Selectionfree screening protocol for plant transformation: an opensource platform for plant biotechn

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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
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 10165-174 )
• Recombinases, such as Cre/lox recombination
  system (Hajdukiewicz, P.T., Gilbertson, L.A. and
  Staub, J.M. (2001) Plant J. 27 161170.)

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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 4111-117)
• 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. 191069-1075
  Zhang et al., 2001 Mol. Biotechnol. 17109-117)

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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
35S
35S
CAT intron
GUSPlus
Hyg (R)
pCambia1305.1
35S
35S
GRP
CAT intron
GUSPlus
Hyg (R)
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 CAMBIAs BIOS Initiative