CPC Best Plant Conservation Practices

to Support Species Survival in the Wild

See Topic In The Forum

Seed Banking Alternatives

Collecting and Maintaining Exceptional Species in Tissue Culture and Cryopreservation

Basiphyllaea-corallicola-photo-by-JE-Possley

rocks-at-Matheson-covered-with-A-dentatum

Key Messages about Tissue Culture and Plant Cryopreservation Methods

Tissue culture and plant cryopreservation are alternative storage methods for exceptional species that produce few seeds or seeds that are intolerant to drying or freezing.
Adequately storing exceptional species requires specialized expertise, infrastructure, and greater resources than conventional seed storage.
Because species-specific protocols are unknown for many rare plant species, this area of plant conservation is a pioneering investigative field. CPC practitioners have opportunities to contribute significantly to the field of tissue culture and plant cryopreservation methods.

Why Plant Cryopreservation Methods Are Important to Endangered Native Plants

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Exceptional plants are those that cannot be conserved long-term using conventional seed banking methods. This includes species with few or no seeds available for banking, species with seeds that are intolerant of desiccation and freezing, or seeds that can tolerate drying, but not freezing, or species that may only tolerate storage at –20°C for less than 10 years.

For ex situ conservation, such species require methods alternative to conventional storage, such as plant cryopreservation and in vitro methods. Many of the world’s plant species may fit these storage categories (see Seaton et al. 2018).

The primary purpose of a conservation collection of an exceptional species is to support the species’ survival and reduce its extinction risk, therefore accurate records of provenance, differentiated maternal lines, and diverse genetic representation are prerequisites.

Plant cryopreservation or tissue culture are alternative storage processes to conventional storage, but these alternatives are only real conservation solutions if the practitioner is able to restore a tissue cultured or cryopreserved plant part back to a rooted plant that is capable of being transplanted into the wild.

As is true for seed research, much more has been done on agricultural or commercially important species than for rare wild species. As more CPC practitioners conduct tissue culture and plant cryopreservation research, the more will be known about the specific needs of rare species and the more possible it will be to examine patterns.

At this stage in the development of the science, we provide recommendations based upon FAO guidelines and recent research noting that a large difference between these bodies of research stems from the fact that because our species are rare, we usually have few propagules to initiate studies, and we are thrilled to have any survival!

Space and staffing capacity will always impact an institution’s conservation program. Exceptional species require a greater commitment of staff time, specialized expertise of staff, and greater infrastructure than conventional seed banking.

In this light, we encourage practitioners to reach out to the experienced in vitro (tissue culture) and plant cryopreservation specialists in the CPC network if they are planning to create internal programs or if they desire to collaborate on research projects for exceptional species.

What do bananas, avocados and vanilla have in common?

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Several authors have examined patterns in seed storage behavior (see references) that can help collectors. Begin with a literature review to check if any previous research has been done on your taxon. You can check congeners, but beware that this is not always reliable or conclusive.

Our Hawaiian colleagues have found quite varying storage behavior within a single genus (Walters, Weisenberger and Clark, personal communications). Many factors determine variation in seed tolerance to desiccation or freezing. The following are some general patterns observed in seeds that tend to withstand orthodox storage or not.

Trait	Likely to Be Orthodox (Desiccation and Freezing Tolerant)	Questionable Tolerance to Orthodox Storage
Habitat	Arid is especially likely; If it is not growing in a wetland, it is likely	Wetland, riparian
Conditions in nature	Seeds normally experience dry down and/or hard freezes	Seeds normally remain moist and do not experience hard freezes
Season of seed production	Not spring	Spring
Life form	Not tree	Trees
Seed bank	Persistent	Not persistent
Dormancy	With dormancy	No dormancy
Seed moisture content at time of maturation	Dry when it is naturally shed from plant	High (30%–70%)
Seed size		Very large (avocado seeds aren't desiccation tolerant) or very small (orchid seeds and fern spores require storage in liquid nitrogen)

Plant Groups with High Proportion of Desiccation Sensitive Seeds	Plant Groups with Predominantly Orthodox Seeds
ANITAGrade, Arecales, Ericales, Fagales, Icacinales, Laurales, Magnoliales, Malpighiales, Myrtales, Orchidaceae, Oxalidales, Santalales, Salicaceae, Sapindale	Solanaceae, Poaceae, Asteraceae, Brassicaceae

In vitro, symbiotic germination of Platanthera species for conservation efforts

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Making Collections of Exceptional Species

Make preparations before making collections.

