Collections Standard Operating Protocol, Plant group: Vascular Plants

• Ensure collecting permits are in place and you are familiar with the Darwin Tree of Life (DToL) Code of Conduct document.
• Ensure all H&S documentation including institutional risk assessment documents have been completed and approved.
• Ensure the risk assessments for the use of silica gel has been completed.
• Ensure the risk assessment for Fieldwork Plant Health work has been completed and you are familiar with methods
• of footwear and tool decontamination.

Genome sequencing pathway, vascular plants

Sampling equipment ready for field work

• Marker pen/pencil/pen
• Trowel
• Secateurs
• Sterilising wipes
• 5 inch forceps
• Fine watchmaker forceps
• 150 mm x 150 mm white dissecting tile
• 1.5 ml eppendorf tubes with attached lid
• GPS
• Cool box and ice packs/sheets with insulation (to prevent direct contact with leaf material)
• Field version of sample manifest
• Packets of silica gel - for DNA barcode sample (if collecting sample in field)
• Ziplock bags - for genome size sample (if collecting sample in field)
• Damp absorbent paper (kitchen roll)
• FluidX tubes (if collecting straight to sample tube)
• Barcode reader (if collecting straight to sample tubes)
• Liquid Nitrogen shipper (if collecting straight to sample tubes)
• Herbarium press

Collection of samples in the field

• Where possible, multiple specimens should be sampled from the same individual. For example, for large plants, all specimens (material for the genome sequencing, DNA barcoding, flow cytometry and voucher) should be from the same individual.
• Where this is not possible, due to the size of the sample, care should be taken to make specimens from the same part of a clump/population. For example, where individuals are not sufficiently large, all genome specimens should be from the same individual, but material for RNA, DNA barcoding, flow cytometry and the voucher specimen can be taken from other individuals within the same clump or population.
• Note that if the plant has developing seeds, these cannot be assumed to be the same genetic individual as the parent plant and should not be included in the material sampled for genome sequencing, although they can be included in the other samples.
• Where the plants are very small, each tissue collection for genome sequencing may be a different individual and specimens for DNA barcoding, flow cytometry and the voucher may be different individuals or a pool of multiple individuals.
• For dioecious plants, if flowers are present, note whether the sample is male (anther-bearing) or female (ovary-bearing); when both sexes are present in the population, collect samples from both for genome sequencing and for genome sizing, so that the decision about which to sequence may be made later. If fruits are present, note that the specimen is a female plant. Otherwise record sex as unknown.

Relative amounts of tissue: c. 0.15 g of fresh plant tissue from the angiosperm Agrimony (centre), the fern Equisetum (left) and the moss Polytrichum (right), with a lab Sharpie pen and a 7.6ml Fluidx tube for comparison.

Collecting plant material for genome sequencing: flash freezing tissue in liquid nitrogen, sampling from the RBGE living collection

Shipping samples to the Sanger Institute.

1. Prepare and label 50 mm zip-lock bags with 10 g of silica gel OR prepare and label a teabag. Label with the COLLECTOR_ID number.
2. Processing procedure is the same as sampling for genome sequencing - use a clean 1.5 ml eppendorf to prepare leaf discs; use sterilised fine forceps to remove small leaves; use sterilised fine forceps to break any stems up into 5 mm fragments.
3. Place the tissue to be barcoded in the labeled bag of silica gel/tea bag.
4. All tissue must be dried as quickly as possible. Please ensure there is either sufficient silica gel in the zip-lock specimen bag to totally submerge the plant tissue or sufficient DRY silica gel in the storage tub to cover tea bag specimens.
5. Record the COLLECTOR_ID and register the sample in the DToL Manifest. Email the completed manifest to edtolnumbers@rbge.org.uk, cc lforrest@rbge.org.uk.
6. Post the sample to the address given below, or bring it to the DToL silica gel collection at RBGE.

Samples for Genome size estimation (flow cytometry)

1. Prepare and label zip-lock bags with water-dampened kitchen roll/white roll. Label with the COLLECTOR_ID number.
2. Remove intact leaves/whole stems.
3. Place the specimen in a labelled grip seal bag and store in the cool bag - avoid direct contact between the leaf material in the specimen bag and ice pack (wrap ice pack in tissue).
4. Record the COLLECTOR_ID in a Sample Processing Sheet and register the sample in the DToL Manifest.
5. Store the specimens at 4°C (refrigerator) prior to shipping to Kew (N.B. Do not freeze samples at any point as this will degrade the nuclei and reduce the quality of the genome size estimate).
6. Ship the sample, in a padded envelope, to the address given below. Use Royal Mail first class post; also include a print-out of the manifest details for the samples that are contained in the envelope.

Voucher specimens

• Select material from the plant.
• For larger plants take all material from a single individual – do not mix individuals.
• For smaller plants, where multiple individuals are collected, any material from the individual used for genome sequencing must be processed separately.
• Include fertile material (e.g. sporangia, cones, flowers, fruit) if possible.
• Include extra material (e.g. leaf, flower, fruit) for later dissection
• Include below-ground parts (e.g. roots, bulbs, corms) if possible.

• Remove soil and any obvious invertebrates.
• Ensure the material will fit a standard herbarium sheet (42 x 26.4 cm). Either trim excess or divide into two or more sheets, clearly marked as such (e.g. sheet 1 of 2, sheet 2 of 2...)
• Remove any excess material to reduce bulkiness and ensure important features are visible.
• Make all cuts obvious, e.g. keep leaf base or section of petiole on specimen.
• For fertile angiosperms, prepare a flower to show internal structure.
• Cut bulky tissues (e.g. fruits and seeds, fleshy roots/bulbs/corms) in half.
• Any bulky fruits or seeds need a separate label.

• Lay out on a folded sheet of newspaper, leaving space for the label and a capsule (envelope) to contain loose material.
• Leave loose – do not attach to the newspaper with tape.
• Mark newspaper with COLLECTOR_ID number on outside bottom left hand corner – clearly mark any specimens which need more than one sheet.
• Ensure that both sides of the leaf can be seen by turning at least one leaf the other way up. If there is only one leaf, fold it to show both sides.
• Fold any long stems or leaves into ‘N’, ‘V’ or ‘Z’ shapes, as appropriate.
• Place newspaper between blotting paper in a plant press.

• Dry specimens using a drying frame over a source of heat, ensuring the heat source is not too hot, to prevent the specimens becoming brittle.
• Alternatively place the plant press in a warm, dry place (e.g. a drying room).
• Keep the number of specimens in the press to a minimum to aid drying.
• Change the blotting paper daily until the specimens are dry.
• Corrugated metal sheets can be used at intervals in the press, to increase heat and air flow.

Mounted specimen ready to be laid away in the RBGE herbarium