Plant Inventory Protocol

ampling of leaves for DNA isolation

These guidelines are excerpted, with slight modifications, from Caujapé-Castells et al (2011) Manual del Banco de ADN de la Flora Canaria. Departamento de Biodiversidad Molecular y Banco de ADN, Jardín Botánico Canario “Viera y Clavijo”-Unidad Asociada CSIC (Cabildo de Gran Canaria).

Following these indications, the samples are generally kept in a good condition for a long time.

 

GENERAL GUIDELINES

 Provide a minimum geo-referencing of the accessions (GPS, Google earth, UTM, clear descriptions of the location…). If necessary for your objectives, map/geo-reference the plants within the accessions.

 Collect as many leaves as possible per sampled individual, to have a surplus of leaf material.

 Only if leaves are not available, you may take other parts of the plant, but at the risk of not obtaining a good yield in the subsequent DNA isolation.

 The number of individual samples to collect per accession depends to a large extent on the objectives of the research, but always try to collect as many as you may (a minimum of three individuals per accession, and separated from each other on the population space (if possible).

 Place each individual sample within a zippered plastic bag (we use bags of 10×15 cm) with an exclusive code (we print numerical labels and stick them on the external side of the bag). Add a small-moderate amount of silica gel (proportional to the number and size of the leaves). You may add the silica gel by night, or next day in your lab.

 If the leaf samples are quite humid at the moment of collection, it is preferable that they sleep overnight on a dessication oven (btw. 50-60 ºC) prior to depositing them in the plastic bags with silica gel.

 Before sealing the plastic bags, remove most of the air they may contain, to eliminate environmental humidity that could accelerate deterioration, and to reduce storage space.

 When the silica gel within the plastic bag gets humid, replace it for a dry one.

 Keep a database/Excel file with all the info associated with the samples

 

OTHER CONSIDERATIONS

 There are also some other simple rules that we need to observe (when possible) to guarantee an optimal extraction and further PCR amplification:

 1. Make sure that all collected leaves do belong to the target species

 2. If taking several leaves per specimen, make sure they belong to the same individual.

 3. Do not sample exclusively the biggest plants, try to represent all morphological forms present in the population space

 4. Take always the greener and cleaner leaves from those available in each target plant.

 5. Whenever possible, take a herbarium voucher, and deposit it on an institutional herbarium.

 6. Always try to collect adult leaves that have not started senescence.

 7. Do not collect succulent leaves unless you may readily dessicate them and extract their DNA soon after collection.

 8. Collect the whole leaves, without cutting them or wounding them. If they are a bit bigger than the sampling bag, fold them carefully to make them fit in.

 9. Take the leaves to pieces only if they are extremely big.

 10. Always keep a sample of the dry leaves on a refrigerated chamber. In any case, store the leaf samples in a fresh place, away from the direct light.