INSTRUCTIONS FOR SENDING PLANT MATERIAL FOR DNA ANALYSIS

General Collecting Information. For most projects, we do not require more than one sample per species. If a voucher (dry, typical herbarium specimen) can be sent, this would be appreciated. If it cannot be sent, an indication of where the voucher will be housed is needed. When collecting smaller parasitic plants, clip the host branch keeping the parasite as intact as possible. The plants must be clean and free of soil, insects and macroscopic fungi. Avoid "steaming" the plant such as during transit from the field to the lab. If some time must elapse before shipping, refrigerate but do not freeze the plant. I will reimburse mailing costs, but I must approve this prior to shipping - Send an email to DLN.

A number of methods can be used to ship plant samples for later DNA extraction. Use the method that is most applicable to your collecting conditions.

Silica Gel Dried Material

How Does it Work? High molecular weight DNA can be obtained from most plants by drying the tissue on silica gel. The concept here is that the sample should dry quickly but not at high temperature. This means that for especially large or succulent material it is necessary to tear or cut the leaves with a sharp razor blade to allow more rapid drying at room temperature.

Where Do I Get Silica Gel? Silica gel can be obtained from many sources (even locally ­ it's used for flower drying, to keep electronics dry, etc.). I purchase it through Fisher Scientific:

Silica gel, non-indicator type, 28-200 mesh, grade 12, 2.5 kg, S157-212 . Ca. $134.00
Silica gel, indicator type, 10-18 mesh, grade 47, 500 g,  S161-500. Ca. $46.00.
Mix these two types together for use.

How Much Tissue and Silica Gel? Collecting on silica is very straightforward. A 1 to 10 ratio of plant material to silica gel is commonly used. Thus, to dehydrate 1 g of tissue (wet weight), use 5 g of silica. Simply place the silica gel in a ziplock style plastic bag, add the plant tissue, and seal. Small (sandwich size) heavy gauge (freezer style) ziplock plastic bags are best because they do not tear. The material should be bone dry within 24 hours. If it is not, the silica gel may not have been fully dehydrated or the weight ratio was incorrect. I generally use grade 12 silica with a bit of indicator silica mixed in. The indicator silica "reports" when the silica is dehydrated (blue) or hydrated (pink). Some labs reuse silica gel (after baking it in an oven), but I worry about cross contamination, so I always use new silica.

Labeling and Shipment. Label information is important, so please provide the following:

The label should be placed inside the ziplock bag with the specimen. For shipping - simply use the most reasonable postal rate available (no need for overnight or express service). Also, no special permits are required for dry material. Mark outside of package "For Scientific Study - No Commercial Value".

Fresh Material

Fresh (or fresh-frozen on liquid nitrogen) plant material is the ideal starting material for DNA extractions. Logistical reasons, of course, prevent workers from using this option. For many plants (especially xerophytically adapted or succulent ones), shipment at ambient temperature within plastic bags is possible IF the time in transit is short. Within the United States, express mail services are available nearly everywhere and this guarantees delivery overnight or within one business day. International express shipments are usually quite expensive and may involve custom brokers, etc., hence are infrequently used. International shipments of fresh plant material also requires importation permits from the USDA Animal Plant Health Inspection Service, Plant Protection and Quarantine. Because some parasitic plants are listed as noxious weeds, this group may also require additional paperwork. For these reasons, I recommend against shipping fresh parasitic plant material.

Herbarium Material

DNA can be extracted from dry herbarium specimens, however, it is generally inferior to material dried on silica gel. Several factors appear to affect the quality of DNA present in herbarium specimens:

Herbarium samples can be shipped via regular mail ("book rate") and usually do not require special permits to enter the United States. Note they may need permits to be shipped from particular countries. Mark the outside of the box "Herbarium Samples For Scientific Study - No Commercial Value". For more on extracting DNA from herbarium specimens, see Savolainen et al. (1995).

Preserved (Ethanol Fixed) Material

Plant samples preserved in ethanol can be used to obtain genomic DNA (see Flournoy, Adams and Pandy 1996), but extraction methods must be modified (specifically, use of a protease). Samples preserved in FAA or any other fixative that contains formaldehyde are generally degraded. For other "wet methods" of preserving plant tissues in the field, see Nickrent (1994) and Storchová et al. (2000).

References

Flournoy, L. E., R. P. Adams, and R. N. Pandy. 1996. Interim and archival preservation of plant specimens in alcohols for DNA studies. BioTechniques 20: 657-660.

Nickrent, D. L. 1994. From field to film: rapid sequencing methods for field collected plant species. BioTechniques 16: 470-475.

Savolainen, V., P. Cuénoud, R. Spichiger, M. D. P. Martinez, M. Crèvecoeur, and J.-F. Manen. 1995. The use of herbarium specimens in DNA phylogenetics: evaluation and improvement. Plant Systematics and Evolution 197: 87-98.

Storchová, H., H. Hrdlicková, J. J. Chrtek, M. Tetera, D. Fitze, and J. Fehrer. 2000. An improved method of DNA isolation from plants collected in the Field and conserved in saturated NaC1/CTAB solution. Taxon 49: 79.