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Protocol

Plants are grown outdoors in lysimeters, i.e., PVC tubes or either 25 cm in diameter and 2.0 m length or 20 cm diameter and 1.2 m length that are filled with a sandy clay loam Alfisol at a bulk density of approximately 1.35 g cm-3, which is a standard value for Alfisols. A set of 20cm diameter and 1.2 m length tubes are also available with Vertisol. Before filling the tubes, the soil is sieved to particles smaller than 1 cm to ensure homogeneous packing of the soil into the tube. Plant spacing and soil volume available for water extraction in the lysimetric system are similar to those in field conditions.

The cylinders can be lifted and weighed with a block-chained pulley and an S-type load cell of either a 100-kg or 200-kg capacity (Mettler-Toledo, Geneva, Switzerland)and an accuracy of 10 or 20 g. The lysimeters are separated from one another by a distance of approximately 3-5 cm; thus, the plant density is approximately 10 plants m-2 in the large tubes and 20-25 plants m-2 in the small tubes, and then is similar to typical field plantings densities. This allows an accurate assessment of the water extraction pattern of a crop cultivated under conditions similar to those in the field.

Seeds are sown in 3-4 hills in each cylinder. The seedlings are thinned to 2-3 seedlings per cylinder at 10-14 days after sowing (DAS) and later thinned to one plant per cylinder at 15-20 DAS. Plants are initially fully irrigated and this period of time is used to bring up the soil at field capacity. Plant transpiration assessments usually start at about 3 weeks after sowing. At this stage, the cylinders are covered with a plastic sheet (except in the case of groundnut) and a 2-cm layer of low-density polyethylene beads to prevent soil evaporation. Weighing is usually done every week or fortnight. Water treatments are specific to crops and desired stress scenarios, the simplest one being a terminal stress consisting of stopping irrigation at some stage (e.g. 3 weeks after sowing in chickpea, 5 weeks after sowing in sorghum).

Transpiration is calculated from cylinder weight differences and water additions. At harvest, above ground biomass is separated into the grain and vegetative biomass parts. Transpiration efficiency (TE) is then calculated as the ratio of the total biomass by the cumulated transpiration (multiplied by 1000 to express it in g biomass kg-1 water transpired). The biomass at 21 DAS is quite small and still leads to a slight over-estimation of TE. However, at 21 DAS the biomass differences between genotypes are small so that genotypic differences in TE are unlikely to be related to growth differences in the initial stages. Roots are not extracted from the cylinders at harvest to keep the lysimeter soil undisturbed. While not including the root biomass in the TE calculation would, in turn, lead to a slight under-estimation of TE, we discussed earlier that omitting the roots was unlikely to alter the genotypic differences in TE (Vadez et al., 2011a Crop and Pasture Science, 62 (8) 1-11, Vadez et al.,2011b Functional Plant Biology 38, 553-566). Water extracted from the cylinders (as a proxy for the root depth or root water extraction capacity) is then computed (under water stress conditions) as the difference between the initial lysimeter weight (which represent the cylinder weight at field capacity) and the final cylinder weight at harvest. The total water used to support plant transpiration throughout the crop life cycle is the sum of all transpiration data taken in the course of the crop. Pre- and post-anthesis water extraction can be computed as the sum of the transpiration values until and after anthesis respectively.

Data Analysis

An excel file with the following sheets: Details (date of different operations, germplasm used, any detail), Weight (cylinder weights), Transpiration (calculated from weight differences and water additions), Water To Add (a theoretical calculation of how much water needs to be added based on the treatments that are decided upon – see formulae – Essentially, water is added back to the cylinder on the basis of the initial weight at field capacity, deducting 2 kg to ensure that the added water does not drain), Water Added (a rounding to usually the nearest 500 g of how much water should be added – see formulae), NTR (a sheet where all data on transpiration, phenological recording, harvest measurements, and where computation of indices such as TE are made). This file is used during the experiment to store the cylinder weight data, to calculate the re-watering quantities for each cylinder, and to record any experimental detail.

Step 1 – Calculate transpiration, cumulated transpiration and water extraction

Transpiration is the difference between consecutive weights plus water added since previous cylinder weighing (T = Weight dayn-1 – Weight dayn + Water Added between dayn-1 and dayn). Cumulated transpiration is the sum of all transpiration values (see ‘Transpiration’ sheet for Excel formulae). Water extracted is the difference between the first cylinder weight and the last cylinder weight (see ‘Weight’ sheet).

Step 2 – Perform additional calculations in the ‘NTR’ sheet

Copy the values of the ‘Transpiration’ sheet into the NTR sheet. This is to avoid working with Excel formulae. Harvest and phenological observation data can be added in the sheet (making sure data are merged by unique cylinder identifiers). Total biomass is calculated as the sum of vegetative and reproductive biomass (eg Leaf DW + stem DW + panicle DW for cereals, leaf DW + stem DW + pod DW for legumes). In the case of groundnut, pod weights are multiplied by a factor of 1.65 to reflect the higher energy requirement of oily content of groundnut pods. TE is then computed as the ratio of total biomass divided by cumulated transpiration (multiplied by ‘1000 to express it in g biomass kg-1 water transpired) – See ‘NTR’ sheet for formulae.

Step 3 – Enjoy playing with data!

Run regressions and relationship, discover new trends, test crazy ideas!!!

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