SUPPLEMENTARY METHODS
Aeroponics system details and optimizations
Materials used
Each customized aeroponics system (Fig. 2a,b) was composed of six large 133 L
(90 cm deep) black plastic barrels deep enough to allow roots to grow to a maximum of
45 days in the dark without twisting at the bottom. Since nutrient salts are corrosive, we
used plastic pipes, fittings, pump impellers and tanks. An Epoxy-encapsulated feedback
pump was chosen which had a mesh screen over the inlet to reduce debris intake. The
main submersible pump distributing the nutrient solution to the sprinklers was made of a
rugged thermoplastic body and a garden hose adapter to ease the connection with the
main pipe. Black plastic pipes [1.5 cm outer diameter (OD)] were used for the main
distribution line and feedback system. Drip tubes (6 mm outer diameter) were used to
connect the main line to the sprinklers via pressure regulators which were chosen to
match the flow rate of the microjets (98 litres per hour). Drip tubes were used at the
bottom of each barrel to allow recycling of the nutrient solution to the feedback pipe. All
building components were cleaned with antibacterial detergent and bleach; before the
first usage, pipes were flushed with high pressure water to eliminate construction dust
and microbes.
Closed recirculating system
It was critical to have a constant rate of refilling of the nutrient tank without
solution accumulation at the bottom of each barrel. This was achieved through a
combination of optimal feedback pipe diameters, gravity-return slope, pump flow rates
and pressure regulation. The slope of the feedback pipe was optimized to maintain the
flow rate of the feedback pump; in our case, a 2% slope was optimal for the feedback
pipe. The pipe connecting the feedback pump to the nutrient tank was 50 cm long
terminating 10 cm below the maximum height that the pump was capable of pushing
liquid against gravity; this ensured constant pressurized feedback flow.
Plant suspension design
Our plant suspension system was designed to ensure uniform darkness inside
the chamber verified by the fact that no root anthocyanin pigmentation was observed.
The bottoms of 13.8 cm plants pots were cut at 5.8 cm from the top, and bottoms were
replaced by nylon nets (0.6 cm x 0.3 cm) allowing roots to hang down from the top. Two
13.8 cm holes were cut per lid. The mesh size of the nylon net was chosen to hold the
seeds and white aquarium stones, while allowing the crown roots to go through. When
crown roots were blocked by too small a net mesh size (e.g. 0.2 cm x 0.2 cm), they split
into multiple small-diameter branches below the net leading to artefacts. White
aquarium stones were chosen to avoid nutrient accumulation in the crown region; in an
earlier experiment, calcium phosphate and sulphate were observed to precipitate when
we used Rockwool (Grodan®).
Optimization of root misting
In aeroponics, oxygen delivery is not a problem but delivering the nutrient
solution to the roots homogeneously and at optimal time intervals are crucial. Nutrient
delivery must be optimized for each genotype according to the root size and in some
cases developmental stage. It is also critical to have uniform misting among barrels.
This was achieved by matching the number of barrels with the pressure of the
submersible pump (e.g. 6 barrels were chosen instead of more). Two microjets were
used in each barrel, one flanking each of the two root systems. The flow rate at each
microjet was measured to be 16.5 mL s-1 ± 0.8. In order to ensure that each root
system was constantly moistened and to meet the plant transpiration demand at 30
days after planting, the frequency of misting was optimized. Various misting frequencies
were tested (10 s, 20 s, 25 s, 30 s per minute, and continuous); 10 s of misting per
minute was optimal. Another optimization parameter was the positioning of each
microjet relative to each root. We found that the best spray uniformity was created by
placing the microjets at the height equivalent to the bottom of the seed net, resulting in
spraying roots from above. Sprinklers with a 5-20 micron droplet size and 180°C
spraying pattern were cost effective, efficient and allowed uniformity. Using these
optimized conditions, plants exhibited no signs of water stress, accumulation of salts on
the root surface, and had low plant-to-plant variability. It is important to note that if
plants failed to be misted for >15 min, for example due to a power failure, the roots were
permanently damaged.
Nutrient solution optimization
The nutrient solutions were optimized for maize growth using aeroponics. We
first chose Hoagland’s solution (Hoagland & Arnon, 1950) as a base as it has been
empirically used in many maize nutritional studies using open semi hydroponic and
closed hydroponic systems (Wang et al., 2005, Liu et al., 2008, Vamerali et al., 2003).
Plant grown with Hoagland’s solution displayed symptoms of micronutrient deficiencies
and potentially calcium deficiencies. To increase calcium, the original nitrate component
was replaced by Ca(NO3)2 and NH4NO3, and to maintain the potassium concentration,
KNO3 was replaced by KCl. A ratio of 75% nitrate: 25% ammonium ions was maintained
in both high and low nitrogen nutrient solutions. The initial nutrient deficiencies observed
in aeroponics compared to other soil-less techniques, may have been related to
increased aerobic conditions in the rhizosphere which might increase root respiration
and growth, creating greater ion demand and absorption (Jones, 1997). Indeed
aeroponically-grown maize plants were large and grew fast. Rather than adjusting the
micronutrient content of the original Hoagland’s solution, we supplemented with a
commercial micronutrient mix. As iron deficiencies were observed, Fe-EDDHSA (Grabi
chemical) was added after testing various chelating agents.
Water quality was an important factor for pH management, to control ion
concentrations and prevent algal growth. Filtered deionised water was used to prepare
nutrient solutions which avoided contamination by various organic and inorganic
chemicals. As pH is critical for nutrient uptake, it was measured daily and maintained in
the 5.7-6.3 range using strong acids or bases; multiple chelators further buffered
against changes in pH. Finally, the temperature of the nutrient solution was checked
daily to ensure that the solutions were kept at ± 2°C from room temperature. Utilisation
of black barrels as nutrient solution tanks in a greenhouse is not recommended as the
nutrient solution temperature rose up to 5°C above room temperature in the summer. To
avoid this temperature rise, the solution tank covered with a white plastic garbage bag.
Nutrient solution replacement
The need for individual nutrients and their uptake were expected to change
dynamically throughout development. Unpredictable water loss (system leaks, evapotranspiration, seasonal variation) was expected to cause further variability in the nutrient
solution concentration. To address these issues, the nutrient solution was initially
monitored daily using Electrical Conductivity (EC) as a means to determine the renewal
frequency. The EC was observed to be constant (2.8 µs/cm ± 0.4) for up to only 6 days
when feeding twelve 30-day old plants. Hence, the nutrient solution was changed every
six days even when plants were younger. However, EC measurements do not indicate
which elements to replenish and at which concentrations. Furthermore, as our closed
aeroponics system was constantly recirculating, the nutrient solution was expected to
be altered by root decay and exudates, adding suspended precipitates, microorganisms
and algae. To overcome all of these issues, we decided to replace the entire 100 L
nutrient solution every 6 days throughout the experiment. Some growers consider
filtering, ozone or UV treatments to prevent biotic contamination. However, these
strategies are costly and labour intensive; keeping the nutrient solution in the dark,
extensive cleaning of the system prior to start the experiment and short-term use of
each nutrient solution helped cope with these problems.