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@versailles.inra.fr
Infiltration Medium:
1/2 X Murashige & Skoog salts
1 X B5 vitamins
5.0% Sucrose
...044 uM Benzylamino Purine (10 ul per liter of a 1 mg/ml stock in DMSO)
Selection Plates:
1/2 X Murashige & Skoog salts
0.8% Agar
Autoclave, cool, then add:
1 X B5 vitamins
Antibiotic (such as Km 50 ug/ml)
The overall rate of positives was about one
transformants per 2,500 seed plated for selection.
Note however that the time required to go from seed to seed is still
months.
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, Life Sciences, 316 : 1188-1193
Bechtold N, Ellis J, Pelletier G (1993). In planta Agrobacterium
gene transfer by infiltration of adult Arabidopsis thaliana plants.
C. R. Acad. Sci. Paris, Life Sciences, 316 : 1194-1199.