Use of biotechnologies for the conservation of plant biodiversity

In vitro techniques are very useful for conserving plant biodiversity, including (a) genetic resources of recalcitrant seed and vegetatively propagated species, (b) rare and endangered plant species and (c) biotechnology products such as elite genotypes and genetically engineered material. Explants from recalcitrant seed and vegetatively propagated species can be efficiently collected under field conditions using in vitro techniques. In vitro culture techniques ensure the production and rapid multiplication of disease-free material. Medium-term conservation is achieved by reducing growth of plant material, thus increasing intervals between subcultures. For long-term conservation, cryopreservation (liquid nitrogen, −196°C) allows storing plant material without modification or alteration for extended periods, protected from contaminations and with limited maintenance. Slow growth storage protocols are routinely employed for a large number of species, including numerous endangered plants, from temperate and tropical origin. Cryopreservation is well advanced for vegetatively propagated species, and techniques are ready for large-scale experimentation in an increasing number of cases. Research is much less advanced for recalcitrant species due to their seed characteristics, viz., very high sensitivity to desiccation, structural complexity and heterogeneity in terms of developmental stage and water content at maturity. However, various technical approaches should be explored to develop cryopreservation techniques for a larger number of recalcitrant seed species. A range of analytical techniques are available, which allow understanding physical and biological processes taking place in explants during cryopreservation. These techniques are extremely useful to assist in the development of cryopreservation protocols. In comparison with crop species, only limited research has been performed on cryopreservation of rare and endangered species. Even though routine use of cryopreservation is still limited, an increasing number of examples where cryopreservation is used on a large scale can be found both in genebanks for crops and in botanical gardens for endangered species.     

Importance of <Emphasis Type="Italic">in vitro</Emphasis> technology to future conservation programmes worldwide

The latest IUCN statistics show that of over 12,000 plant species, 70% are threatened, 19% are critically endangered and 28 species are extinct in the wild. Target 8 of the Global Strategy for Plant Conservation (GSPC) highlights the importance of ex situ conservation of critically endangered plants. Long-term germplasm storage for species with recalcitrant seeds needs alternative measures. In vitro methods complement seed banking and other ex situ measures and are vital for long-term conservation. Conservation Biotechnology at RBG Kew is currently working on a number of rare and threatened recalcitrant species from biodiversity-rich areas of the world to develop good quality in vitro propagules for cryopreservation, recovery and restoration projects. The importance of successful in vitro propagation methods, transplantation technologies, cryopreservation and international networking for the integrated conservation of these species are discussed in detail.     

Conservation of medicinal plants of Western Ghats,India and its sustainable utilization through <Emphasis Type="Italic">in vitro</Emphasis> technology

Climate change, alien species, and use of land for intensive farming and development are causing severe threat to the plant genetic diversity worldwide. Hence, conservation of biodiversity is considered fundamental and also provides the livelihoods to millions of people worldwide. Medicinal plants play a key role in the treatment of a number of diseases, and they are only the source of medicine for majority of people in the developing world. The tropical regions of the world supply the bulk of current global demand for “natural medicine,” albeit with increasing threat to populations in the world and its genetic diversity. India is a major center of origin and diversity of crop and medicinal plants. India poses out 20,000 species of higher plants, one third of it being endemic and 500 species are categorized to have medicinal value. The Western Ghats is one of the major repositories of medicinal plants. It harbors around 4,000 species of higher plants of which 450 species are threatened. Currently, the number of species added to the red list category in this region is increasing, and the valuable genetic resources are being lost at a rapid rate. Demand for medicinal plants is increasing, and this leads to unscrupulous collection from the wild and adulteration of supplies. Providing high-quality planting material for sustainable use and thereby saving the genetic diversity of plants in the wild is important. During the last 25 years of intensive research, Tropical Botanic Garden and Research Institute has developed in vitro protocol for rapid regeneration and establishment of about 40 medicinally important rare and threatened plants of Western Ghats. In situ conservation alone would not be effective in safeguarding these important species. Thus, utilizing the biotechnoligical approach to complement ex situ conservation program is becoming vital. Propagating biotechnology tools in plant conservation program is a prerequisite to succeed in sustainable use and to complement the existing ex situ measures. In addition to propagation, storage of these valuable genetic resources is equally important. In vitro slow growth of 35 species and cryopreservation using embryo/meristem/seed in 20 different species of rare medicinal plants of this region is accomplished. Plants developed in vitro of ten medicinal plants, which have restricted distribution, were reintroduced in the natural habitat as well.     

