9 Vacuum Transformation

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RANSFORMATION OF ARABIDOPSIS BY VACUUM INFILTRATION

osted to the Arabidopsis bulletin board; 11 Jan 1994

ndrew Bent e-mail address: [email protected]

his protocol is based on the work of Nicole Bechtold, Jeff Ellis an

eorges Pelletier. My modifications were incorporated to streamlin

heir procedure. The most significant changes eliminate the need t

proot and re-plant infiltrated plants. - Andrew Bent

lant Growth:

. Grow plants of the appropriate genotype to a stage at which bol

re just emerging.

have found that it works well to grow 12-15 plants in a 3.5" pot.

f pot is covered with nylon window screen after planting, plants ghrough the screen and when pot is inverted for infiltration less d

alls out. If plants are grown for the first four weeks in short d

ou will get larger plants and a greater seed yield (transfer plant

o long days to induce bolting). Success may also depend on freque

ertilization and strong light intensity.

. Clip off emerging bolts to encourage growth of multiple seconda

olts.

nfiltration will be done four to eight days after clipping.

acuum Infiltration:

. Grow a large liquid culture of Agrobacterium carrying the

ppropriate construct.

tart a 25 ml overnight (LB + antibiotics) two to three days ahead

ime. Add this culture to 400ml of LB + antibiotic the day before

nfiltration. My experiments were done using A. tumefaciensV3101pMP90 (C. Koncz and J. Schell, 1986,

ol. Gen. Genet. 204:383-396).

. Harvest cells by centrifugation (5K 10min. in GSA rotor,

referably at room temp.) and resuspend in 3 volumes infiltration

edium (OD600 approx. 0.8).

arvest cells at an OD600 of >2.0. A 400 ml culture will give enou


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ells for infiltration of at least six pots.

. Add Agrobacterium (in infiltration medium) to a dish or beaker

nvert plants (pot, soil, and all) into liquid solution. Be sure

olts and entire rosettes are submerged.

one liter beaker filled with >200 ml of solution fits well with ou

.5" pots. Bacterial solution can be extended by reusing for at le

ne additional pot.

. Place beaker into bell jar. Draw a vacuum until bubbles form o

eaf and stem surface and solution starts to bubble a bit, then

elease vacuum very rapidly.

he necessary time and vacuum pressure will vary lab-to-lab. Pract

n a few dispensable plants first. Good infiltration is visibly

pparent as uniformly darkened, water-soaked tissue. Be sure to ha

ood traps in your vacuum system or you will quickly saturate the puil.

. Remove plants from beaker, lay them on their side into a plasti

lat and cover with plastic wrap or a dome to maintain humidity. T

ext day, uncover plants and set upright.

. Grow approximately four weeks, keeping bolts from each pot

ogether and separated from neighboring pots.

. When siliques on plants are very dry, harvest seed (all seed fr

ne pot together).

election of Putative Transformants:

anamycin selection protocol: (Note that Basta selection is much le

abor intensive - but your present binary vector system is more lik

o encode antibiotic resistance.)

0. Pour selection plates.

lastic 150 x 15 mm petri dishes are convenient.

1. Sterilize seed.

variety of sterilization protocols are appropriate.

place seed in 15 ml plastic orange cap tubes and then treat:

minute in ethanol or isopropanol


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minutes in 50% Bleach/50% water/0.05% Tween.

rinses with sterile water.

t is advisable to add one or two control seeds from a known

ransformed plant onto a marked location on at least a few of the

election plates. Sterilize these seed also.

2. Plate seed by resuspending in sterile, room temperature 0.1%

garose and spreading onto selection plates. Dry plates in laminar

low hood until seed no longer flows when plate is tipped.

se one ml agarose for every 500-1000 seed. Plate 2000 to 4000 see

er 150 x 15 mm plate. Higher densities can make antibiotic select

ess effective.

3. Vernalize plates for two nights in cold room. Move plates to

rowth chamber.

4. After about 7 days, transformants should be clearly identifiab

s dark green plants with healthy green secondary leaves and roots

hat extend over and into the selective medium.

5. Transplant plantlets to soil, grow, and collect seed.

ransplanting success is improved by breaking up agar around root

rior to pulling, by removing any adhering chunks of agar from root

efore planting, by saturation of soil with water after transplanti

nd by growing plants under a dome (for high humidity) for the first

ay or two. If you break the root, put plantlet onto a new selecti

late for a few days before transplanting.

nfiltration 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)

election 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)


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he citation for the above protocol is:

ent,A.F., Kunkel,B.N., Dahlbeck,D., Brown,K.L., Schmidt,R.,

iraudat,J., Leung,J. and Staskawicz,B.J. (1994). RPS2 of Arabidop

haliana: A leucine-rich repeat class of plant disease resistance

enes. Science 265:1856-1860.

t would also be good to cite the inventors of this approach, Becht

t al.:

echtold, N., Ellis, J., and Pelletier, G. (1993). In planta

grobacterium mediated gene transfer by infiltration of adult

rabidopsis thaliana plants. C. R. Acad. Sci. Paris, Life Sciences

16:1194-1199.

ood Luck!

Andrew Bent

------------------------------------

ndrew Bent

ssistant Professor

epartment of Agronomy

niversity of Illinois

rbana, IL 61801

hone 217-244-6308

ax 217-333-4777

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