Guidelines for Tissue Collection and Storage Related to Genetic Studies of Rare Plants

The collection and preservation of plant tissues in DNA banks can serve as a long-term repository for the genetic material of a species, facilitating a range of genetic studies (phylogenetic, population genetic, and conservation genetic research) into the future. To maintain the integrity of a DNA collection, proper long-term storage is required to prevent degradation by nucleases, oxidation, hydrolysis, and ionizing radiation1,2. The quality of plant tissues or DNA extracts may be best maintained through cryopreservation, where materials are stored in the vapor phase of liquid nitrogen (below –180°C), a temperature at which molecular motion significantly slows. Because liquid nitrogen is cold in and of itself, mechanical failures and electrical outages are less of an issue with liquid nitrogen vats than ultracold freezers3. However, cryopreservation may not be financially or logistically feasible for many institutions.

As an alternative to cryopreservation, institutions may opt to maintain tissue collections in a dried or frozen (–20°C or –80°C) state. Silica gel is a commonly used desiccant that can be used to quickly and easily dry plant material for the purpose of extracting DNA6. If silica gel is used, plant tissues should be dried down as quickly as possible to mitigate DNA degradation. While desiccated tissues can be maintained at room temperature with silica in air-tight containers, at least for short periods5, Hodkinson et al. (2007) recommend maintaining dried plant materials in a –20°C freezer, a practice followed by the Missouri Botanic Garden7. The Smithsonian Institution, on the other hand, stores dried tissues at –80°C. Studies assessing the long-term effects of storing desiccated tissues at various temperatures are lacking, although “the colder the better” is generally the recommendation for fresh tissues and DNA extracts. DNA quality is further maintained by avoiding long-term storage of tissues in newspapers or other acidic materials, in humid conditions, and in contact with light, all of which may degrade DNA5. Storing tissues in air-tight containers is particularly important, as storage in containers with poor seals can reduce the quality of DNA5.

Maintaining fresh plant material in freezers is another viable method for the long-term preservation of tissues. Frost-free freezers that cycle through temperatures should be avoided, as temperature fluctuations can lead to DNA damage2 or protein deterioration8. Many plant tissues should be stable in long-term storage at -70°C to -80°C for the extraction of DNA, RNA, and proteins, while degradation may occur at -20°C9. However, Neubig et al. (2014) found that plant material frozen for 24 years maintained high quality regardless of whether tissues were stored at -20°C or -80°C in a pulverized or intact state5. Storage at -80°C may be the prudent choice to increase molecular integrity of the samples. Because -80°C freezers are prone to mechanical or electrical failures3, appropriate alarm systems and back-up storage space should be in place.

If time, space, and resources allow, lab facilities may choose to store DNA extracts in addition to tissue specimens5, as DNA samples are more stable than tissues9. DNA extracts are often stored in TE (Tris EDTA)2,9 buffer which prevents contamination by bacteria and degradation via nucleases10. To reduce the number of freeze-thaw cycles and thus a deterioration of DNA quality, frequently accessed samples can be stored in aliquots at -20°C, while archival extracts can be preserved at -80°C1,2,11. In a study investigating optimal storage of orchid leaf samples originally dried with silica, DNA extracts of the tissue stored at -20°C for 7 – 12 years had slightly higher quality than DNA newly extracted from the tissue that was stored in desiccated form in the dark without silica for the same period of time5. DNA extracts may be suitable for PCR for 4 – 7 years when stored at -18°C and for over 4 years when stored at -80°C1. After evaluating various storage buffers and temperatures, Smith and Morin (2005) found that the highest DNA yields were achieved with storage at -80°C or dried at room temperature with trehalose12. The highest DNA quality, on the other hand, was achieved with trehalose either dried or at -80°C, while quality deteriorated with storage at 20°C and 4°C. As is the case with plant tissues, storage at liquid nitrogen temperatures is optimal for preserving the highest quality extracts2. While, DNA extracts may better preserve the integrity of DNA, it is still worthwhile to maintain tissues in cold storage which will allow a wider variety of downstream molecular studies. Most DNA banks store tissues (or cells) and only extract DNA as requested1.

RNA-Seq is a burgeoning field in genomics that allows one to examine the gene-coding portions of genomes and to assess differential expression of genes among treatment groups of interest. Thus, labs may elect to store tissues for RNA in addition to DNA extractions. Tissues are typically flash frozen in liquid nitrogen and either stored at -80°C or at liquid nitrogen temperatures.

