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From freezing to the field—in vitro methods assisting plant conservation

Volume 9 Number 1 - January 2012

Valerie Pence


Off the main public path of the Cincinnati Zoo & Botanical Garden, in the corner of the park, one can find the Carl H. Lindner Family Center for Conservation and Research of Endangered Wildlife (CREW).  From this small, two-story building, plant and animal research is being conducted that is impacting rare species across the country, and beyond.  CREW’s Plant Research Division (PRD)’s particular focus is using in vitro (tissue culture) methods for propagating and preserving endangered plants.  The work is directed primarily at species for which traditional approaches are not adequate.  Thus, when seeds are few and cuttings are not workable, tissue culture can provide an alternative method for propagation.  When seeds are lacking or are “recalcitrant” (sensitive to the drying necessary for traditional seed-banking), freezing tissues from in vitro cultures can be an additional tool for banking valuable genetic material from rare taxa.  The PRD has developed protocols for the in vitro propagation and/or preservation of over 50 rare species, providing materials for restoration projects and tissues for long-term storage in liquid nitrogen in CREW’s CryoBioBank.

Propagation for restoration

Propagation is a key element in providing plants for recovery projects.  For many endangered species, traditional propagation by seed or cuttings can meet propagation needs, but for species with few or no seeds or few individuals, plant tissue culture can be used to supplement these methods (Sarasan, et al., 2006). The PRD collaborates with a number of other institutions, including botanical gardens in the Center for Plant Conservation network (Center for Plant Conservation 2012), government agencies, NGOs, and research stations.  These institutions often identify plants for which in vitro approaches could be useful and then provide the starting material (seeds or shoot tips) to CREW for propagation.

Over 100 botanic gardens around the world have tissue culture and micropropagation facilities” (BGCI GardenSearch database, 2012)

The Autumn buttercup (Ranunculus aestivalis) is one such species, known from one site in south central Utah in the Sevier River Valley.  When it became obvious that the population was declining, The Nature Conservancy bought the property in order to protect the plant. Over the past decade, five organizations, including CREW, The Arboretum at Flagstaff, The Nature Conservancy, U.S. Fish & Wildlife Service, and Utah State University, have come together in a partnership to work on studying and restoring this species (Pence et al., 2008).  A small number of seeds were collected at the site and sent to CREW where they were germinated in vitro, and shoot propagating cultures were initiated from each seedling.  As with most tissue culture systems, the shoots can be maintained and multiplied in culture by dividing and sub-culturing the shoots onto fresh medium every 6-8 weeks.  By changing the components of the medium, the shoots can be stimulated to form roots, and once rooted, the shoots are then ready to be moved back into soil.  This is a clonal method of propagation, but each genetic line initiated from a separate seed is maintained separately, creating a collection of genetically distinct lines in tissue culture. 

When the plants were rooted, they were sent to the Arboretum at Flagstaff, where they were carefully removed from their culture tubes and acclimatized to soil and to real-life conditions in a greenhouse.  At that point they were ready to face life out in the wild.  In 2007, a group of buttercups, originally propagated at CREW, was planted back out at the Sevier Preserve, and, while none of the plants from the original population have been seen in recent years, a group of the CREW-propagated plants are surviving and flowering. 

Another such species is the Cumberland sandwort (Minuartia cumberlandensis), a small plant that grows only in sandstone rockhouse habitats in the Daniel Boone National Forest of southern Kentucky and northern Tennessee.  Like the Autumn buttercup, CREW initiated several genetically different tissue culture lines from seeds that had been collected by researchers at the Missouri Botanical Garden.  Shoots were produced, rooted, and plants transferred back to soil. 

In order to demonstrate the feasibility of using tissue culture propagated plants for the recovery of this species, an experimental out-planting was made at a rockhouse site in southern Kentucky in collaboration with the U.S. Forest Service. Plants were planted in different areas of the rockhouse, in order to evaluate the microhabitats within the rockhouse, which differed in light and moisture levels. Over the course of six years, plants at several of the microsites have grown and reproduced well, indicating the viability of these methods, should they be needed to help preserve the species in the future.  A genetic study is also underway to analyze the genetic diversity of this new, experimental population.

By combining the laboratory strengths of CREW with the field expertise of collaborators in botanical gardens, governmental agencies, and non-profit organizations, the work at CREW is being integrated into conservation efforts for species recovery and demonstrating that tissue culture propagation can be an important tool for conservation and restoration. 

Resources for the Future

In a similar fashion, the PRD is utilizing in vitro methods to provide tissues for long-term liquid nitrogen storage, when seed banking is not a workable option for a species (Engelmann, 2004).  This work centers on the CryoBioBank (CBB), CREW’s liquid nitrogen storage facility.  The plant collections, or “Frozen Garden.” within the CryoBioBank include seeds, spores, and tissues of about 150  plant species in four distinct collections, all stored at -196oC (-320oF). 

