Valerie C. Pence

Biodiversity conservation and conservation biotechnology tools

This special issue is dedicated to the in vitro tools and methods used to conserve the genetic diversity of rare and threatened plant species from around the world. Species that are on the brink of extinction because of the rapid loss of genetic diversity and habitat come mainly from resource-poor areas of the world and from global biodiversity hotspots and island countries. These species are unique because they are endemic, and only a few small populations or sometimes only a few individuals remain in the wild. Therefore, the challenges to support conservation by in vitro measures are many and varied. The editors of this invited issue solicited papers from experts from Asia, Africa, Europe, Australia, and North and South America. This compilation of articles describes the efforts in these diverse regions toward saving plants from extinction, and details the direct application of in vitro and cryopreservation methods. In addition, these contributions provide guidance on propagation of rare plants, including techniques for large-scale propagation, storage, and reintroduction. The in vitro techniques for conserving plant biodiversity include shoot apical or axillary-meristem-based micropropagation, somatic embryogenesis, cell culture technologies and embryo rescue techniques, as well as a range of in vitro cold storage and cryopreservation protocols, and they are discussed in depth in this issue.

Evaluating costs for the in vitro propagation and preservation of endangered plants

In vitro methods provide opportunities for propagating and preserving endangered plant species when seed-based methods are not adequate. Such species include those that produce few or no seeds, as well as species with recalcitrant seeds. Tissue culture propagation methods can be used to produce such plants for reintroduction, research, education, display, and commerce. They can also be the basis for tissue banking as a way to preserve genetic diversity when seeds cannot be banked. With some recalcitrant species, embryo banking, a method which also utilizes in vitro culture for recovery germination, is possible. The number of endangered species that will require in vitro methods is estimated to be at least 5,000 worldwide. Further information is needed to identify these species, and the ongoing collection of information into databases on endangered species and recalcitrant species will help provide this. The costs of these methods are higher than for traditional propagation and preservation, but they may be necessary for species under higher threat. The multiplication rate of a culture, as well as the rates of rooting and acclimatization, has a major effect on the number of transfers needed for producing plants or tissue for banking, and improvements that will increase the efficiency of these steps can help lower costs. Further research into factors affecting the growth of tissues in vitro, as well as coordination of efforts among institutions with infrastructure for in vitro work, should facilitate the application of in vitro methods to the endangered species that cannot be propagated or preserved using seeds.

Cryopreservation of immature embryos of Theobroma cacao.

Immature, white zygotic embryos of Theobroma cacao L. (cacao) retained the ability to produce callus and to undergo somatic embryogenesis after slow hydrated freezing and desiccated fast freezing in liquid nitrogen. The highest rate of somatic embryogenesis occurred in embryos which were precultured on a medium containing 3% sucrose, frozen slowly with cryoprotectants before exposure to liquid nitrogen, and recovered on a medium containing 3 mg/liter NAA. Embryos precultured on media containing sucrose increasing to 21% had a higher rate of survival but were less embryogenic after freezing. These results suggest that immature embryos might be used for long-term germplasm storage of T. cacao germplasm.

The possibilities and challenges of in vitro methods for plant conservation

SummarySeed-based methods are generally the most efficient for propagating and storing plant germplasm, but these methods are not always adequate, and some species can benefit from in vitro methods for conservation. For species that produce few or no seeds in the wild, plants may be propagated in vitro, and in vitro shoot tips can provide material for cryostorage when seeds are not available or are recalcitrant. In vitro propagated plants may also serve as subjects for research, without depleting the genetic resources of the species. Clonal plants can be used to test out suitable habitat and can be used for basic research on endangered species, without disturbing the wild population. Despite the effectiveness of widely used techniques, however, there are still species that resist initiation into culture or that may be difficult to root or acclimatise. Similarly, tissue cryopreservation methods may be restrained by cost, particularly in maintaining multiple genotypes of many species. Maintaining such genotypes in vitro is also costly and runs the risk of loss or change over time. Examples of the successful use of in vitro methods will illustrate the variety of applications of these techniques, but costs and specific challenges will also be discussed to help define areas where further research is needed to realise the potential of in vitro methods as a tool for conservation.

IN VITRO COLLECTING (IVC). I. THE EFFECT OF COLLECTING METHOD AND ANTIMICROBIAL AGENTS ON CONTAMINATION IN TEMPERATE AND TROPICAL COLLECTIONS

SummaryIn vitro collecting is the process of initiating tissue cultures in the field. In order for in vitro collecting to be broadly available as a technique for collecting plant germplasm, the levels of contamination in such cultures must be controlled. Two techniques for in vitro collecting were compared: leaf punch and needle collecting. The effectiveness of these methods for collecting leaf and stem tissues from plants at tropical and temperature sites was compared. Stem tissue collected by the needle collecting method gave cultures with an average contamination percentage of 31% and 16%, from the tropical and temperate sites, respectively, while with the leaf punch method, average contamination percentages were 90% and 69%. The effectiveness of antimicrobial agents in reducing contamination in leaf punch cultures was evaluated. Addition of the fungicide benlate and the antibiotics cefotaxime and vancomycin, to the leaf punch collections reduced contamination to an average of 30% in the tropical collections and 35% in the temperate collections. Over 90% of both tropical and temperature species collected in multiple samples of 10 or more had at least one clean sample using this medium. The use of either the leaf punch method in combination with a fungicide and antibiotics or the needle collecting technique yielded a high percentage of clean tissues for study and growth.

