Correction to vacuum infiltration protocol

David Bouchez bouchez at tournesol.versailles.inra.fr
Wed Nov 3 08:52:46 EST 1993


Hello everyone,




There was a error in the protocol for transformation by vacuum
infiltration that we mailed to the newsgroup two weeks ago.
The composition of the infiltration medium is :




Murashige and Skoog macro and micronutrients containing 10 ug/l
benzyladenopurin and 5% sucrose.




and no abscissic acid !




We are sorry about this unfortunate mistake that slipped our
attention and that it won't be too much of a trouble.


Here is the correct protocol :




Protocol for vacuum-infiltration transformation of 
Arabidopsis thaliana.






Laboratoire de Biologie Cellulaire
Station de Genetique et Amelioration des Plantes
Institut National de la Recherche Agronomique
78026 Versailles Cedex FRANCE




This protocol is based on excerpts of the following papers : 


Bouchez D, Camilleri C, Caboche M (1993). A binary vector 
based on Basta resistance for in planta transformation of 
Arabidopsis thaliana. C R Acad Sci Paris, Sciences de la 
vie/Life sciences, 316 : (in press, October issue).


Bechtold N, Ellis J, Pelletier G (1993). In planta 
Agrobacterium mediated gene transfer by infiltration of 
adult Arabidopsis thaliana plants. C R Acad Sci Paris, 
Sciences de la vie/Life sciences, 316 : (in press, October 
issue).






Arabidopsis thaliana (L.) Heyn., ecotype Wassilevskija (WS) 
was used for all the experiments and to generate a 
collection of T-DNA insertional mutants. The C58C1 (pMP90) 
strain of Agrobacterium tumefaciens, containing the pTiC58-
based helper plasmid pMP90 and the binary vector pGKB5, was 
used for transformation by vacuum infiltration.




Construction of pGKB5


A new plant transformation vector was designed, that could 
be used for T-DNA tagging in Arabidopsis. The T-DNA region 
of this plasmid is flanked by fragments containing the right 
and left borders of the TR-DNA of pRiA4. The GUS-nos3' 
reporter cassette from pBI101.1 is inserted 40 bp away from 
the right border. This region has been checked by 
sequencing, and no stop codon is present in frame with the 
GUS coding sequence. As the GUS gene possesses its own ATG 
initiation codon, the T-DNA should be  able to produce 
active transcriptional or translational gene fusions upon 
its insertion in the genome. The T-DNA also contains two 
plant selectable markers derived from pGSFR280 that confer 
resistance of plant cells to kanamycin and to the herbicide 
Basta (phosphinothricin).
This binary plasmid derives from a pBGS plasmid, 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, confers a very high stability in 
Agrobacterium under non selective conditions : Agrobacterium 
strains containing pGKB5 show no detectable loss of the 
plasmid after repeated subcultures (25 generations) in 
medium lacking kanamycin. The binary vector was introduced 
into several Agrobacterium disarmed strains by 
electroporation : C58C1 (pMP90), C58C1 (pGV2260), LBA4404 to 
give the strains MP5-1, GV5-2, LB5-1 respectively.




Selection procedure


We have tested the possibility of selecting transformed 
plants in the greenhouse under non-sterile conditions, using 
the herbicide Basta as a selective agent. Basta resistant 
seeds were obtained from a transformant generated by 
conventional root transformation. Reconstruction experiments 
were performed, where 10 Basta resistant seeds were mixed 
with 100-10,000 wild-type seeds and subjected to different 
selection protocols. Poor recovery of resistant plants was 
obtained when spraying the herbicide solution on the 
plantlets. Efficient recovery was achieved using the 
following protocol : seeds (up to 1 g : approximately 50,000 
seeds) are mixed with 4-5 volumes of fine sand and sown on 
30 cm X 30 cm trays containing medium sand. The trays are 
then placed in growth chambers and sub-irrigated with a 
nutrient solution containing the herbicide Basta (5-10 mg/l 
phosphinothricin). Sensitive plantlets germinate, but 
cotyledons fail to expand and turn yellow rapidly, whereas 
the resistant plantlets look normal. The latter can be 
distinguished from the sensitive as early as three days 
after germination. Resistant plants can then be transferred 
to soil and allowed to set seeds.
Resistant plantlets can be isolated in horticultural 
conditions at high density (up to 100-150 seeds/cm2). 
Therefore the entire progeny of inoculated T0 plants can be 
sown and screened at low cost. The seed output of primary 
transformants (T1) is as high as normal greenhouse plants 
and enough seeds are produced to allow multiple screening 
procedures without multiplication.




Plant transformation


Using this vector/selection system, transformed plants were 
obtained by the seed imbibition transformation technique 
(Feldmann) and the inoculation of decapited plants (Nam).




Transformation by vacuum infiltration


Six mg of WS seeds (about 300 seeds) were sown on compost in 
40x30 cm sowing flats . The germination was synchronized by 
cold treatment for 48 h at 40C, and the flats were placed in 
greenhouse (16 hours day photoperiod, 150C night/25 0C 
minimum day temperature cycle), sub-irrigated with the 
standard nutrient solution of Coic and Lesaint.MP5-1 
Agrobacterium were grown with rifampicin 50mg/l, gentamycin 
100 mg/l and kanamycin 50 mg/l, for 14 hours at 28 0C in LB 
medium (final OD600 = 0.8). After centrifugation, the 
bacterial pellet was resuspended in the infiltration medium 
(IM) , at one third of the initial culture volume (IM = 
Murashige and Skoog macro and micronutrients containing 10 
ug/l benzylaminopurin and 5% sucrose ).Batches of 100 to 500 
plants, 3 to 4 weeks old and well developped, were taken out 
of the soil, rinsed with water, and immersed in 2 l of 
Agrobacterium -containing IM medium in a vacuum chamber (10 
l volume). Plants were put under vacuum (10^4 Pa) for 20 min 
with occasional swirling and the vacuum was then broken. All 
port handling of treated plants until harvested were with 
latex gloves. Treated plants were planted on new compost in 
sowing flats, 54 plants par flat and incubated for 2 days 
under plastic wrap to prevent their dehydration and to 
facilitate their rooting. Four to six weeks after planting, 
T1 seeds were harvested in bulk. Transformants seeds were 
selected in the greenhouse on sand, sub-irrigated with water 
containing Basta herbicide (5-10 mg/ml phosphinothricin) as 
described. Two months later, T2 seeds were harvested 
individually and kept for further analysis.


We are in the process of generating a collection of T-DNA 
insertion lines according to this procedure. The first lines 
from the collection will be made available to the community 
during the coming year 1994, through the Arabidopsis stock 
centers. Seed stocks from newly generated T-DNA lines will 
be sent periodically as they become available.


For any information, contact :


David Bouchez or Nicole Bechtold
Laboratoire de Biologie Cellulaire
INRA-Centre de Versailles F-78026 Versailles Cedex FRANCE
tel. (33) (1) 30.83.33.94 - fax (33) (1) 30.83.30.99


e-mail : bouchez at versailles.inra.fr



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