in planta answers (I)

Jimmy Botella J.Botella at botany.uq.edu.au
Fri Jun 7 09:10:46 EST 1996


Hi everyone!

Thanks for the overwelming response to my post. Here are the messages 
recieved.
(FIRST HALF)

In planta transformation by vacuum infiltration
                                                  version 4
                                                  (2/24/95)
                                               Takashi Araki

The following is an adaptation of the procedure described by
Andrew Bent (UC, Berkley).  It works very well for ecotypes
Columbia and Nossen and reasonably well for Landsberg erecta.  

Plant growth
1. Prepare pots with soil and cover them with a window mesh. Do
not pack soil too tightly. Make sure that the mesh is in
contact with the soil surface, otherwise germinating seedlings
will have a hard time to get through the mesh. Sow seeds. 

I use a 21/4 in. square pot and plant seeds to five spots
(several seeds per spot). One week after germination I choose
one seedling at each spot and remove the rest. 

2. Grow plants to a stage at which bolts are just emerging
(about 1 cm tall).

In my experience older plants with >5 cm bolts could still be
used. 

3. Cut off the tip of the emerging bolt to induce the growth of
secondary inflorescences. Do not cut the whole bolt. Cut should
be made above the top most cauline leaf. (This is to leave
axillary inflorescence meristems at the base of cauline
leaves.) 

Infiltration should be done about 4 days  after decapitation. I
usually remove developing siliques and fertilized flowers (if
any).

The soil should have enough water at the time of infiltration
(otherwise it will absorb much of the Agro. suspension).  It is
better to add some water to the holes on the bottom of a pot
(holding it upside down) just before infiltration.

Vacuum infiltration
4. Grow a large liquid culture of Agrobacterium carrying the
appropriate construct. Prepare 10 ml preculture and inoculate
200 ml medium with antibiotics the day before infiltration.

5.  Harvest cells by centrifugation (5k, 15 min., preferably at
room temp.) and resuspend in infiltration medium (see below) to
an OD600 of approx. 0.8.

OD600 of overnight culture will be 1.2 to 1.6. A 200 ml
suspension will be required for an infiltration. You can use
the same suspension as many as three times. This will give you
an idea of culture volume of your Agrobacterium.

6. Add 200 ml of Agrobacterium suspension to a beaker and 
invert a pot with plants into the suspension.  Be sure that
entire plants are submerged and no large air bubbles are
trapped at the center of a rosette.

I use a 400 ml beaker and a wire loop to hold a pot .

7. Place beakers inside a bell jar. A 10 in. diameter jar can
hold as many as 4 beakers in it.  Be sure to have a trap.
Vacuum should be pulled and released rapidly after a specified
time.

In our lab. 10 to 15 minutes by a house vacuum gives a good
result. This is a weak vacuum (ca. 15 in. Hg or weaker) and no
air bubbles from leaves are observed.  (Recently, I use vacuum
pump. The strength of vacuum is 15 in. Hg or 50 kPa for 15
min.) After infiltration leaves are only partly darkened.

8. Remove pots from beakers, and lay them on their side into a
plastic flat and cover with a plastic dome to maintain
humidity. The next day (or later), uncover the flat and set the
pot upright.

Do not water for several days. After this time, a small amount
of water should be added to the plastic flat (not directly to
the pot). The first flowers should set seeds.

9. Grow 3 to 4 weeks, keeping bolts from each pot together and
separated from neighboring pots.

10. Harvest seeds (all seeds from one pot together).

Selection of transformants
11. Prepare selection plates (see below). 150 x 15 mm petri
dish is convenient. Plates should be dried well in the hood
before planting seeds.



12. Surface-sterilize seeds.
Treat seeds:

2 min. in 70% ethanol
15 min in 5% bleach/95% water/1% SDS.
3 rinses with sterile water.

13. Resuspend seeds in 1 ml sterile water in a 15 ml sterile
tube. Add 8 ml cool "top agar" (see below) and plate seeds like
phage. Pour the mixture onto the plate and let the liquid
spread over the media. Let them sit for 30 min., then rotate
them 180  and leave them for additional 30 min. The media will
absorb the liquid and seeds will not move any more. Seal the
side with Parafilm.

I plant up to 100  l dry seeds per plate. This will be the
upper limit of density to have an efficient screening.

"Plate-like-phage" method will result in uniform plating. Do
not shake the plate hard. This will result in clumping of
seeds.

14. Leave plates for 2-7 days in cold room. Move plates to
growth room.

15. After 5 to 7 days, transformants can be easily identified
as dark green plants with long roots. Transfer these seedlings
onto "second selection" plates. After several days, plants will
have several foliage leaves and good roots. If you picked
pseudo-resistant plants, they will be killed by now. Transfer
plants to soil. Keep covered for several days.

Survival of plants after transferring to soil should be about
100%. 

