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USDA Hemp Descriptor and Phenotyping Handbook, Version 3

ABOUT

Objectives

The USDA Hemp Descriptor and Phenotyping Handbook was undertaken with the following objectives:

  • To assist breeders and researchers in identifying accessions with specific traits to facilitate germplasm selection within hemp (Cannabis sativa L.) improvement programs.
  • To identify gaps in the existing hemp collections and help formulate strategies for future collection and conservation efforts.
  • To designate and maintain a core collection of critical materials.
  • To increase NPGS user utility and accessibility to hemp germplasm and associated data.
  • To identify duplicate accessions and reduce costs of hemp genetic resource conservation.

The methods and protocols are based on peer-reviewed literature and/or crowd-sourced from the hemp community. Robust, reliable, and high-dimensional data generated from these phenotyping efforts will empower conservation of hemp genetic diversity and aid selection of materials with unique trait combinations for breeding programs.

We have attempted to compile a list of standardized characterization and evaluation methods to capture passport information and to quantify morphology, horticultural and agronomic quality, pathogen resistance, and metabolic profile. This document can be used a reference to standardize phenotypic data collection across the broader pool of hemp germplasm and will be updated periodically as better methodologies emerge.

The information gained from these phenotyping efforts will be digitally stored and made publicly available within GRIN-Global alongside the hemp germplasm held within the Plant Genetic Resources Unit (PGRU) in Geneva, NY. An example of the germplasm that is held at PGRU can be seen here. Phenotypic summaries of PGRU hemp genetic resources can be accessed on GRIN here.

PGRU coordinates hemp germplasm collection and exchanges from domestic and foreign sources. Information related to plant genetic resources increases usefulness to diverse stakeholders. Phenotypic data can be collected either by the curator during routine multiplication or by collaborators during collection, germplasm screening, or breeding experiments. PGRU asks germplasm recipients or donors to provide as much data associated with these materials as possible.

Collected data can be stored in a spreadsheet using the trait_name as column headings and PUID as row names. Our lab prefers the conventions of “Tidy Data” 1,2. This document can then be emailed to the hemp germplasm curator () for inclusion into GRIN-Global. Please do not hesitate to reach out with any questions, comments, suggestions!

Versioning

This is version 3.0 of the USDA Hemp Descriptor and Phenotyping Handbook published in June 2023. Here, we try to draw on new experience, publications, conversations since the publication of version 1.0 in September 2021.

Archived versions of the Hemp Phenotyping and Descriptor Manual can be found here:

Editors

  • Zachary Stansell (Hemp Curator, USDA-ARS, Plant Genetic Resources Unit; Project Lead, corresponding author 📧: )
  • Tyler Gordon (Hemp Breeder, USDA-ARS, Plant Genetic Resources Unit; Lead Geneticist)
  • Anthony Barraco (Biol Sci Technician, USDA-ARS, Plant Genetic Resources Unit; Protocol Development)
  • Daniel Meyers (Biol Sci Technician, USDA-ARS, Plant Genetic Resources Unit; Protocol Development)
  • [Anthony Rampulla] (Biol Sci Technician, USDA-ARS, Plant Genetic Resources Unit; Protocol Development)
  • [Tori Ford] (USDA-ARS ORISE Fellow; Editor)
  • Anya Osatuke (Cornell Cooperative Extension; Editor/Literature Summary)

The authors gratefully acknowledges the critical review, editing, and the numerous suggestions for improvements made by Ademola Aina, Olivia Aldin, Masoume Amirkhani, Anthony Barraco, Craig Beil, Gary Bergstrom, Mark Berhow, Peter Bretting, Charlie Brummer, Mark Bridgen, Kadie Britt, Korey Brownstein, Zachary Brym, Carlyn Buckler, Ali Cali, Brian Campbell, Craig Carlson, Jeffrey Carstens, Ernst Cebert, David Chalkley, Chengci Chen, Alyssa Collins, Whitney Cranshaw, Randy Crowl, Heather Darby, David Dierig, Jorge de Silva, Chris Delhom, Sadanand Dhekney, Shelby Ellison, David Fang, David Gang, Nicholas Genna, Heather Grab, Jason Griffin, Kelly Gude, Joshua Havill, Yu Jiang, Nick Kaczmar, Joanne Labate, Michael Loos, Jessica Lubell-Brand, Tyler Mark, Victoria Meakem, Virginia Moore, Maylin Murdock, Jay Noller, Luis Alberto Monserrate Oyola, Dániel Pap, Bear Reel, Andrew Ristvey, Moira Sheehan, Savanna Shelnutt, Chris Smart, Larry Smart, Faith Sparks, George Stack, Jeffrey Steiner, Conor Stephen, Alan Taylor, Jacob Toth, Daniela Vergara, and Don Viands.