Acquire legal permission to make collections.
Research the seed storage requirements of a taxon, as this will determine how rapidly the seed or tissue will need to be processed after collection and the tools and containers needed for collection. (See “Determining Storage Requirements”) FAQ – How do I know my species storage requirements?
To avoid over-collection and/or to learn from other collectors, survey other ex situ collections to determine what related species and/or populations have already been collected. You can check the database on the Botanic Gardens Conservation International website.
You can search for priority species for your region in the “Rare Plant Finder”.

Collecting and Working with Orchid Seeds

David Remucal, University of Minnesota Landscape Arboretum While many seedbanks avoid working with orchids, more groups are taking them on, or collecting them for groups that... Read More

While collecting, do no harm to the collecting site or the rare population.

Strive to collect plant material at the appropriate stage of maturity.

Note maturity of seed in your accession. In general, it is best to collect mature spores and seeds. However, immature seeds may be able to be stored in liquid nitrogen or have embryos extracted for in vitro culture.
Collect plant tissue that is in condition appropriate for your method. (See Figure 2.1.)
For shoot collection, collect young growing or newly grown plant tissue. Generally, plant tissue that is too old will not take to traditional propagation by cuttings or tissue culture.
If plant tissues cannot be transported quickly to a lab, consider making in vitro field collections. (See Pence et al. 2002.) This will require collecting plant tissue with instruments sterilized with 70% ethanol.
For stem tissue, document the maturity or developmental state of the shoot (soft/hardened, leaf expansion stage, degree of expansion, color, etc.)
Take photos of the seed pods or shoots to document aspects of maturity.

When appropriate, collect associated soil mutualists.

For terrestrial orchids, gather soil close to the plant to capture mycorrhizae (Batty et al. 2002).
Mycorrhizae may be important for germination and cultivation of some species.

Transport to seed banking or tissue culture facility in the shortest time possible and in the best condition possible.

The more rapid the processing, the better chance of survival. Before shipping seeds, converse with the recipient scientist at the seed bank or propagation facility to be sure that someone will be present to receive and process the accession promptly.
Ship seeds or plant tissues overnight to seed bank or propagation facility. Provide special care to specimens while transporting to banking or culture facility. Wrap stem cuttings in barely moist (not sopping wet) paper towels and enclose each maternal line in a labeled Ziploc™ bag. Use a Styrofoam container to minimize temperature fluctuation during transport. Don’t ship with ice or dry ice. If overnight shipping is not possible, consider in vitro field collection.

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Figure 2.1 – Meristem architecture. Though meristem architecture varies across species, understanding it for the target rare species will inform what propagation options are available.

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Figure 2.2 – Diversity of Propagule Types and Options for In Vitro Culture. In vitro culture may be initiated with spores, seeds or embryos extracted from seeds. Growing tips from meristems at apex or nodes or cells extracted from leaves or roots can be sources for in vitro culture.

Capture representative genetic diversity.

In the absence of genetic data, capture the breadth of diversity of the species by collecting seeds or plant tissues from spatially separated individuals with different appearances, collecting from multiple populations, and keeping all accessions separated by maternal line.
- Consider the genetic diversity represented by the propagule you are collecting/preserving. Vegetative material (roots or shoots) represents the same genotype as the parent. Seeds or spores are the product of fertilization potentially from multiple parents, thereby they have potentially greater genetic diversity than vegetative material. (See Figure 2.2.)
- For some exceptional species (ferns and orchids), it is very easy to obtain hundreds of genetically related seeds or spores from a single maternal plant. Take care to collect seeds or spores from many individuals to increase the potential diversity represented in your accession. (See CPC Best Practice Section “Collecting Seeds from Wild Rare Plant Populations.”)
If possible, collect parallel leaf samples for DNA banking while making seed collections.

Plan your sampling strategy for the collection.

