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- 1 (2009-11-19 (Thu) 13:55:07)
Plant sample collection for genetic diversity studies †
Field collection †
For each population †
- Take a GPS point
- Collect a voucher specimen (representative of the whole population) for the herbarium
Requirements for the voucher specimen for herbarium: †
- Includes a reproductive organ, either flower or fruit. For example, the voucher specimen for Sonneratia alba included a flower bud (Figure 1)
- Shows clearly the branching patterns of the species
Figure 1. Herbarium voucher specimen for S. alba
Sample collection †
- For each individual, collect a small branch with fresh, young leaves
- ATTENTION: Preferably, collected leaves should not contain any damage caused by parasites or herbivores (Figure 2)
- Place branch into a big bag. You don't have to mark the branches at this stage. Samples will be sorted later on.
- Collect at least 30 samples for each population
Figure 2. Leaves sample collected for one individual.
Post-collection processing
Post-collection processing comprises of two main stages: 1) Sample sorting, 2) Pressing and Drying.
1) Sample sorting
Empty all branches from the bag. You should have one branch for each individual. †
Each sample (a branch) will be divided into two parts: †
- For DNA extraction
- As a voucher specimen for future references
For DNA extraction †
- Choose the youngest leaf with the least damage from the branch. For example, in Figure 2, the leaf showed by the white arrow will be suitable
- Cut the leaf from the branch and tear it into small pieces of approximately 1cm x 0.5cm (Figure 3)
- Insert 5-6 pieces into a tea bag (Figure 4). Label the tea bag accordingly. Refer to section on labeling for the recommended practice
- ATTENTION: leaf pieces will be dried with silica gel later. To ensure thorough drying, reduce the number of pieces inserted into the tea bag for thick, succulent leaves.
For voucher specimen †
- Keep a voucher specimen for each sample so to enable us to reference back to it if there are any doubts of misidentification. The voucher specimen for individual sample will be a mini version of the voucher specimen for herbarium.
- Cut a small section of the branch for the voucher specimen (Figure 5).
- ATTENTION: The section should contain at least a leaf and a clear depiction of the branching pattern. As pointed out by the white arrow in Figure 5, all other leaves can be removed, but the petiole should remain to represent the branching pattern.
- Label the voucher specimen accordingly. Refer to section on labeling for the recommended practice.
Figure 3. Pieces of leaf sample for DNA extraction.
Figure 4. leaf pieces inserted into tea bag.
Figure 5. Voucher specimen for individual sample
Labeling †
- For herbarium voucher specimen, labeling is done on the outer side of the newspaper used for pressing (protocol for pressing is described in the following section) (Figure 6). Three important information that has to be included are:
Specimen number. Normally it will be the date of collection followed by the number of the voucher specimen. For example, in Figure 6, "091112" refers to date of collection (12th Nov 2009) and "01" is the number for the voucher specimen. †
Species name. Example: Sonneratia alba †
Location. Example: Sungei Buloh, Singapore †
- As for individual sample, each will be assigned a number. This number will be written on the tea bag (which contains leaf samples for DNA extraction) and the individual voucher specimen (Figure 7).
Figure 6. Labeling for herbarium voucher specimen.
Figure 7. Labeling for each sample. The sample number is written on the voucher specimen and the tea bag.
2) Pressing and Drying
Pressing and drying for all voucher specimens (herbarium and individual)
Herbarium voucher specimens †
- For herbarium vouchers, place the branch onto a sheet of newspaper (Figure 8).
- ATTENTION: Do not use many sheets of newspaper. This might prevent thorough drying.
- ATTENTION: Arrange the specimen properly. Ensure that there are some leaves facing upward and some facing downward (as shown by the two white arrows in Figure 8). Ensure that the flower or fruit is not blocked. If the specimen seemed too crowded with leaves, cut away some of the leaves/branches. Once the specimen is dried, the arrangement cannot be changed.
- Fold the newspaper and press onto it to flatten the specimen (Figure 9). Label the outer side of the newspaper.
Figure 8. Arrange specimen properly before pressing.
Figure 9. Fold the newspaper and press downwards.
Individual voucher specimen †
- Place a few voucher specimens flat onto a sheet of newspaper.
- Fold the newspaper and press downwards.
- Label the outer side of the newspaper (Figure 11). The labeling should include:
The number of the herbarium voucher specimen that all these individual voucher specimens should refer to (Eg. "09111201"). †
The sample numbers that are included in this sheet of newspaper (Eg. "11-20") †
Figure 10. Arrangement of individual specimens on newspaper.
Figure 11. Labeling of the newspaper containing individual voucher specimen.
Drying †
- All voucher specimens pressed in newspaper will have to be dried. Follow the below protocol for drying:
Figure 12. Put a wood plank at the very bottom of the stack. Then place a variegated cardboard on top.
Figure 13. Place a sheet of newspaper containing specimens onto the cardboard.
Figure 14. Place another cardboard, followed by another sheet of newspaper containing specimens.
Figure 15. Alternate this arrangement until all specimens have been placed.
Figure 16. Place another wood plank on top of the stack.
Figure 17. Tie the stack firmly together with a rope.
Figure 18. If an oven is available, place the stack in 65oC for 4 hours. If not, you may follow this set up to dry the specimens. Insert the stack into a bag for drying. Preferably the bag should be wind-proof. You may choose a bag with similar material as a wind-breaker.
Figure 19. This is an overview of the set up. Hot air is provided by the hair dryer, channeled into the bag by an open-bottom plastic bottle. The stack is placed a distance from the inlet of hot air, so to avoid excessive heating. A belt is tied around the stack externally to ensure that the hot air is channeled through the stack. One end of the bag is cut open to allow air to flow through.
Drying of samples for DNA extraction
The tea bags are placed into plastic zip-lock bag (Figure 20). You may place all tea bags from the same population into one zip-lock. †
The zip-lock should be labeled (Figure 21). Information which should be included are: †
- The location (Eg. Singapore)
- The herbarium voucher specimen that these samples should be referred to (Eg. "09111201")
- The sample numbers that are in the zip-lock (Eg. "DNA 1-32")
Insert sufficient amount of silica gel into the zip-lock bag before sealing it. Shake to mix well (Figure 22). †
To ensure thorough drying, place the zip-lock bag into a bigger zip-lock bag and fill the bigger bag with silica gel before sealing it tight. †
ATTENTION: Replace the silica gel when it has turned pink. †
Figure 20. Tea bags are inserted into a zip-lock bag.
Figure 21. Labeling on the zip-lock bag.
Figure 22. Silica gel is inserted into the zip-lock bag before sealing it.
Figure 23. The zip-lock bag is placed into a bigger zip-lock bag.
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