The drawing on the front cover is used with permission by Anya Osatuke. Kadie Britt provided many primary source images and text for the invertebrate section. Craig Carlson provided original figures, methods, and many ideas. Jacob Toth, Joshua Havill, Savanna Shelnutt, Brian Campbell, Shelby Ellison, and Jeffrey Carstens provided many helpful comments, references, protocols, and edits. We have tried to acknowledge everyone who’s helped with this work, but any omissions are solely Zachary Stansell’s fault.

This work has drawn heavily on input from the Cornell Hemp Stakeholder Survey. Please take the survey if you have not already done so.

Please contact zachary.stansell@usda.gov with any questions, comments, remarks, or ideas.

The United States Department of Agriculture (USDA) prohibits discrimination in its programs on the basis of race, color, national origin, sex, religion, age, disability, political beliefs, and marital or familial status. Not all prohibited bases apply to all programs. Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact the USDA Office of Communications at (202)720-5881 (voice) or (202)720-7808 (TOO). To file a complaint, write to the Secretary of Agriculture, U.S. Department of Agriculture, Washington, DC 0250, or call (202)720-7327 (voice) or (202)720- 1127 (TOO). USDA is an equal employment opportunity employer.

Boxes

Throughout this document there are special colored text boxes:

Phenotype/Descriptor

trait_name [datatype; units]

elevation_meters [decimal; m]
Elevation of collecting site above sea level.

🧪Phenotyping Protocol🧪

Seed germination

  • 10 waterproof trays
  • Sterile water-holding material (cotton wool, paper towels)
  • 200 seeds, stored between 4 to 56 weeks…

📐Equation📐

Percent moisture may be calculated as:

\(\frac{(wet - dry)}{wet} \times 100\%\)

📜List📜

Invertebrate pests

  • Acherontia atropos
  • Aculops cannabicolus
  • Aecidium cannabis

📚Additional References📚

One of the earliest publications lauding hemp was “The Praise Of Hemp Seed” by John Taylor -Taylor (1620).

Keywords

  • hemp
  • Cannabis sativa L.
  • germplasm
  • phenotype
  • trait
  • characterization
  • evaluation
  • USDA-ARS
  • NPGS
  • PGRU

Language

GRIN-Global supports displaying data in multiple languages for system-level data. That is, if the system requires text to be displayed that is not actual GRIN-Global data, that text should be in the appropriate language for the current user. This is accomplished by using a table ending with _lang as a child table.

Data types & units

Units
All units are SI unless otherwise indicated.

datetime
A datetime data type that can handle time in nanoseconds and has a year range extending from the year “0001” to “9999.”

decimal
The decimal data type can store a maximum of 38 digits, all of which can be to the right of the decimal point. The decimal data type stores an exact representation of the number; there is no approximation of the stored value.

int
The integer data type is stored as a 4-byte integer; numeric values can range from \(-2^{31}\) through \(2^{31} – 1\).

nvarchar
An nvarchar field can store a string of text characters (maximum 4,000). The “n” in nvarchar means uNicode. “varchar” is an abbreviation for variablelength character string. Essentially, nvarchar is variable text field that supports two-byte characters, therefore capable of handling non-English symbols.

PASSPORT

An accession consists of seed or plant material representing a sample of a single species, collected at a single time and location. An accession may be a sample of multiple plants found at the same location at the same time, or it may be collected from a single individual. By default, NPGS will retain different samples of a putative cultivar/population as discrete inventories nested within the Plant Introduction accession.

Accession

taxonomy_species_id [nvarchar]
Scientific name of accession linking the accession record to its taxonomy parent (genus / species). Modified from GRIN-Global. Subtaxon may be included:

  • ‘subsp.’ (subspecies)
  • ‘var.’ (variety; not the same as the breeder’s named variety [uniform & stable product of breeding] or cultivar.)
  • ‘f.’ (form)
  • ‘group’ (botanical variety not cultivar name )

PUID [nvarchar]
If persistent, unique identifier has been previously assigned, report. Assigned to one accession to be unambiguously referenced at the global level, with associated information aggregated via automated means. Genebanks not applying a true PUID should use a combination of Institute Code, Accession Number, and the Genus as a globally unique identifier. Modified from Bioversity International, FAO (2015).

improvement_status [nvarchar]

Short paragraph. If known, elaborate on material improvement status, e.g., wild, landrace, breeding material, hybrid, founder stock, colonal selection, mutant, polyploid, mapping population, transgenic, etc.