Use population size and fecundity to plan your sampling strategy for the collection.
The ultimate number targeted for collection will likely depend on an institution’s capacity to process and maintain the stored and living plants. Take into account labor, space, and facilities capacity as you make your collection (Pence 2011).
Attempt to capture as many unrelated individuals from each population as possible. Strive to collect from 50 maternal plants (see CPC Best Practice Chapter “Genetic Guidelines for Acquiring, Maintaining, and Using a Conservation Collection;” and maintain maternal lines separately. FAQ – Why collect from 50 maternal plants?
- If collecting seeds from a population with less than 100 individuals, attempt to capture seeds from all reproductive individuals whether you are storing seeds or embryos cryogenically. For larger populations, subsamples from 50 maternal plants are sufficient.
- If collecting shoots for tissue culture, base numbers to collect on the architecture of the plant and the number of axillary buds present at nodes. For species with multiple branches, collect one to five stem cuttings from each mother plant. For species with a single meristem, be sure the plant architecture supports axillary buds before removing the apical meristem. If buds are taken from the base of the plant or at soil level, they will likely require stringent decontamination with surface disinfectant agent and antimicrobial agents.
- Genetic studies can help determine the representation of the wild genes in the ex situ conservation collection (Griffith et al. 2015).

Seek to capture at least five populations of a species.

If they exist, seek to capture at least five populations of a species across space and time. See “Collecting Seeds from Wild Rare Plant Populations.”

Document the collection appropriately.

Essential accession information includes: institution name, accession number, collector, collection date, species name, family, locality information, georeferenced latitude and longitude, site ownership, permit documentation, and population information (the total number of individuals in the population, number of reproductive individuals, and number of individuals sampled for seeds that were harvested). (See Example Field Collection Form from California Botanic Garden.)
- Providing habitat information may provide clues to germination or tissue culture requirements of the species. Recommended fields include light and moisture conditions, soil type, slope orientation, and associated species. Provide photos of habitat and plant in its habitat.
- Be sure to document any associated collections (for example, leaf litter, soil, mycorrhizal fungi) and maintain the link through processing of samples.
- Gather and report additional accession data according to your institutional protocols. Complying with International Transfer Format for Botanic Garden Plant Records and/or Darwin Core standards will allow easy transfer of information to partners.
Complete one field form per accession. Multiple accession numbers and field forms only need to be created for collections made from populations that are differentiated by at least 1 kilometer.
Transmit accession data to CPC and NLGRP via online form provided to Participating Institutions through the CPC member resources. Once inside this resources tab, the accessions submission form can be found on the “NLGRP submission” tab.

Laboratory Procedures for Exceptional Species

At the seed bank or cryopreservation laboratory, follow steps for each material type to maximize its survival.

Maintain maternal lines for any of these procedures. For highest conservation value, maintain material in a form that will allow for recovery of whole plants that can be used for reintroduction to the wild.

Steps for Maximizing Survival of Seeds (Suspected of Exceptional Storage Behavior)

Test the viability of fresh seeds by conducting a germination trial. For example, conduct an experiment with 10 replicates of 10 seeds. Reduce sample size of your test based upon how many seeds you have and how many you would ultimately like to store. Use seedlings for in vitro culture.
Determine whether seeds fit categories for conventional storage. See “Determining Storage Requirements” box.
If they fit orthodox categories, see CPC Best Practice Chapter “Conventional Seed Banking to Support Species Survival in the Wild.”
If seeds are suspected to be exceptional and you have enough seeds, test five or more seeds to determine whether seeds can withstand desiccation. Desiccate seeds over salt solutions or silica using methods described in CPC Best Practice Section, “Cleaning, Processing, Drying, and Storing Orthodox Seeds.”
If you have a precise scale, test individual seed water content of five to 10 seeds. If you do not have a scale of the precision needed for the seed size, this test can be done at NLGRP. Weigh, dry in oven to drive off water until there is no weight change, and reweigh. You can calculate moisture content. If your seed has high moisture content when it is shed, then it is likely to be exceptional. (See Kew Information Sheet, Measuring Seed Moisture Status.)
Once you have determined that seeds are exceptional, the next required steps will differ depending upon seed size. For large seeds (oaks and larger), extract embryos and follow steps below for in vitro and cryopreservation. FAQ – Do I need to go through the in vitro step if I think long-term cryo-preservation is necessary?
For small seeds, such as orchids, seeds less than 5 mm in diameter, and fern spores, desiccate using air-dry methods, place seeds into a small container (a DSC pan or a microcentrifuge tube), load into cryogenic storage straw and place into liquid nitrogen.

