TITLE: Closed-chamber hydroponics for whole-plant phenotyping 
AUTHORS: Daniel Ginzburg, Jack Cox, Seung Yon Rhee

[DESCRIPTION]
Noninvasive phenotyping can quantify dynamic plant growth processes at higher temporal resolution than destructive phenotyping and can reveal phenomena that would be missed by end-point analysis alone. Additionally, whole-plant phenotyping can identify growth conditions that are optimal for both above- and below-ground tissues. However, noninvasive, whole-plant phenotyping approaches available today are generally expensive, complex, and non-modular. We developed a low-cost and versatile approach to non-invasively measure whole-plant physiology over time by growing plants in isolated hydroponic chambers. We demonstrate the versatility of our approach by measuring whole-plant biomass accumulation, water consumption, and water use efficiency every two days on unstressed and osmotically-stressed sorghum cultivars. Our system can be implemented using cheap, basic components and requires no specific technical expertise. It can, in theory, be used for any non-aquatic vascular plant species.

[GUIDELINES]
This protocol has been designed for Sorghum bicolor (sorghum) seedlings at the third leaf stage. All aspects of the protocol (tube size, nutrient concentration, age of switching from open- to closed-hydroponics, frequency of measuring biomass accumulation and water consumption, etc.) can be modified to suit different species, developmental stages, or biological questions.

[STEPS]
SECTION: Closed-system hydroponic growth apparatus construction
9. Using a 21/64 inch drill bit, drill holes into the centers of 50mL Falcon tube caps.
SECTION: Pre-phenotyping hydroponic growth conditions
16. Ensure that the drainage height of the ebb & flow system reaches the bottom ~2-3cm of the cut pipette tips during irrigation.
SECTION: Transitioning samples from open- to closed-hydroponic conditions
22. Once 50% of samples reach a pre-determined stage of development (i.e. the third true-leaf stage), prepare a bulk nutrient solution (EC of 1200 mS/cm) such that 40 mL of solution can be allocated for each sample in 50mL Falcon tubes.

SECTION: Quantifying biomass accumulation, water use, and change in media pH
30. Every two days, prepare fresh nutrient solution and measure pH as described above for both control and osmolyte-supplemented solution.
SECTION: Determination of evaporative loss in closed-hydroponic system:
39. Grow plants hydroponically in soil-filled pipette tips as described above.
SECTION: Closed-system hydroponic growth apparatus construction
11. To prevent unnatural water retention and pressure buildup in the filled tips, create pinholes with a needle or small pair of scissors into the tips ~25% from the bottom.

SECTION: Closed-system hydroponic growth apparatus construction
10. Using scissors, cut 1250 uL pipette tips ~3 cm from the tip (narrower) end and insert cut tips into the pre-drilled Falcon tube caps so that the tips fit snugly into the holes in the caps and rest almost flush with the top of the cap.
SECTION: Closed-system hydroponic growth apparatus construction
12. Using scissors, cut Rapid-Rooter starter plugs (General Hydroponics, Santa Rosa, CA, USA) into rectangular slices of ~0.5cm length and ~0.9cm width and insert 1 slice into the top of each tip and then gently push slices to the bottom of the pipette tip. 

SECTION: Closed-system hydroponic growth apparatus construction
13. Fill caps with tips with potting soil. Ensure the soil is not overly compact as this will inhibit seedling growth. Additionally, ensure that there are no clumps in the soil which would prevent water from reaching the soil from below due to capillarity.  

SECTION: Closed-system hydroponic growth apparatus construction
14. Place soil-filled caps with tips into tube racks, and leave to soak overnight in tap water which reaches the bottom of the tips but does not submerse the caps.
SECTION: Closed-system hydroponic growth apparatus construction
15. The next day, plant individual sorghum seeds into soil-filled tips and then put all samples into the ebb & flow system.

SECTION: Construction and operation of ebb &amp; flow system for &quot;open&quot; hydroponic cultivation
1. Drill a ¾ inch diameter hole into opposite corners of an 18 gallon plastic tote (reservoir).
SECTION: Construction and operation of ebb &amp; flow system for &quot;open&quot; hydroponic cultivation
2. Place the reservoir with holes drilled onto a second, 18 gallon reservoir without holes.

SECTION: Construction and operation of ebb &amp; flow system for &quot;open&quot; hydroponic cultivation
3. Insert a ¾ inch threaded and barbed bulkhead fitting (Botanicare, Vancouver, WA, USA) from above into one of the drilled holes and fasten from below with a rubber washer. 

SECTION: Construction and operation of ebb &amp; flow system for &quot;open&quot; hydroponic cultivation
4. Water in an ebb and flow system needs to reach, but not submerge, the seedlings in the upper reservoir. Thus, to increase the irrigation height the water reaches in the upper reservoir before draining back down, screw a threaded height extender (Botanicare, Vancouver, WA, USA) into this first hole from above.

SECTION: Construction and operation of ebb &amp; flow system for &quot;open&quot; hydroponic cultivation
5. Fit a threaded, plastic debris screen (Botanicare, Vancouver, WA, USA) into the second hole which was drilled into the upper reservoir to allow water to gently pump up into the top reservoir from below. 
SECTION: Construction and operation of ebb &amp; flow system for &quot;open&quot; hydroponic cultivation
6. Place an 8W, 800 liter/hour submersible pump in the bottom reservoir and connect the pump to the barbed end of the bulkhead fitting with PVC plastic tubing.  

SECTION: Construction and operation of ebb &amp; flow system for &quot;open&quot; hydroponic cultivation
7. In order for water to constantly recirculate during irrigation, sufficient water must be added to the ebb & flow system to allow for filling the top reservoir up to the drainage height and to ensure enough water is in the bottom reservoir to be pumped back up.
 