There are a multitude of nucleic acid storage methods, many of which are beyond the scope of this document. The long-term preservation of plant tissue samples via desiccation by silica gel and storing flash frozen tissue at -80°C are two methods that are commonly employed. The following recommendations for preserving plant material are based largely on guidelines from the Smithsonian Institution13 and the Global Genome Initiative14, which provide an excellent workflow for the collection and storage of plant tissues intended for DNA or RNA extractions. These guidelines include preserving silica-dried and flash-frozen plant tissues in long-term storage at -80°C. I summarize and expand on these guidelines below and refer the reader to these sources for further reference.

Tissue Desiccation and Storage

Working with Silica Gel Desiccant

While there are many potential reagents for desiccating plant tissues4, silica gel is a commonly used desiccant that can easily and rapidly dry down plant tissues for the purpose of DNA extractions4,6. Silica gel can act as a skin, eye, and respiratory irritant, and thus appropriate personal protective equipment (gloves, facemask, and goggles) should be worn when working with silica (consult the safety data sheets for your particular product). Silica gel is available in various mesh sizes, where finer grades (28 – 200 mesh size) can more quickly dry down tissues due to the larger surface area to volume ratios. However, larger grades (2 – 4 mm bead size) should be sufficient for drying, and are less expensive and less likely to be inhaled than smaller mesh13.

Indicating silica gel changes color when saturated and can thus be used to signal when a change of silica gel is required. A ratio of 1:10 indicating:non-indicating (white) silica gel is suggested13. Three types of indicating silica gel are available (colors change from orange to clear/white, orange to green, or blue to pink when saturated). The orange to clear/white indicating beads are recommended, as they are the least toxic13. Once saturated, the silica gel can be dried for reuse by heating in a plant dryer. Follow the manufacturer’s instructions for re-drying the beads.

Collecting, Desiccating, and Storing Plant Tissue

What to collect. Young, actively growing leaf tissue is best for DNA extractions9, although extremely young tissue that is fragile should be avoided13. Young tissue is preferable to older tissue because it has a more cells per volume13, is less likely to be colonized by fungal endophytes13, and is less likely to have secondary compounds that could interfere with DNA extractions and inhibit downstream PCR reactions. It is also possible to extract DNA from young shoots, roots, seeds, pollen, or gametophytes9.

How much tissue to collect. From 15 spatially distant individuals (see section on Sample Size), collect 3 – 5 leaves per plant7 (or 10 – 25 cm2 of leaf material from species with large leaves13), provided it is not detrimental to the plant. Because leaf thickness can vary by species, another useful guideline may be to collect at least 100 mg fresh tissue, if not harmful to the plant. DNA extractions generally require 50 mg to less than 100 mg wet tissue per sample (or less than 20 mg dry tissue)15, and it is ideal to have extra material with which to work should optimization of the extraction protocol be required. Thus, 100 mg wet tissue may provide enough material for two extractions. This amount can be adjusted depending on the starting tissue amount recommended by the DNA extraction protocol in use.

How to collect tissues. To avoid DNA degradation, it is advisable to dry down tissues as quickly as possible, preferably with 12 – 48 hours13, although within 12 hours is most ideal6. This can be achieved by storing samples with silica, in a ratio of at least 10:1 of silica gel to total leaf tissue5,6, although more silica may be required for species with thick leaves or high water content. Drying should occur in a cool location at ambient temperature13. Leaf tissue can be placed in separate coin envelopes (2 ¼ x 3 ½ inches) by sample13, properly labeled with sample name, date, species, collection location, and collector. White coin envelopes are recommended, which tend to be less acidic (and thus less degradative to DNA) than brown envelopes13. Envelopes are then placed in a sealable Ziploc plastic bag with the appropriate ratio of silica gel desiccant for the amount of tissue stored. Multiple envelopes can be stored in the same Ziploc bag, preferably grouped by collection location, and archival paperclips can be used to secure envelopes if needed. Alternatively, samples can be collected into small, sealable polyethylene bags (2 ¼ x 3 ½ inches) for each sample, with an individual aliquot of silica13. Although tissues may dry more quickly, the drawback is that silica is time-consuming to remove when samples are ready for permanent storage and silica cannot be re-used (to prevent contamination)13. See Funk et al. (2017) for a list of products and suppliers.

