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@versailles.inra.fr

Plant Growth:

• Grow plants of the appropriate genotype to a stage at which bolts are just emerging. I have found that it works well to grow 12-15 plants in a 3.5" pot. If pot is covered with nylon window screen after planting, plants grow through the screen and when pot is inverted for infiltration less dirt falls out. If plants are grown for the first four weeks in short days you will get larger plants and a greater seed yield (transfer plants to long days to induce bolting). Success may also depend on frequent fertilization and strong light intensity.
• Clip off emerging bolts to encourage growth of multiple secondary bolts. Infiltration will be done four to eight days after clipping.

Vacuum Infiltration:

• Grow a large liquid culture of Agrobacterium carrying the appropriate construct. Start a 25 ml overnight (LB + antibiotics) two to three days ahead of time. Add this culture to 400ml of LB + antibiotic the day before infiltration. My experiments were done using A. tumefaciens GV3101.
• Harvest cells by centrifugation (5K 10min. in GSA rotor, preferably at room temp.) and resuspend in 3 volumes infiltration medium (OD600 approx. 0.8). Harvest cells at an OD600 of >2.0. A 400 ml culture will give enough cells for infiltration of at least six pots.
• Add Agrobacterium (in infiltration medium) to a dish or beaker and invert plants (pot, soil, and all) into liquid solution. Be sure bolts and entire rosettes are submerged. A one liter beaker filled with >200 ml of solution fits well with our 3.5" pots. Bacterial solution can be extended by reusing for at least one additional pot.
• Place beaker into bell jar. Draw a vacuum until bubbles form on leaf and stem surface and solution starts to bubble a bit, then release vacuum very rapidly. The necessary time and vacuum pressure will vary lab-to-lab. Practice on a few dispensable plants first. Good infiltration is visibly apparent as uniformly darkened, water-soaked tissue. Be sure to have good traps in your vacuum system or you will quickly saturate the pump oil.
• Remove plants from beaker, lay them on their side into a plastic flat and cover with plastic wrap or a dome to maintain humidity. The next day, uncover plants and set upright.
• Grow approximately four weeks, keeping bolts from each pot together and separated from neighboring pots.

Selection of Putative Transformants:

• When siliques on plants are very dry, harvest seed (all seed from one pot together).

For Kanamycin selection:

(Note that Basta selection is much less labor intensive - but your present binary vector system is more likely to encode antibiotic resistance.)

 

• Pour selection plates. Plastic 150 x 15 mm petri dishes are convenient.
• Sterilize seed. A variety of sterilization protocols are appropriate. I place seed in 15 ml plastic orange cap tubes and then treat:
  
  1 minute in ethanol or isopropanol
  5 minutes in 50% Bleach/50% water/0.05% Tween.
  3 rinses with sterile water.
  
  It is advisable to add one or two control seeds from a known transformed plant onto a marked location on at least a few of the selection plates. Sterilize these seed also.
• Plate seed by resuspending in sterile, room temperature 0.1% agarose and spreading onto selection plates. Dry plates in laminar flow hood until seed no longer flows when plate is tipped. Use one ml agarose for every 500-1000 seed. Plate 2000 to 4000 seed per 150 x 15 mm plate. Higher densities can make antibiotic selection less effective.
• Vernalize plates for two nights in cold room. Move plates to growth chamber.
• After about 7 days, transformants should be clearly identifiable as dark green plants with healthy green secondary leaves and roots that extend over and into the selective medium.
• Transplant plantlets to soil, grow, and collect seed.

Transplanting success is improved by breaking up agar around root prior to pulling, by removing any adhering chunks of agar from root before planting, by saturation of soil with water after transplanting, and by growing plants under a dome (for high humidity) for the first day or two. If you break the root, put plantlet onto a new selection plate for a few days before transplanting.

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.

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