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)

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.

Development of garlic regeneration system

(Zheng et al., 2003. In Vitro Cell. Dev. Biol. Plant 39: 288-292)

Development of a garlic genetic transformation system

GUS transformation system destructive

GFP transformation system non-destructive

Genetic transformation: analysis of transgenic garlic via GUS

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)

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

Genomic DNA blot : 802 bp fragment of Cry1Ca PCR

product as a probe 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

N 1 2 3 4 M 5 6 7 8 M 9 10 111213

Genetic transformation: overview

Cultivar Explant ConstructFunctionalgene

Number ofplants orclusters

Printanor root pPB34 uidA;hpt;Ho4

42

Printanor root pPB36 uidA;hpt;Cry1Ca

44

True seed embryo pPB36 uidA;hpt;Cry1Ca

3

Bulbil bulbil pCAMBIA1301 uidA;hpt 5Printanor root PC1300IntA-

GFPgfp;hpt; 9

Printanor root pCAMBIA1301 uidA;hpt 2Messidrome root pPB36 uidA;hpt;

Cry1Ca5

Genetic modification of S metabolism

ATP sulfurylase isolated from Arabidopsis thaliana and Allium

cepa (shallot)

cDNA fragment generated from RT-PCR

Fusion constructs used for garlic transformation

Arabidopsis APS1-GFP GFP-APS1

Shallot SAPS-GFP GFP-SAPS

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 1 2 3

Stable expression of fusion protein in garlic leaf

APS1-GFP GFP GFP-APS1

Transgenic shoot with APS1-GFP fusion protein

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)