Conservation In vitro of threatened plants—Progress in the past decade

Summary In vitro techniques have found increasing use in the conservation of threatened plants in recent years and this trend is likely to continue as more species face risk of extinction. The Micropropagation Unit at Royal Botanic Gardens, Kew, UK (RBG Kew) has an extensive collection of in vitro plants including many threatened species from throughout the world. The long history of the unit and the range of plants cultured have enabled considerable expertise to be amassed in identifying the problems and developing experimental strategies for propagation and conservation of threatened plants. While a large body of knowledge is available on the in vitro culture of plants, there are limited publications relating to threatened plant conservation. This review highlights the progress in in vitro culture and conservation of threatened plants in the past decade (1995–2005) and suggests future research directions. Works on non-threatened plants are also included wherever methods have applications in rare plant conservation. Recalcitrant plant materials collected from the wild or ex situ collections are difficult to grow in culture. Different methods of sterilization and other treatments to establish clean material for culture initiation are reviewed. Application of different culture methods for multiplication, and use of unconventional materials for rooting and transplantation are reviewed. As the available plant material for culture initiation is scarce and in many cases associated with inherent problems such as low viability and endogenous contamination, reliable protocols on multiplication, rooting, and storage methods are very important. In this context, photoautotrophic micropropagation has the potential for development as a routine method for the in vitro conservation of endangered plants. Long-term storage of material in culture is challenging and the potential applications of cryopreservation are significant in this area. Future conservation biotechnology research and its applications must be aimed at conserving highly threatened, mainly endemic, plants from conservation hotspots.     

Conservation <Emphasis Type="BoldItalic">in vitro</Emphasis> of rare and threatened ferns—case studies of biodiversity hotspot and island species

The importance of in vitro tools to complement other ex situ methods for saving plants from extinction is more relevant than ever before. More than 50% of the world’s plant species are endemic to the 34 global biodiversity hotspots (GBHs), each holding at least 1,500 endemic plant species. In addition, a large number of small islands hold a number of endemic species on the brink of extinction. Conservation support concentrating more on these hotspots and small islands would significantly reduce the loss of species that is currently occurring. In the majority of these cases, the resources are either locally scarce or difficult to access for in vitro conservation to support other ex situ measures. Most island countries are small, and their geographical position is a stumbling block to initiate active partnerships with other countries when they need to use in vitro tools to rescue plants that produce recalcitrant seeds/spores or propagate only by vegetative means. However, many biodiversity hotspot countries have facilities and expertise, and they concentrate on their own flora for in vitro conservation programmes. For decades, because of the grave threat these plants face, the Conservation Biotechnology Unit, previously known as the Micropropagation Unit, at Royal Botanic Gardens Kew (RBG Kew) has been at the forefront of assisting countries to save their valuable biodiversity through both in situ and ex situ methods. Approaches mentioned here highlight work on recalcitrant ferns from GBHs and small islands. Source materials from recalcitrant species, either spore or seed and in some cases vegetative material, need to be used immediately after collection for tangible results in vitro. This becomes more difficult when only a few plants or small populations are left in the wild. The task becomes harder when available material is small in quantity, and there is greater restriction on the use of available genetic diversity in the wild. This paper highlights the importance of proper collection measures, in vitro culture procedures and cryopreservation and methods for the integrated conservation of threatened ferns from both GBHs and small islands. The importance of international networking to achieve these conservation goals also will be discussed.     