plant_name [nvarchar]

Top name assigned to display (sometimes referred to as the top name), typically given by farmer, breeder, seed-saver. Cultivar name is a possible type of top name. If in non-Latin alphabet, provide original spelling alongside a Latin-alphabet transliteration in remarks. Modified from GRIN-Global and Bioversity International, FAO (2015).

accession_pedigree [nvarchar]

Description of plant pedigree, if known, e.g.:

  • “Selection from ‘Carmagnola’”
  • ‘Beniko’/‘Carmagnola’//‘Futura 75’///‘Carmaleonte’/‘Felina 32’//‘Futura 75’ * “Mutation found in ‘Beniko’”

ploidy [int]
Record ploidy if known. If mixoploid or other, elaborate in passport_remarks. See Adriel Garay and Sabry Elias (1998).

accession_ipr [nvarchar]
State PVP registration status, if applicable. U.S. Link.

Varieties may also be protected by a U.S. Plant Patent (e.g. CW2A) or Utility Patent and/or Plant Breeder’s Right from a UPOV country; e.g., Canada.

crop_use [nvarchar]

Explain crop use(s); e.g., oil, fiber, secondary metabolite, ecosystem services.

Germplasm source

source_cooperator_id [nvarchar]
Field associating the cooperator (person or organization) who was the source of the germplasm. See Appendix.

collector_cooperator_id [nvarchar]
Indicating the individual collecting sample. See Appendix.

developer [nvarchar]
List the name of the organization (or person) that bred the material.

Sampling & location

Modified from S-1084 Collection Protocols, GRIN-Global, personal conversations with hemp researchers (Shelby Ellison and Jeffrey D. Carstens), and Bioversity International, FAO (2015) standards. See Appendix.

number_plants_sampled [int]
Number of plants sampled to collect the accession material ( S-1084 Collection Protocols). See Appendix.

source_date [datetime]
Date when germplasm is collected from source material ( S-1084 Collection Protocols).

geography_id [nvarchar]
The internal geographic identifier indicating the cooperator’s country and state ( S-1084 Collection Protocols).

elevation_meters [decimal; m]
Elevation of collecting site above sea level ( S-1084 Collection Protocols).

latitude & longitude [decimal]
Latitude and longitude in decimal degree format. The format is 10 integers and 8 decimals. Positive values are east of the Greenwich Meridian; negative values are west of the Greenwich Meridian ( S-1084 Collection Protocols).

coordinate_method [nvarchar]
Georeferencing method used (e.g.; GPS, map, estimated). Modified from Bioversity International, FAO (2015).

uncertainty [decimal; m]
Maximum coordinate uncertainty radius.

georeference_datum [nvarchar]
Geodetic datum/spatial reference system; WGS84 datum is preferred.

accession_inv_voucher_note [nvarchar]
If applicable, include additional voucher information.

ARCHITECTURE

Unless stated otherwise, measure plant architecture traits as the mean of 10 unpruned plants during week of sampling. Samples submitted to NPGS will be evaluated by a USDA-ARS laboratory using similar protocols as described below.

Morphology

Architectural traits modified from Carlson et al. (2021).
Architectural traits modified from Carlson et al. (2021).

ht [decimal; cm]
Height of the stem from the ground to tip apical inflorescence, modified from (2021).

mcd [decimal; cm]
Maximum canopy diameter (mcd) as width of plant at widest set of branches (2021). Measured from widest tip to tip without stretching branches. Include flowering tissue in measurement.

mcdh [decimal; cm]
Height evaluated at maximum canopy diameter (mcdh) from ground to max canopy diameter (2021).

trkl [decimal; cm]
Trunk length (trkl) is evaluated as distance from ground to first branch (2021).

inl [decimal; cm]

Average internode length (inl) is calculated between internodes along the primary stem (50 cm max, see diagram) (2021).

\(inl = \frac{ht-trkl}{branches}\)

hyp_a and hyp_b [decimal; cm]

Calculated (2021).

\(hyp.a = \sqrt{(ht - mcdh) ^ 2 + \frac{mcd}{2}^2}\).

\(hyp.b = \sqrt{mcdh^2 + \frac{mcd}{2}^2}\)

kite_perimeter [decimal; cm]

Calculated (2021).

\(kite.perimeter = 2 \times (hyp.a + hyp.b)\)

kite_area [decimal; \(cm^2\)]

Calculated (2021).

\(kite.area = \frac{ht \times mcd}{2}\).

ba [decimal; (0-180°)]

Kite branch angle ba is calculated from the lower kite triangle, using the difference of maximum canopy diameter height and trunk length (2021).

