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VALVEKENS'S PROTOCOL: A GUIDE TO HIGH EFFICIENCY
(Department of Molecular Genetics, The Cambridge Laboratory, The
John Innes Centre for Plant Science Research, Colney Lane, Norwich
* The protocol described here is mainly intended as a guide for
newcomers, but I hope that people with expertise in transformation will
also benefit. I have tried to stress details, tips, and any pieces of
information that are important for obtaining transformants. Some of
these points come from my own experience, others from Renate
Schmidt or Caroline Dean. They, George Coupland and June
Swinburne have critically read the protocol -- many thanks.
* Before doing any transformation experiments it is advisable to acquire
some background on this topic. The following references are useful
reading: Lewin, B. 1990. Interaction of Ti plasmid DNA with the plant
genome. Genes IV. Oxford Univ. Press. pp744-749; Zambryski, P.
1988. Basic processes underlying Agrobacterium-mediated DNA
transfer to plant cells. Ann. Rev. Genet. 22, 1-30; and Valvekens, D.,
Van Montagu, M. and Van Lijsebettens, M. 1988. Agrobacterium
tumefaciens- mediated transformation of Arabidopsis thaliana root
explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA 85,
5536-5540. I am closely following, and unless specified reproducing,
the original protocol of Valvekens et al. The Valvekens' protocol, as
followed in Caroline Dean's lab, appears in this book (Transformation),
the media and the protocol described here are essentially identical.
However, there are some differences in the practicalities of the process.
A variation of Valvekens's protocol, by Nigel Kilby and Ian Furner, also
appears in this book (Transformation: modification). It achieves root
formation in the transformed, regenerated plants, with the aim of moving
them to soil. Nevertheless, I find it more convenient to continue the
transformation process in tissue culture, thereby obtaining sterile seed.
* If you do not have experience in transformation according to
Valvekens's protocol, you are advised to work with only one construct
and not to embark on a large scale experiment. When the method is
working, try to obtain all the independent transformants you need with a
particular construct from the same experiment. If a few constructs (up to
four) are to be introduced separately into Arabidopsis, then it is
advantageous to carry out the transformations concurrently. In this case,
handling of plates, pots and so on is greatly facilitated by assigning one
colour to each construct. It is very helpful to write a list including: the
number of the individual transformed callus, date when it is moved to
GM, date when it is moved to a pot, date of collection of seed, and
* I emphasise that I have used this protocol for transforming A. thaliana
Landsberg erecta, using Agrobacterium tumefaciens C58C1 containing
the plasmid pGV2260. Variations in growth times, regeneration
efficiencies and so on can be expected with other strains.
* Use Difco Bacto Agar. It is advisable to store 1 litre conical lasks with
0.5 l of GM agar in the cold room. When you need the agar, melt it in the
* Dissolve all the components with a magnetic stirrer and place the
solution in 400 ml bottles. Autoclave and store them in the cold room.
* Prepare IM + gel rite agar 0.25%. It is advisable not to put more than
0.5l of medium in a single flask (of 1 l). Otherwise the flask becomes too
heavy for pouring the medium properly. Autoclave, cool down to
~45oC, add the hormones and pour the medium on petri dishes. In my
experience, gel rite gives better callus regeneration than Bacto Agar.
(iv) PREPARATION OF ANTIBIOTIC STOCK SOLUTIONS
In the flow hood, add 5 ml of sterile distilled water to the vial containing
500 mg. Shake and use. Leave any remaining solution in the original
vial on ice and store it at -20oC as soon as possible until next used.
Weigh the right amount into a weighing boat. In the flow hood, add
sterile water, and filter sterilise using an Acrodisk. You can filter pouring
the solution in the flask with the medium, or in a tube. Never leave the
augmentin stock solution on ice or freeze it. It will go off (becomes
Prepare a few ml. Sterilise through an Acrodisk and make aliquots of 50
(v) PREPARATION OF HORMONE STOCK SOLUTIONS
* Hormones are the most important factor for good regeneration of
transformed root cells. Take all necessary precautions to ensure proper
preparation and handling of your stock solutions. Use high quality
dimethyl sulfoxide (DMSO) as the solvent. Sigma supplies it in 5 or 10
ml aliquots in ampoules (Catalogue No. d 2650). Once open, use the
whole contents of the ampoule. It is advisable to weigh at least 5 mg of
any hormone in order to minimise errors.
