T-DNA information
Origin
The
Versailles T-DNA lines collection was initiated in 1992 through the
use of the vacuum infiltration transformation method (Bechtold et al.1993).
Transformation
method
For
the in planta transformation method by vacuum infiltration
we used the Arabidopsis ecotype Wassilevskija (WS-4) and the binary
vector pGKB5, (Bouchez et al. 1993). This vector contains a promoterless
GUS reporter gene fused to the right border, and the genes conferring
kanamycin and Basta resistance as plant selection markers
Four-week
old plants, well developed, with the first fruits formed and secondary
inflorescences appearing, were infiltrated for 20 minutes with an Agrobacterium
culture (OD600nm= 0.8) resuspended in one-third of the initial volume
in infiltration medium. Plants were then transferred to soil and 4 weeks
later the seeds were harvested. The T1 transformants were then selected
on sand sub-irrigated with water containing Basta (5-10 mg/l phosphinothricin),
transferred to soil, and isolated with plastic tubes to prevent seed
contamination. The T2 seeds were harvested 6 weeks later.
pGKB5
The
T-DNA region of this plasmid is flanked by fragments containing the
right and left borders of the TR-DNA of pRiA4 (Jouanin et al. 1989).
The GUS-nos3' reporter cassette from pBI101.1 (Jefferson et al. 1987)
is inserted 40 bp away from the right border. As the GUS gene possesses
its own ATG initiation codon, it is able to produce active transcriptional
or translational gene fusions upon its insertion in the genome. The
T-DNA also contains two plant selection markers derived from pGSFR280
(De Blok et al. 1987) that confer resistance of plant cells to kanamycin
and to the herbicide Basta (phosphinothricin).
This
binary plasmid derives from a pBGS plasmid (Spratt et al. 1986), harbours
a bacterial kanamycin resistance gene, and is able to replicate both
in Escherichia coli and in Agrobacterium. The origin of replication
of pRiA4, cloned as a large 8 kb BamHI fragment from pLJbB11 (Jouanin
et al. 1985), confers a very high stability in Agrobacterium under non
selective conditions. The binary vector was introduced into several
Agrobacterium disarmed strains by electroporation : C58C1 (pMP90) (Koncz
and Schell 1986), C58C1 (pGV2260) (Deblaere et al. 1985), LBA4404 (Hoekema
et al. 1983) to give respectively the strains MP5-1, GV5-2, LB5-1. Strain
MP5-1 was used for obtaining all the T-DNA lines generated in this collection.
Figure
1. Functional map of the binary vector pGKB5.
Arrows
indicate coding sequences, and black boxes promoter / terminator regions.
The right and left border fragments derived from the TR-DNA of pRiA4
from Agrobacterium rhizogenes strain A4 3 ; the black strips
correspond to the 24 bp border sequences that serve as signals for T-DNA
transfer. The chimeric kanamycin and Basta resistance genes originate
from pGSFR280 (De Blok et al (1987). Sites for EcoRV are indicated above
the T-DNA. The hatched area corresponds to the probe used in hybridization
experiments. A large origin of replication from pRiA4 (ori pRiA4) (Jouanin
et al 1985) has been used to insure a good stability of the vector in
Agrobacterium even without selection pressure. uidA : coding region
of the beta-glucuronidase from E. coli ; nos 3' : 3' region of the nopaline
synthase gene from pTiC58 ; ocs 3' : 3' region of the octopine synthase
gene from pTiAch5 ; nptII : neomycin phosphotransferase II ; P nos :
promoter region of the nopaline synthase gene ; P 35S : promoter of
the 35S transcript of the Cauliflower Mosaic Virus ; bar : coding sequence
of the basta resistance gene from Streptomyces hygroscopus ; 3' g7 :
3' region of the gene 7 from the T-DNA of pTi15955.
