Vacuum Infiltration for Landsberg Erecta

Msantos at uga.cc.uga.edu Msantos at uga.cc.uga.edu
Thu Feb 1 16:02:02 EST 1996


Hello net.	

Thanks to all who responded thoughtfully to my question regarding v.i.
for Le. Here is a compilation of some of the responses I got. At the
bottom-most of the list of responses is a detailed protocol from Beth
Krizek (Meyerowitz lab). Again, many thanks. - Mike Santos	

MSANTOS at UGA.CC.UGA.EDU	
Crop and Soil Sciences
University of Georgia

-----

Hi Mike-



We've had no trouble with vacuum infiltration of Ler,  but there don't
seem   to be any references available.  There's a protocol posted on
the arabidopsis electronic conference database.



Anne Britt

Assistant Professor

Section of Plant Biology

University of California, Davis

Davis, CA 95616

fax: (916) 752-5410

______________________________________________________________________________


Hy Mike,

we have been (moderately) successful with he vacuum infiltration
method, using Landsberg erecta. The protocol we used is a modification
of the original protocol from Bechtold, Ellis and Pelletier, we got
the modified protocol from Andrew Bent (Staskawicz Lab , Berkeley). We
grew 10-20 plants in pots under short days. The pots were covered with
a nylon screen after planting, allowing the plants to grow through the
screen. The primary bolts were cut off, and when secondary bolts
developed (about 10 days later) , we used the plants for
transformation. Our Agro strain is C58, vectors came from Jonathan
Jones lab (pSLJ1711), carrying a Kan resistance. Plants were submerged
upside down in a beaker with the Agro-solutionn. I tried different
infiltration times, without seeing much of a difference. However, I
got only a small number of transformants, about 2 per pot. There are
more people here at the Cambridge Lab (John Innes Centre, Norwich) who
have been successful with Landsberg, I'll ask them to contact you.

Best regards

Ruediger Simon
John Innes Centre
Colney Lane
Norwich NR4 7UH
United Kingdom
e-mail: simon at bbsrc.ac.uk
-----

I put the same query up about a month ago. The general response was
that landsberg works fine with the infiltration protocol. One group
indicated that they get 10-fold fewer transformants, while a second
group said that Landsberg gave them about the same. I'm going for it
with my usualk  protocol, that gives about 1 in 1000-5000 transformed
seeds. I normally use Columbia, but I need to try my constructs in the
GA-deficient lines that are in the Landsberg background.

Andy
------------------------------------------------------------------------------
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>I am enclosing a procedure which we have used quite successfully with L-er.
>If you have any questions, feel free to contact me.
>
>Beth Krizek (Meyerowitz lab)
>818-395-8438
>krizeke at starbase1.caltech.edu
>
>>
>Transformation of Arabidopsis by Vacuum Infiltration
>
>(This is basically the procedure that was posted on the network with
>suggested changes from Marty Yanofsky and Takashi Araki (postdoc in Marty's
>lab 619-534-7298))
>
>Plant Growth
>1. Plant seeds of appropriate genotype (I have only used Ler) on top of
>cheesecloth covered soil, making sure soil fills pot. I use a rubber band
>to secure the cheesecloth.
>
>2. After plants have bolted, clip off the primary bolt to encourage the
>growth of secondary bolts. Perform infiltration 4-8 days after clipping.
>
>Vacuum Infiltration
>1. Grow a large liquid culture of Agrobacterium (I have used ASE strain)
>carrying the appropriate construct. Start a 25ml overnight (LB+
>antibiotics) two to three days ahead of infiltration. One day prior to
>infiltration use this culture to inoculate a 400ml culture.
>
>2. After 24 hours of growth, cells are usually at a density of at least 1
>OD. Harvest cells by centrifugation and resuspend to an OD of .8 in
>infiltration media.
>
>3. Invert the pot into container with infiltration solution and put into
>vacuum oven at RT. Infiltrate for 10-15 mins at 10-15 in3 Hg.
>
>4. Release vacuum and remove pot , lay it on its side in tray and cover
>with Saran wrap (to keep humid). Remove cover the next day and stand
>upright.
>
>5. The Agro infiltration solution can be reused for multiple pots.
>
>Selection of Transformants
>6. Pour selection plates.
>
>7. Sterilize seeds in 50% water/50% bleach/drop of Tween. Rinse 3-4 times
>with sterile water.
>
>8. Plate seeds by resuspending in room temp .1% agarose and pipeting onto
>selection plates. Dry plates in laminar flow hood until set. Use 1ml
>agarose for every 500-1000 seed. Plate 2000-4000 seed per 150x15 mm plate.
>Put a small number of control seeds from a known transformed plant on one
>of the plates for comparison.
>
>9. Vernalize plants for two nights in cold room. Move plates to growth chamber.
>
>10. After about 7 days, transformants should be clearly visible at dark
>green plants with roots which extend over and into the selection media.
>
>11. Transfer seedlings to soil.
>
>Infiltration Media                                      Selection Plates
>.5X MS salts                                            1X MS salts
>1X B5 vitamens                                  .3% sucrose
>5% sucrose                                              8g TC agar
>.044uM benzylamino purine
>.03% Silwet L-77
>                                                        Autoclave and add
>appropriate antibiotics
>(for pCGN add Kan 50ug/ml ; I also add
>timentin 200 ug/ml)
>
>
>Silwet L-77 is sold by OSI Specialty 800-523-5862. Speak with Linda Ayers
>at ext 3. 1 gallon costs approximately $240 and it is not sold in smaller
>amounts.
>
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