* Once you have prepared the stock solution, make aliquots of 55 ul,
except for the 2-iP, where they should be of 550 ul. Store them at -
20oC for up to two months. Once an aliquot has been thawed, use the
right volume and discard the rest. Never reuse remaining stock
(vi) SIM AGAR (with and without antibiotics)
* The amounts of hormones and antibiotics given are for 0.5 l.
(i) Do exactly the same as if you were preparing CIM-agar. However,
(ii) Antibiotics added to the plates to be used in the first two weeks after
* Augmentin is probably not necessary when the Agrobacterium strain
is kanamycin sensitive (C58C1 is kanS). In this case, both vancomycin
and kanamycin act upon agrobacteria. However, if you infect with a
strain that is kanamycin resistant, add augmentin because vancomycin
alone is likely to result in agrobacteria taking over.
(iii) Prepare 2 plates (aiming for ~25 mlplate-1), to be used for the +
regeneration control (1 for each week). To the rest of the medium, add:
(iv) Plates for the following weeks: decrease by 1/3 the concentration
of vancomycin, adding 2.5 ml0.5 l-1, and omit augmentin.
* I prepare sets of plates for two weeks at a time. After having poured
the medium, leave the lids open for ~20 minutes and, once the medium
has set, place the plates in the original plastic bags and store them in the
cold room. Always keep the plates horizontal as gel rite is "soft" and
your plates can be ruined if you incline or invert them.
* To obtain high quality roots, sow ~100 seeds/GM agar plate, leaving
(i) You can sprinkle the seed on the medium (separate any clumps of
seed). However, I sow the seed one by one, using forceps, with a
template underneath (it only takes about 10 min per plate). Four plates
with seedlings/construct may be enough to obtain 10-30 independent
transformants. Use deep petri dishes (e.g., Falcon 3003, 20 mm deep),
which allow better growth of the seedlings than conventional ones and,
are, therefore, likely to improve the quality of the roots.
(ii) Place the plates at 4oC for 4 d, to break seed dormancy. Then move
the plates to a growth room. After 2-3 weeks, condensation can appear
on the inner side of the lid. Try to avoid this by placing two short pieces
of Micropore 3M tape vertically on opposite sides of the plate (instead of
one long piece horizontally). This allows better gas exchange (Renate
Schmidt, pers. comm.) and helps to keep the inside of the plate less
moist. On the other hand, you can always remove the tape occasionally
and dry the lid with sterile filter paper.
* It is advisable to sow seed on a few plates every week, thereby
providing a constant supply of roots, until you are confident that the
transformation experiment has worked. This will save you a lot of time if
you have to repeat a transformation.
* The best time for preparing roots is after an incubation period of
approximately four weeks in the growth room. Use the plates when the
plantlets have not yet started to bolt, the roots are white, and the
medium is not too dry. Discard old plates, as the efficiency of
transformation is likely to drop substantially.
(i) Remove all the aerial part of the seedlings, taking groups of a few
stems with forceps and cutting them out, at the medium surface, with a
scalpel. Then completely remove any leaves or aerial parts still
remaining. Add 15-20 ml of CIM. Mix it gently with the medium, and pull
the roots out with forceps. Sometimes it is possible to take all the roots
together, as a large root mass, instead of the roots of individual plants. If
possible, do it this way because it is quicker.
(ii) Transfer the roots to a petri dish containing ~25 ml of CIM to wash the
roots. Remove the original medium as much as possible, but try not to
damage the roots. Repeat this step. Make 3-5 bunches of roots and
place them on the surface of a CIM-agar plate. Spread the roots with
forceps and a spatula, so that they occupy a larger area and are in close
contact with the medium, but without burying them. Remove the excess
liquid with a Gilson pipette. Leave the surface of the plate dry, in order to
prevent hormone dilution. Seal the plates with 3M tape and leave them
for 3 d in the growth room. Use one CIM agar plate per GM with
* Before you start, check the roots carefully for contamination (see
Transformation). If you discover any, either discard that plate or if it is
localised to a single spot, use the roots which are furthest from the
(i) Take individual bunches of roots and transfer them to an empty petri
dish. Cut them first longitudinally, making root strips ~0.5 cm wide. After
this, cut the roots perpendicularly, every ~0.5 cm. To make the actual
root explants, 2 or 3 of these root pieces may be necessary. An average
of 30-40 root explants can be obtained from each CIM agar plate with
roots. Use a fresh empty petri dish per CIM agar plate with roots.