\(ba = \arctan \frac{mcd}{2(mcdh- trkl)}\)

nodes | nodes_opp | nodes_alt [int]
Number of internodes (nodes) per plant; nodes are by definition \(2 \times\) branches (2021). Number of opposite internodes (nodes_opp) per plant. Number of alternate internodes (nodes_alt) per plant. When grown from seed, branching is initially opposite, transitioning to alternate as the plant matures. Plants propagated from cuttings generally have alternate branching in the whole plant Stack et al. (2021).

branches [int]

Number of branches per plant (2021). When grown from seed, branching is initially opposite, transitioning to alternate as the plant matures. Plants propagated from cuttings generally have alternate branching in the whole plant Stack et al. (2021).

\(branches = n.opp * (2 + n.alt)\)

kite.circularity [categorical]

A continuous scale of apical dominance can be derived (2021):

\(kite.circularity = \frac{4\pi\cdot kite.area}{kite.perimeter^2}\)

dia [decimal; mm]
Diameter of the stem at soil level using calipers, forestry or fabric measuring tape, modified from Carlson et al. (2021).

pith_diameter [decimal; mm]
Diameter of the pith in the stem cross section at stem midpoint, modified from International Union for the Protection of New Varieties of Plants (2012).

Uncrewed aerial vehicle evaluation

uav_xxx [TBA]
See Carlson et al. (2021).

Remarks

architecture_remarks [nvarchar]
If possible, report date of measurement [days from sowing], sex average, minimum, and maximum height and width observed in a planting (cm).

📚Additional References📚

  • Anderson (1980)
  • Werf, Haasken, and Wijlhuizen (1994)
  • Werf et al. (1995)
  • Meijer and Keizer (1996)
  • Ranalli (1999)
  • Mishchenko and Lajko (2016)
  • Magagnini, Grassi, and Kotiranta (2018)
  • Backer et al. (2018)
  • Spitzer-Rimon et al. (2019)
  • Carlson et al. (2021)
  • Danziger and Bernstein (2021)
  • Stack et al. (2021)
  • Vergara et al. (2021)

LEAF

Unless otherwise noted, gather leaf data from the uppermost set of mature leaves, as mean of 5 leaves gathered from each of 10 different plants immediately before onset of flowering.

Morphology

petiole_length [decimal; cm]
central_leaflet_length [decimal; cm] central_leaflet_width [decimal; cm]

Leaf is flattened and measured from tip until start of rachis; petiole is flattened and measured from base of rachis until petiole base, modified from -International Union for the Protection of New Varieties of Plants (2012) and -Anderson (1980).

(A) central_leaflet_length and (B) petiole_length measurement.
(A) central_leaflet_length and (B) petiole_length measurement.

Imaging

leaf_color_L [decimal]
leaf_color_a [decimal]
leaf_color_b [decimal]

The average color of uppermost set of mature leaves, collected before flowering, measured with a colorimeter, modified from -International Union for the Protection of New Varieties of Plants (2012). A RHS color chart may also be used, but values should be converted to (L*a*b*) before addition to GRIN. From Wikipedia (accessed 2023-01-11): The CIELAB color space, also referred to as L*a*b*, is a color space defined by the International Commission on Illumination […] in 1976. It expresses color as three values: L* for perceptual lightness and a* and b* for the four unique colors of human vision: red, green, blue and yellow. CIELAB was intended as a perceptually uniform space, where a given numerical change corresponds to a similar perceived change in color.

There are many programmatic solutions to convert colors (I use R for everything: 1,2,3) as well as many online tools (e.g., Colormine).

Consider printing a label to include in the scan as well. PGRU germplasm imaging and scans typically include accession ID, species, and plant id name (e.g. ‘FIN-314’). PGRU uses a small color wheel in the corner of our templates (DOWNLOAD), but that might not be necessary for you since you are measuring color with a colorimeter.

leaf_variegation [nvarchar; Y/N]

Indicate whether not variegation has been noted or is present.
Variegated leaves are most likely not virus based, but it might be worth investigating.

leaf_scan_protocol [.jpg or .png]

🧪Scan protocol🧪

Equipment

  • Flatbed scanner
  • Desktop monitor
  • Black cloth, ideally velvet, of equal dimensions to scanner bed.

Protocol

  1. Gather one mature leaf from a representative sample of 10 plants a week before the onset of flowering. Retain petioles. Keep on ice if wilting is a concern.
  2. Scan leaves within the hour of collection using a scanner with the lid open, draping black fabric over the leaves to absorb background light. Include scale or ruler (cm) and puid.
  3. Convert the scanned leaf image into .png file and save.