List of traits - ARG component of SAM project 1.Days to flower 2.Floral pigmentation (disks, stigmas, interfloral bracts) 3.Plant height 4.Stem diameter 5.Stem specific gravity 6.Stem biochemistry (NREL analysis) 7.Branch # 8.Plant biomass Traits 3-8 should be recorded after a plant has flowered – if possible Iowa – 16 plants per accession, BC – 9 plants per accession “Randomly” select healthy, well spaced plants for phenotyping. Do not select border row plants in Iowa or plants at the very end of the row in BC. Also avoid off-types or plants where the central stem/leader has been broken off.

Plant Height First, cut the plant as close to the ground as possible. We used a Sawzall to do this – but a nice hand saw works well too. Then record plant height (in cm) from the soil line to the point on the stem that connects to the terminal flower. Top point Bottom point

Plant Height Plant height for this study is actually the length of the primary stem. In most ARG accessions the main or primary stem is obvious. In some of the smaller accessions the branches are often much longer than the main stem. For this study the main stem (shown in purple) is defined as the “straightest” stem that usually flowers first. The tip of the main stem will often die off early while the rest of the plant continues to flower.

# of Branches Next, count the number of branches that extend from the main stem. A branch is defined as a branch that makes a flower bud. Don’t worry about counting secondary and tertiary branches. Not a branch

Stem Sampling for NREL Examine the stem and identify where to cut the stem sample to submit to NREL. In GA the plants all had a clear defining line in both upright and lodged plants) between the brown, highly branched, “gnarly” base of the stem and the a more consistently green stem. We cut our stems at this defining line (in red). It will be different for Iowa and BC.

Stem Sampling for NREL Based on the what I saw in Iowa you will sample your stems directly from the ground up in most cases. But not all

Stem Sampling for NREL Stem sections should be ~ 11” long. You don’t need to make angled cuts as outlined in the first set of instructions that I sent in the Spring The orientation of the stem (top and bottom) is more obvious in ARG (vs. annuus) so it’s unnecessary to indicate the orientation of the stem with an angled proximal cut. You can cut the stems flat at the top and bottom. Then remove all off the branches/leaves and label the stem. Put the stem in a ziploc bag and transport it back to the lab on ice. If necessary the stems can be stored for 1-2 days in the fridge before doing density measurements – see later slide. Please remove the branches as close to the stem as possible – I went back later and cut branches flush to the stem – we did not have the correct hand pruner to do this in the field on the day I took pictures

Plant Biomass After the stem section has been removed, put the rest of the plant (including the end piece if you didn’t cut your stem section from the bottom) into large paper bags (we used lawn and leaf bags) and then dry the plant until it is completely dry. Record the dry weight of the entire plant in grams. The dried plant can then be discarded. No need to separate the plant into different components (flowers, leaves, stem, etc. We only need a single measurement of the total dry biomass of the plant. FYI - we needed 1-3 large lawn and leaf bags per plant.

Stem Diameter Measure stem diameter at the middle of the NREL stem section (N/S orientation and E/W orientation at 90 degrees – 2 measurements will be averaged). Avoid the branch scars and nodes. This measurement can be done back in the lab. E N W S Stem cross-section

Stem Specific Gravity Place the stem section into a beaker of water and hold it with a small needle or pointer so that it is completely submerged for a brief period of time and record the weight of the water displaced by the stem. The stem section should then be placed into a paper bag and dried in a forced air oven at 60C for at least 3-4 days. Once the stem section has reached a constant weight record the dry weight in grams. Dried stem samples should then be shipped to UGA and I will then ship all of the samples together to NREL for MBMS analysis. Dan – if you have any additional questions about this let me know. I know Brooke did this for another study last year so she should be able to help you set up a “density” apparatus for measuring this. I can also send you pictures of the apparatus that we are using for you to duplicate if necessary.