Table 2.

Troubleshooting table.

Procedure	Step	Problem	Possible reason	Solution
Box 2	6	Poor germination rate	Sterilization is too harsh.	Reduce exposure to chlorine gas or reduce the amount of HCl added to the Erlenmeyer flask. Verify germination rate on 1/2X LS plates without sucrose.
			Seeds storage suboptimal.	Ensure seeds are stored in the dark with low humidity. Verify germination rate on 1/2X LS plates without sucrose.
Box 4	1	Seeds are contaminated	Seeds were not properly decontaminated.	Use fresh bleach during sterilization and ensure adequate sterilization time. Alternatively, increase the exposed surface area of seed aliquots by reducing the number of seeds in each tube. It may be necessary to empirically determine the duration of sterilization based on the volume of seeds in individual aliquots. Alternatively, surface sterilize seeds with Ethanol solution (50% Ethanol (v/v) +0.01% Triton X-100) for 5 min, followed by fresh bleach solution (10% commercial bleach+ 0.01% Triton X-100) for 5 min and wash 4 times with sterile Milli-Q water.
	3	Plants or substrate are contaminated	Substrate was not properly sterilized.	Ensure sufficient time between autoclave cycles (24-48 h) during substrate sterilization. The rest provides an opportunity for dormant spores to germinate, which can then be killed during the second autoclave cycle.
			Tissue culture box not sealed.	Check filters and seals of Microbox. Discard if damaged.
			Water or media was contaminated during irrigation or inoculation.	Ensure clean, undamaged glassware and proper sterile technique are being used.
1 - FlowPot	12	Microbox becomes deformed after autoclaving.	Lid was sealed on the Microbox while autoclaving.	Do not seal lids while autoclaving and follow manufacturer’s instructions for Microbox sterilization.
	15	Liquid is unable to be passed through the FlowPot.	Glass beads or debris are obstructing the irrigation port.	Insert a sterile needle into the bottom of the FlowPot to clear the irrigation port.
	15	The mesh retainer is dislodged from the FlowPot.	Glass beads or debris are partially obstructing the irrigation port, resulting in increased backpressure.	Insert a sterile needle into the bottom of the FlowPot to clear the irrigation port.
			Substrate is packed too tightly into the FlowPot, resulting in increased back pressure.	Assemble FlowPots using less substrate and do not pack.
			The nylon cable tie relaxed during autoclaving.	Do not fully tighten cable ties until after autoclaving or use a cable tie gun. Alternatively, change the material of the cable ties and avoid nylon.
	20	Plants appear chlorotic	Recalcitrant byproducts of sterilization may be deleterious to plant growth.	In our experience, using 50-60 °C water to flush the substrate improves performance if plants appear chlorotic.
	20	Excess condensation builds up on the walls of the Microbox during plant growth.	Too much moisture is contained within the system.	The depth filter of the described Microbox is hydrophobic, so water is generally retained within a sealed box for quite some time. Decrease the amount of FlowPots per box to reduce excess moisture. We recommend no more than four FlowPots per Microbox.
			Temperature fluctuations within the Microbox are increasing the rate of evaporation from FlowPots.	Maintain constant temperatures within the Microbox. Adjusting day/night temperatures can help account for the radiant energy absorbed by the Microboxes from the light source. Alternatively, we found that placing growth chamber temperature probes into sealed Microboxes with humidity levels corresponding to those of boxes growing plants helped mitigate temperature fluctuations. Additionally, insulating Microboxes from metal surfaces by placing them on matte black ceramic tiles may also help reduce thermal fluctuations and the build up of condensation.
	20	Plants are water stressed.	Substrate is drying out.	Aseptically apply ~8 mL sterile water with a needle and syringe to the center of each FlowPot, avoiding damage to plant roots. We have found that watering plants after sowing is generally unnecessary and that the substrate sustains plant growth for at least 8 weeks without intervention. In our experience, the drying of substrate is usually due to temperature fluctuations increasing the rate of evaporation. The water is usually retained within the system on the bottom or sides of the Microbox as the filter is hydrophobic.
	20	Plants are nutrient stressed.	Nutrients are depleted from the substrate.	Apply ~8 mL 1/2X LS media with a needle and syringe to the center of each FlowPot as needed. We have found that supplementing FlowPots with nutrients after sowing is generally unnecessary and that the substrate sustains plant growth for at least 8 weeks without intervention.
	20	Plants fail to germinate.	Seeds sterilized for too long.	Decrease sterilization time. It may be necessary to empirically determine the duration of sterilization based on the volume of seeds in individual aliquots. We found that 6 h sterilization time had no adverse effect on germination rate. Alternatively, liquid bleach can be used to sterilize seeds.
2 - GnotoPot	2	Occasionally, the dry pellets do not fully expand.	Variance in manufacturing.	Visually inspect the dry pellets before placing inside the 2 inch pots to make sure that the mesh covering peat is not ruptured. Also after full expansion make sure that the central part of the Jiffy-7® is accessible for placing the seed at the time of seed sowing. If the central hole is not accessible use a pair of tweezers to manually open up the mesh.
	26	Too much algal growth inside the box.	Excess of microbial community inoculum from soil.	Use a smaller amount of the soil for preparing the soil tea. Also make sure that the soil inoculum is only added to the central area of peat pellet
	26	The peat pellet looks dry after 6 weeks.	Excessive water loss	Although the GnotoPots perform very well in retaining the moisture, depending on the changes in the seasonal ambient humidity levels in different geographical locations some pots might have a drier peat pellet later in the growth phase. To hydrate these pots simply raise one side of the Microbox to about 10 degree angle to move the sitting solution to one side of the box and wait for 1-2 minutes. Perform this step for all the relevant controls as well.