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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