1. Workpackage 2: Breeding Systems specific objectives The development of a reliable transformation protocol of garlic using Agrobacterium tumefaciens as a vector persons involved Si-Jun Zheng, Betty Henken, Frans Krens, Chris Kik (Plant RI, Wageningen, the Netherlands)

2. Short overview activities 2000/2001 development of garlic regeneration protocol

3. Short overview activities 2000/2001 callus formation was induced from the cutting edges of an explant

4. Short overview activities 2000/2001 callus was induced along the entire length of the explant

5. Short overview activities 2000/2001 conclusion: an efficient callus induction and plant regeneration system not only from root- tip but also from root segments could be developed, which can be used for garlic transformation

6. Short overview activities 2000/2001 paper was written entitled: ‘The development of an efficient plant regeneration system from callus derived from root segments of garlic (Allium sativum L). accepted by In Vitro Cell. Dev. Biol. Plant

7. Overview activities 2001/2002 development of a garlic transformation protocol

8. Development of transformation protocol callus with transient expression of uidA gene after co- cultivation 4 days with A.tumefaciens

9. Development of transformation protocol garlic callus line from cv. Printanor after selection for 5 months (pPB36)

10. Development of transformation protocol garlic shoots were regenerated after selection for 5 months and regeneration for 3 months (cv. Printanor, pPB36)

11. Development of transformation protocol garlic shoots were obtained after selection for 5 months and regeneration for 3 months (cv. Printanor, pPB36) in total 4 callus lines produced plants origin: root, seed, bulbil

12. Analysis of transgenic garlic GUS and GFP assay PCR (standard and adaptor ligation)

13. Analysis of transgenic garlic: GUS GUS expression in the leaves and roots of a garlic plant

14. callus line with GFP expression after selection for 4 months (cv. Printanor ) Analysis of transgenic garlic: GFP

15. callus line with GFP expression after selection for 4 months and regeneration for another 2 months (cv. Printanor)

16. Analysis of transgenic garlic: standard PCR uid A primers resulting in a 710 bp fragment lane 1-3: transgenic garlic lane 4: negative control lane 5: positive control lane 6: 1kB ladder 1 2345 6

17. Cry-1Ca primers resulting in a 802 bp fragment lane 1: 1 kB ladder lane 2-6: transgenic garlic Analysis of transgenic garlic: standard PCR 1 21 3456

18. Analysis of transgenic garlic: AL- PCR the method AP1, AP2 LB1, LB2 Adaptor Genomic DNA T- DNA -------------------- Zheng et al. (2001) Transgenic Research 10, 237-245

19. PCR amplification of genomic DNA flanking the T-DNA right border after Ssp I digestion lane 1: 1kb DNA ladder marker lane 2: untransformed garlic DNA lane 3: transgenic garlic plant transformed with pCAMBIA1301- Ho4 lane 4: transgenic garlic plant transformed with pCAMBIA1301- Cry1Ca 123 4

20. Overview activities 2001/2002: conclusions transgenic garlic can be produced within 6-8 months 4 callus lines produced transgenic garlic plants (root: Printanor/pPB34 and Printanor/pPB36; true seed/pPB36; bulbil/pCAMBIA1301)

21. Next steps transformation research writing a paper on garlic transformation regeneration of transgenic plants stably expressing genes from the sulphur pathway (starting with the alliinase gene(s)??)