CPC Best Plant Conservation Practices

to Support Species Survival in the Wild

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Key Messages about Orthodox, Intermediate and Recalcitrant Seed

Orthodox seeds are capable of being dried to internal seed moisture of less than 12% water, stored at freezing temperatures, and surviving.
Recalcitrant seeds cannot be stored in a conventional freezer as they cannot survive after drying and/or freezing at -20°C.
Intermediate seeds tend to age faster than orthodox seeds and may have only a 5 year lifespan when stored at -20°C. They have greatest longevity when dried between 45 and 65% RH.
There is much to learn about seed traits that contribute to storage longevity.

Similarities and Differences Among Seed Storage Guidelines

Conventional seed banking is a fundamental plant conservation practice within the CPC network and around the world. In 1995, CPC published Guidelines for the Management of Orthodox Seeds (Wieland 1995). These guidelines not only presented practical advice for preserving the genetic diversity of seeds of the rarest plant species in North America based upon the best science of the period, they blossomed from a partnership with the ARS-USDA National Laboratory for Genetic Resources Preservation (formerly the National Seed Storage Laboratory) in Fort Collins, Colorado. The dedicated NLGRP staff, their excellent facilities, and collaborative research with CPC conservation officers have been central to our growing understanding of how to store seed while maintaining viability. While much has stayed the same, this updated version details some of the technological advances that have emerged over the last 25 years.

In addition to using recommendations from our previously published guidelines (Wieland 1995), these updated guidelines incorporate protocols from FAO’s Genebank Standards for agriculture (FAO 2014), MSBP Seed Conservation Standards (MSB 2015), our research, as well as published research from around the world. Note that the international guidelines from FAO and MSBP are broad, encompassing advice for economically important and common species, the CPC guidelines pertain to collections of rare wild species and assuring that seeds can live long enough in storage to support a conservation mandate.

Prior to making seed collections, there are several considerations a practitioner can make depending upon the type of plant material that is available. (See “Questions to Ask before Acquiring a Conservation Collection” and “Questions to Ask to Determine the Most Efficient Way to Preserve the Plant Tissue Long-Term.”) In some cases, you may need to do a reconnaissance trip to a population before you will be able to answer the questions about whether seeds are present or not. In other cases, you may need to conduct preliminary laboratory trials to determine whether seeds are viable or not, or desiccation tolerant or not.

Seeds capable of conventional freezer storage (temperatures –18°C + 3°C) are called orthodox (see “What is an ‘Orthodox’ Seed?” below). Conventional freezer storage is relatively inexpensive and highly accessible to many institutions. In most cases, the aim is to store long-term (> 20 years). If seeds are not desiccation tolerant (also known as exceptional species, recalcitrant, or intermediate; see “What Is a ‘Recalcitrant’ Seed?” and “What Is an ‘Intermediate’ Seed?” below) or you are only able to collect shoots, rather than seeds, see CPC Best Practice Chapter “Alternatives to Conventional Seed Banking”.

Several steps are required to bring seeds from the wild into the seed laboratory for processing before placing into cold storage. We cover each of these steps in this chapter which is intended to help practitioners keep orthodox seeds alive for as long as possible so that the seeds may be used for future reintroductions to the wild. We urge practitioners to follow the NEW practices to increase the longevity of seeds in storage.

Imperiled Palmer's rabbitbrush, Ericameria palmeri var. palmeri has orthodox seeds and is known from San Diego County and Baja California **Photo credit:** Stacy Anderson, San Diego Zoo Wildlife Alliance

What Is an “Orthodox” Seed?

A seed’s physical and physiological states determine whether it can be stored by conventional means or not. Seed water content, relative humidity and temperature are key interacting factors contributing to longevity (see Figure 1.1). In order for a seed to be stored and survive freezing temperatures, a prerequisite is that it is capable of surviving desiccation or removal of most of the water in its cells.

