The following protocol provides step-by-step instructions to set up a hydroponic system in a laboratory. This protocol has been optimized for Arabidopsis thaliana. One of the major factors that contributes to a successful establishment of a hydroponic culture is the health of seedlings used for the experiment.
Therefore, sterilization of instruments, seeds and culture media play an important role in reducing the risk of contamination and provide a good start for the plants before they are transplanted to the hydroponic system. In this step the chlorine gas released from the reaction between bleach and hydrochloric acid sterilizes the surface of the seeds. Before transferring seeds into a 1.5 milliliter centrifuge tube, use a pencil to label the tube and identify the seeds.
Pencil is preferred because ink may fade away during sterilization. Also mark the cap of the tube with a Sharpie. This mark should go away during the course of the sterilization process due to the chlorine gas released.
The recommended seed volume per tube is around 100 microliters. Place the labelled tube, cap open into the desiccator or vacuum chamber. Place the desiccator in an active chemical fume hood.
Pour 100 milliliters of bleach into a 250 milliliter beaker, place it in the desiccator and close the desiccator's valve. Quickly add 3 milliliters of 12%hydrochloric acid to the bleach using a burette. Close the lid immediately.
Allow the sterilization to proceed for four hours. Here is the desiccator in an active laminar flow hood after four hours of sterilization and the bleach was removed. The red mark fade away indicates a sufficient amount of chlorine gas has been generated.
Open the lid of the desiccator widely and aerate the sterilized seeds for at least 30 minutes. After this, seeds can be used immediately or stored in a dry place. This step helps to provide healthy seedlings prior to the transplanting phase.
These are the items needed to start the seed plates. Sterile seeds, 1/4 MS media plates, sterile filter paper, millipore tape, sterile toothpicks, marker pen, aluminum foil. First transfer seeds from the centrifuge tube to a sterile filter paper.
Slightly wet one end of the toothpick and use this moisturized end to pick up the seeds from the filter paper and lay them on the agar surface. Spread the seeds across the plate at a density of approximately one seed per square centimeter. Here we use only a few seeds to demonstrate the step but you can make a plate full of seeds.
Write the date on the plate's lid to keep track of the plant age. Use micropore tape to keep plate lid attached to the plate body. This tape also allows moisture exchange between the air and the microclimate inside the plate.
Use aluminum foil to cover the plate and block light before placing the plant in the cold room for two days. Longer times may be used to aid the germination process. Like the 1/4th MS, the hydroponic solution also needs to be sterilized after preparation.
We have successfully used the following nutrient solution to grow Arabidopsis thaliana but it could be modified to accommodate specific experiments or different plant species. It is suggested to prepare a stock solution of each macronutrient in different bottles and all micronutrients in one bottle, except for iron-EDTA. A 10x nutrient solution could be prepared in advance of the experiment, but needs to be autoclaved and stored in the cold room.
The following are materials you need to prepare plant holders. Hydroponics container, foam board, paper knife, cork borer, sterile foam tube plugs, tweezers. First measure the size of the container.
Then cut the foam board according to the dimensions needed. A paper knife is helpful in this case. Check of the size of the foam board fits well to the the container.
Use small tweezers to hold the board. Adjust the size of the foam board if needed. Use a pencil to mark positions where the plants will be anchored.
Use a cork bore to create holes in the foam board. Recommended density:One plant per 10 centimeters squared. Next make a cut running along each foam plug using a razor blade.
Use extreme caution when using razor blades. Each hole will be filled with a foam plug. Thus the size of the holes need to be proportional to the plugs.
Soak the two plugs in deionized water and liquid autoclave them prior to the hydroponics set up. Now transfer seedlings and set up the air pump. This is hydroponic solution at working concentration.
These are the sterile foam tube plugs. And these are the medium plate 12 day old seedlings. Open the plate.
Use small tweezers to gently pull a seedling out of the medium plate and lay the root along the incision of the foam plug. Carefully plug the foam tube holding the seedling into the foam board and place the board into the container with hydroponic solution. Keep transplanting to get desired number of plants.
Make sure the foam tube plugs are soaked with the hydroponic solution. Next we will show how to set up the aeration system, using small aquarium type air pumps, plastic tubing, aquarium bubble stones, valve system, and an air pump are needed. First connect the short, cut off part of the air line tubing to the bubble stones.
Then connect the stones to the valve system. Next connect the valve system to the pump. After activating the pump, the person in the scene is adjusting the valve to control the air flow rate.
The air flow should be just enough to provide oxygen without causing damage to the root system. Now place the foam board with seedlings into the hydroponic set up. Cover container sides to limit the light reaching the solution and limit the growth of algae.
Also cover the container tops with transparent caps to prevent intense light which could desiccate newly transplanted seedlings. Once the plants become bigger remove the caps. For researchers interested in plant nutrients hydroponic experiments provide an ideal system to test plant responses to different nutrient conditions by manipulating the concentrations of the elements of interest.
Researchers can set up nutrient sufficient, deficient, or toxic conditions. These are Arabidopsis seedlings grown in the hydroponic system as described in the protocol. Plants were allowed to grow for three weeks in the hydroponic system before changing the composition of the hydroponic solution.
The person in the scene is renewing the hydroponic solution before giving treatment to the plants. A stock solution of the toxic element cadmium was used to spike the hydroponic solution. The cadmium concentration used in this experiment was 20 micromolar.
After the solution is prepared, plants are then returned to their containers. After six days of treatment plants exposed to cadmium showed delayed growth and chlorosis symptoms. Reduction in both shoot and root biomass is also observed.
This figure shows a different experiment for Arabidopsis plants grown in the hydroponics system where treated with different concentrations of zinc. Plants grown at zinc concentrations higher than 42 micromolar, showed delayed growth due to high zinc concentrations. While plants without added zinc also showed delayed growth due to zinc deficiency compared to plants grown with 7 micromolar of zinc.
In conclusion, this hydroponic system is both inexpensive and scalable. It allows us to test plant sensitivity to toxic elements or study plant nutrient requirements. Both roots and shoots can be observed and collected separately for elemental analysis or gene expression analysis.