* Always include the appropriate antibiotics in Agrobacterium cultures.
Also make sure you already have a recent culture before obtaining the
roots. 24-36 h. before the infection of root explants, start a 5-10 ml
Agrobacterium culture by inoculating with 50-100 ul of a recent
saturated culture. Once the final culture is saturated, keep it always at r.t.
until infecting the roots. Storage of cultures at low temperatures results
* It is wise to remove the antibiotics before infecting the root explants.
To this end, spin the cells down and resuspend them in the original
volume of fresh YEP. You can use an Eppendorf tube with 1 ml of
culture (keep the conditions sterile).
(i) Transfer 1 ml of cell suspension into 20-25 ml of CIM in a petri dish.
Soak the root explants for 2-10 min, trying not to disrupt them. Longer
times could be detrimental as Agrobacterium is toxic to plant cells.
Transfer the root explants to a sheet of sterile filter paper 8.5 cm in
diameter (place the filter on an upside down lid of a petri dish). Take one
explant, dry it incompletely on 2-3 stacked sheets of fresh filter paper
and place it on the CIM-agar plate. Do the same with the remaining
explants without overdrying them. Use 2 CIM agar plates per original
petri dish of roots, thus placing 15-20 infected root explants per CIM-
* Make sure the agrobacteria in CIM do not sediment, by shaking the
plate before transferring each set of root explants. This will facilitate a
more uniform infection of the roots.
6. CO-CULTURE OF THE ROOT EXPLANTS WITH AGROBACTERIUM
* In my opinion, after 2 d incubation in the growth room the presence of
agrobacteria must be evident. Each root explant should be contained in
a slightly whitish area. If this is not the case, prolong the culture for up
to 24 h. This should allow for enough time to have a good agrobacterial
growth. If you are uncertain of what to do, you can always wash the explants in
two sets, allowing different co-culture times.
* On the other hand, other people with experience in transformation
believe that Agrobacterium growth must not be evident. If it is, the same
plant cell can be transformed several times, thus carrying the construct
in more than one locus. However, around 70% of my transformants
* If the growth of agrobacteria is insufficient, there will be very few
transformation events, resulting in too few green calli. On the other
hand, agrobacteria overgrowth can produce several transformants per
root explant, leading to several, very close green calli in most root
explants. This makes it difficult to distinguish between individual
regenerating calli and even chimaeric regenerating plants can be
formed, coming from different transformation events. If the agrobacteria
overgrowth is very severe, root cells can be killed and no transformants
will be obtained. Both extremes are, therefore, undesirable. Aim for an
average of 1-2 regenerating calli per root explant.
* It is also possible to infect the cut roots and make the explants
* In Valvekens's protocol, washes are done in CIM + vancomycin.
