workpackage 2: breeding systems
DESCRIPTION
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.TRANSCRIPT
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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)
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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.
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Development of garlic regeneration system
(Zheng et al., 2003. In Vitro Cell. Dev. Biol. Plant 39: 288-292)
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Development of a garlic genetic transformation system
GUS transformation system destructive
GFP transformation system non-destructive
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Genetic transformation: analysis of transgenic garlic via GUS
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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)
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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
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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
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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
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Genetic modification of S metabolism
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ATP sulfurylase isolated from Arabidopsis thaliana and Allium
cepa (shallot)
cDNA fragment generated from RT-PCR
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Fusion constructs used for garlic transformation
Arabidopsis APS1-GFP GFP-APS1
Shallot SAPS-GFP GFP-SAPS
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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
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Stable expression of fusion protein in garlic leaf
APS1-GFP GFP GFP-APS1
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Transgenic shoot with APS1-GFP fusion protein
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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)