Mutation
screening
The
T2 families, for which enough seeds were produced, were screened in
vitro on medium as described by (Estelle and Somerville 1987)
and modified according to (Santoni et al. 1994). At least 100 seeds
were sowed on medium containing 100 mg/l kanamycin for the segregation
analysis of the KanR trait (in order to estimate the number of T-DNA
insertions and to screen for gametophytic mutations). The screening
of the hypocotyl development mutations was carried out both in light
and darkness conditions 10 days after sowing. About 50 seeds of each
T2 families were also sown in the greenhouse for the screening of developmental
and fertility mutations.
GUS screening
The
GUS activity of 2,000 first T2 lines was tested histochemically on 15-
days-old seedlings grown in vitro either under light or dark conditions
(Mollier et al. 1995). For all the families we tested the GUS activity
on flowers and siliques taken from 6 weeks old plants grown in the greenhouse.
Seed
production and conservation
Because
of the low number of T2 seeds it has been necessary to multiply them.
T3 seeds come from the bulk of about 50 T2 plants for each family.
Seeds are stored in a room with controlled temperature(4°C) and
humidity (20 % RH). The seed aliquots contain hemizygous and homozygous
for the T-DNA insertions and wild type individuals.
FST
production
A protocol for FST production based on "gene-walking" (Devic
et al.. 1997), was optimized for a large scale of an amplification and
a systematic sequencing (Balzergue S, Dubreucq B et al.. 2001). All
these genomic data are managed in the data base FLAGdb/FST (now FLAGdb++
link http://193.51.165.9/projects/FLAGdb++/HTML/index.shtml) developed
by the Bioinformatique team of INRA-URGV (Samson F et al.. Nucleic AcidsRes.
2002 Jan). Produced FSTs, as well as the corresponding lines, are accessible
for the international scientific community via the FLAGdb++ Web site.
To date, a total of 41313 FST were generated and a deposit of approximately
30 000 FST in the EMBL web site was carried out. Moreover, 2025 lines
were distributed to French laboratories and 2716 outside. A study on
the first 9000 FSTs brought informations on the mechanism of integration
of the T-DNA in the A. thaliana genome (Brunaud V et al.. 2002).
The requests for T-DNA lines corresponding to your favourite FST is
possible through the pages "Ask for line" of the FLAGdb++
Web sites or through the publiclines interface (link http://dbsgap.versailles.inra.fr/publiclines/)
Available data
For all FST requested, we send the current data available (only in french)
in the Agrobact+ database, with the seeds.
These phenotypes are recorded through analysis of T2 seedlings in
vitro and flowering plants in the greenhouse. This information
includes:
* in vitro segregation on about 100 seeds sown on kanamycin
100 mg/l (corresponding to 3:12:1, 1 :1, 15:1, 63:1 … , segregation).
* GUS expression at seedling stage in vitro for a limited number of
lines.
* GUS expression in flowers and siliques in the greenhouse.
* seedling observations in vitro
* plant observations in the greenhouse.
The
INRA Versailles T-DNA lines are available to profit and non-profit organization.
Databases and information for ordering the biological material are available
from the Arabidopsis thaliana Resource Centre for Genomics
web site :
http://www-ijpb.versailles.inra.fr/fr/cra/cra_accueil.htm
Important
notes
* The segregation of the kanamycin marker was estimated on about 100
seedlings. It is necessary to confirm the segregation with more seeds
(about 300).
* For several lines no resistant progeny were recovered on kanamycin,
but in Southern analysis some of them do contain the T-DNA. These lines
are marked "?" in the segregation data.
* We have observed differences in GUS expression between the seedling
stage in vitro and in greenhouse conditions. GUS expression in flowers
and siliques is variable with greenhouse conditions.
* The germination rate of the lines may depend on growth and harvest
climatic conditions.
* In this collection, about 60% of the lines have a single locus T-DNA
integration. 75 % of T-DNA integrations are in tandem (Bechtold et al.
1993).
* About 1/4 of the mutations are tagged.
