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 2002

Plant regeneration from non-apical root segment

Short overview activities 2002

Effect of explant sources on overall callus induction andregeneration in three different garlic cultivars, Messidrome,Morasol and Printanor. Means and their standard errors (SE) areindicated. Means with the same letters are statistically equal (Linearscale, student test, = 0.05)

Rootsegments

Callus induction %(total root segments)Mean SE

Shoot regeneration %(callus lines with shoot)Mean SE

Apical 88.33 (720) 2.74 a 4.42 (29) 0.81 aNon-apical 61.25 (720) 4.16 b 11.27 (50) 1.49 a

Short overview activities 2002

Paper with new title: The development of an efficient cultivar independent plant regeneration system from callus derived from both apical and non-apical root segments of garlic (Allium sativum L.) accepted by In Vitro Cell. Dev. Biol. Plant

Development of transformation

protocol

Transgenic garlic in the greenhouse

Overview activities 2002

A reliable genetic transformation protocol has been developed in garlic

Callus line with GFP expression after selection for 4 months (cv. Printanor)

Analysis of transgenic garlic: GFP

Analysis of transgenic garlic: GFP

Small garlic shoots with GFP expression after selection for 4 months and regeneration for another 3 months (cv. Printanor)

Analysis of transgenic garlic: GFP

Garlic shoot with GFP expression after selection for 4 months and regeneration for another 4 months (cv. Printanor)

First GFP plant produced in Plant RI

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

Cry1Ca primers resulting in a 802 bp fragment

lane 1: 1 kb DNA ladder marker

lane 2-6: transgenic garlic

Analysis of transgenic garlic:

standard PCR

1 21 3 4 5 6

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

1 2 3 4

Analysis of transgenic garlic: bio-

assay

Transgenic garlic with Cry1Ca gene is highly resistant to beet armyworm (Spodoptera exigua)

Overview activities 2002Summary of successful callus lines in garlic transformation

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

Next steps transformation

research Detailed molecular characterisation of the

transgenic garlic plants (Southern blot etc.)

Writing a paper on garlic transformation Transfer, via genetic modification, a

(key)gene from the sulphur metabolic pathway into garlic

Shallot ATP sulfurylase is being cloned