Seeds and spores are prepared for freezing by simple drying, but tissues, such as shoot tips and embryos, require more elaborate procedures.  Concentrated solutions of sugar and other chemicals, known as cryoprotectants, remove water from the tissues and protect the cells from damage during the freezing and thawing processes.  The extremely low temperatures of liquid nitrogen provide stability for the tissues, maintaining them in a state of suspended animation for decades.

The CryoBioBanks's Frozen Garden is made up of four distinct collections.  The Regional Seed Bank includes seeds of state and federally endangered species from Kentucky, Indiana, and Ohio, including such species as Short’s goldenrod (Solidago shortii) and Running buffalo clover (Trifolium stoloniferum).  Because not all endangered plants are seed plants, there is a Pteridophyte (Fern) Bank and a Bryophyte (Moss) Bank, which contain spores, as well as tissues of gametophytes and sporophytes. The Endangered Plant Tissue Bank contains samples of tissues from in vitro cultures of endangered plants that are grown at CREW.  Material is continually added to the collection, and some samples are over 20 years old.  Recently, samples of pollen from the American chestnut (Castanea dentata) were shown to be viable after 15 years of storage in liquid nitrogen, while shoot tips of the Cumberland sandwort were recovered after 10 years in liquid nitrogen.

Perhaps one of the most important uses of the CryoBioBank is to preserve tissues from endangered species that fall into the category of “exceptional” species.  These are plants that either produce few or no seeds, or their seeds are sensitive to drying and cannot be put through the rigors of normal seed-banking (Pence, 2011).  For these plants, banking tissues from in vitro cultures in liquid nitrogen can provide an alternative method for long-term germplasm storage. At CREW, multiple genotypes of several species are being collected, put into culture, and then cryopreserved, in order to maintain a back-up to the genetic diversity of these species, should they decline or be lost in the wild.  These include three endangered pawpaws endemic to Florida, the Four-petal pawpaw (Asimina tetramera), Beautiful pawpaw (Deeringothamnus pulchellus), and Rugel's pawpaw (Deeringothamnus rugelii), all with desiccation-sensitive seeds (Pence, 2006).  Samples from a species from the mountains of New Mexico, Todsen's pennyroyal (Hedeoma todsenii), which produces no seeds, are being collected, cryopreserved, and also analyzed genetically at CREW to document the diversity of the population (Pence et al., 2009).  There appears to be some diversity, even though no seed production has been documented in this species, suggesting that reproductive deficiency is a recent development in this species. Todsen’s pennyroyal is found on the grounds of White Sands Missile Base, and this work has been funded, in part, by a grant from the U.S. Army.  Again, partnerships with those working in the field have been an important part of this work. 

Tissue banking of exceptional species, as well as the in vitro propagation of plants for reintroduction, supports Target 8 of the Global Strategy for Plant Conservation (Botanic Gardens Conservation International, 2011), which sets goals for ex situ conservation and restoration.  While these methods are more labor intensive and, thus, more expensive than traditional methods, they do provide tools for dealing with exceptional endangered species, and for maintaining precious plant diversity into the future.


Botanic Gardens Conservation International.  Plants 2010.  The Global Partnership for Plant Conservation.  Supporting the worldwide implementation of the Global Strategy for Plant Conservation.  Cited 12 Nov 2011.

Center for Plant Conservation. 2012.  Participating Institutions. Cited Jan 2012.

Engelmann, F. 2004.  Plant cryopreservation:  Progress and prospects.  In Vitro Cellular and Developmental Biology Plant 40: 427-433.

Pence, V.C.  2006.  Propagating and preserving pawpaws (and other rare species) from Florida.  The Palmetto 23 4: 8-11.

Pence, V.C.  2011.  The possibilities and challenges of in vitro methods for conservation.  Kew Bulletin 65: 539-547.

Pence, V.C, Murray, S., Whitham, L., Cloward, D., Barnes, H.and Van Buren, R.  2008.  Supplementation of the autumn buttercup population in Utah, USA, using in vitro propagated plants. In: Global Re-introduction Perspectives.  Soorae PS, ed., IUCN/SSC Re-introduction Specialist Group, Abu Dhabi, UAE.  Pp. 239-243.

Pence, V.C., Winget, G.D., Lindsey, K.L., Plair, B.L. and Charls, S.M.  2009.  In vitro propagation, cryopreservation, and genetic analysis of the endangered Hedeoma todsenii (Lamiaceae).  Madrono 56: 221-228.

Sarasan, V., Cripps, R., Ramsay, M. M., Atherton, C., McMichen, M., Prendergast, G., and Rowntree, J. K.  2006.  Conservation in vitro of threatened plants--progress in the past decade.   In Vitro Cell. Dev.  Biol.--Plant 42: 206-214.

Valerie Pence
Center for Conservation and Research of Endangered Wildlife (CREW)
Cincinnati Zoo & Botanical Garden
3400 Vine Street
Cincinnati, OH 45220