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OSSSU PROTOCOLS

SPECIES IDENTIFICATION

    1. Find collection records of both pollen and seed parents, and extract relevant data.
    2. Determine whether a herbarium specimen has been made.
    3. Where no herbarium specimen has been made, a pressed specimen should be made where practical.  This should include flowers and leaf as a minimum, and be lodged in an appropriate institution herbarium.
    4. Failing the above a flower should be preserved in spirit and lodged within a herbarium.
    5. As a bare minimum, a photographic record (or drawings) should be made of the flower (front and side views) and the whole plant.
    6. Link records within your database to the herbarium specimen and original collection records.

FIELD COLLECTION

    1. Harvesting of seed should only proceed with the approval of appropriate bodies.
    2. Take photographs of the habitat, and record appropriate field data (e.g. altitude, temperature data, phorophyte if possible if it is an epiphyte, soil conditions, light levels).

POLLINATION

    1. Endeavour to cross-pollinate different clones, as this will produce progeny with as broad a genetic base as possible.  However, where only a single plant of a rare species is available, self pollination is acceptable.
    2. Choose healthy, well-grown plants.  These are generally more likely to produce capsules containing large quantitities of viable seed.
    3. When available, pollinate a number of flowers on each plant.  Where a species carries multiple flowers, pollinate 10% of available flowers. 
    4. Pollinate freshly opened flowers.  They are more likely to be successful than older blooms i.e. pollen is likely to begin losing its viability as flowers and pollen age.
    5. To maximise the number of full seeds per capsule, a full pollen load (i.e. all of the available pollen from one individual flower) should be applied to the stigma of the seed parent.
    6. Label the pod parent with the date of pollination, and record.
    7. Record how many flowers were pollinated, and how they were pollinated (by hand or naturally-occurring pollinators).

HARVESTING SEED

    1. Regular observation of developing capsules is recommended, with daily observations as they approach maturity. Generally, seed capsules will change colour from green to yellow towards the end of their development.  However, judging the maturity of seed capsules of individual species can be a matter of experience. Development times of seed capsules will vary somewhat according to environmental conditions.
    2. Records environmental conditions as seed capsules mature (e.g. greenhouse temperature).
    3. Do not place bags around developing of seed capsules (to prevent loss of seed), as the seed capsules tend to 'sweat', and this can lead to loss of the whole capsule and its contents due to bacterial or fungal infections. 
    4. Record date of seed collection and whether the capsule had begun to split (dehisce).  Photograph the capsule at the time of harvest if possible.
    5. Calculate and record time to maturity of the seed capsule so that this can be related to seed quality.
    6. Capsules which have not yet split, and have been removed from the parent plant, can be allowed to dehisce 'naturally' in a small beaker or similar vessel in a cool, dry room.

PROCESSING SEED

    1. To reduce contamination and debris, trim both ends of seed capsules.
    2. To remove seed from capsule, gently squeeze the capsule from both ends.
    3. We would like to know how many seeds are present in the capsules of all 250+ species in the project.  In order to achieve this, we suggest that you weigh all of the seeds from 3 capsules (or more where available) separately.  A small sub-sample of seed can then be weighed on a 7 place balance, where available, and the number of seeds counted in that sub-sample.  This information can then be used to calculate the numbers of seeds in each of the individual capsules.


You will be provided with small seed storage tubes (grey top) which can be placed within the larger Universal tubes (blue top), together with a sachet of silica orange indicator.

The universal tubes (blue top) can then be stored within a larger sealed jar.


SEED STORAGE
    1. Aim to obtain a good sample size (20,000 seeds, or more where possible).
    2. Pool samples and mix thoroughly (this will assist in maintaining the maximum genetic diversity in each flask).  Only store seed separately for individual plants where sufficient material is available to give good sample sizes (to fill a flask).
    3. Dry/equilibrate seed over a saturated solution of lithium chloride (LiCl).  The relative humidity will be approximately 12% at 20ºC.
    4. To make a saturated solution, dissolve the salt in warm water until excess crystals remain in the bottom of the vessel.  Bring the solution to a gentle boil to dissolve the crystals.  On cooling some undissolved salt should remain in the bottom of the desiccator.
    5. Place seed as a thin layer in the desiccator (seal with vaseline) for seven days at approximately 150C.
    6. Rapidly transfer seeds (to avoid change in seed moisture content) to hermetically-sealed tubes.  Tubes should be as full of seed as possible, so that the seed dominates the air space.
    7. Small vials of seed can be stored within larger tubes, within a larger, air-tight, storage jar.
    8. Individual storage tubes and their containers should all be labelled.
    9. Storage vessels should contain sachets of silica orange as an indicator (not as a desiccant) to demonstrate that seals have not been broken and moist air has not entered the storage vessel.  Prior to use, the sachets of silica gel should be equilibrated over the saturated LiCl solution.  Orange silica gel begins to turn green when the relative humidity approaches 22%.
    10. Store the tubes of seed in a freezer at around -20ºC.

QUANTIFICATION OF GERMINATION

    1. Draw (or photograph) your criteria for germination.  A key criterion is the splitting of the seed coat (testa), but criteria may vary between species.  For example, the criteria for epiphytic species may be the appearance of rhizoids and the greening of the protocorm.  Terrestrial species may not develop rhizoids, and are normally germinated in the dark and therefore do not turn green at this stage.
    2. Score all seeds and record as the following 3 categories: empty, potentially incompetent (i.e. an abnormally small embryo), potentially viable (i.e. with a full embryo).
    3. Record germination of full (i.e. viable) seeds.
    4. Sow seeds on at least two different media.  The media should have contrasting chemical compositions.  We suggest that everyone uses Knudson C as one of the chosen media, as this would enable us to carry out a comparison of all 250+ species conserved by project members on one medium.
    5. Sow a minimum of 100 to 200 seeds per Petri dish.  NB Lower numbers (e.g. 50 seeds) are acceptable where insufficient seed is available e.g. where species have small capsules.
    6. Spread seed evenly over the surface of the medium.
    7. Sow a minimum of 3 replicate Petri dishes for each species.
    8. Score germination at one monthly intervals for a minimum of 3 months, and up to 9 months, or longer where germination continues to increase.  The aim is to produce complete germination progress curves until the germination has reached its maximum (ideally 100% of viable seeds)
    9. Where too many seeds have been sown, the dish can be divided into quarters which are marked with a permanent pen.  The same quarter should be counted on each occasion.  A piece of graph paper should be place behind the dish to assist counting.
    10. Temperate terrestrial species should be germinated in the dark.  Where appropriate, germination scoring can take place under a green 'safe light'.
    11. Tropical epiphytic species should be allowed to germinate under cool white fluorescent lights using a 12 hour photoperiod.
    12. Temperate terrestrial species should be germinated at 20±20C.
    13. Tropical epiphytic species should be germinated at 250C.
    14. Monitoring orchid seed during the storage period should be carried out according to the following schedule: During the OSSSU period: immediately after drying, 1 month, 1 year, 2 years; Post OSSSU: 4 years, 8 years, beyond.
    15. Three randomly selected containers should be removed for each test.

    NB. Record everything, and keep good records.
    Difficult to germinate species should be assessed early on.