TITLE: A custom-made hydroponic culture system to study plant roots during root infection with Plasmodiophora brassicae
AUTHORS: Susann Auer

[DESCRIPTION]
Plant roots are fascinating organs that anchor the plant in the ground, take up water and nutrients and are the interaction zone for plants with their surrounding microbial rhizosphere. In contrast to easily accessible leaves, the study of these belowground organs is more difficult as harvesting the tissue is time-consuming and - when sampled as whole - kills the entire plant. The time-consuming and often disruptive process of root harvest can hamper molecular studies that require for samples to be harvested in a narrow time frame in order to rule out negative sample bias such as circadian gene regulation or effects that stem of the sample handling rather than the actual assay.  
For our studies of the soil-borne pathogen Plasmodiophora brassicae in interaction with soil microbes in roots we needed a plant cultivation method that allows quick and easy harvest of root tissue while at the same time allowing the roots to form the pathogen-specific clubbed root structures. 
We therefore came up with an easy custom-build setup, that can be easily implemented in other laboratories using similar materials. Our setup requires clear square plastic containers high enough to keep our desired plant species (Arabidopsis and Brassica) and a custom-build rack with holes that fit 10ml pipette tips. This setup allows easy cultivation of plants in sand and hydroponic medium and largely damage-free root harvest within several seconds for each plant. This method allowed us to harvest samples for a large microarray experiment as well as countless experiments for root transcript studies. 
Since the height of the box and the extend of the pipette tips is a limiting factor we suggest to use this system only for relatively small plants such as Arabidopsis or young stages of larger plants such as Brassica rapa or Brassica napus.   

[BEFORE_START]
We study the interaction of plants (Arabidopsis, Brassica) with the clubroot pathogen, a soilborne protist that causes swelling of the roots and other microbes. A normal experiment in our setup contains 4 treatment groups so we use four boxes with 93 pipette tips each, amounting to roughly 400 plants in total. For our studies we need large amounts of root material that need to be harvested quickly to allow for comparisons across all treatment groups. We therefore came up with this hydroponic cultivation system that allows complete, non-destructive root harvesting within seconds for each plant. 

The system described here was made from materials that should be available in hardware stores. We used hardware store plastic storage boxes that are 31-36x26-33x31cm (LxWxH) in dimension and have a loosely-fitting lid that allows some air circulation, see details under "Materials". 
The pipette racks were built to fit inside these boxes and each can hold 93 pipette tips. They consist of a durable plastic board (32x28cm) with holes drilled in it and 4 feet to stand inside the box. The holes have a diameter of 1.2 cm and hold a 10 ml pipette tip. When the pipette tip rack is placed inside the box, each pipette tip reaches to the bottom of the box which allows low levels of medium, as low as 1 l total volume, and thus reduces the weight of each box during the experiment. 
In our setup we can fill 4 l of medium into the containers with no problem. However since the boxes are moved throughout the experiment we recommend to use smaller amounts of volume to ease handling.  



[GUIDELINES]
Read the "Before start" to get instructions on how to build a hydroponic culture system for cultivating Arabidopsis and Brassica in sand with liquid medium. 

[STEPS]
SECTION: Setup of the system
1. Place tip rack in the clean container and fill 10 ml pipette tips with sand to the top. 
SECTION: Setup of the system
1.1. Tap the tip lightly on a hard surface to make sure the sand does spread evenly in the tip. Fill tip up again to the top if necessary. You don´t want the sand to be packed too tightly as this might cause problems with ventilation of the roots later. 
SECTION: Setup of the system
2. Place pipette tip in the rack of the container and continue with the next tip until you have all pipette tips filled with sand. 
SECTION: Setup of the system
3. Fill liquid sterile medium in the box containing the tips and let the medium be soaked up by the sand. Since soaking up medium will lower the level of medium in the box make sure you fill in enough medium to cover at least the lower third of your pipette tips that reach into the medium. 
SECTION: Setup of the system
3.1. The soaking will take a few seconds or minutes. Make sure all tips are fully soaked and the medium reaches to the top of the sand surface. If not, lightly tap the tip to spread the sand better and continue soaking. You want to make sure that all tips soak up the medium so that your plants have access to water and nutrients. 
SECTION: Sowing of seeds
4. Sow seeds on the surface of the sand. Before sowing, make sure the sand surface is wet with medium so the seed has enough liquid to germinate. 
SECTION: Sowing of seeds
4.1. When using Arabidopsis seeds you can seed 2 seeds per pipette tip a few millimeters apart from each other. After germination you remove one of the seedlings. When using Brassica seeds one seed per tip is enough. Alternatively use Brassica seeds that are pre-germinated with the radicle already emerging. Place these seeds gently on the sand and make sure the radicle points downwards. 