Research from the 1950s and 1960s demonstrated that drying and cooling crop seeds increased survival time (i.e., longevity). This response became known as “orthodox seed behavior” and is described by Harrington’s Thumb Rules, which state: (1) For each 1% decrease in moisture content, the storage life of the seed is doubled and (2) For each 10°F (5.6 °C) decrease in storage temperature, the storage life of a seed is doubled. Harrington also coined the Hundred Rule, which indicates that the storage temperature (°F) and Relative Humidity (%) should add up to less than 100 to achieve safe storage (Harrington 1960). Later Ellis and Roberts (1980) developed viability equations that predict the proportion of seeds that are viable after storage under a variety of conditions. These have been fundamental to seed conservation predictions for orthodox seeds.

https://saveplants.org/wp-content/uploads/2020/09/decline-in-vigor-germination-of-seed.png

Figure 1.1 – The decline in vigor and germination of a seed accession over time. The amount of time a seed is viable in storage will be a function of seed characteristics, storage temperature and seed moisture. (Redrawn from Harrington 1960.)

Oak species like Quercus havardii have recalcitrant seeds **Photo credit:** Sean Hoban, The Morton Arboretum

What Is a “Recalcitrant” Seed?

Eric Roberts (1973) coined the word ‘recalcitrant’ to refer to seeds that are not orthodox. While orthodox seeds can be stored conventionally, recalcitrant seeds cannot be stored conventionally.

The term “recalcitrant” anthropomorphizes seed responses to water loss. A recalcitrant seed tolerates some water loss, but not the extreme level survived by orthodox seed. Water remaining in recalcitrant seeds forms lethal ice crystals during conventional storage.

Removing embryos from recalcitrant seeds and storing them at liquid nitrogen temperatures is a possible solution to the problem (Al-Zoubi and Normah 2015). Protocols developed for several North American species are labor-intensive but give high survival (Reed 2008). Examples of North American recalcitrant seeds that survive cryopreservation are: Zizania texana, Howellia aquatilis, Acer saccharinum, and Quercus sp. (although species native to Mexico have not yet been tested). Hawai’i and Florida may be “hotspots” within the US for seed recalcitrance, and yet we see a low incidence (<5% of species) (for example, Salazar et al. 2017).

“Recalcitrant” is also used to describe a seed that is difficult to germinate, which happens when seeds lack embryos (that is, “empty” seeds), have fastidious germination requirements (that is, dormant seeds or those with rudimentary embryos), or age quickly (possibly intermediate-type seeds). Some examples of U.S. natives that have been called recalcitrant but survive considerable desiccation are Helonias bullata, Actaea racemosa, Asimina tetramera, Castanea ozarkensis, and Magnolia ashei.

US endangered Chamaesyce deltoidea var. deltoidea grows in Dade County, Florida, and is suspected to have intermediate seeds **Photo credit:** Joyce Maschinski, Center for Plant Conservation

What Is an “Intermediate” Seed?

In the 1990s, it became apparent that some seeds did not fit either orthodox or recalcitrant seed categories. These seeds, known as intermediate, share functional characteristics with recalcitrant seeds and can be categorized as non-orthodox or exceptional. The viability of intermediate seeds is not maintained in the freezer and they need to be cryopreserved in liquid nitrogen. Intermediate seeds can be dried to lower water contents than recalcitrant seeds, and this means that they can be cooled at 30° to 100°C/min (compared to hundreds of degrees per sec for recalcitrant seed tissues) and still avoid lethal ice formation. Unlike recalcitrant seeds where the embryonic axis must be surgically excised to achieve required cooling rates, intermediate seeds can be placed whole into liquid nitrogen, and this process requires less labor and there is usually no impact on survival from the initial exposure to liquid nitrogen.

An early estimate of the incidence of intermediate behavior, about 10%–15% of angiosperms world-wide (Dickie and Pritchard 2002), was based on only one of several syndromes now recognized for the category (Walters 2015). Now we believe there are many more seeds that could be classified as intermediate. Moreover, the tendency to produce intermediate seeds might be a characteristic of a population and its ecology, rather than a species. Intermediate seeds were originally characterized in crop seeds—papaya (Ellis et al. 1991) and coffee (Eira et al. 2006)—but U.S. natives such as several Cuphea and Salix species also exhibit intermediate traits (Crane et al. 2006; Ballesteros and Pence 2017). We believe between 25% and 50% of endangered species from Hawai’i, especially in Campanulaceae and Gesneraceae, produce intermediate-type seeds (Walters, Weisenberger and colleagues, unpublished data).

