Manual Field Data Protocols

Data were collected manually using standard field methods. These measurements are used to calibrate and validate phenotypes derived from sensor-collected data.

Abstract

Materials

• barcode scanning protractor

• barcode scanning ruler

• ceptometer (Decagon AccuPAR LP-80)

• digital caliper

• drying oven

• forage chopper

• hand shears

• infrared thermometer

• juice extractor

• leaf area meter (Li-Cor 3100, Li-Cor Inc.)

• leaf porometer (SC-1 Leaf Porometer, Decagon Devices)

• leaf punch

• meter stick

• paper bags

• portable photosynthesis system (Li-Cor 6400, Li-Cor Inc.)

• scale

• SPAD Meter (SPAD 502 Plus Chlorophyll Meter, Minolta)

• spray paint

Equipment

Procedures

Variable
Canopy Height	Canopy height for single row of central 2 data rows of 4-row plot. Measured in cm using meter stick, taken at the height representing the plot 'potential', ignoring stunted plants. The canopy height was measured as the height of the foliage (not the inflorescence) at the general top of the canopy where the upper leaves bend and/or establish a canopy surface that would support a very light horizontal object (imagining a light sheet of rigid plastic foam), discounting rare or exceptional leaves in the upper-most 2 or 3 percentile.
Panicle Height	Height of the top of the inflorescence panicle for single central data row of 4-row plot, when panicle extends notably above canopy height.
Seedling Vigor and Emergence	Count the number of emerging seedlings at about 20% emergence, and then repeat every other day until final stand is achieved. A seedling is defined as emerged when the coleoptile is visible above the soil surface. Final stand is defined as when a similar count +/- 5% is achieved on successive counts 1-2 days apart. Count seedlings in the entire plot. Two Alternatives 1. Explicitly count number of plants emerged 2. For each plot, assess % germination in categories (e.g. [0,20], [20,40], …) This is the standard method
Canopy closure and leaf area index	Sunfleck ceptometer readings will be taken at least monthly to determine radiation interception and canopy closure. Using e.g. Decagon AccuPAR LP-80. Leaf area index will be calculated using Beer's Law for light extinction. A total of 5 readings will be taken per plot and averaged. Readings will be taken on clear days. Incident light will be measured at least once per rep. NDVI will also be measured weekly using a tractor mounted unit until the tractor can no longer navigate through the field due to the height of the crop. References:Prometheus Wiki http://prometheuswiki.publish.csiro.au/tiki-index.php?page=Canopy+light+interception+assessment+-+from+DC20
Leaf Architecture / Leaf erectness	Barcode scanning protractor is used to measure youngest fully emerged leaf
Leaf Width	Barcode scanning ruler measured at the widest part of the leaf
Stem number	Manually count the total number of stems in the plot will be counted bi-weekly after thinning for all plants in the plot.
Stem diameter	Stem diameter for each of 10 plants per plot will be measured with a digital caliper at 10 and 150 cm every month. For each plant take a few diameter samples and record the most common value. Use a black sharpie to mark the location at which the sample was taken.
Canopy Height	An "eyeball" estimate of plant height for the entire plot will be taken weekly beginning at the 5-leaf stage. Canopy height, view the canopy horizontally with a measuring stick, taking the height where a light piece of foam would rest on the canopy. Estimate the median height of healthy standing plots, ignoring plants that look really bad (e.g. are lodged). For method development: on subset of plots (10), capture the distribution of heights, e.g. max, min, median, upper and lower quantiles.
Lodging	There are three measures: 1. Percent lodging 0-100 scale 2. Lodging severity 0-100 scale 3. Lodging score 0-100 scale 4. Whether this is stalk or root lodging (categorical 'root', 'stalk') A lodging score will be taken weekly once lodging is observed. The lodging score will be recorded as a percentage and is a combination of the fraction of the plants lodged and the severity of lodging. For example, if 50% of the plants are 50% lodged, then the lodging score would be 25%. The severity of lodging is determined by how far the plants are leaning from vertical. If a plant is laying on the ground the severity of lodging is 100%. If a plant is leaning 45 degrees from vertical, then the severity of lodging is 50%. How to differentiate between stalk lodging and root lodging: scoring 'lodging' implies diagnosing a cause of inclined stems. A better approach may be a visual estimate of a range, with an optional note for root or shoot lodging. Done as deflection from vertical, this might look like:Min_angle Max_angle Loding_type0 1010 4530 60 R20 40 S…Where R = root lodging, S = stem lodging. Since stems are usually curved, the question remains of what reference height to consider?
Above-ground yield	Alleyways will be trimmed by hand with a weed whacker with a blade to accommodate space required between plots for a 2-row forage chopper. Actual plot length will be measured from the first to last stalk cut by the forage chopper. The stalks trimmed by hand will be spray painted to delineate them from stalks in the harvest area. The chopped forage will be weighed in a bag and a 2-quart sample removed for moisture and quality analysis. The sample will be dried in an oven at 65 C until constant weight is achieved. The dried forage will be ground and submitted for quality analysis. Sorghum Checkoff provided 1.5 pg protocol
Total biomass and tissue partitioning	Plants will be (destructively?) sampled (from west of gantry plots?) five times during the season from the 5 leaf stage through final harvest. The area sampled will be 1 meter of row. The plants will be cut off at ground level and immediately placed in a cooled ice chest for transport from the field to the laboratory where they were stored at 5°C until processing.
Allometry	Plant height will be measured from the base of the plant to the point where the top leaf blade is perpendicular to the stem. The number of stems and their average phenological stage will be recorded. Leaves will be removed from the stem at the collar and separated into green and brown leaves.
Leaf Area Index (LAI)	Leaf area of green leaves will be measured with a leaf area meter (Li-Cor 3100, Li-Cor Inc., Lincoln, NE, USA). Heads will be separated from the stems. Stem area will be estimated from stem length (without the head ) x diameter. The stems, brown and green leaves, and heads will be dried separately in an oven at 65°C for 2–4 d and weighed. Leaf area index and stem area index will be calculated.
Specific Leaf Area (SLA)	Specific leaf area will be calculated by dividing green leaf area by green leaf weight.
Phenology	Phenology will be determined according to Vanderlip (1993). Before heading, developmental stages were based on the appearance of the leaf collars. After heading, phenological stages were determined based on the development of the grain. Numbers ranging from 1 (50% of plants heading) to 7 (50% of plants at physiological maturity) were assigned to designate growth stage after the vegetative period. Before heading, growth stages represent mean leaf number of all plants and not the most advanced 50% as was done after headingReference: https://www.bookstore.ksre.ksu.edu/pubs/S3.pdf
Days to flag leaf emergence
Days to spike emergence
Days to anthesis/flowering	Once anthesis begins, anthesis will be noted 3 times per week until anthesis ends. Anthesis is defined as when 50% of the plants have one or more anthers showing.
Maturity pattern	Once maturity begins, maturity will be noted 3 times per week until maturity ends. Maturity is defined as when 50% of the plants have reached black layer.
Moisture content	Forage moisture content will be determined at final harvest and from the biomass samples by weighing the forage before and after drying in an oven at 65 C for a minimum of 48 h. How large is the sample? ~ 1 pound in a lunchbag, 2 samples per plotHow will it be packaged / labeled?Subsamples?
Lignin content	Determined by NIRS from the moisture sample at final harvest.
BTU/DW	Determined by NIRS from the moisture sample at final harvest.
Juice extraction	Juice will be extracted from stalks from the biomass samples at final harvest using a sweet sorghum mill. The juice will be weighed and brix measured. Brix concentration in the juice – Brix will be measured in the juice extracted as described above.
Plant temperature	A hand-held infrared thermometer will be used to measure plant temperature bi-weekly. A total of 5 readings will be recorded per plot within 2 hours of solar noon.
Plant color	A Minolta SPAD meter will be used to record plant color on plants using the most recently fully expanded leaf on a bi-weekly basis.
Photosynthesis	Using LiCOR 6400, measure A-Ci and A-Q curves to estimate parameters of Collatz model of C4 photosynthesis coupled to the Ball Berry model of stomatal conductance. One reading from the youngest fully expanded leaf. These readings will be taken monthly within 2 hours of solar noon.
Transpiration/stomatal conductance	Stomatal conductance was assessed using a leaf porometer (Decagon Devices, Pullman, WA) by taking 5 readings per plot on most recently fully expanded leaves. Readings will be taken on the 12 photoperiod sensitive lines in the biomass association panel. These readings will be taken bi-weekly and within 2 hours of solar noon at least two times during the season.