Steps for Maximizing Survival of Tissues in In Vitro Culture

Review any previous in vitro studies on your species or genus to guide details of your procedures.
If no other work has been done on your species or genus, conduct basic protocols for in vitro culture. Document steps at each stage. If tissues do not respond to the basic protocol, modify the protocol and test again. (See Figure 2.2 and Table 2.1.)
Take steps to minimize contamination.
Treat incoming material with surface disinfectant agent under ventilation hood. FAQ – For Stage one of micro-propagation, surface sterilization, how do I know what disinfectants and concentrations will be effective without damaging my material?
Include anti-microbial agents (for example, fungicide, PPM, etc.) in medium especially for material received from the wild.
Monitor regularly for growth and contamination. Refresh medium as needed.
Multiply the number of shoots in cultivation.
Maintain enough material from each maternal line to guard against attrition of the line over time.
Acclimatize plants away from direct sunlight slowly.
- Remove from culture, transfer to appropriate soil mixture, and maintain in high humidity either under plastic bags or glass. Slowly add ventilation. If plant tissue wilts, replace protective cover and proceed with hardening at a slower pace. This process may take months.
- Arid-adapted plants, such as those from deserts, tend to have more difficulty acclimatizing from tissue culture to ambient conditions than species from wet or humid ecosystems. While still in culture, some pre-acclimatization hardening can be considered, such as providing greater aeration to the cultures, higher light, or lower nutrients, as appropriate for the species. Replace sealed cap with vented cap to harden the plantlet in the tube. Remove from culture, transfer to an appropriate soil mixture, and maintain in high humidity.

The Potential of Shoot Tip Plant Cryopreservation for the Ex Situ Conservation of Oaks.

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Table 2.1 Stages of Micropropagation

Stage	Operations	Comments
1. Establish and stabilize	Handle and pre-select target plants to reduce the potential of microbial contamination. Choose explant type: shoot tips, flower buds, axillary buds, leaves, or embryos. Stimulate axillary or adventitious shoot formation on appropriate medium.	Pretreat explants to prevent browning. Examine microbial colonies to determine if contamination is coming from fungi or bacteria living within plant tissues or from agar surface that was not effectively sterilized.
2. Multiply shoots	Divide the multi-branched explant. Place small shoots onto new media with hormones to stimulate more shoot development.	Check for contamination.
3. Roots form	Induce rooting by transferring well-developed shoots to media containing auxins.	Plantlets are less susceptible to accidental microbial contamination at this stage.
4. Acclimate	Deflask rooted plantlets and acclimatize to greenhouse conditions.	Remove agar from roots. Use pasteurized potting mix to remove a potential source of contamination.

Tissue Culture lab in Kunming Botanic Garden houses many rare species from China. **Photo credit:** Joyce Maschinski.

https://saveplants.org/wp-content/uploads/2020/09/figure-2.3.jpg

Figure 2.3 – In vitro and Plant Cryopreservation. From in vitro donor plants, shoot tips can be excised, sterilized, and placed onto a sterile nutrient medium. Application of cytokinin will encourage multiple shoots to grow. With the proliferated shoots, tests of plant cell cryopreservation can ensue options.

Shoot tips are placed onto a high sucrose solution for osmoprotection, followed by immersion, incubation, or exposure to a cryoprotectant solution (depicted is encapsulation dehydration), exposed to liquid nitrogen, and then stored. Experimental steps along the process are advised for species with unknown plant cryopreservation tolerance.

Experimental components may include molarity of osmoprotection solution, type of and duration of exposure to the cryoprotectant, the rate and duration of exposure to liquid nitrogen, and the duration of long-term storage. After storage, tips are warmed and recovered using in vitro methods.

