1. Gather Seed from Multiple Species or Controlled Crosses of a Single Species

Note: This example used seed from 15 cross types within the species Helianthus annuus (sunflower) using wild, hybrid, and crop types as the maternal (seed producing) parent.

1. At the end of the growing season, collect mature seed heads in labeled bags. Clean seed from chaff and place seed in envelopes labeled with parental cross type in standard format (i.e., maternal x paternal).
2. Bulk seed together in large envelopes using equal amounts of seed from an equal number of identical parental cross types. For example, 100 seeds from 10 cross types with the same maternal and paternal parents. Label the envelopes with identifying (cross type) information. These large envelopes are seed masters for use in filling burial strips in Section 2.

Table 1. Parental cross types produced from hand pollination. Sunflower crop-wild hybrid cross types were produced with hand-pollination for use in seed burial experiment. For all cross types, the maternal parent listed first and the paternal parent listed second. Cross types marked with † are part of reciprocal cross type pairs with the same % crop alleles but different maternal parents. Table has been previously published in: Pace, B. A. et al. (2015)¹⁵.

2. Create Custom Seed Burial Strips

NOTE: For this example, we had three removal date treatments and 15 replicates, so we required 45 strips total. This example uses 15 compartments per strip so, we required enough fine-meshed polyester fabric or mosquito netting to house 15, 7 x 10 cm compartments. See Figure 1.

1. Start by cutting a 20 x 105 cm strip of fabric for each seed strip.
2. Measure and mark compartments, then fold the fabric in half. Using a high-temperature glue gun, seal one short (10 cm) end. Once the glue cools slightly, but not completely, press the fabric sides together to create a seal.
   CAUTION: High-temperature glue and glue gun tips can cause burns if contact with skin is prolonged.
3. Glue lines from the folded edge to the open short end at each compartment mark. Press the two fabric sides together along the glue lines to ensure seals without holes between compartments. Seeds migrating between compartments contribute to experimental error.
   NOTE: Now that the compartments have been created, the unfolded edge should remain open and ready to receive seed and their identifying information (cross type name).
4. Randomly assign seed cross types to compartments for each strip.
   1. Fill seed compartments with a uniform number of seeds (20 for each compartment are used in this example). For labels, use industrial permanent markers on cut-to-fit plastic garden darts so that they are able to withstand burial.
      NOTE: Alternative labeling methods may be used.
   2. After each compartment is filled with seed and its label, seal the compartment immediately with the high-temperature glue gun. Proceed until the entire seed strip is completely sealed. Label overall strip in the first compartment with replicate, block and/or treatment information.
      NOTE: For our study, since we had temporal removals, blocks, and replicate strips within blocks, we included all of this information.
   3. Check for gaps between compartments and spot glue as needed before moving on to the next strip.

Figure 1. Burial seed strip schematic. Example of burial strip showing cross type identification for individual compartments. Maternal parent is listed first with the paternal parent listed second. Colors indicate different maternal parentage, with yellow for wild, blue for F₁ hybrid, and red for crop. Please click here to view a larger version of this figure.

3. Bury Seed Strips in the Field

1. Dig plots to the specified depth to house strips. Assign strips to individual blocks in which strips are randomly arranged to allow for removal date to be used as the main plot and compartment identity (in this example, cross type) as the subplot in analysis.
2. Cover strips with earth. Cut metal mesh hardware cloth to be 10 cm larger than plots and place over buried strips. Use U-pins to secure edges and cover cloth edges with earth.
   NOTE: This covering is for excluding burrowing seed predators. As another option, Mercer et al.¹³ constructed and buried boxes of hardware cloth that surrounded the seed strips due to intense gopher pressure in their field location.)
3. Place colored surveyor's flags to distinguish strip location and removal dates to complement clear written records.

4. Excavate Strips and Evaluate Seed

1. Dig up seed strips assigned to a given removal date. Maintain the strips in a cool moist environment until they reach the laboratory where seed evaluation will take place. This can be accomplished by wrapping the strips in wet newspaper and placing them in a cooler for transport or overnight shipping.
2. Rinse mud and soil from strips in water until strips are clean enough that external debris will not obscure seeds.
3. Use scissors to cut open one compartment at a time. Count germinated seeds and remove them. Count clearly dead seeds (e.g., rotting, or putrefied seeds). Place ungerminated seeds on labeled, wet Petri-sized blotter paper to further distinguish ungerminated, dormant, and dead seeds.
   1. Place Petri dishes with ungerminated seeds into a growth chamber set for standard germination conditions: 25 °C /10 °C 12 hr day/night. Allow seeds one week to germinate. Any that do, count these as viable, but ungerminated since they did not germinate under field conditions.
4. After the growth chamber treatment, test remaining ungerminated seeds for viability using Tetrazolium chloride (TZ)¹³. To accomplish this, make up a percent solution of TZ appropriate for the study species using the AOSA Tetrazolium Testing Handbook¹⁴. For this example, a 1% solution is used.
   1. Cut the ungerminated seeds in half and place one half were the embryo and endosperm are visible in a Petri dish containing enough TZ solution to cover the seed (seeds may float, this is okay).
   2. Place TZ Petri dishes into an incubator set at 30 °C and wait 3 hr. Note: Higher temperatures will result in a faster reaction, if desired, but overstaining is a risk.
5. Remove Petri dishes from the incubator and evaluate. Seeds that have stained red in their embryos are germinable, while unstained seeds are dead. Consult the AOSA Tetrazolium Testing Handbook for more detail on distinguishing marginal cases and particular species.