Intermediate seeds are characterized by at least one of the following symptoms:

Longevity is highest if seeds are dried to between 45 and 65% RH compared to the 15 to 20% RH optimum observed for orthodox seeds.
Seeds age faster when stored at conventional freezer temperatures compared to refrigerated temperatures. Faster aging might be detected within days, months or years, making it difficult to identify which species’ seeds are intermediate
Longevity of seeds increases with drying and cooling (as with orthodox seeds), but seeds still age rapidly during conventional storage and will die within about 5 years.

The Physics of Seed Storage Behavior

It is helpful to look at the categories of seed storage physiology for orthodox, recalcitrant, and intermediate seeds in the context of water content and temperature. The interaction between water content and temperature that affects the physical structure of cytoplasm is portrayed in a phase diagram, which includes a vitrification-plasticization curve (Figure 1.2). All seeds have a threshold water content, below which they are damaged by further drying (Walters 2015). At room temperature, orthodox seeds can be dried safely to water contents between 0.03 and 0.08 g H2O/g total mass (3 to 7%), while safe water content for recalcitrant seeds is above 0.20 g H2O/ g total mass (> 20%). Intermediate seeds lie in between these two levels (Figure 1.2). Below the lower thresholds, some seeds may experience increased aging and die (Walters 2015).

Examining seeds in the context of how much cell volume is occupied by water, dry matter and void space is a promising direction for research. Consider the contents of cells within a seed. As seeds mature they accumulate food reserves which replace water and reduce the water content. However the water potential (availability of water to participate in reactions) stays about the same until seeds mature and separate from the maternal plant. No longer having a supply of water, orthodox seeds dry, causing cells to shrink and molecules within the cells to compress. The proportion of cell volume occupied by dry matter at the onset of dehydration can help predict the amount of water loss that can be tolerated by a seed. See Dr. Chris Walters describe the importance of understanding the phase of water inside a cell in her 2018 keynote at the CPC National Meeting:

Why Seeds and Spores Die in the Freezer and Sometimes Faster That Anticipated

Chris Walters, Research Leader of the Plant Germplasm Preservation Research team at USDA-ARS National Laboratory for Genetic Resources Preservation Knowing how long storedgermplasmsurvives is critical... Read More

To preserve viability during storage, the molecules within a cell must compress until a glass forms (also called vitrification). Orthodox seeds survive to very low water contents and therefore tolerate glass formation; hence, they can be safely preserved in the freezer under conventional storage conditions. In contrast, threshold water contents for recalcitrant and intermediate seeds are higher than the water content required to form a glass. The only way to achieve a glassy state is to cool the cytoplasm profoundly. But, cooling cells containing water is dangerous because lethal ice crystals will likely form. Therefore, preservation methods must cool seeds rapidly to avoid ice formation. For recalcitrant seeds, this can be on the order of hundreds of degrees per second, and it may require excising the embryo to make the sample small enough to cool quickly enough. Once the glass forms, the material must be stored at liquid nitrogen temperatures to avoid warming and inducing ice formation. Because rare species’ seeds come in all shapes, sizes, and physiologies, we present this physical chemistry perspective to help practitioners understand challenges to preservation and make wise decisions for their seeds.

https://saveplants.org/wp-content/uploads/2020/09/figure1.2_0.png

Figure 1.2 – Orthodox, recalcitrant and intermediate seeds and the vitrification-plasticization curve. a) Phase diagram showing boundaries between fluid, ice and glass in relation to temperature and water content. b) Seeds categorized as orthodox, intermediate, or recalcitrant are dried to different water contents. To preserve viability, seeds must be dried and cooled. Note that at conventional freezer temperatures (–20°C), orthodox seeds are in glass, however intermediate and recalcitrant seeds must be cooled rapidly to liquid nitrogen temperatures to be in glass and avoid ice formation.

Probing Life and Death in the Glassy State of Cryopreserved Seeds, Fern Spores and Pollen: Fundamental Research to Optimize Ex Situ Preservation

Dani Ballesteros, Christina Walters, Hugh W. Pritchard Royal Botanic Gardens Kew, Wellcome Trust Millennium Building, United Kingdom, United States Department of Agriculture., United States Often... Read More

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The Difference between Orthodox, Intermediate, and Recalcitrant Seed