Arabidopsis Hydroponics (September, 2008)

1. Prepare microfuge tubes by cutting off caps and tip. Cut tips off about half-way down the cone section. You want to leave a little shoulder to hold the rockwool plug. See photo for microfuge preparation.

2. Cut rockwool plugs with a No. 4 cork borer. The rockwool plug should be the length of the microfuge tube from the top to the shoulder. Grain direction does not seem to make a difference. You can use a hacksaw to cut pieces of the appropriate length and then use the cork borer to cut out plugs.

3. Place the rockwool plug in the microfuge tube. The key is NOT to compress the rockwool or pack it in. This will inhibit or can completely block root growth through the plug. What I do is to tap the bottom end of the tube on the counter to cause the plug to sit down to the shoulder. Then I turn it upside down and tap the cap to stretch out the plug so that it is not compressed. The shoulder naturally compresses the rockwool. This is a problem and is a MAJOR difference from our previous method where the rockwool plug was stretched out by gravity. Nonetheless the shoulder is important to hold the rockwool and keep it from falling into the solution. The top of the rockwool should be level with the top of the microfuge tube. If the levels are uneven, so will be your germination. This method is an improvement on our previous method if it is done correctly.

4. Place microfuge tubes in a foam floatie with the bottom of the microfuge tubes just touching the surface solution of Gibeaut’s nutrient solution. Substitute Fe EDDHA in place of Sprint (Sequestrene) 330. We recently found inhibitory problems on germination with Sprint 330. They should naturally wet. If they don’t, check to see that they are touching the solution. If they are, then wet the rockwool by pouring nutrient solution over them. Floating the tubes on the surface insures constant and uniform wetting, which will allow for uniform germination.

5. To insure good germination and inhibit algae build up (not full proof) we pipette 200 µL of 0.8% phytoagar (bactoagar is inhibitory) on to the top of each rockwool plug.

6. Sprinkle dry Arabidopsis seeds (they do not have to be cold stratified as commonly believed. I have tested this and found no difference in germination rate or uniformity.) in a Petri dish filled with water. Let seeds settle to the bottom and select these seeds as they are denser, probably due to greater starch content. Pipette three seeds to the top of every microfuge tube. Seeds do NOT have to be sterilized.

7. To insure germination in very dry climates like Reno, we cover the floaties and their tubes with plastic wrap (saran wrap) for a few days until germination starts. Floaties are covered with black plastic covers to inhibit algae growth. See photo of young seedlings germinating.

8. Seeds will take about 3 days to germinate.

9. One week after planting, thin seedlings to one seedling. Later thinning is more difficult and can damage or impair the remaining seedling, slowing down its growth.

10. Roots should grow through the rockwool by 10 to 14 days after planting.

11. After two weeks, plants are ready to have their tubes placed in the final hydroponic setup in a 50 mL centrifuge tube. The tube cap has a hole in it to fit the microfuge tube perfectly (use a bit that is 6/16”) and the bottom of the tube is cutoff. The cap should also have a small hole drilled in it (use a bit that is 3/32”) to allow air from below to pass out and let the tube settle properly in the hydroponic container lid. The hydroponic container lid should have holes cut in it to allow the 50 mL tubes to fit through but not the cap so that the cap sits on the upper surface of the lid. The lid should be thin, dark Plexiglas or other rigid plastic to allow the rockwool plugs to reach the solution surface below. The centrifuge tube acts as a guard to help prevent root entanglement with other roots in the hydroponic solution. The solution surface should be brought up close to the lid. We use two aeration stones, one at opposite corners and bubble air continuously. See hydrosetup photo.

9. By about 2 weeks, algae will start to build up on the top of the rockwool. The plant leaves will soon cover this small surface area and block light and inhibit the algae. One can place sterile dark artificial soil mix on the surface of the rock wool to help block the light but this is really not necessary and only makes for an extra step. Once roots are growing past the rockwool into the solution, the solution levels can be lowered to encourage drying out of the upper surface of the rockwool and further inhibit algae growth.

10. Plants will grow rapidly after this stage.

I have found very little loss of roots in the rockwool when harvesting plants grown in this manner!

Gibeaut’s solution (former post-doc of mine)

1.5 mL/L of 1 M Ca(NO3)2

1.25 mL/L of 1 M KNO3

0.75 mL/L of 1 M MgSO4

0.5 mL/L of 1 M KH2PO4

1.0 mL/L of micronutrient stock

1.0 mL/L of FeEDDHA stock

The pH should be about 6.0 without adding any acid or base. I try to maintain the pH between 5.0 and 7.0. If the pH should change then add a little KOH or HNO3 to bring the pH back to 6.0.

Solutions are usually good for at least two weeks.

I have found (and Brent Kaiser at University of Adelaide has confirmed this) that adding a small amount of NH4SO4 improves growth significantly. I call this solution, Cramer’s solution, with the additional 1 mM NH4SO4 added to the Gibeaut’s solution.

Cramer’s solution