Protocol

Author

Gemma Hoyle

Overview

Standard protocol for the collection of native alpine seeds for ex situ conservation and general (unspecific) research purposes.

Materials/Equipment

• Standard Checklist for assessing a potential seed collection (example below)
• Standard Collection Data Sheet (example below)
• Calico bags/paper bags/envelopes for collected seed, depending on seed size
• Hand lens x10 magnification
• Identification books
• Labels for herbarium specimen
• Large plastic bags for herbarium specimens
• Camera
• GPS

Procedure

1. Locate plants setting mature seed
   • This may be an opportunistic find or a targeted population of a specific species.
   • Plants need to be at or very close to the point of natural seed dispersal to ensure seed maturity and maximised longevity in storage.
   • To assess seed maturity, consider:

     -Seed colour change

     -Seed hardness

     -Fruit development, colour, texture, odour (e.g., ripe fleshy fruits)

     -Detachment of seed within the fruit (e.g., rattling)

     -Detachment of the dispersal unit from the mother plant

2. Allocate someone to fill out the FIELD DATA SHEET (example below)
   • For assessing a potential seed collection.
   • This checklist is completed regardless of whether or not a collection is made.
3. Attempt to identify family, genus and species – Field name
   • Use expertise, photos, herbarium specimens, publications.
   • If species ID is not certain but collecting seed is deemed worthy, take special care to collect seed from exactly the same species.
   • A herbarium can determine whether or not the Field name is correct using the herbarium specimen (see below).
4. Assess population size
   • Allow 2 or 3 people to independently estimate the number of individual plants within the visible population and agree on a rough total.
   • At least 20 individual plants will constitute a population.
   • Keep collections made from separate -clumps’ of the same species separate.
   • In the case of sparse / dispersed populations of < 20 individuals, seeds from individual plants should be kept separate. This allows max. genetic variation to be maintained in future multiplication steps by ensuring the next generation are harvested from every parent plant originally sampled in situ.
5. Assess safety and practicality of making collection
   • Can all plants be sampled from safely
   • Is there enough time left in the day
   • Consider weather conditions.
   • Are there enough people
6. Assess seed quality
   • Carry out a cut test in order to assess the proportion of empty, damaged, infested and immature seeds.

     -Section a total of at least 10 seeds from a range of several well-spaced individuals across the population.

     -Use scalpels, scissors, nail clippers or finger nails to cut seeds in half and examine embryo/endosperm contents.

     -Ascertain what a healthy, full, mature seed looks like (i.e. full, with firm and fresh endosperm / embryo).

     -Tiny seeds can be held on adhesive tape during sectioning and examined using a hand lens.

7. Average number of dispersal units per plant
   • Allow 2 or 3 people to quickly estimate number of dispersal units (flower heads/capsules/fruits etc.), per plant and agree on an average.
8. Average number of potentially viable seeds per dispersal unit
   • Estimate the number of seeds per dispersal unit.
   • Based on cut test results (# 6), estimate the proportion of viable seeds per dispersal unit.
9. Estimate number of potentially viable seeds available for collection
   • Estimate the total number of potentially viable seeds ready for collection.
   • Calculate 20% of this available seed.
   • Minimum number of seeds required per collection: 25 potentially viable seeds from 20 parent plants (total 500 seeds) without sampling more than 20% of available seed.
10. Decide to make a collection and decide sampling technique.
    • Proceed if a minimum of 500 seeds can be collected without sampling more than 20% of available seed.
    • Agree on number of plants to be sampled from by each person.
    • Agree on how many seeds/dispersal units are to be collected from each plant.
    • Decide between calico bags/paper bags/envelopes depending on seed size.
    • Agree upon a sampling technique that minimises the presence of any empty, damaged or immature seeds.
    • Each person to sample from different plants, therefore decide on most appropriate approach e.g. tagging/staking plants, walking transects etc.
    • Remember that seed collecting must be carried out randomly and evenly – each individual plant must have an equal chance of being selected.
    • Record the total number of plant sampled from on the data sheet.
    • Agree to stop collecting when either 20% of available seed or 10,000 seeds have been collected – assuming it is time efficient to collect up to 10,000 seeds.
    • Do not collect seeds from the ground.
11. Collect a herbarium specimen
    • Select vigorous, typical specimens. Avoid insect-damaged plants.
    • Specimens should be representative of the population, but include the range of variation of the plants.
    • Specimens should include flowers and/or fruits. Different types of foliage, flowers and fruits should be collected from the same plant.
    • Roots, bulbs, and other underground parts should also be dug up, and soil removed.
    • Tie a numbered tag onto the specimen. Assign specimens the same corresponding # as the seed collection. All duplicate specimens should bear the same number.
    • Collect sufficient material to fill a herbarium sheet (c. 450 x 300 mm), leaving enough room for the label. Plants too large for a single sheet may be divided and pressed as a series of sheets.
    • Bark and wood samples are desirable additions when collecting woody plants.
    • Tall rosette plants and grasses may be pressed complete by bending them once or more into the shape of a -V’, -N’ or -M’.
    • Dioecious plants should be represented by both sexes.
    • Press specimens ASAP. If pressing is not possible, specimens may be stored in plastic bags, preferably wrapped in damp (not wet) papers. Keep cool and moist.
    • Place each specimen in a fold of several sheets of newspaper within the press.
    • Add a sheet of corrugated cardboard to act as a ventilator if necessary.
    • Replace newspaper with dry sheets each day until specimen is dry and brittle.
12. Allocate someone to:

    -Fill out Collection Field Data Sheet (example provided below)

    -Check everything is appropriately labelled

    -Take photos

    -Take responsibility for additional necessary handling (e.g. seed drying etc.) prior to returning from field

Notes and troubleshooting tips

This standard protocol is expected to be revised for particular experimental needs as necessary.

Links to resources and suppliers

Standard checklist for assessing potential seed collections (can be modified)
Download Checklist

Standard collection field data sheet, seed collections (can be modified)
Download Data Sheet

References

See attached forms: ‘Standard Checklist for assessing a potential seed collection’ and ‘Standard Collection Data Sheet’

Health, Safety & Hazardous Waste Disposal Considerations

• Weather and site conditions should be carefully considered and planned for prior to undertaking any field expedition.