Summary of agar to soil transplant methods

Russell Malmberg russell at DOGWOOD.BOTANY.UGA.EDU
Mon Apr 5 10:22:08 EST 1993


SUMMARY OF REPLIES TO GETTING ARABIDOPSIS OUT OF AGAR


Thanks to those who replied to my request for information.
I received many suggestions very quickly.
Attached is a summary in case others are interested.


Russell Malmberg
russell at dogwood.botany.uga.edu


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I have found that I can get seedlings of any age onto soil by
making sure they do not dry out.  This is done by covering the
flat with Saran Wrap for several days.  Use forceps to pull them
off-not by grabbing them-by closing them so that the tips come
together off of the plant and then pulling upon the cotyledons.

Bill Kubasek

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To transplant from agar plates to soil mix, I follow two
alternative paths. If it is a small seedling, you can pluck it
out of the agar fairly simply without loosing roots. If that
appears hard, I cut the smallest agar plug possible that still
contains enough of the root system to support growth (about one
third) and move the plant with the attached agar plug to a 1.5%
phytagar medium plate (.5X MS salts is adequate) and grow the
plant in the dish positioned vertically. In about a week you
should get good root growth on the surface of the agar. You can
then remove the plant with the whole root system and drop it into
a hole or fissure in a pot with wet mix. Re-close the mix around
the root system delicately and you are in business. In Seattle we
do not bother to adapt the plant to the greenhouse, but, in
Georgia you may want to place the plant in a high humidity
environment for a couple of days.  To get as much seed as
possible in tissue culture, we grow the plant in a 2.5 cm thick
petri dish on 0.5X MS salts or AGM (see Valvekens). The dish is
sealed with porous tape. Usually, seed shatters as it ripens, so
we harvest pods as they turn yellow with small scissors and
forceps.

Luca Comai

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If they have roots we have had good luck with transferring them
directly to soil.  Just take a pencil, poke a hole in moist soil
(we use a mixture of promix:soil (missouri"dirt":sand (3:1:1)),
gently lift the plants out of the agar, put the roots in the hole
and gently pack the hole. Water, cover for two to three days.

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I've just been doing that very thing you asked about. I
transplanted seedlings which were 14 days old from 1.2% agar
plates onto compost very successfully. Make compost tray but
don't water it. Then the compost will fall around the seedling
roots better. Disrupt the agar a bit with forceps, and holding a
leaf or cotyledon gently pull the seedling and you should be able
to pull out the whole length of root from the agar. Then simply
make a hole in the compost and plant the seedling. When you've
finished the whole tray, spray it well with a fine spray.  Put on
a plastic cover with the inside well wetted to maintain high
humidity. This is very important for the first few days. With
luck you should be successful. Even the ones where the root
breaks a bit seem to do ok.

kevin pyke (university of york, UK)

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At a young stage it is very easy to transplant seedlings (up to 5
days) to soil. I normally use a soil mixture of compost
(sterilized by irradiation or steaming) : riversand : vermiculite
(4:1:1). If the plants are kept contained after transplanting to
prevent to much evaporation there is normally no loss of plants. 
At later stages the plants have a lot more roots and it is more 
difficult to pull the seedlings out of the agar without
destroying part of the root system. In such cases I cut the agar
around the roots and  take the plantlet out with some agar stuck
to the roots. If the plant has a lot of branches with leaves, I
normally cut these to prevent evaporation.  Survival rate is
mostly around 50%. Of course you can also try to complete the
life cycle in vitro by transplanting them to large, high
containers which permit as much gas exchange as possible without
contaminating the medium. You can grow them on half MS agar
medium without sugars. This also reduces the contamination risk.

Mark Aarts

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The seedlings survive better the older they are when transferred
to soil. I've had best luck with survival when the roots are
longest just before bolting. For some reason (which I and others
in my lab have observed empirically) the seedlings don't seem to
grow as well and take longer to grow if you wait until past the
time of bolting.  My only suggestions for the actual
transplantation to soil are:
a.) make sure the soil has been heavily watered with Arabidopsis
growth salts,
b.) when you actually remove the seedling, make sure you've
removed every bit of agar from the roots (otherwise you will
probably have a great deal of trouble with fungal growth).
If you would like more info, I'd be happy to send you a copy of
our lab protocol for transplantation.

                         Jim Campanella

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Time:  the earlier the better.
How:  Fill small pots (Aracon system is fine. 
Another solution is to use small pots and make tubes to go over
them if you want to keep the plants separate) with 2:1 mixture of
vermiculite and potting soil.  Moisten well with distilled water. 
Open your agar plates and put some distilled water in to soften
up the agar. If roots are large, you may have to let it soak for
a while and gently break up the agar with forceps.  Lift out the
plants very gently with forceps -- if you squeeze, you kill --
and put in soil.  Cover flat containing pots with plastic cover 
to keep moisture in.  Put at moderate light level for a couple of
days and spray the plants to keep them moist.  Remove cover after
a few days and fertilize.

