Guide to Growing Arabidopsis

To grow Arabidopsis in the lab requires several steps which will be described in this guide (the steps are usually performed in this order):
A. seed preparation
B. soil preparation
C. tray preparation
D. saturating the soil
E. sowing seeds onto soil
F. germination and plant growth
G. irrigation and nutrient solution
H. dealing with disease and pests
I. harvesting of seeds

A. Seed preparation
Take packet of wild-type seeds out of refrigerator and allow it to warm up to room temperature before opening it (if you open the packet while it’s cold, condensation may form inside and ruin the seeds). Into a scintillation vial (or any small vial) weigh out about 1.2mg of seeds per tray of plants you want to sow. Fill the labeled vial with dH2O and incubate the seeds at 4°C for 2-4 days, but no more than a week. Once the seeds develop a radicle and shoot at 4°C their viability in soil decreases greatly. The “cold-snap” step is necessary because Arabidopsis seeds will preferentially germinate after experiencing a period of cold; you can also do the cold-snap after you’ve sown the seeds by placing the tray with seeds in the cold room for at least 3 days (5-7 days is optimal).
Using a Pasteur pipette pull up about 50 seeds (no need for accuracy here) and place these into another vial filled with 0.1% agar solution. This technique makes it easier to sow the seeds but several other methods may be just as effective.

B. Soil preparation
Into a large tub place approximately 4 scoops of potting soil, 3 scoops of vermiculite and 2 scoops of perlite; mix the soil with the scoop (i.e. a Nalgene 1L beaker). The vermiculite and perlite are necessary to aerate the soil and retard fungus growth. Considering that each plant tray requires about 1L of soil, mix up only the amount of soil you plan to use. You can try adding some water to the mixture to minimize the dust formed; wearing a dust mask is highly recommended.
Cover the tub with aluminum foil and sterilize the soil in the autoclave in the ‘wrapped’ cycle two times. Once the soil has been sterilized it should be used within a week else algae begin to form on the surface in which case the soil should be re-sterilized.

C. Tray preparation
Obtain planting trays which are clean: any algae, fungus, old seeds, and dead plants should be scrubbed off. Make sure the trays have drainage holes at the bottom. Label your trays with the date the seeds were sown as well as the seeds’ ecotype (i.e. Landsberg or Columbia) and your initials.

D. Saturating the soil
Place the plant trays in the sink in PS525 or in the cafeteria-style trays on a counter. Fill the trays with sterile soil to about 2-3cm from the top, approximately 1L of soil for the medium-sized plant trays.
Saturating solution is necessary to wet the soil before seeds can be placed on it and also contains a powerful fungicide (Daconil-2787) and larvacide (Knock-Out Gnats) to protect against these plagues. Using a Nalgene 4L beaker, fill it with water (not necessarily dH2O) and add 1-2ml of Daconil and 15ml larvacide. Pour this solution onto the soil, wetting it thoroughly. It typically takes at least an hour for the solution to drain through the soil, so it might be convenient to saturate the soil the day before you plan to sow the seeds, or else you could wait. It’s best if the solution has drained from the soil since this makes it easier to transport the trays to the growth chamber without risking spilling water everywhere.
Before sowing the seeds it is sometimes helpful to spray the surface of the soil with water to make sure the soil’s surface is very moist: this will increase the germination rate of the seeds since they must not dry out after they are sown.
Note that if you want to use the 4.5² circular pots to allow for longer root growth use 25ml larvacide and 5ml fungicide per 4L of saturating solution.

E. Sowing seeds onto soil
The general rule is, the fewer plants in a tray, the better they will grow. Planting 6 or fewer seeds in a medium-sized tray will lead to healthy plants but is somewhat inconvenient if you desire large amounts of leaf mass. Eighteen plants per tray (3 rows of 6) will yield reasonably good growth; a higher plant density may result in adverse interactions between the plants.
Several methods can be used to get the seeds onto the soil in as sparse a distribution as possible. From a 0.1% agar solution the seeds can be picked up with a Pasteur pipette and set down on the soil one at a time. You can also just squeeze out the seeds in a straight row onto the soil and later when the seeds have germinated, pull out the excess plants with tweezers to achieve the desired density. Or, you can hand-plant each seed with tweezers and then cold-snap the seeds right on the tray. In any case, the seeds should not be covered over with more soil since the seeds require light to germinate.
In some instances it may be useful to imbibe the seeds on Whatman filter paper in petri dishes placed at 4°C for 3-7 day, then transferred to soil using tweezers, one at a time. Though labor intensive, this method leads to high germination rates, and tweezer-handling ensures proper spacing.

