Procedures for Measuring Dry matter, Nutrient uptake and Yield in Soybean

Achim Dobermann

Dept. of Agronomy and Horticulture

University of Nebraska-Lincoln

Objective

· To quantify dynamics of dry matter and aboveground plant nutrient accumulation, yield and maximum plant nutrient accumulation of soybean.

Definition of Growth stages

Determine the actual growth stage by examining about 10 randomly selected plant per plot. Leaf senescence is sometimes hard to see. For that reason, seed size within pods at R 6.5 and pod color at R 6.75 are perhaps easier signs to use than leaf color. To discern between normal plant senescence and premature plant death: leaf petioles of plants that have died due to stress (e.g. heat, drought, frost, etc.) remain attached; they do not drop off easily.

VE	Emergence
VC	Cotyledon (lowest stem node - identifiable via two opposing abscission scars)
V1	First node: fully developed leaves at unifoliate nodes (two opposite scars)
V2	Second node: fully developed trifoliate leaf at node above the unifoliate leaf
V3	Third node: three nodes on the main stem with fully developed leaves
….
R1	Beginning bloom: one open flower at any node on main stem
R2	Full bloom: open flower at one of the two uppermost nodes on main stem
R3	Beginning pod: pod 5 mm long at one of the four uppermost nodes on main stem
R3.5	Mid-pod elongation: pod 10-12 mm long at one of the four uppermost nodes on main stem. Vegetative stem and lead development ceases in indeterminant cultivars.
R4	Full pod: pod 20 mm at one of the four uppermost nodes on main stem
R5	Beginning seed: seed 3 mm in a pod at one of the four uppermost nodes on main stem
R6	Full seed: One pod in the top four nodes contains a seed that fills the entire seed cavity. (15% dry matter accumulated, about 18 d to R7)
R6.5	All the normal pods on the top four nodes have pod cavities completely filled. Leaf senescence (yellowing) begins in the lower canopy just before this and progresses upward. (50% dry matter accumulated, about 9 d to R7)
R 6.9	Maximum dry matter and nutrient accumulation (2-4 d to R7). Pods on all positions on the stem are losing green color. At least one normal pod on the main stem has turned yellow, but only few leaves and petioles have dropped. The membrane between pod wall and seed easily separates from the seed. When the membrane easily separates from the seed, transfer of C and N to the seed has slowed or ceased.
R7	Physiological maturity (PM, as used by breeders): one normal pod on main stem has reached its mature pod color, i.e., turned brown.
R8	Full, harvestable maturity (HM): 95% of the pods have turned brown.

typical Sampling stages for biomass and nutrient uptake studies

V3 Three nodes ~ late May

V6 Six nodes ~ mid June

R1 Beginning bloom: one open flower at any node ~ early July

R3.5 Mid-pod elongation: pod 10-12 mm ~ mid August

R6.9 Maximum nutrient uptake ~ early September

R8 Full maturity (Harvest) ~ late September

Materials

Paper bags, plastic, cloth or nylon mesh bags for harvest

Harvest: Cloth harvest bags for seed; paper sacks for residual dry matter

Wire tags with plot labels

Twine for bundling plants

Balance with 0.01 or 0.1 g accuracy for fresh and dry weight determination

Drying oven, plant grinder

Seed counter

Grain moisture meter

25 ml plastic (HDPE) bottles or containers for sample storage

Labels

Sampling principles

Composite samples of two 1 m row segments (2-m sample) within an experimental plot are used to estimate dry matter (DM) and plant nutrient accumulation during vegetative and early reproductive growth (Fig. 1). A composite sample of two 0.5 m row segments at R6.9 stage (close to physiological maturity) is used to determine nutrient concentrations in seeds, podwalls, and vegetative parts and to obtain the harvest index at the stage of maximum biomass accumulation. At R8 stage (maturity), plot seed and haulm (stems, leafs, podwalls) yields are measured from a larger harvest area (default: two 30 ft row segments).