References

• Pérez-Harguindeguy N., Díaz S., Garnier E., Lavorel S., Poorter H., Jaureguiberry P., Bret-Harte M. S., CornwellW. K., Craine J. M., Gurvich D. E., Urcelay C., Veneklaas E. J., Reich P. B., Poorter L., Wright I. J., Ray P., Enrico L.,Pausas J. G., de Vos A. C., Buchmann N., Funes G., Quétier F., Hodgson J. G., Thompson K., Morgan H. D., ter Steege H., van der Heijden M. G. A., Sack L., Blonder B., Poschlod P., Vaieretti M. V., Conti G., Staver A. C.,Aquino S., Cornelissen J. H. C. (2013) New handbook for standardised measurement of plant functional traits worldwide. Australian Journal of Botany 61 , 167–234. https://doi.org/10.1071/BT12225

• Vanderlip RL. 1993. How a sorghum plant develops. Manhattan, KS, USA: Kansas State University Cooperative Extension. Field Experiments in Crop Physiology. 2013, Jan 13. In PrometheusWiki. Retrieved 15:03,June 21, 2016, from http://www.publish.csiro.au/prometheuswiki/tiki-pagehistory.php?page=Field Experiments in Crop Physiology&preview=41

Photosynthesis / leaf chemistry from hyperspectral data references:

• Shawn Serbin et al - Leaf optical properties reflect variation in photosynthetic metabolism and its sensitivity to temperature 2011 J Exp Bot

• Mapping biochemistry and photosynthetic metabolism in ecosystems using imaging spectroscopy (Presentation) - Remotely estimating photosynthetic capacity, and its response to temperature, in vegetation canopies using imaging spectroscopy 2015 Remote Sensing of the Environment

• Spectroscopic determination of leaf morphological and biochemical traits for northern temperate and boreal tree species 2014 Ecological Applications

• Additional Draft Protocols are available at https://docs.google.com/document/d/1iP8b97kmOyPmETQI_aWbgV_1V6QiKYLblq1jIqXLJ84/edit#