1. Workpackage 2: Breeding Systems
Specific objectives transformation research
Development of a reliable transformation protocol for
garlic using Agrobacterium tumefaciens as a vector
The production of transgenic garlic with an altered S metabolism
Persons involved
Si-Jun Zheng, Betty Henken, Frans Krens, Chris Kik
(Plant RI, Wageningen, the Netherlands)

2. Short overview of research 2002-2003
In vitro: callus induction and plant regeneration on non-apical parts of roots.
Genetic transformation: two transformation systems via GUS and GFP; molecular analysis on transgenic plants.
Genetic modification of sulphur metabolism: gene isolation and fusion, transform APS1 gene into garlic.

3. Development of garlic regeneration system
(Zheng et al., In Vitro Cell. Dev. Biol. Plant 39: )

4. Development of a garlic genetic transformation system
GUS transformation system
destructive
GFP transformation system
non-destructive

5. Genetic transformation: analysis of transgenic garlic via GUS

6. Genetic transformation: analysis of transgenic garlic via GFP
Garlic plants with GFP expression after selection for 4 months and regeneration for another 3 months (cv. Printanor)

7. 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 DNA ladder marker 1 2 3 4 5 6

8. Genomic DNA blot : 802 bp fragment of Cry1Ca PCR product as a probe
N M M
lane 1-13: individual transgenic garlic plant transformed with with pCAMBIA1301- Cry1Ca
lane M:  DNA digested with Hind III
lane N: untransformed garlic DNA as negative control

9. Genetic transformation: overview

10. Genetic modification of S metabolism

11. ATP sulfurylase isolated from Arabidopsis thaliana and Allium cepa (shallot)
cDNA fragment generated from RT-PCR

12. Fusion constructs used for garlic transformation
Arabidopsis
APS1-GFP
GFP-APS1
Shallot
SAPS-GFP
GFP-SAPS

13. ATP sulfurylase (APS1) fused with GFP
Lane 1: N-terminus fusion fragment APS1-GFP
Lane 2-3: C-terminal fusion fragment GFP-APS1
M: 1kb DNA ladder marker M

14. Stable expression of fusion protein in garlic leaf
APS1-GFP GFP GFP-APS1

15. Transgenic shoot with APS1-GFP fusion protein

16. Next steps in transformation research
Finalising manuscript on garlic transformation (for Molecular Breeding)
Molecular and functional analysis of transgenic plants with APS1 gene (if there is enough time)