Maximum plot dry matter yield is estimated from the seed yield measured at harvest and the harvest index obtained from the biomass sample collected at R6.9 stage. Maximum plant nutrient accumulation in seed, podwalls, and vegetative parts is then calculated from nutrient concentrations measured in the R6.9 samples and the estimated maximum dry matter fractions. The default values in the spreadsheet template refer to typical experimental plots (Fig. 1) with 8 rows planted at 30” (0.762 m) row spacing, but different row spacing and sample sizes can be entered.

Fig. 1. Sampling areas within a typical experimental plot (50’ x 20’, 8 rows @ 30’’ row spacing).

Sampling for measuring dry matter and nutrient accumulation at V3 to R3.5 stages

Identify the appropriate sampling date (growth stage) by regularly checking plants. At the time of sampling, collect all aboveground biomass from a pooled 2-m row biomass sample (B). Twice throw a 1 m metal bar or wooden stick along a row (typically rows 2 or 3 in an experimental plot, Fig. 1) to select two 1-m row segments with homogeneous and representative crop stand. Avoid areas close to the plot border or areas skipped by the planter or sampled earlier. Cut plants in each 1-m segment below the cotyledon node scars. Pool the two 1-m samples into one composite 2-m sample per plot and place all plants head down in large paper or cloth bags. Avoid leaf losses, long exposure of samples to sun, or contamination by soil by processing the samples immediately.

Count and record the number of plants in the 2-m sample (nPlant). Weigh the whole sample to obtain the total fresh weight (FW_B, g). Tare the bag before weighing. Take a representative subsample of 400 to 500 g immediately after weighing the total fresh weight to avoid moisture loss and record the fresh weight of the subsample (FW_Sub, g). Dry subsamples at 70 ºC to constant weight. Avoid overpacking the drying oven so that good air circulation is maintained. After 10 days check the weight of 3 samples daily to identify when a constant weight is achieved. Remove samples from drying oven and weigh immediately to record the final oven-dry weight of the subsample (DW_Sub, g). Grind samples and transfer ground material to pre-labeled 25-ml HDPE bottles.

Sampling for measuring the harvest index and plant nutrient concentrations at r6.9 stage (maximum nutrient uptake)

Identify the appropriate sampling date (growth stage) by checking plants on a daily basis. Harvesting this sample at the correct maturity stage and before significant leaf loss has occurred is critical for obtaining good estimates of the harvest index and maximum nutrient uptake.

Collect all aboveground biomass from a pooled 1-m row sample. Twice throw a 0.5 m metal bar or wooden stick along the row bordering the final harvest area (typically row 5 in an experimental plot, Fig. 1) to select two 0.5-m row segments with homogeneous and representative crop stand. Avoid areas close to the plot border or areas skipped by the planter or sampled earlier. Carefully cut plants in each 0.5-m segment below the cotyledon node scars. Pool the two 0.5-m samples into one composite 1-m sample per plot and place all plants head down in large paper or cloth bags. Caution: leaf losses may occur easily at this stage or may have already occurred due to senescence. Collect all dropped leaves from the soil surface within the 0.5-m sampling areas and add them to the sample, but avoid contaminating the sample with soil. Avoid long exposure of samples to sun by processing the samples immediately.

Count and record the number of plants in each 1-m biomass sample (nPlant_B). Clip pods from the stems and separate each sample into two samples: one containing all vegetative plant parts (stems, petioles, leaves) and one containing pods.

Vegetative sample: Measure the total fresh weight of the vegetative plant parts [V] in the 1-m biomass sample (FWV_B, g). Take a representative subsample of about 500 g immediately after weighing the total fresh weight of the vegetative sample. Record fresh weight of the subsample (FWV_sub, g), and dry it at 70 ºC to a constant weight. Avoid overpacking the drying oven so that good air circulation is maintained. After 10 days check the weight of 3 samples daily to identify when a constant weight is achieved. Remove samples from drying oven and weigh immediately to determine the final oven-dry weight of the subsample (DWV_sub, g). Grind samples in a plant mill and transfer ground material to pre-labeled 25-ml HDPE bottles.

