From Plant Press, Vol. 24, No. 1, January 2021.
By Gabe Johnson.
Countless Ph.D. research botanists sincerely believe such misconceptions as: ethanol immediately destroys DNA in leaf tissue collections, leaves in both fresh and spent silica gel are equally dry, desiccated tissues rehydrate in absolute ethanol, and that EDTA inhibits all deoxyribonuclease (DNase) activity. Ethanol treatment of plant tissue for molecular studies is especially misunderstood by researchers in recent times, even though it can be a useful tool to improve DNA preservation for molecular studies. In this Information Age with the ability to sequence and process trillions of nucleotides of data in a single study, the capacity to isolate high quality DNAs must advance to utilize these new technologies to their fullest potential. Greater DNA quality and quantity are required for many of these next-generation sequencing methods than were necessary for traditional PCR-based Sanger methods. Obtaining sequenceable DNAs from a more complete taxonomic sampling will require an equally diverse set of DNA extraction tools, including ethanol preservation and pretreatment.
Biological tissues typically undergo a “treatment” such as freezing or drying prior to DNA extraction. The degree to which this treatment preserves the nucleic acid content of the leaf depends on the needs of the researcher and the logistical limitations of the field work. Such treatments can be as simple as allowing harvested leaves to dry in ambient conditions (usually with poor results) or the use of one of an array of desiccants, solvents, buffers, or cryogens (see Funk et al., Biodiversity Data J. 5: e11625; 2017). While a diversity of tissue treatment methods has been used to preserve DNA (i.e., Hamilton et al., Anal. Biochem. 49: 48-57; 1972), by the mid 1990’s silica gel desiccation was the default method for plant systematics studies (Chase et al., Taxon 40: 215-220; 1991). This preservation method has many virtues by being simple, inexpensive, non-toxic, and satisfactory for most major lineages of plants from algae to angiosperms, as well as fungi. However, the resulting DNA quality has limitations, and in particular is more fragmented than if extracted directly using fresh or frozen tissues.
Tissue from a collard leaf, Brassica oleracea L., is preserved in 96% ethanol to preserve DNA. (A) Since vascular tissue contains much less DNA per unit area, the large veins are removed. (B) Leaf lamina is quickly torn into ~1 cm2 pieces by hand. The ruler to the right is in cm. (C) Leaf fragments very easily fit into the mouth of a 50 mL conical vial. (D) The name of the plant and collection information is written in pencil on card stock and inserted into the vial; pencil writing does not dissolve away in ethanol. (E) To prevent excessive evaporation or accidental spills, the cap is sealed with parafilm. (F) The outside of the plastic vial is labeled with a special solvent resistant permanent marker. (G) Leaf fragments sink into the ethanol and chlorophyll leaches from tissues, leaving an off-white color. If the leaf fragments float and remain green after a couple hours, cut tissue into even smaller pieces. Adding isopropanol to the ethanol can also help. (photos by G. Johnson)
Just as DNA in herbarium specimens is better preserved for some lineages more than others, better DNA is extracted from silica dried tissues for certain taxa more than others. Tissue preservation media, dry or liquid, have a dual purpose: to protect the DNA within the tissue and to prepare the tissue for homogenization and efficient DNA isolation. Upon separation from the plant, a leaf undergoes a physiological stress response that initiates many cellular processes related to senescence: wound formation, polyphenol oxidation, and apoptosis. These cellular changes can rapidly degrade the DNA through programmed exonuclease activity and oxidative stress from reactive chemical species used in wound defense.