However, vancomycin is very expensive and can be omited (see
* You will need several plates with 25-30 ml of CIM (SIM can also be
used), hereafter called A, B, and so on. In addition you will need a "root
collector." This is a portion of metalic sieve (a triangle of approx. 2
cm/side) in which a long, thin spatula has been inserted. Sterilise it by
autoclaving or by immersion in 90% ethanol. Do not flame it as this will
(i) Wash together the 15-20 infected root explants contained on the
same CIM-agar plate. Transfer them to plate A and separate them
thorougly using forceps so that the roots are properly washed. Transfer
the roots to plate B. Collect the remaining roots in plate A with the root
collector and transfer them to plate B (with forceps). Wash the roots in
plate B as before and transfer them with forceps to plate C placing them
in 15-20 groups. Collect the remaining roots in plate B and add them to
a few of those groups of roots. Transfer the clumps of roots to three
stacked sheets of Whatman No.1 filter paper, 8.5 cm in diameter, and
allow them to dry. Collect the remaining roots in plate C and add them to
a few of the drying root clumps. Following this procedure, the roots are
properly washed. This avoids the possibility of agrobacteria taking over
in the SIM agar plates. Incidentally, if this happens it is very difficult to
get rid of the agrobacteria and your chances of successful transformation are
(ii) Transfer the reconstituted root explants to SIM agar plates
containing the appropriate antibiotics and leave the plates in the growth
room. It is advisable not to reuse any of plates A, B or C for further
washes. In additon, the root collector should be sterilsed, by immersion
in 90% ethanol, after washing each set of explants coming from the
same initial CIM agar plate. This will avoid a general contamination if there
was a previous one, which had been masked by growth of
* Hormones (especially IAA) and antibiotics are likely to lose activity after
several days in the growth room. From now on, transfer your root
explants into fresh SIM agar plates weekly. Five to 6 weeks after the
washes, discard your root explants. You are likely to obtain only a few
* After approximately 3 weeks, green calli will be regenerating into
shoots-leaves (hereafter called regenerating shoots). Wait until they
form a structure of ~5-8 mm in length, because regenerating shoots that
are too small tend to die in GM. However, do not leave the regenerating
shoots in SIM agar for too long as they may show poorer regeneration or
even die. Cut the regenerating shoot at its base, removing callus as
much as possible, and transfer it to a GM agar plate. Deep plates are
better, as they allow more space for the regenerating shoot to grow and
bolt. Deep plates are, however, 4 times the price of conventional plates.
You can place up to 10-12 regenerating shoots per plate.
* From now on, regeneration continues with neither kanamycin
selection nor plant hormones in the medium. Number each
regenerating callus that you transfer to GM and, obviously, give the
same number to anything developing from that callus. Check your SIM
agar plates every few days; more regenerating shoots will appear.
Transfer as many regenerating shoots as possible to GM. You are likely
* When the regenerating callus produces many "leaves" but does not
bolt, keep teasing the material apart, maintaining the meristem(s) until
the plant normalises and forms a proper rosette.
9. HANDLING OF THE REGENERATING TRANSFORMED PLANTS
* Be extremely careful with serrated forceps and any metal instruments
with grooves. Normal sterilisation with ethanol and flaming is sometimes
not enough to ensure sterility. The best system is to wash thoroughly
the serrated area with a cleaning brush, using detergent, and proceed
normally afterwards. Autoclaving of instruments is equally effective, but
* Bear in mind that not all regenerating plants in Magentas set seed and
you can lose some of them as a result of contamination. It is therefore
advisable to have 2-3 regenerating plants from the same callus (Renate
* When the regenerating shoot is bolting, it has to be transferred to a
pot with GM. Do it quickly, as a delay of a few days can mean that it goes
back to a callus-like shape. Put the regenerating shoot on an empty
petri dish. Sometimes two or more regenerating plantlets develop from
the original regenerating shoot and only one of them is actually bolting.
In this case, carefully separate them and transfer the non-bolting ones
into a GM agar plate. Remove any callus or dead leaves at the base of
the regenerating shoot. If necessary, cut healthy leaves out so that the
base of the bolting shoot can be introduced into the GM medium.
Transfer it to a pot (Magentas are the best in my experience, but some
people like cylindrical pots). Keep the pot closed. If you lift the lid at
this stage, the plant is likely to wilt and die. If more regenerating plants
coming from the same callus bolt, transfer them into pots (up to 3 per
* Sometimes, rosette leaves lift and detach the base of the plant from
the medium. Obviously, if this happens, push the plant into the medium
and if necessary, remove some of the leaves first.