Steps for Maximizing Survival of Cryopreserved Plant Tissue

After seed tests (above) have been conducted, place into an appropriate airtight container for storage in liquid nitrogen.
For shoots that have been growing in vitro or for seed embryos, isolate shoot tips or embryos. This may require a dissecting microscope. Best cryopreservation of plant tissue culture usually occurs on small materials. For example, tips with size less than 2 mm × 2 mm are best to use. For shoots that have been growing in vitro or for seed embryos, isolate tips or embryos (Figure 2.2 and 2.3). Learn more about the circumstances for which shoot tip cryopreservation is appropriate.
Surface sterilize isolated seed embryos either before or after cryopreservation of plant tissues.
Follow cryopreservation protocols . For each step in the cryopreservation protocol, test a control group to determine if the plant tissue has survived that step.
Strive for 40% survival after liquid nitrogen exposure. If survival is lower than 40%, conduct more experiments with cryopreservation to improve survival, or bank more material.
Strive to have replicate vials of at least 10 shoot tips per vial of each maternal line preserved.
Recover material by warming in a 40°C water bath.
Plate onto in vitro medium (see Saad and Elshahed (2012) for media descriptions). Use antioxidants to minimize tissue browning. The kind of antioxidant may vary across species.
Take steps to avoid contamination.
Document all steps in process.
Once roots have formed, acclimate as above.

Maintaining Exceptional Species in Collection

Duplicate accessions to maintain in one or more than one living collection (Fant et al. 2016).

Consider all attempts to store exceptional species as experimental.

Because we have much to learn about the optimal ways to store exceptional spe-cies, all trials should be recorded using best scientific method.
To maximize our ability to learn the best way to keep exceptional species alive in storage, do not store exceptional species in

Monitor the viability of the accession after 5, 10, 15, and 20 years in cryostorage if enough material is available.

Document the experimental protocols carefully.

Whenever steps of protocol are compared to controls, report survival of controls and treated groups. Remember that steps that are NOT successful will help future practitioners.
Note the age of the material used.
Take photos of your shoot tips in vitro culture.
Note average shoot tip size and photograph this.
Note condition of tips and any appearance differences across treatments (for example, hairy, shape, leaves present or not, etc.). Note survival of phenotypes.
Track the type of medium used for pre-culture, stock, and recovery culture; note any additives used (for example, ABA, antibiotic, etc.); cryoprotectant used; cold hardening treatment; cooling rate and vitrification method used; and any modifications in standard procedures.

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International Standards

Reference for CPC Guidelines

FAO Genebank Standards for Plant Genetic Diversity (FAO 2014)

Standards for Acquisition of Germplasm

6.1.1 All germplasm accessions added to the genebank should be legally acquired, with relevant technical documentation.

6.1.2 All material should be accompanied by at least a minimum of associated data as detailed in the FAO/ Bioversity multi-crop passport descriptors.

6.1.3 Only material in good condition and of consistent maturity status should be collected, and the sample size should be large enough to make genebanking a viable proposition.

6.1.4 The material should be transported to the genebank in the shortest possible time and in the best possible conditions.

6.1.5 All incoming material should be treated by a surface disinfectant agent to remove all adherent microorganisms and handled so that its physiological status is not altered, in a designated area for reception.

Standards for Testing for Non-orthodox Behaviour and Assessment of Water Content, Vigour and Viability

6.2.1 The storage category of the seed should be determined immediately by assessing its response to dehydration.

6.2.2 The water content should be determined individually, on separate components of the propagule, and in a sufficient number of plants.

6.2.3 The vigour and viability should be assessed by means of germination tests and in a sufficient number of individuals.

6.2.4 During experimentation, cleaned seed samples should be stored under conditions that do not allow any dehydration or hydration.

Standards for Hydrated Storage of Recalcitrant Seeds

6.3.1 Hydrated storage should be carried out under saturated RH conditions, and seeds should be maintained in airtight containers, at the lowest temperature that they will tolerate without damage.

6.3.2 All seeds should be disinfected prior to hydrated storage and infected material should be eliminated.

6.3.3 Stored seeds must be inspected and sampled periodically to check if any fungal or bacterial contamination has occurred, and whether there has been any decline in water content and/or vigour and viability.