Mulberry biodiversity conservation through cryopreservation

A 238 mulberry germplasm accession collection from diverse regions maintained under tropical conditions was identified from an ex situ field gene bank. The purpose was to prioritize the in vitro conservation and cryopreservation to develop long-term biodiversity conservation for ensuring sustainable utilization of these valuable resources. Reliable cryo techniques using desiccation and slow freezing of winter-dormant buds were used. Storage potential of bud grafts of different Morus species at −1.5°C for 90 d indicated species-specific variation, and most of the wild species were found sensitive. In vitro regeneration and cryopreservation (−196°C) protocols using differentiated bud meristems, like axillary winter-dormant buds, were worked out for a wide range of landraces, wild, and cultivated varieties of Morus. Buds maintained under subtropical location are also amenable for cryopreservation. Successful cryopreservation of winter-dormant buds belonging to Morus indica, Morus alba, Morus latifolia, Morus cathayana, Morus laevigata, Morus nigra, Morus australis, Morus bombycis, Morus sinensis, Morus multicaulis, and Morus rotundiloba was achieved. Among wild species, Morus tiliaefolia and Morus serrata showed moderate recovery after cryopreservation. Survival rates did not alter after 3 yr of cryopreservation. Inter-simple sequence repeat markers were used to ascertain the genetic stability of cryopreserved mulberry germplasm accessions, which showed no difference detected among the plantlets regenerated from frozen apices in comparison to the nonfrozen material.     

The possibilities and challenges of <Emphasis Type="Italic">in vitro</Emphasis> methods for plant conservation

Seed-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.     

Ex situ conservation of plant germplasm using biotechnology

Conservation of plant genetic resources attracts more and more public interest as the only way to guarantee adequate food supplies for future human generations. However, the conservation and subsequent use of such resources are complicated by cultural, economical, technical and political issues. Over the last 30 years, there have been significant increases in the number of plant collections and in accessions in ex situ storage centres throughout the World. The present review is of these ex situ collections and the contribution biotechnology has made and can make to conservation of plant germplasm. The applications and limitations of the new, molecular approaches to germplasm characterization are discussed. In vitro slow growth is used routinely for conserving germplasm of plants such as banana, plantain, cassava and potato. More recently, cryopreservation procedures have become more accessible for long-term storage. New cryopreservation techniques, such as encapsulation-dehydration, vitrification and desiccation, lengthen the list of plant species that can not only tolerate low temperatures but also give normal growth on recovery. Extensive research is still needed if these techniques are to be fully exploited.V.M. Villalobos is with the Food and Agriculture Organization of the United Nations, Viale delle Terme di Caracalia, 00100 Rome, Italy. F. Engelmann is with the International Plant Genetic Resources Institute (IPGRI), Via delle Sette Chiese 142, 00145 Rome, Italy.     

Plant cryopreservation: a continuing requirement for food and ecosystem security

This issue of In Vitro Cellular and Developmental Biology—Plant is dedicated to current developments in liquid-nitrogen cryopreservation methods and their use in plant biology and germplasm preservation. The development of cryopreservation for storage of plant cells, tissues, and organs began in the 1960s and continues to this day. Long-term storage of in vitro cultures of secondary metabolite cell cultures, embryogenic cultures, clonal germplasm, endangered species, and transgenic products remains an important requirement for many scientists, organizations, and companies. The continued development of cryopreservation techniques and their application to new plants is the subject of this issue.     

The distribution,diversity, and conservation status of Cycas in China

As ancient gymnosperm and woody plants, cycads have survived through dramatic tectonic activities, climate fluctuation, and environmental variations making them of great significance in studying the origin and evolution of flora biodiversity. However, they are among the most threatened plant groups in the world. The principal aim of this review is to outline the distribution, diversity, and conservation status of Cycas in China and provide suggestions for conservation practices. In this review, we describe the taxonomy, distribution, and conservation status of Cycas in China. By comparing Chinese Cycas species with its relatives worldwide, we then discuss the current genetic diversity, genetic differentiation of Cycas, and try to disentangle the potential effects of Quaternary climate changes and topographical events on Cycas. We review conservation practices from both researchers and practitioners for these rare and endangered species. High genetic diversity at the species level and strong genetic differentiation within Cycas have been observed. Most Cycas species in southwest China have experienced population retreats in contrast to the coastal Cycas's expansion during the Quaternary glaciation. Additionally, human activities and habitat fragmentation have pushed these endangered taxa to the brink of extinction. Although numerous efforts have been made to mitigate threats to Cycas survival, implementation and compliance monitoring in protection zones are currently inadequate. We outline six proposals to strengthen conservation measures for Cycas in China and anticipate that these measures will provide guidelines for further research on population genetics as well as conservation biology of not only cycads but also other endangered species worldwide.     

