Workpackage 2: Breeding Systems specific objectives The development of a reliable transformation protocol of garlic using Agrobacterium tumefaciens as.

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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)

Short overview activities 2000/2001 development of garlic regeneration protocol

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

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

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

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

Overview activities 2001/2002 development of a garlic transformation protocol

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

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

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

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

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

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

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

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

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

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

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

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

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)

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)??)