Growth on "second selection" plates is to eliminate
pseudo-resistants and to enhance the development of a good root
system and early bolting. You can leave your plants on plates
for up to 10 days. Some plants will bolt during the growth on
the plates.

Use non-selecting plates (i.e. MS media w/o. antibiotics), if
your seedlings are really resistant (as evidenced by long
roots) but look weak or unhappy. There is no need of continuous
selection. Growth in the absence of selection will help them to
be strong enough to survive subsequent transfer to soil.

If you have contamination, rinse plants with water and transfer
to soil without respect to the growth stage. This will save
your plants.

Materials
Infiltration medium:
1/2 x Murashige-Skoog salts
1 or 1/2 x B5 vitamins
5% sucrose
0.5 g MES
pH 5.7 with KOH
0.044  M benzylaminopurine (10  l of a 1mg/ml stock in DMSO per
liter) 0.02% Silwet L-77 (Union Carbide Chemicals and
Plastics)*

*I have not tried other surfectants or detergents.
Do not change the composition of the Infiltration medium.

"Top agar":
Of course, it must be at room temperature.
0.1% tissue culture agar
Autoclave 15 min. Do not mind if solution does not look uniform
after cooling.

Selection plate: 
1 x Murashige-Skoog salts
1 x B5 vitamins
1% sucrose
0.5 g MES
pH 5.7 with KOH
0.8% agar
antibiotics 

in 150 mm dish.

For kanamycin selection, 30mg/l works well for Col or Ler.
Higher concentration (50mg/l) is required for No.

Addition of carbenicilin (100 mg/l, final) and benomyl (10
mg/l, final) is necessary to reduce contamination and/or
prevent growth of surviving Agrobacterium.

"Second selection" plates:
Same as selection plates in 90 mm dish.



AGL1: C58 recA/pTiBio542DT-DNA 
Lazo X, ..., and R. A. Ludwig (1991). Biotechnology 9, 963-967.
                                info. from Alejandra M. Mandel


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From: Greenberg Jean <greenbej at beagle.colorado.edu> To: Jimmy
Botella <J.Botella at botany.uq.edu.au> Subject: Re: vacuum
infiltration with AGLI In-Reply-To:
<31A3A503.6175 at botany.uq.edu.au> Message-ID:
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When I did vacuum infiltration, I had a similar problem to what
you described UNLESS I was careful not to infiltrate the
leaves.  I grow the plants so that there is a layer of bridal
veil over the pot and the leaves and strems grow above the
veil.  That way I can invert the plants into a beaker full of
agrobacteria and then put the whole thing in a dessicator and
pull the vacuum.  If you do it this way you can adjust the
level of agro slurry so that the leaves do not get inoculated
but the stems do.  I think the main thing is not to get agro in
the soil and leaves in high concentrations.  Good luck,

Jean Greenberg
Assistant Professor
U of Colorado, Boulder


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Date: Thu, 23 May 1996 13:01:31 +0000 From: Qingshun Li
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Dear Jimmy:
    I had the similar problem, although I use Columbia and
    pKYLX 71. 
What I found was that some larvae of Fungus gnat grew in the
soil.  There is sucrose in the infiltration buffer.  That
stimulates fungus growth, then the larva, which somehow hurt
the roots of Arabidopsis.
    My solution is to control fungus gnat adults, a kind of
    little 
fly, before they lay eggs in the soil.  Yellow sticky paper is
good enough to catch them.  After transfermation, control
humidity and water.  I don't water them untill very dry, that
somehow prohibit the fungus--larvae.
    If you don't mind, could you send me a copy of compiled
    related 
information you collect from the net?    
    Good luck!

Qingshun Li, Ph.D.
Dept. of Agronomy
University of Kentucky
Lexington, Kentucky 40546-0091
Phone 606-257-2544
E-mail agr022 at pop.uky.edu

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J.Botella at botany.uq.edu.au (Jimmy Botella) From: George Haughn
<haughn at unixg.ubc.ca> Subject: Re: vacuum infiltration with
AGLI X-PMFLAGS: 35127424 10

Hi:

We have had better success with Columbia and WS than Landsberg
erecta.  We had an experience similar to yours even with the
original in planta treatment (Katavic et al., 1994, MGG 245:
363).  We guessed that the reason stemmed from the fact that
Ler is not as vigourous a plant as some of the other ecotypes
and is therefore more susceptable to abuse.  I suspect that if
one treats Ler more gently then it could be transformed the
same as other ecotypes. 