Potential contribution of cryopreserved germ plasm to the preservation of genetic diversity and conservation of endangered species in captivity.

Demographic and genetic objectives of captive propagation programs for endangered species focus on establishing demographically secure populations that maintain adequate levels of genetic diversity. Long-term storage and utilization of cryopreserved germ plasm could extend the population's generation length and allow higher levels of genetic variation to be maintained in smaller populations. Since fewer breeding animals would be needed, more species would be "rescued" from extinction using the cage facilities currently available at existing institutions. Doubling generation lengths for callitrichid primates through use of cryopreservation could almost triple the number of species that could be rescued in world zoos. Additionally, long-term cryopreservation would allow for a third population, that of the frozen zoo. Three-way exchange of germ from germ plasm banks to captive and wild populations would increase genetic diversity at reduced risk and expense. Advances in reproductive technology and better understanding of the reproductive physiology of these animal populations are necessary to permit routine application of artificial insemination and embryo transfer using frozen-stored germ plasm.     

<Emphasis Type="Italic">In vitro</Emphasis> conservation of European bryophytes

The use of in vitro techniques for conservation has been rising steadily since their inclusion in The Convention on Biological Diversity and The Global Strategy for Plant Conservation. Unfortunately, bryophytes are often overlooked in conservation initiatives, but they are important in a number of large-scale ecosystem processes, i.e. nutrient, water and carbon cycling. There is a long history of the use of tissue culture in cultivating bryophytes, and many species respond well to in vitro techniques. For 6 yr (2000–2006), The Royal Botanic Gardens, Kew and the UK statutory conservation agencies supported a project for the ex situ conservation of bryophytes. Living and cryopreserved collections of UK threatened species were successfully established and the cryopreserved collection continues to be maintained. Other in vitro conservation collections are maintained over Europe, at botanic gardens, museums and by individual university researchers, but there is no coherent European collection of bryophytes for conservation, or standardisation of techniques. A major issue for many in vitro collections is the maintenance of within species genetic diversity. Such diversity is considered to be important, as it is the basis by which populations of species can adapt to new conditions and evolve. We are proposing to establish a European network for in vitro conservation of bryophytes. We envisage that this will include living collections, cryopreserved collections and spore collections. Conservation of genetic diversity would be a priority and the collections would provide a valuable resource for conservation initiatives and support research into rare and threatened species.     

Development of an in vitro culture technique for conservation of Saccharum spp. hybrid germplasm

An in vitro method for the establishment and storage of over 200 Saccharum spp. hybrid clones was developed that involved only 1 medium for shoot development and multiplication, and no decontamination procedures. Apical buds, from the leaf axils of developing leaves surrounding the apical meristem, were cultured on medium containing the plant growth regulators 6-benzylaminopurine (BAP) and 6-furfurylaminopurine (kinetin), and regenerated multiple shoots. Shoots transferred to medium containing naphthaleneacetic acid (NAA) developed roots. In vitro plants transferred to a medium containing half strength salts and vitamins without plant growth regulators were placed in storage at 18°C. After 12 months of storage plants were transferred to fresh medium and returned to storage. The genetic integrity of clones (based on phenotype assessment) was not affected by the in vitro culture method and up to 14 months of low-maintenance storage conditions. These in vitro plants will be further tested for genetic stability using biochemical and molecular techniques.     

In vitro propagation of Paphiopedilum orchids

Paphiopedilum is one of the most popular and rare orchid genera. Members of the genus are sold and exhibited as pot plants and cut flowers. Wild populations of Paphiopedilum are under the threat of extinction due to over-collection and loss of suitable habitats. A reduction in their commercial value through large-scale propagation in vitro is an option to reduce pressure from illegal collection, to attempt to meet commercial needs and to re-establish threatened species back into the wild. Although they are commercially propagated via asymbiotic seed germination, Paphiopedilum are considered to be difficult to propagate in vitro, especially by plant regeneration from tissue culture. This review aims to cover the most important aspects and to provide an up-to-date research progress on in vitro propagation of Paphiopedilum and to emphasize the importance of further improving tissue culture protocols for ex vitro-derived explants.     

