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

1. 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

2. 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).

3. 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

4. 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:

5. 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)

6. GusPlusTM approach • Three model crops: Arabidopsis, rice and tobacco. • Mono- and dicotyledonous species • Three different transformation systems: • Floral dip • Leaf disc • Callus

7. GUSPlusTM vectors Hyg (R) 35S 35S CAT intron GUSPlus pCambia1305.1 Hyg (R) 35S 35S GRP CAT intron GUSPlus pCambia1305.2

8. 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

9. 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).

10. Summary of GUSPlusTM selection for transgenic tobacco plants

11. 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.

12. 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.

13. Summary of GUSPlusTM selection for transgenic rice plants

14. 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.

15. 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.

16. Summary of GUSPlusTM selection for transgenic Arabidopsis plants

17. 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.

18. 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

19. 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

20. 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.

21. 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