  

SECTION: Maintenance during experiment
5. Cover the plastic box with a lid and place the box with your tips in a suitable environment for germination. We use a greenhouse or climate chamber with longday conditions: 16 hours light at 23 °C, 8 hours dark at 18 - 23 °C. Keep the lid on during the whole experiment except for medium changes and harvests. 
SECTION: Maintenance during experiment
5.1. For Arabidopsis and Brassica the best results were obtained under somewhat natural light conditions in a greenhouse that has an average temperature of 20 °C and is located on the top of the Biology building at TU Dresden, Germany. Average light intensity was 120 µmol/s×m² on cloudy days and up to 700 µmol/s×m² on sunny days. 

SECTION: Maintenance during experiment
6. Monitor the development of your plants in the first few days closely. To avoid drying out of the sand surface within the first few crucial days of germination it helps to daily spray the tips from above with sterile nutrient solution until the sand surface is thoroughly wet. The spraying should be done until the plants have reached the 2 true leaf stage or when they appear stable and large enough to reach with their roots deeper in the sand. 
SECTION: Maintenance during experiment
6.1. Germination rate on sand is usually worse than on soil and you might lose one third of your plants within the first days. To avoid this, check on them daily and make sure the sand stays moist with nutrient solution. Once the plants are large enough to reach the nutrient solution and do not flood up when you spray the sand from above they will likely survive. To prevent microbial contamination of your plants always use gloves, open the box only as long as necessary and always use autoclaved fresh nutrient solution. Wiping down the insides of the box with ethanol from time to time can prevent contamination. 

SECTION: Maintenance during experiment
7. Start your pathogen treatment at the desired growth stage. We inoculate Arabidopsis plants 10 to 14 days after sowing and Brassica plants 3 to 5 days after sowing with 200µl of 107 spores/ml of Plasmodiophora brassicae in 50 mM KH2PO4, depending on the developmental stage of the plants.  

SECTION: Maintenance during experiment
8. Maintain your cultures throughout the experiment by changing the medium every week to fresh, autoclaved medium. Should plants struggle to reach the medium, carefully pipette a small volume of medium up to 200 ml on top of the sand surface taking care to not uproot the plant. 

SECTION: Harvesting of plants
9. To harvest your plants first prepare two shallow containers and fill them with tap water at room temperature. Fill the first container with water almost to the top and the second with a small amount of water, just enough to cover the bottom. Take each individual tip carefully out of the box and submerge in water of the first container. Then carefully, under water, pull at the plant on top to slide the whole plant out of the tip taking care to not break off the roots. Use a paint brush to remove residual sand from the surface of the roots and then put this plant in the second container, making sure the roots are in the water and leaves float on the surface. Repeat this process with all tips. Each harvest should only take a few seconds.  

  

SECTION: Maintenance during experiment
8.1. After pathogen inoculation we continue cultivation of Arabidopsis in this setup up to day 40 after sowing and of Brassica up to day 24 after sowing. 

   

SECTION: Optional adjustments and hacks
10. 
Aeration of medium: If necessary, you can aerate the liquid medium using an aquarium pump. We did not observe better growth with aerated medium so we don´t do it. 

  

Backup seedlings: Germination rate on sand is in our experience often lower than on soil. We therefore prepare extra tips with seeds in a similar manner as described above. Three tips are then placed together in one 50 ml falcon tube that is filled with 5 ml of medium. Several falcon tubes are placed in a falcon rack and covered with a transparent hood and located at the same growth conditions as the larger containers. These backup tips replace tips in the larger container where no seeds germinated on the surface. For a small scale experiment to test out medium and growth conditions this approach can work as well. Medium should be exchanged twice a week. Also, watch the falcons closely for contamination with algae and microorganisms.