<Emphasis Type="Italic">In Vitro</Emphasis> Preservation of Spanish Biodiversity

Spanish territories contain many of the hot spots of plant biodiversity among European countries. Most of the Spanish territory is found in the Mediterranean basin and in the Canary Islands, a region of great floral singularity and diversity (Macaronesian bioregion). Therefore, an important effort must be made to contribute to its conservation. Several strategies can be considered, but seed conservation under standard conditions is the most resource-efficient method. However, the application of this methodology is not always possible for recalcitrant seeds or species for which vegetative propagation is necessary or convenient under some circumstances. Micropropagation is one of the measures suggested for preserving endangered species. During the 1990s, several in vitro culture protocols for Spanish endemics were established. The main purpose of this strategy was to obtain a considerable number of individuals to reduce the loss of natural populations. Likewise, diverse slow growth protocols were developed for this material. However, these efforts usually did not lead to the establishment of in vitro collections. The advantages and disadvantages of the in vitro conservation strategy will be reviewed for some cases. The establishment of the in vitro protocols together with the development of cryopreservation techniques created the ideal conditions to generate cryogenic collections. In this paper, we review the knowledge and experience accumulated during the last decades in micropropagation, slow growth, and cryopreservation for Spanish plant wild species. Their application in the development ex situ collections and their contribution toward an integrated system to conserve threatened species will be discussed.     

Genetic and epigenetic stability of cryopreserved and cold-stored hops (Humulus lupulus L.)

Conventional cold storage and cryopreservation methods for hops (Humulus lupulus L.) are available but, to our knowledge, the genetic and epigenetic stability of the recovered plants have not been tested. This study analyzed 51 accessions of hop using the molecular techniques, Random Amplified DNA Polymorphism (RAPD) and Amplified Fragment Length Polymorphism (AFLP), revealing no genetic variation among greenhouse-grown controls and cold stored or cryopreserved plants. Epigenetic stability was evaluated using Methylation Sensitive Amplified Polymorphism (MSAP). Over 36% of the loci were polymorphic when the cold and cryo-treated plants were compared to greenhouse plants. The main changes were demethylation events and they were common to the cryopreserved and cold stored plants indicating the possible effect of the in vitro establishment process, an essential step in both protocols. Protocol-specific methylation patterns were also detected indicating that both methods produced epigenetic changes in plants following cold storage and cryopreservation.     

Cryopreserved storage of clonal germplasm in the USDA National Plant Germplasm System

The US Department of Agriculture-Agricultural Research Service (USDA-ARS), National Plant Germplasm System (NPGS) plant collections are a critical source of genetic diversity for breeding and selection of improved crops, including vegetatively propagated plants. Information on these collections is readily accessible to breeders and researchers on the internet from the Germplasm Resources Information Network (GRIN). The clonal collections are at risk for loss due in part to their genetic diversity that makes growing them in one location a challenge, but also because it is difficult to have duplicate collections without incurring great expense. The development of cryopreservation techniques during the last two decades provides a low maintenance form of security backup for these collections. National plant collections for vegetatively propagated crop plants and their wild relatives are maintained by the USDA-ARS, NPGS at 15 sites across the country. These sites include various combinations of field, greenhouse, screenhouse, and in vitro collections. Cryopreserved backup collections in liquid nitrogen storage were instituted in the 1990s, increased greatly in the 2000s with the advent of new techniques, and are continuing today. Collections of dormant buds of temperate trees, shoot tips of in vitro cultures of many crops, and embryonic axes of some large seeded or recalcitrant seeded plants are all part of the clonal backup storage system.     

<Emphasis Type="Italic">In vitro</Emphasis> conservation of Malaysian biodiversity—achievements,challenges and future directions