Ex Situ Conservation of the Federally Endangered Plant Species Clematis socialis Kral (Ranunculaceae)

ABSTRACT: The Center for Plant Conservation (CPC) has created sampling guidelines for the ex situ conservation of rare plant species. These guidelines estimate the number of individuals needed to maximize the genetic diversity of the collection according to population genetic theory. For many clonal plant species, knowledge of the number of unique individuals is not easily discerned and application of these guidelines must be based on molecular genetic data. In this paper, we discuss the steps taken in order to meet CPC guidelines for the conservation of a rare clonal plant, Clematis socialis. Due to limited seed availability, methods were developed for successful in vitro propagation and cryopreservation of C. socialis shoot tips. Inter-simple sequence repeat (ISSR) analysis identified fifteen unique genotypes in the ex situ in vitro collection. One genotype in this collection has been conserved from a population that is now presumed extinct. Although the initial sampling protocol managed to capture considerable genetic diversity, an additional 97 genotypes are needed to meet CPC guidelines. The information and experience gained through the initial C. socialis ex situ conservation efforts form the basis for a strategy to improve ex situ conservation activities for this endangered species. We recommend that additional in vitro collections be made from each of the five extant populations and placed in cryostorage.

In Vitro Propagation, Cryopreservation, and Genetic Analysis of the Endangered Hedeoma todsenii (Lamiaceae)

Abstract Todsens pennyroyal (Hedeoma todsenii R. S. Irving, Lamiaceae) is a federally endangered species from the mountains of south central New Mexico that rarely produces seed. In vitro propagation methods were developed to provide material for cryostorage and for reintroduction, if that becomes necessary. Cultures were initiated from shoot tips taken from the ex situ collection at The Arboretum at Flagstaff, resulting in 12 genetic lines that were maintained on MS medium with 0.1 mg/L BAP and 0.01 mg/L NAA. Tests with other media commonly in use in CREWs Endangered Plant Propagation Program indicated that MS medium with 0.5 mg/L BAP increased shoot production and MS medium with 0.5 mg/L IBA increased root production over the maintenance medium. Other concentrations of IBA tested did not improve rooting, and a pulse of IBA followed by culture on charcoal-containing medium did not increase rooting significantly above the control. Approximately half of the plants moved to soil survived acclimatization, regardless of previous treatments. Survival through cryopreservation averaged 35% with no significant difference between the encapsulation dehydration and encapsulation vitrification procedures, and shoot tips from all 12 lines have been banked in liquid nitrogen for long-term storage. RAPD analyses indicated that there was less diversity among plants that exist in close proximity in situ than among genotypes that are separated by more distance. These propagation, cryopreservation, and genetic analysis protocols are all methods that can be used as tools to provide support for the long-term conservation of this species.

Survival and genetic stability of shoot tips of Hedeoma todsenii R.S.Irving after long-term cryostorage

Endangered and rare species for which seed banking is not possible require alternative methods of ex situ conservation for long-term preservation. These methods depend primarily on cryopreservation methods, such as shoot tip cryopreservation, but there are few datasets with information on the long-term survival of shoot tips stored in liquid nitrogen. In this study, survival and genetic stability of shoot tips of the endangered species, Hedeoma todsenii, banked over multiple years were examined. In vitro cultures cryopreserved with both the encapsulation dehydration and the encapsulation vitrification methods showed good average survival after up to 13xa0yr of storage in liquid nitrogen. The application of droplet vitrification to this species increased survival significantly, with an average of 72%, compared with 24–45% survival obtained with other methods. As measured with microsatellite and sequence-related amplified polymorphism (SRAP) markers, the genetic stability of the same genotypes stored over different periods of time typically did not change. However, there was an average of 10.4% band loss between replicate samples that did indicate a potential change in DNA composition. These results demonstrate the use of shoot tip cryopreservation as an effective ex situ conservation tool for this species, but genetic stability of the cryopreserved tissues should be closely monitored.

Tools for the ex situ conservation of the threatened species, Cycladenia humilis var. jonesii

This study developed tissue culture and cryopreservation methods for the threatened Cycladenia humilis var. jonesii as tools for propagation and ex situ conservation, since it produces few seeds for banking. The cultures initially displayed abnormal (hyperhydric) growth, but more normal characteristics were promoted by venting the cultures.

Fern Conservation: Spore, Gametophyte, and Sporophyte Ex Situ Storage, In Vitro Culture, and Cryopreservation

Many species of ferns and lycophytes are threatened by habitat destruction, over-collecting, invasive species, and climate change, and plans to secure their in situ and ex situ conservation are urgently needed. However, there has not been a development of standard methods for fern ex situ storage and conservation, as there has been for seed plants. This is likely due to the fact that ferns have been less studied and are less understood than seed plants and that there has been less funding and institutional focus on them for conservation purposes when compared with domesticated or wild seed plants. Tissues from different stages of the fern and lycophyte life cycle can be used as a ready source of germplasm for ex situ conservation. Spore storage is generally the most efficient method for ex situ fern conservation, but there are also situations where storing gametophytes or sporophytes can be extremely useful. The longevity of spores at −20 °C, the standard seed banking temperature, may vary with the species. Storage in LN should improve longevity in spores and is likely necessary for non-spore tissues. Recent results of a study on the viability of spores, gametophytes, and sporophytes after 20 years of storage in liquid nitrogen is used as a case study to illustrate the potential of these methods. The state of current methods and needs for further research and application are discussed. While there is much more to learn, these tools are available for fern ex situ conservation and should be implemented on a broader scale to preserve rare fern taxa and provide materials for future restoration and research.