To determine whether samples have sufficiently dried, samples can be checked after the desired drying time has passed (e.g. 12 hours). Samples are sufficiently dried if they break cleanly when bent6. At this point, most silica except a small amount can be removed6, as excessive storage of tissues with silica gel can lead to DNA degradation9. To optimize the drying process, multiple practices can be followed, as suggested by Funk et al. (2017). It is advisable to move the beads around periodically to maximize their contact with leaf tissues. When placing leaves in envelopes, spread leaves throughout the envelope, as stacking can prevent drying of the interior leaves. Cut particularly thick or waxy leaves into smaller pieces to maximize surface area for drying5. For very large thick mid-ribs, remove with scissors to dry easily and avoid tearing the leaf, which could release enzymes that degrade DNA7. Clean scissors with 70% ethanol between samples to prevent cross-contamination.

Long-term storage. Once tissues are properly dried, most silica can be removed and the samples can be placed under long-term storage conditions. Store samples at -80°C13, if available, or if ultra-cold freezers are not available store at -20°C7,10 or room temperature4 in airtight opaque containers. Coin envelopes or Ziplocs can be stored in boxes with small amounts of silica to absorb any excess moisture9,13. The Smithsonian Institution recommends Lock & Lock boxes (HPL 836) and including a relative humidity indicator card (Sorbent Systems) in each box to monitor humidity, which should not exceed 30%13.

Flash Freezing and Storing Tissue Samples

Flash freezing tissue samples in liquid nitrogen and preserving them in -80°C freezers is another method of preserving high quality DNA or RNA. Tissues can be collected in 8 mL cryogenic tubes (externally threaded with o-rings)13, labeled appropriately with sample and collection information. Cryovials are place in liquid nitrogen-filled storage Dewars at the collection location and are transported to the lab where they are stored in cryoboxes in -80°C freezers. If freezing samples for RNA preservation, apply extra cleanliness procedures, including wearing laboratory gloves and properly disinfecting all tools between samples to minimize nuclease exposure. Because RNA degrades quickly and gene expression changes rapidly, samples should be placed into the liquid nitrogen as quickly as possible after collection. Be sure to wear appropriate PPE (goggles, cryo gloves, long pants, close-toed shoes, lab coat) when working with liquid nitrogen. As an alternative to working with liquid nitrogen, samples can also be preserved in RNA later.

SAMPLE SIZE

DNA. Collecting multiple samples per population opens the possibility of future population genetic analyses (e.g., assessing genetic diversity, gene flow, population structure, population assignment, cryptic species, signals of selection, etc). Traditionally, researchers have suggested collecting as many samples per population as possible. However, some recent studies suggest that with the larger number of single nucleotide polymorphisms (SNPs) that can be assayed through next generation sequencing, it is possible to sample fewer individuals per population16–18. Through simulations of empirical data, Nazareno et al. (2017) found that 6 – 8 samples were sufficient to obtain accurate diversity estimates for a self-incompatible, outcrossing Amazonian plant species when 1000 SNPs were considered17. Similarly, Willing et al. (2012) found that 4 – 6 samples and greater than 1000 SNPs could sufficiently estimate genetic differentiation for a simulated dataset, but suggested that greater that 10 samples are required for outlier analyses16. Nevertheless, a recent simulation comparing trade-offs in sample size versus sequencing depth encouraged sampling as many individuals as possible at the expense of lower sequence coverage, in order to estimate genetic diversity accurately and calculate population differentiation statistics19.

For institutions interested in population genetic studies, we suggest beginning with a sampling target of 15 individuals per population, provided that there is a sufficient number of individuals from which to collect and that such collections would not harm the population. However, the ideal size sample will depend on the specific goals of the study (e.g. the type of genetic estimates desired) and the life history of the species of interest (demographic history, mating system, pollen and seed dispersal syndrome, etc)17. Individuals collected should be spatially distant and thus more likely to be representative of the population’s genetic composition.

RNA. Collecting samples for RNA is both effort- and time-intensive. Labs may opt to collect one sample per population or species for RNA preservation to begin, and collect additional samples per population as specific studies require. Having at least one sample per species would allow the creation of a reference transcriptome, which could be useful for identifying genes and variants and may facilitate future studies.