Pod sample: Dry the whole pods sample at 70 ºC. Avoid over packing the drying oven so that good air circulation is maintained. After 5 days, check samples daily to identify when podwalls can be easily separated from seeds. Separate into podwalls [PW] and seeds [S] using a small soybean thresher. Re-dry PW and S samples at 70 ºC to constant weight. Remove samples from drying oven and weigh immediately to determine the final oven-dry weight of podwalls (DWPW_B, g) and seeds (DWS_B, g) in the 1-m biomass sample. Grind the podwalls in a plant mill and the seed samples in the grain mill, transfer ground material to pre-labeled 25-ml HDPE bottles for storage and chemical analysis.

sampling for measuring final yield at R8 (Harvest)

Carefully gather all plants from the harvest area (e.g., 2 x 30ft of rows 6 and 7 in Fig. 1). Cut plants at soil surface, count and record the number of plants (nPlant_H), and place plants into nylon mesh harvest bags. Avoid losses of stems and leaves.

Thresh the plant bundles to separate the dry matter into seeds and other aboveground biomass (haulm = chaff, podwalls, stems etc.) using a soybean thresher. Be careful to minimize seed loss from the thresher. High moisture content of the bundle is the biggest concern. Therefore the bundle may need to be left to dry for a few days in a covered area before threshing. Measure the fresh weight of the haulm sample (FWH_H, g). Take a haulm subsample of about 500 g, determine its fresh weight (FWH_sub, g), dry it at 70 ºC and measure the dry weight (DWH_sub, g). Determine fresh weight of seeds (FWS_H, g) and measure the field seed moisture content (MC_harv) using a grain moisture meter. Take a seed subsample of about 100 g seed using a small cup measurer and record the fresh weight of the subsample (FWS_sub,g). Count the number of seeds in the subsample using a seed counter (nS_sub). Dry seed subsamples at 70 ºC to constant weight and record dry weight (DWS_sub,g). If chemical analysis is needed, grind the seeds subsample using a grain mill, transfer sample to a pre-labeled 25 ml HDPE bottle and submit for analysis with the other plant samples.

Chemical analysis

Submit all samples for chemical analysis at the end of the season when all plant samples have been processed. Submit all vegetative samples collected at V3 to R3.5 stages and V, PW, and S samples collected at R6.9 for chemical analysis of plant nutrient concentrations. Include three anonymous duplicate samples to assess repeatability and two anonymous standard plant samples to assess accuracy of the chemical analysis.

Calculations

Dry matter and nutrient uptake at V3 to R3.5 stages:

Default values: Row width RW = 0.762 m (30”)

Biomass sampling area: B = 2 m (two 1 m row segments)

Oven-dry weight of the biomass sample (DW_B):

DW_B (g) = (DW_sub/FW_sub) x FW_B

Plant population density (POP_v, at different growth stages):

POP_v (1000 plants/ha) = (nPlant/1000) x (10000/RW/B)

Total aboveground dry matter (TDM):

TDM (kg/ha) = (DW_B/1000) x (10000/RW/B)

Total nutrient accumulation (UN):

UN (kg/ha) = TDM x N/100 N = nutrient concentration in biomass (%)

Harvest index at R6.9 stage:

Default values: Row width RW = 0.762 m (30”)

Biomass sampling area: B = 1 m (two 0.5 m row segments)

Oven-dry weight of the vegetative biomass sample at R6.9 (DWV_B):

DWV_B (g) = (DWV_sub/FWV_sub) x FWV_B

Seed dry matter yield based on biomass sample (SDM_B, oven-dry, about 3% residual moisture):

SDM_B (kg/ha) = (DWS_B/1000) x (10000/RW/B)

