2.1. Plant Material and Growth Conditions
Five wheat cultivars with apparent differences in total root biomass and cumulative root length at the onset of tillering (Z2.1) from a previous semi-hydroponic phenotyping study were used in this study. The cultivars were Ghurka and Bahatans-87, released in 1916 and 1924, respectively; Hartog, released in 1983; and Tincurrin and Harper, released in 1978 and 2010, respectively (Table 1
The five cultivars were grown in 24-L glass-walled rhizoboxes (0.24 m long × 0.10 m wide × 1.0 m deep) filled with soil. Plyvinyl chloride (PVC) rhizoboxes have been previously described in root studies [12
]. Briefly, the acrylic glass side was covered with a removable, black PVC sheet to avoid light exposure to the roots. The rhizoboxes were placed on steel stands at a 30° angle to force the roots to grow along the acrylic glass side as described elsewhere [12
]. The visibility of the root system does not differ between rhizoboxes angled of 30 or 45° when seeds are sown in contact with the acrylic glass side [17
]. The 30° inclination facilitates root system mapping when using large rhizoboxes [24
Top soil (0–15 cm) was collected from a field site at Cunderdin (31°64′N, 117°24′E), Western Australia, and was classified as a reddish-brown sandy clay loam: Red Calcic Dermosal [25
]. The soil consisted of 63.5% sand with a particle size between 0.010–0.020 inches, 8.3% silt and 28.3% clay with a pH, measured in a 1:5 suspension of soil in 0.01 M CaCl2
, of 6.0. The soil contained 6 μg g−1
of nitrate–N, 4 μg g−1
of ammonium–N, 46 μg g−1
of Colwell P and 691 μg g−1
of Colwell K [15
]. Air-dried soil was sieved to 2 mm and mixed uniformly with 25% in (vol.) of washed and air-dried river sand to improve drainage [15
]. The soil was packed to a bulk density of approximately 1.56 g cm−3
]. Compound fertiliser equivalent to 65 kg ha−1
N, 79 kg ha−1
P, 71 kg ha−1
K and trace amounts of micronutrients (S, Cu, Zn, Mo and Mn), optimal amounts for wheat grown on Cunderdin soils [26
], was mixed homogeneously into the top 0.1 m of soil in each rhizobox before sowing.
The experiment was conducted in an evaporatively cooled glasshouse at The University of Western Australia, Perth, Australia (31°93′S, 115°83′E) from May to July 2017 with an average air temperature of 16 °C, maximum temperature of 22 °C, minimum temperature of 9 °C, relative humidity of 61% and 10–11 h of natural light. The five cultivars were grown in a completely randomized block design with three replicates.
Four homogenous pre-germinated seeds were sown equidistant from each other at a depth of 2 cm in each rhizobox, ensuring that the seeds were in contact with the acrylic glass wall. This corresponded to a field sowing density of 160 plants m−2
]. The plants were hand-watered as required with tap water to maintain the soil water content close to field capacity and to avoid excessive drainage. Plant phenology was monitored regularly using the scale of Zadoks and Chang [29
]. The experiment ended at 63 days after sowing (DAS) when some roots had reached the bottom of the rhizoboxes.
A separate, but simultaneous experiment was conducted to monitor, in detail, the phenological development in each cultivar. The experiment was conducted in pots (25 cm diameter, 30 cm height), with eight plants per pot, corresponding to a field sowing density of 160 plants m−2
], using the same five cultivars, the same soil and similar a plant density as those used in the rhizobox study and grown in the same glasshouse with similar temperature, light and watering conditions. Time to tillering (Z2.1), time to booting (Z4.9) and time to anthesis (Z 6.1) were recorded [29
] (Table 2
). Differences in the phenological development between plants grown in pots and rhizoboxes were not expected to be due to the differences in soil volume between the pot experiment (15L) and the rhizoboxes (24L) [30
], since phenology in wheat is driven by temperature and natural light [32
2.2. Root Traits: Non-Destructive Measurements
Root growth was monitored weekly from seven DAS through the glass wall of each rhizobox by removing the black PVC cover sheet. Visible new roots were marked on transparency films using a black waterproof, permanent pen. Immediately afterwards, the visible new roots were also marked on the acrylic glass of the rhizobox so that new root growth could be recognized at the next measurement. The glass wall was covered with the black PVC cover sheet immediately after the glass was marked. The transparent film was scanned at 600 pixels per mm using a portable scanner (Jenkins PS4100: East Bentleigh, Vic, Australia) and root images were analyzed for root length using WinRhizo Pro software (v2009, Regent Instrument, Quebec, QC, Canada).
2.3. Destructive Measurements
When the experiment ended at 63 DAS, destructive above-and below-ground measurements were taken. The plants in each rhizobox were harvested by cutting the shoots from the roots at the crown. Aboveground measurements included shoot biomass, leaf area (LA), leaf biomass, specific leaf area (leaf area per unit leaf weight, SLA), tiller number, and plant height. Leaf area was measured using a portable leaf area meter (LI-3000, Li-COR Biosciences, Lincoln, NE, USA). Shoots, spikes and leaves were separated and oven-dried at 70 °C (Heratherm OMS 100, Thermo Scientific, Langenselbold, Germany) before being weighed on a precision balance (Voyager®, Ohaus Corporation, Parsippany, NJ, USA).
Destructive belowground measurements included root length, root biomass, root length density (root length per unit of soil volume; RLD) and specific root length (root length per unit of biomass; SRL). Each rhizobox was opened by removing the glass wall, and the soil profile was sampled in 0.2 m sections from the top by cutting the soil with a carbon steel filling blade. The roots in each section were recovered from the soil by washing through a 1.4-mm sieve to produce a clean sample Palta and Fillery [34
]. The recovered roots from each 0.2-m soil section were placed in plastic bags and stored at 4 °C until being scanned at 400 dpi per mm (Epson Perfection V800, Long Beach, CA, USA) to quantify root morphological traits. The root samples were dried and weighed after scanning as per the shoot samples. Root images were analyzed using WinRHIZO Pro software (v2009, Regent Instrument, Quebec, QC, Canada) [35
]. The total root length density was calculated as the total root length divided by the soil volume. The distribution of RLD in the soil profile was calculated as the root length in 0.2 m sections from the top to the bottom of each rhizobox divided by the soil volume of the corresponding section (0.0024 m3
). The specific root length (SRL), an indirect measure of the thickness of the root system, was estimated as the total root length divided by the total root biomass [8