F. Germination and plant growth
Successful germination and plant growth requires appropriate soil moisture, nutrient levels, light intensity, humidity, and temperature. If any of these are compromised, Arabidopsis will respond by flowering early and dying prematurely, producing little leaf mass for experiments. The plants can also be stressed by overcrowding, fungus infestation, or insect infestation. All these factors must be conscientiously monitored to ensure reliable data obtained from experiments using these plants.
Maintenance of soil moisture is imperative for successful seed germination. This can be ensured by covering the tray with plastic wrap taped to the tray, without any airholes. This will provide a high level of humidity for the seeds. During the first two weeks, make sure that the trays are adequately irrigated by maintaining about 1cm of standing water under the trays. Remove the plastic wrap when the seeds have germinated, usually 3-5 days. After those two weeks the plants are not as sensitive to drought and should not remain flooded with water for long periods as it can be a stress to the plants.
The optimal temperature for plant growth is 25°C, with lower temperatures being allowable; higher temperatures can be very detrimental, particularly in the first two weeks of growth. Following the procedure of the Bleecker lab, we grow our plants at 23°C with a 16 hour photoperiod (8 hours dark) under fluorescent illumination supplemented by incandescent light yielding an intensity of 100-150mE/m2×s.

G. Irrigation and nutrient solution
Up to the 4-5 leaf stage, the soil should be kept moist, but after that the soil should be allowed to dry out periodically. The plants grow better and fungus and fungus gnat growth will be retarded. If algae begins to cover the soil, allow the tray to dry out before watering again. After the 4-5 leaf stage, the plants should be irrigated with nutrient solution approximately every two weeks. Be careful not to overfertilize. The first sign of underfertilization is when the rosette leaves or sepals of young flowers turn purple. The signs of overfertilization are leaves and inflorescences that turn yellow, discolor, weaken, flop over, and die. It is better to underfertilize than overfertilize, since overfertilized plants are unrecoverable.
To make 50L of nutrient solution dissolve the reagents one at a time in about 4L of ddfH2O and pour these into the 50L Nalgene container. When all of the reagents have been dissolved add ddfH2O up to the 50L mark.

0.005M KNO3	25.28g
0.0025M KH2PO4	17.01g
0.004M MgSO4 × 7H2O	49.30g
0.002M Ca(NO3)2 × 4H2O	23.62g

H. Dealing with disease and pests
Fungus gnats and aphids are our most common insect problems. Fungus gnat larvae can be killed by the bacteria found in Knock-Out Gnats but these bacteria are killed by the nutrient solution and the larvacide does not control adult flying fungus gnats. Since younger plants are the most vulnerable to being chewed on by fungus gnat larvae, it is useful to use larvacide in the soil saturating solution even though it will be inactivated once nutrient solution is applied. To keep fungus gnat levels down for a longer time span requires watering with larvacide diluted 1:200.
Aphids left untreated will kill an infested plant within a matter of days. Use a fresh 1:50 dilution of Orthene in a spray bottle and spray the plants with a fine mist, covering every surface, repeating the next day. Orthene is very effective against aphids, but overspraying will seriously hurt the plants. It is important that not any new plants be introduced into the growth room until all those plants are dead and the room has been thoroughly decontaminated.
An important aspect of insect control is detection and identification before populations multiply. Traps (yellow cards with adhesive) are vital in this regard. Fresh traps should be placed in plant growth rooms frequently and monitored continuously.

I. Harvesting of seeds
As plants mature, they will need less water, but watering should continue while seeds are setting. It is always best to collect seeds promptly, as soon as the seliques break with little or no applied pressure. For certain stocks, especially the Ler ecotype, seliques will often break open prematurely. Seeds left too long on the plants fail to dry properly and are very susceptible to fungus, severely lowering germination rates. Similarly, while the plants are growing, try to keep maturing and mature seliques out of the bottom of the tray, where they are especially vulnerable to fungus.
Seed collection can be accomplished by rubbing your fingers around the selique, and allowing the seeds to fall onto a piece of paper placed below. Filter the seeds through a section of cheese cloth onto another piece of paper, to remove the seliques. If seliques are stored along with the seeds, fungus growth can result. Seeds can be stored in Eppendorf tubes.
Collected seeds should be left in the tubes at room temperature for approximately one week, then a hole should be poked in the top of the tube, and the tube should then be placed in a sealed container with Drierite. After another week or so, the seeds can be planted. Note that seeds can be planted earlier than this, but the germination rate goes down the earlier you plant them after collecting them. Seeds meant for long-term storage should remain under continued desiccation at 4°C.

References
This guide has been developed mainly by slight modification of the techniques described by M. Running (Meyerowitz lab) and R. Scholl and their description of protocols has been very liberally duplicated here. Reference has also been made to Bleecker et.al. whose work in this field we are also respectfully emulating.
The methods specified here are continually being improved: if you have any suggestions or comments please submit these to be included in upcoming versions of this guide.

Running, M. 1995. Plant growth guide: How we grow plants. C.I.T.
Scholl, R. 1996. Handling Arabidopsis plants and seeds. A.B.R.C.
Bleecker, A. et. al. Development and age-related processes that influence the longevity and senescence of photosynthetic tissues in Arabidopsis. Plant Cell. 5: 553-564.

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