Pod harvest index (podHI):

podHI = DWS_B/(DWS_B+ DWPW_B)

Harvest index (HI):

HI = DWS_B/(DWV_B + DWS_B+ DWPW_B)

Plant population density and yield at harvest:

Default values: Row width RW = 0.762 m (30”)

Harvest area: H = 18.288 m (two 30 ft row segments)

Final plant population density:

POP_f (1000 plants/ha) = (nPlant_H/1000) x (10000/RW/H)

Plot seed yield (plotSY, grams per harvest area, adjusted to 13% moisture content):

plotSY (g) = FWS_H x [(100 – MC_harv)/87]

Seed yield adjusted to 13% moisture content (SY):

SY (kg/ha) = (plotSY/1000) x (10000/RW/H)

SY (bu/acre) = (plotSY/1000/67.25) x (10000/RW/H)

Plot haulm yield (plotHY, oven-dry):

plotHY (g) = (DWH_sub/FWH_sub) x FWH_H

Haulm yield (HY, oven-dry):

HY (kg/ha) = (plotHY/1000) x (10000/RW/H)

Apparent harvest index at full maturity (aHI):

aHI = (plotSY x (87/97))/(plotHY + (plotSY x (87/97)))

100-seed weight (HSW, oven-dry):

HSW (g) = (DWS_sub/nS_sub) x 100

Maximum dry matter and nutrient accumulation:

Default values: Row width RW = 0.762 m (30”)

Biomass sampling area: B = 2 m (two 1 m row segments)

Harvest area: H = 18.288 m (two 30 ft row segments)

Seed dry matter (SDM, oven-dry, about 3% residual moisture):

SDM (kg/ha) = SY x 87/97

Podwalls dry matter (PWDM):

PWDM (kg/ha) = SDM/podHI – SDM

Vegetative dry matter (VDM, stems, leaves, petioles):

VDM (kg/ha) = SDM/HI – SDM – PWDM

Maximum total dry matter (TDM):

TDM (kg/ha) = SDM + PWDM + VDM

Maximum plant nutrient accumulation (UN, N shown as example):

sUN (kg/ha) = SDM x sN/100 seed

pwUN (kg/ha) = PWDM x pwN/100 podwalls

vUN (kg/ha) = VDM x vN/100 vegetative

UN (kg/ha) = sUN + pwUN + vUN total

comments

· All calculations are done in a spreadsheet template containing all equations needed. Use metric units (g) for weighing and basic calculations. English units are only used for final yield reporting. All nutrients are reported on elemental basis.

· Grain yield is adjusted to a standard moisture content of 13%. Dry matter yields of grain, cobs, and stover refer to oven-dried dry matter, which typically contains about 3% residual moisture after drying at 70 ºC. Oven-dry matter is used for calculation of total nutrient uptake because plant nutrient concentrations obtained from tissue analysis are always expressed on oven-dry matter basis as well.

· If sampling areas or row width differ from the default values, change RW, B and H in the spreadsheet.

· Exclude 1 m from sampling at each end of the experimental plot.

· In experiments with large plot sizes (>200 m²), collect samples from two separate sampling areas within each plot and process them separately.

· Collect the R6.9 sample at the peak of biomass production and nutrient uptake. The soybean plots should be checked daily when R6.9 nears to insure this can be done before plants start shedding many leaves.

· As a minimum, plant nutrient analysis is required for seeds and vegetative parts sampled at R6.9. Nutrient concentrations in podwalls can be estimated by regression once a large enough database has been established.

· Plot the two estimates of seed yield (SDM_B at R6.9 and SDM at harvest) as a quality control measure (see default graph and regression estimates in worksheet “R6.9 and R8”). SDM_B tends to be 10-15% larger than SDM, but both estimates should be highly correlated with a slope close to 1 and a high R² value. Poor correlation (small R²) may be due to individual outliers in the graph. Significant deviation of the slope from 1 indicates a systematic bias in sample processing. Reasons for both must be clarified.