Standards for In Vitro Culture and Slow Growth Storage

6.4.1 Identification of optimal storage conditions for in vitro cultures must be determined according to the species.

6.4.2 Material for in vitro conservation should be maintained as whole plantlets or shoots, or storage organs for species where these are naturally formed.

6.4.3 A regular monitoring system for checking the quality of the in vitro culture in slow-growth storage, and possible contamination, should be in place.

Standards for Plant Cryopreservation

6.5.1 The explants selected for plant cryopreservation should be of highest possible quality, and allow onward development after excision and cryopreservation.

6.5.2 Each step in the cryo-protocol should be tested individually and optimized in terms of vigour and viability in retention of explants.

6.5.3 Means should be developed to counteract damaging effects of reactive oxygen species (ROS) at excision and all subsequent manipulations.

6.5.4 Following retrieval, explants should be disinfected using standard sterile procedures.

Batty, A. L., K. W. Dixon, M. C. Brundrett, and K. Sivasithamparam. 2002. Orchid conservation and mycorrhizal associations in K. Sivasithamparam, K. W. Dixon, and R. L. Barrett, editors. Microorganisms in plant conservation and biodiversity. Kluwer Academic Publishers, London.

Ballesteros, D., and V. C. Pence. 2017. Survival and death of seeds during liquid nitrogen storage: a case study of seeds with short lifespans. CryoLetters 38:278–289.

Bunn, E., and B. Tan. 2002. Microbial contaminants in plant tissue culture propagation in K. Sivasithamparam, K. W. Dixon, and R. L. Barrett, editors. Microorganisms in plant conservation and biodiversity. Kluwer Academic Publishers, London.

Exceptional Plant Conservation Network, accessed May 29, 2018

Fant, J. B., K. Havens, A. T. Kramer, S. K. Walsh, T. Callicrate, R. C. Lacy, M. Maunder, A. Hird Meyer, P. P. Smith. 2016. What to do when we can’t bank on seeds: what botanic gardens can learn from the zoo community about conserving plants in living collections. American Journal of Botany 103: 1–3.

Food and Agriculture Organization of the United Nations (FAO) 2014. Genebank standards for plant genetic resources for food and agriculture. Rome, Italy.

Fernandez, H., A. Kumar, and M. A. Revilla, editors. 2011. Working with ferns. Springer, New York.

George, E. F. 1993. Plant propagation by tissue culture. Part 1: the technology. Edington, England, Exegetics Ltd.

George, E. F. 1996. Plant Propagation by tissue culture Part 2: in practice. Edington, England, Exegetics Ltd.

Griffth, M.P., M. Calonje, A. W. Meerow, F. Tut, A.T. Kramer, A. Hird, T.M. Magellan, and C.E. Husby. 2015. Can a botanic garden cycad collection capture the genetic diversity in a wild population. International Journal of Plant Sciences176: 1-10.

Panis, B., B. Piette, and R. Swennen. 2005. Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Science 168:45–55.

Pence, V. C. 2011. Evaluating costs for the “in vitro” propagation and preservation of endangered plants. In Vitro Cellular & Developmental Biology – Plant 47:176–187.

Pence, V. C., J. A. Sandoval, V. M. Villalobos, and F. Engelmann, editors. 2002. In vitro collecting techniques for germplasmconservation. IPGRI Technical Bulletin No. 7. International Plant Genetic Resources Institute, Rome, Italy.

Reed, B. M., S. Wada, J. DeNoma, and R. P. Niedz. 2013. Mineral nutrition influences physiological responses of pear in vitro. In Vitro Cellular & Developmental Biology – Plant 49:699–709.

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Sakai, A., D. Hirai, and T. Niino. 2008. Development of PVS-based vitrification and encapsulation – vitrification protocols. Pages 33–57 in Reed, B. M., editor. Plant cryopreservation: a practical guide. Springer, New York.

Seaton P.T., S.T Hosomi., C.C., Custódio, T.R. Marks, N.B. Machado-Neto, and H.W. Pritchard. 2018. Orchid Seed and Pollen: A Toolkit for Long-Term Storage, Viability Assessment and Conservation. In: Y.I. Lee and E.T. Yeung (eds) Orchid Propagation: From Laboratories to Greenhouses—Methods and Protocols. Springer Protocols Handbooks. Humana Press, New York, NY