References

1. DNA banks – providing novel options for genebanks? (IPGRI, 2006).

2. Lee, S. B., Crouse, C. A. & Kline, M. C. Optimizing Storage and Handling of DNA Extracts. in Forensic DNA Analysis 19–38 (CRC Press, 2010). doi:10.1201/b15361-4.

3. Corthals, A. & Desalle, R. An Application of Tissue and DNA Banking for Genomics and Conservation: The Ambrose Monell Cryo-Collection (AMCC). Systematic Biology 54, 819–823 (2005).

4. Nagy, Z. T. A hands-on overview of tissue preservation methods for molecular genetic analyses. Org Divers Evol 10, 91–105 (2010).

5. Neubig, K. M. et al. Variables affecting DNA preservation in archival plant specimens’. in DNA banking for the 21st century: Proceedings of the US Workshop on DNA Banking 56 (2014).

6. Chase, M. W. & Hills, H. H. Silica Gel: An Ideal Material for Field Preservation of Leaf Samples for DNA Studies. Taxon 40, 215–220 (1991).

7. Missouri Botanical Garden. DNA Bank. Available at: http://www.missouribotanicalgarden.org/plant-science/plant-science/william-l-brown-center/wlbc-resources/wlbc-databases/dna-bank.aspx. (Accessed: 9th May 2018)

8. Florian, M.-L. The effects of freezing and freeze-drying on natural history specimens. Collection Forum 6, 45–52 (1990).

9. Prendini, L., Hanner, R. & DeSalle, R. Obtaining, Storing and Archiving Specimens and Tissue Samples for Use in Molecular Studies. in Techniques in Molecular Systematics and Evolution (eds. DeSalle, R., Giribet, G. & Wheeler, W.) 176–248 (Birkhäuser Basel, 2002). doi:10.1007/978-3-0348-8125-8_11.

10. Hodkinson, T. R. et al. DNA Banking for plant breeding, biotechnology and biodiversity evaluation. Journal of Plant Research 120, 17–29 (2007).

11. Visvikis, S., Schlenck, A. & Maurice, M. DNA Extraction and Stability for Epidemiological Studies. Clinical Chemistry and Laboratory Medicine 36, (1998).

12. Smith, S. & Morin, P. A. Optimal Storage Conditions for Highly Dilute DNA Samples: A Role for Trehalose as a Preserving Agent. Journal of Forensic Sciences 50, 1–8 (2005).

13. Funk, V. A. et al. Guidelines for collecting vouchers and tissues intended for genomic work (Smithsonian Institution): Botany Best Practices. Biodivers Data J (2017). doi:10.3897/BDJ.5.e11625.

14. Gostel, M. R., Kelloff, C., Wallick, K. & Funk, V. A. A workflow to preserve genome-quality tissue samples from plants in botanical gardens and arboreta. Applications in Plant Sciences 4, 1600039 (2016).

15. DNeasy Plant Handbook – QIAGEN. (2018).

16. Willing, E.-M., Dreyer, C. & Oosterhout, C. van. Estimates of Genetic Differentiation Measured by FST Do Not Necessarily Require Large Sample Sizes When Using Many SNP Markers. PLOS ONE 7, e42649 (2012).

17. Nazareno, A. G., Bemmels, J. B., Dick, C. W. & Lohmann, L. G. Minimum sample sizes for population genomics: an empirical study from an Amazonian plant species. Mol Ecol Resour 17, 1136–1147 (2017).

18. Jeffries, D. L. et al. Comparing RADseq and microsatellites to infer complex phylogeographic patterns, an empirical perspective in the Crucian carp, Carassius carassius, L. Mol Ecol 25, 2997–3018 (2016).

19. Fumagalli, M. Assessing the Effect of Sequencing Depth and Sample Size in Population Genetics Inferences. PLOS ONE 8, e79667 (2013).

Suggested Citation

Center for Plant Conservation. Guidelines for Tissue Collection and Storage Related to Genetic Studies of Rare Plants in CPC Best Plant Conservation Practices to Support Species Survival in the Wild. Web Version. https://plantnucleus.com/best-practices/guidelines-tissue-collection-and-storage-related-genetic-studies-rare-plants Accessed: 09/30/2020 - 5:08pm