George  


George Haughn,  Associate Professor
Botany Department,  University of British Columbia
6270 University Blvd,  Vancouver, BC  V6T 1Z4,  Canada
Tel:  604 822-9089
FAX:  604 822-6089
E-mail haughn at unixg.ubc.ca


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From: Lluch Sanfeliu <lluch at far.ub.es>
Subject: Re: vacuum infiltration with AGLI
To: J.Botella at botany.uq.edu.au (Jimmy Botella)
Date: Wed, 22 May 96 19:37:41 METDST
In-Reply-To: <9652204717.~INN-DVJa14669.bionet-news at dl.ac.uk>;
from "Jimmy Botella" at May 21, 96 11:42 pm Mailer: Elm
[revision: 66.25] X-PMFLAGS: 34603136 0

Dear Jimmy,
we have done this kind of plants transformation succesfully
with the Columbia and Wassilevskija ecotypes, using as a
Agrobacterium strain the C58C1 pGV2260 and the vector pBI121.
We would like to know which is the infiltration medium you use
and how do you prepare it, because we think it could be a
problem at this level (a contamination with a plant pathogen
that is co-infiltrated with agrobacterium...). Our IM contains
Murashige and Skoog salt mixture, sucrose, MES, and the pH 5.7.

Cheers!

Angela Masferrer
Unity of Biochemistry
Faculty of Pharmacy
University of Barcelona
e-mail: masferre at far.ub.es


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To: J.Botella at botany.uq.edu.au (Jimmy Botella)
From: abbritt at ucdavis.edu (Anne Britt)
Subject: Re: vacuum infiltration with AGLI
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No one seems to think that the choice of strain is important. 
Maybe there's something nasty in your agro mix- another
bacterium, or something toxic in the medium?


Anne Britt
Assistant Professor
Section of Plant Biology
University of California, Davis
Davis, CA 95616
fax: (916) 752-5410
phone: (916) 752-0699



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To: J.Botella at botany.uq.edu.au (Jimmy Botella)
From: Mahoney at sc3101.med.buffalo.edu (Debbie Mahoney)
Subject: Re: vacuum infiltration with AGLI
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Jimmy,
I do not know the answer to your problem, but I used AGL1 and a
derivative of pBI121 in a vacuum infiltration experiment and
the plants survived just fine. Debbie

        Deborah J. Mahoney
        Department of Molecular and Cellular Biology
        Roswell Park Cancer Institute
        Elm and Carlton Streets
        Buffalo, New York 14263
        lab: 716-845-4404
        FAX: 716-845-8126
        email: mahoney at sc3101.med.buffalo.edu




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19:12:02 -0700 From: Carly Pullen
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J.Botella at botany.uq.edu.au Subject: Re: Vac Inf using AGLI
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Hi Jimmy,

I'm currently transforming Arabidopsis ecotypes Landsberg and
C24 using Agrobacterium GV3101.  I've tried a number of
variations on the basic technique but I don't have my results
with me - they're at Uni.  I can tell you that C24 transforms
much easier than Landsberg although I do have Landsberg
transformants (approx 1-2 per 1000 seed).  I've also found that
Landsberg tends to look quite sick after transformation.  I had
a great run of C24 transformation when the plants were grown at
21 degrees, high humidty after transformation.  

I can send you more specific details of what I've done tomorrow
from university.  What I've told you just know is off the top
of my head after a full day in the lab.

See ya

Carly Pullen
Msc student
Plant Molecular Biology Lab
School of Biological Sciences
University of Auckland
New Zealand



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Date:          Wed, 29 May 1996 11:46:45 GMT+1200 Subject:     
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Hi again,

I hope the message I sent from home last night got to you.

As I said, I'm transforming Ler and C24 using GV3101pMP90.  Ler
look sick cf C24, and the mortality rate is higher but I have
got Ler transformants from this method.  At this stage I only
have half of my results back so far.

RESULTS SO FAR!  

C24 transforms better than Ler.

Infiltration at 30in Hg for 20 mins then growing at 21 degreesC
and high humidity yeilded approx 1 transformant/1000 seed.  

Inf at 15inHg for 20mins plus citowett (a surfactant) then
growing in the glasshouse (conditions not as tightly controlled
as before) resulted in 2/1000 seed.  

I have also tried using Silwet L-77 a surfactant available from
Lehle recommended by Beth Krizek (Meyerowitz lab). I found the
concentration suggested to be toxic (0.03%).  Since then, I
have tried a lower concentration of Silwet and will be
screening for transformants soon.

I found it very useful to search an archived database of the
newsgroup (bionet.genome.arabidopsis).  The archive I searched
is on the Stanford Server 		
http://genome-www.stanford.edu/Arabidopsis/

Good luck,

Please contact me if anything I wrote didn't make sense etc.

Carly Pullen (Msc Student)
Uni of Auckland
New Zealand.


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                         '''''
                         (O O)
            +------oOO----(_)--------------+
            |         Jimmy Botella        |
            |   University of Queensland   |
            |  J.Botella at botany.uq.edu.au  |
            +---------------------oOO------+
                         |_||_|
                          ||||
                         oOOOOo



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