SECTION: Construction and operation of ebb &amp; flow system for &quot;open&quot; hydroponic cultivation
8. To control the duration and number of irrigation events each day, connect the pump to a digital programmable timer.
SECTION: Pre-phenotyping hydroponic growth conditions
17. Before seedling emergence, program the timer to irrigate samples three times daily with tap water for 5 min every 480 min.

SECTION: Pre-phenotyping hydroponic growth conditions
18. Upon root emergence (root tips growing below the bottom of the pipette tip) of 50% of the samples, adjust the programmable time to irrigate seedlings every hour with a single, 15 min irrigation event. Once roots emerge from below the soil-filled pipette tips, the primary source of water for the roots will no longer be water held within the soil but rather the recirculating water of the ebb & flow system. It is therefore important to ensure exposed roots are irrigated frequently to prevent desiccation, but not constantly to encourage downward root growth and to increase root oxygenation.
SECTION: Pre-phenotyping hydroponic growth conditions
19. At this point, add soluble, all-purpose fertilizer to the lower water reservoir using a plastic scoop up to an electrical conductivity (EC) of 1200 mS/cm while the water is being circulated. Measure EC with a waterproof electrical conductivity meter.


SECTION: Pre-phenotyping hydroponic growth conditions
20. Upon root emergence of all samples, increase the duration of irrigation events to be 45 min every hour followed by 15 minof no irrigation to increase root zone oxygenation.

SECTION: Pre-phenotyping hydroponic growth conditions
21. Empty the reservoir every 3-4 days and refill it with fresh nutrient solution.
 



SECTION: Transitioning samples from open- to closed-hydroponic conditions
25. Fill 50 mL tubes with 40 mL of either control or osmolyte-supplemented nutrient solution and then wrap tubes in aluminum foil to prevent algal growth.

SECTION: Transitioning samples from open- to closed-hydroponic conditions
23. If evaluating the effects of osmotic stress on plant growth, split the bulk nutrient solution into two and add an osmolyte (i.e. mannitol) into one of the solutions up to a pre-determined concentration (i.e. 10mM).
SECTION: Transitioning samples from open- to closed-hydroponic conditions
24. Record the pH of both control and osmolyte-supplemented solutions after fertilizer and osmolytes have been added.
SECTION: Transitioning samples from open- to closed-hydroponic conditions
26. Without caps on, weigh foil-wrapped tubes with solution.
SECTION: Transitioning samples from open- to closed-hydroponic conditions
27. Firmly screw caps containing seedlings onto individual tubes.
SECTION: Transitioning samples from open- to closed-hydroponic conditions
28. Weigh tubes with seedlings screwed on to determine initial seedling weight. If the roots of any sample do not reach the nutrient solution when fully closed onto the cap, add additional solution to the tube and reweigh with and without caps. If roots do not reach the water level in the tube, plants will desiccate.
SECTION: Transitioning samples from open- to closed-hydroponic conditions
29. Evenly space out samples into 50mL tube racks and place them in their designated growing environment (i.e. a greenhouse).
SECTION: Quantifying biomass accumulation, water use, and change in media pH
31. Unscrew seedlings and caps from their tubes and place seedlings back into the ebb & flow system (operating with constant irrigation) to keep the roots hydrated while tubes are weighed. Be very gentle when removing plants from tubes and putting them into racks in the ebb & flow system as roots will be exposed and thus vulnerable to mechanical damage.
SECTION: Quantifying biomass accumulation, water use, and change in media pH
32. Screw 50mL tube caps without holes onto each tube to prevent evaporation. One sample at a time, remove the cap and weigh the tube. Before screwing the cap back onto the tube, measure pH of the solution in the tube. Then screw the cap back onto the tube and repeat for all tubes.
SECTION: Quantifying biomass accumulation, water use, and change in media pH
33. Empty nutrient solution from each tube and then refill each with either fresh control or osmolyte-supplemented solution up to 40 mL as described above.

SECTION: Quantifying biomass accumulation, water use, and change in media pH
34. Without caps, weigh tubes again and then screw caps back on to prevent evaporation while seedlings are weighed.
SECTION: Quantifying biomass accumulation, water use, and change in media pH
35. Carefully remove seedlings from the ebb & flow system. Weigh each seedling and then screw it back onto its corresponding tube before returning samples to their growing environment.  

SECTION: Determination of evaporative loss in closed-hydroponic system:
40. When plants reach the stage at which they are transferred from the ebb & flow system to closed tubes, cut off the roots and shoots from the bottom of the tips and the base of the cap, respectively to prevent plant water uptake and transpiration.
SECTION: Determination of evaporative loss in closed-hydroponic system:
41. Fill an equal number of tubes as there are cut-off samples with 1200 mS/cm nutrient solution and weigh the tubes as described above.
SECTION: Determination of evaporative loss in closed-hydroponic system:
42. Screw caps from which roots and shoots were cut off onto nutrient-filled tubes.
SECTION: Determination of evaporative loss in closed-hydroponic system:
43. Place these samples in the same growing environment as all other samples.
SECTION: Determination of evaporative loss in closed-hydroponic system:
44. Each day (for the duration of the growth experiment), remove caps from tubes weigh tubes to determine average daily and cumulative water loss due to evaporation.
SECTION: Quantifying biomass accumulation, water use, and change in media pH
36. Biomass accumulation is calculated as the seedling weight at the current time of measurement minus the seedling weight at the previous time of measurement.
SECTION: Quantifying biomass accumulation, water use, and change in media pH
37. Water consumption is calculated as the tube weight at the previous time of measurement minus the tube weight at the current time of measurement.
SECTION: Quantifying biomass accumulation, water use, and change in media pH
38. Change in media pH is calculated as the pH of fresh media minus the pH of media in tubes after plant growth.