Malaysia is fortunate and proud to contain some of the world’s richest biodiversity. In Malaysia, there are an estimated 185,000 species of fauna and 12,500 species of flowering plants, many of which are endemic to tropical forests in this region. Indeed, such diversity is an important and invaluable national asset to safeguard both present and future generations. In vitro conservation offers possible techniques for the preservation of plant germplasm that at present is difficult to maintain or is maintained with limited success. Research at the Universiti Kebangsaan Malaysia (The National University of Malaysia) focuses on the cryopreservation of woody fruit species with seeds that cannot tolerate cryopreservation (recalcitrant or intermediate). Among the plants with recalcitrant seeds are such traditionally important edible tropical fruits as mangosteen, langsat, and rambai (Garcinia mangostana, Lansium domesticum, and Baccaurea motleyana). Citrus aurantifolia, Citrus suhuiensis, Citrus madurensis, Citrus hystrix, and Fortunella polyandra are among the Citrus and Citrus-related species studied. Cryopreservation studies include the Nepenthes species (pitcher plants) of Malaysia. Fundamental research on desiccation and low-temperature tolerance and on the physiology of desiccation are used to understand seed behavior, a prerequisite for the development of successful conservation techniques. At the same time, cryopreservation protocols for several Citrus and forestry species were developed for embryonic axes and adventitious shoots, mainly using rapid dehydration and PVS2 vitrification techniques. There are no successful standard techniques or protocols for species with highly recalcitrant seeds such as Garcinia species. Modification of existing protocols or development of new methods is required, but this can be accomplished only when a detailed understanding of the recalcitrant nature of the seeds or explants is achieved. While we have considerable knowledge concerning the basics of biochemical processes and some molecular data from work on desiccation-tolerant seeds, a great need remains for understanding the cause of the recalcitrance or desiccation sensitivity of these seeds. It may be necessary to use a systems biology approach that exploits the “omics” technologies to generate global molecular data. In combination with bioinformatics for data integration and analyses, this approach would move toward improved modeling of the biological pathways associated with the development of recalcitrant seeds.     

Cryopreservation by encapsulation-dehydration of ‘Christmas bush’ (<Emphasis Type="Italic">Ceratopetalum gummiferum</Emphasis>) shoot tips

Summary Christmas bush (Ceratopetalum gummiferum Sm) is a shrubby tree species of the east coast of New South Wales in Australia. It is much prized as a cut flower crop because of its bright, pinky red floral calyces. New varieties are being developed, the storage of which is an important issue. In this study, it was shown that shoot tips sampled from in vitro plantlets withstood cryopreservation using the encapsulation-dehydration technique. The protocol leading to optimal regrowth was the following: excised shoot tips were pretreated for 1 d in the dark on hormone-free Murashige and Skoog (MS) medium with 0.3 M sucrose, then encapsulated in 3% calcium alginate and precultured in liquid MS medium with 0.5 M sucrose for 3 d. Precultured beads were dehydrated for 6 h in the air current of the laminar flow cabinet to 24.3% moisture content (fresh weight basis) before rapid immersion in liquid nitrogen. Under these conditions, regrowth of shoot tips after cryopreservation reached 61.4%. Regrowth of cryopreserved shoot tips was not affected by the period of cold acclimation of in vitro mother plants.     

Geographic discrepancies between global and local rarity richness patterns and the implications for conservation

The emerging interest in the biological and conservation significance of locally rare species prompts a number of questions about their correspondence with other categories of biodiversity, especially global rarity. Here we present an analysis of the correspondence between the distributions of globally and locally rare plants. Using biological hotspots of rarity as our framework, we evaluate the extent to which conservation of globally rare plants will act as a surrogate for conservation of locally rare taxa. Subsequently, we aim to identify gaps between rarity hotspots and protected land to guide conservation planning. We compiled distribution data for globally and locally rare plants from botanically diverse Napa County, California into a geographic information system. We then generated richness maps highlighting hotspots of global and local rarity. Following this, we overlaid the distribution of these hotspots with the distribution of protected lands to identify conservation gaps. Based on occupancy of 1 km² grid cells, we found that over half of Napa County is occupied by at least one globally or locally rare plant. Hotspots of global and local rarity occurred in a substantially smaller portion of the county. Of these hotspots, less than 5% were classified as multi-scale hotspots, i.e. they were hotspots of global and local rarity. Although, several hotspots corresponded with the 483 km² of protected lands in Napa County, some of the richest areas did not. Thus, our results show that there are important conservation gaps in Napa County. Furthermore, if only hotspots of global rarity are preserved, only a subset of locally rare plants will be protected. Therefore, conservation of global, local, and multi-scale hotspots needs serious consideration if the goals are to protect a larger variety of biological attributes, prevent extinction, and limit extirpation in Napa County.