* Quite often a regenerating plant in a pot is actually composed of
several regenerating plants kept together. You can separate them (in a
15 cm empty petri dish) and obtain 2-3 plants from the same
transformant. These should be placed in different pots. However, be
very careful when manipulating plants in sterile conditions. The more
that you take them out of the pot or the petri dish, the more likely they
* If the bottom of the plant has formed a mass of callus, this often results
in poor growth, pale colour and no seed formation. It is usually
necesssary to remove this callus. To this end, transfer the plant to a 15
cm empty petri dish (only if the plant is relatively small can conventional
petri dishes be used) and carefully remove the callus and any dead or
wilted leaves. If the plant is big and has many pale stems, prune it,
leaving only a few healthy stems. After this, place the plant in a fresh pot
(you can also use the original one, putting the plant in a different place).
This callus formation is usually associated with moist medium. Use dry
Magentas and keep an eye on the humidity of the medium throughout
the time in which the plants are in them.
* When the medium in the pot becomes contaminated it is sometimes
possible to save the plant. If the contamination has not reached the
plant move it immediately into a fresh pot. If the contamination has
reached the plant, cut the stem and move the upper, uncontaminated
* Closed magentas can be too humid inside and this prevents
pollination, thus resulting in no seed set (see Transformation). To avoid
this, when the flowers reach the lid, lift it and seal with 3M tape. During
the next days, check for any wilting. If this occurs, close the lid as
before. In some cases it is possible to obtain seed in closed Magentas.
However, as a general rule, proceed according to this protocol.
10. HARVESTING AND SOWING THE SEED FROM ORIGINAL
* If you have several plants from the same transformed callus (that
should be in different pots), always collect their seeds separately. Sow
seed from only one of these plants. Although the other plants are
expected to be genetically identical, they could exceptionally be from a
(i) When the pods are yellow-brown (still closed) cut the stems and
place them in an empty petri dish. Before sowing the seed allow them to
* The first thing you must do once you have obtained seed from your
putatively transformed plants is to check that they segregate KanR
individuals, and, if so, determine the segregation ratio, which is
indicative of how many loci harbour the construct.
(ii) Sow ~100 seed on a petri dish containing GM agar + kanamycin 50
ugml-1. Before sowing the seed, they have to be separated from their
pods and the dry stems and pods removed. At this stage, hold the petri
dish containing the seed above the medium. Flick it, so that the seed fall
on the medium, and separate any clumps.
* This is a quick system but it has the following disadvantages:
(a) It is easy to lose seed when separating it from the pods and when
sowing it. You should not lose any transformed seed into the
(b) You can contaminate the medium when manipulating the petri dish
(c) The seed can end up unevenly distributed on the medium.
(d) Due to static electricity, some of the seed remain in the petri dish.
A variation of this method for sowing the seed is described below. It
takes longer, but overcomes (or improves substantially) the drawbacks
(iii) Before collecting your seed, place a sterile Whatman No.1 filter
paper, 8.5 cm in diameter, in a 9 cm petri dish placed upside-down. Cut
the stems containing the pods and place them in the petri dish as
explained above. If necessary, allow the seed to dry. Just before
sowing the seed, add ~2 ml of sterile water and carefully open the pods,
using a long spatula and holding the base of the petri dish as if it were
the lid. The seed will stay on the wet filter paper. Then, using a template
underneath, sow the seed one by one using forceps. If there are some
seed left, allow the filter paper to dry out in the flow hood, seal the plate
with 3M tape (all around) and store it. If you want to sow seed again, add
sterile water and proceed as explained above.
11. EFFICIENCIES THROUGHOUT THE PLANT TRANSFORMATION
* It is difficult to make an estimate of the proportion of individuals that
will pass to the next step. It can vary a great deal and depends on many
factors, among them the construct with which you have transformed. I have used
kanamycin as the selectable marker for Landsberg erecta. My constructs were
cloned into the vectors p0188 and p13019 (Caroline Dean, unpublished) and
introduced in Agrobacterium tumefaciens C58C1 carrying the plasmid pGV2260,
which contains the vir genes. In these conditions, not accounting for any
losses due to contamination, a rough estimate would be:
(a) Green calli that regenerate into shoots 60%
(b) Regenerating shoots that actually bolt 70%
(c) Bolting shoots that develop into a mature plant 80%
(d) Mature plants that set seed 50%.
As stated before, it is possible to improve (d) by having 2 or 3 plants
coming from the same callus. Quite often, for unknown reasons, one of
these plants sets seed while the other(s) do not.
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