Nina Fedoroff

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I assume you mean seedlings and not plants that have gone through
tissue culture.  If the former, I find I have approx a 100%
success rate in getting them to grow in pots.  I dig round the
plant with a spatula, taking a little of the surrounding agar so
as not to damage the roots, then put them in a small hole in the
soil.  Then I cover with saran wrap for a couple of days, then
slit it until the plants look established, then remove it. 
Sometimes I get 2 shoots appearing instead of one.  Plants that
have been on agar plates for 2-3 weeks are at a good stage to
transplant.  If you were asking about plants that have been
through tissue culture, several people sent messages
some time back (in relation to transformation protocols).

Sarah Gilmour

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I routinely transfer plants from agar to soil.  The key parameter
as far as I can tell is humidity.  Plants on plates are very
tender and subjected to easily drying out. What I do is
thoroughly wet the soil and keep the flat in a tray that doesn't
allow the water to drain out.  I gently pull the plant out of
agar (you can leave some agar if you can manage to get the roots
without ripping them) put the plant in the soil and immediately
cover with a plastic dome or saran wrap.  Do not leave your agar
plate open for too long as this can also cause the drying out of
the plants.  After about a week I tilt the cover to acclimate the
plants then after 3 more days remove the cover and take away the
tray.  You might try this first with plants you don't care about,
but it sounds like you want to save the plants you have.  Don't
wait until the plants are too old.

jean greenberg

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What i've usually done is to let them get a bit established under
growth lights while still one the plates (especially if the
screen used was a bit harsh on them). once they begin to put out
their secondary leaves, I transfer them to pots (usually about
four to a four inch pot). i set up the pots with arabidopsis soil
mix (see Lehle's recent catalog), presoaked with nutrient
solution (again, Fred's catalog). poke a hole in the soil with a
sharpie or pencil. Crack open the plate, and grab one of the
expanded cotyledons with a pair of forceps (unless your plants
are pretty sick, the one damaged cot won't hurt them). GENTLY
pull them up out of the agar (i use 0.8% - higher  percentages
will make removal difficult, in which case you'll want to cut out
the agar plug with plant). put the seedling into the hole in the
soil and gently surround it with soil. after you've planted all
four or whatever, cover the pot with plastic wrap. cut a few
small sltis (1 inch long) in the plastic. i then put my plants
straight into the greenhouse. after two days, i increase the size
of the slits. after a couple more days, it is safe to remove the 
plastic entirely. i always water from the bottom. this method has
worked very well for me. if your plants are well-established in
the agar, you'll definately want to remove them by cutting out
the agar as well. this is the only tricky part to the proceedure.
hope this helps. give me a call if you need further 

Brian M. Parks

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If the seedlings have really long roots I usually cut a plug of
agar out (with the roots of course) and gently tease/rinse the
excess agar (and most importantly sugar) off of the roots with a
squirt bottle of water and my fingers.  If the roots are not
terribly long it usually suffices to gently and firmly pull the
plant by the cotyledon or leaf.

Rob Last

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I screen for my mutants at a density of 1000-2000 seedlings per
Falcon 1013(150X25mm) plate containing about 75 ml of 0.75% agar
in PNS medium.  In my case I look for survivors over the
germinated and arrested background of seedlings after 14 and 21
days post plating and growth in high light (100-150 micro
Einsteins).  My transfer survival rate has been about 75-80% for
my M2 seedlings. I use a tweezers to pull the plants out of the
agar and I then lay them on prewetted soil and cover the roots. I
reccreen my M3 under the same conditions, but on smaller (Falcon
1012 100X15mm) plates.  Every screen is different so you will
probably have to try and find the best conditions to see your
phenotype, but it has been my experience that seedlings will
survive the transfer to soil at almost any stage, provided that
you are careful not to damage the roots.  I have transfered 7 day
to 28 day seedlings to soil under selective and nonselective
conditions, and had no problem with survival. Older seedlings are
harder to get out of the agar without damage so you could try
using a lower agar concentration if you need to grow them that
long on plates. I also found that with practice I got better. I
hope this is what you were looking for - happy hunting.

Debbie Mahoney

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In response to your query on the Arabidopsis bulletin board:
If the agar medium didn't contain any toxins (e.g.  to select for
resistance) you can just gently pull the plants out of the agar,
rinse the roots gently in tepid water to remove agar (to minimize
problems with microbial growth) and plant in soil that is
saturated with water.  Pack the soil firmly around the roots. 
(The biggest cause of transplant loss is damage to roots or lack
of contact between roots and potting medium.) Put 


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