusplus tm in the bioforge tm project
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.
slide21

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

ad