Margaret M. Ramsay

CONSERVATION IN VITRO OF THREATENED PLANTS – PROGRESS IN THE PAST DECADE

SummaryIn 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.

Seed fertilization, development, and germination in Hydatellaceae (Nymphaeales): Implications for endosperm evolution in early angiosperms.

New data on endosperm development in the early-divergent angiosperm Trithuria (Hydatellaceae) indicate that double fertilization results in formation of cellularized micropylar and unicellular chalazal domains with contrasting ontogenetic trajectories, as in waterlilies. The micropylar domain ultimately forms the cellular endosperm in the dispersed seed. The chalazal domain forms a single-celled haustorium with a large nucleus; this haustorium ultimately degenerates to form a space in the dispersed seed, similar to the chalazal endosperm haustorium of waterlilies. The endosperm condition in Trithuria and waterlilies resembles the helobial condition that characterizes some monocots, but contrasts with Amborella and Illicium, in which most of the mature endosperm is formed from the chalazal domain. The precise location of the primary endosperm nucleus governs the relative sizes of the chalazal and micropylar domains, but not their subsequent developmental trajectories. The unusual tissue layer surrounding the bilobed cotyledonary sheath in seedlings of some species of Trithuria is a belt of persistent endosperm, comparable with that of some other early-divergent angiosperms with a well-developed perisperm, such as Saururaceae and Piperaceae. The endosperm of Trithuria is limited in size and storage capacity but relatively persistent.

The Role of Botanic Gardens in the Science and Practice of Ecological Restoration

Many of the skills and resources associated with botanic gardens and arboreta, including plant taxonomy, horticulture, and seed bank management, are fundamental to ecological restoration efforts, yet few of the worlds botanic gardens are involved in the science or practice of restoration. Thus, we examined the potential role of botanic gardens in these emerging fields. We believe a reorientation of certain existing institutional strengths, such as plant-based research and knowledge transfer, would enable many more botanic gardens worldwide to provide effective science-based support to restoration efforts. We recommend botanic gardens widen research to include ecosystems as well as species, increase involvement in practical restoration projects and training practitioners, and serve as information hubs for data archiving and exchange.

How bryophytes came out of the cold: successful cryopreservation of threatened species.

The use of ex situ techniques for the conservation of threatened plants has been increasing over the past decades. Cryopreservation is often used for the long-term storage of plant germplasm where conventional methods (i.e. seedbanking) are inappropriate. Simple encapsulation–dehydration protocols were developed for the cryopreservation of bryophytes at The Royal Botanic Gardens, Kew, as part of an ex situ project for the conservation of UK threatened species. The applicability of these methods was tested on 22 species with a broad range of ecological requirements and found to be highly successful across the board. Regeneration rates from frozen material were >68% for all species tested and half had regeneration rates of 100%. The high regeneration rate and broad applicability of the protocols across a range of species was attributed to a combination of the inherent totipotency of bryophytes and the in-built recovery periods in the pre-treatment protocol. In conclusion, bryophytes are well suited to cryopreservation and such techniques would be applicable for the long-term storage of similar conservation collections across the globe.

Which moss is which? Identification of the threatened moss Orthodontium gracile using molecular and morphological techniques

Taxonomic misidentification has potentially serious consequences for the management of threatened species. Closely related moss species are often difficult to distinguish from each other using morphological characteristics. Here we compared the use of molecular (DNA barcoding of the trnL-F intron, AFLPs) and morphological techniques to demonstrate that ex situ cultures, held for re-introduction trails, of the UK critically endangered moss Orthodontium gracile were contaminated with the potentially invasive species O. lineare. Barcoding techniques and AFLPs were both successful in determining Orthodontium species identity. There was some discrepancy between determinations from molecular and morphological techniques and some individuals were misidentified using morphological characteristics alone. When species identity is critical, for example prior to re-establishment or re-introduction programmes, we recommend that identity of mosses and other bryophytes be established by molecular techniques, in particular barcoding of the trnL-F intron.

Chromosome behavior at the base of the angiosperm radiation: Karyology of Trithuria submersa (Hydatellaceae, Nymphaeales)

UNLABELLED • PREMISE OF THE STUDY Hydatellaceae are minute annual herbs with potential as a model system for studying early angiosperm evolution, but their karyology and ploidy levels are almost unknown. We investigated these aspects of Trithuria submersa, a widespread species that we show to be amenable to extended vegetative propagation.• METHODS We cultivated plants of T. submersa in vitro after developing and optimizing culture conditions. We estimated genome size using flow cytometry, counted chromosome numbers using root-meristem squashes after Feulgen staining, and examined meiotic chromosome behavior using microsporocytes.• KEY RESULTS We developed methods to reliably germinate seeds of T. submersa and to propagate them vegetatively in critical thermo- and photoperiod regimes on 1/2 Murashige-Skoog (MS) medium with vitamins and 2% sucrose solidified with 0.7% agar-agar. Seedling growth requires the medium be supplemented with activated charcoal. The mean nuclear DNA content of T. submersa sporophytes is 2C = 2.74 pg (∼2.68 Gbp). The sporophytic chromosome number is 2n = 56 with a bimodal complement, which may suggest an allopolyploid origin. Some of the largest chromosomes lack a recognizable constriction, which relates to a highly unusual and irregular chromosome behavior. Microsporocytes undergo reduced and asynchronous meioses that show a modified intermediate cell division with a nucleus division by fractional postreduction, indicating partially inverted microsporogenesis.• CONCLUSIONS In vitro cultivation and karyological assessment of T. submersa open new opportunities for investigating early-divergent angiosperms. The remarkably different meiotic behavior exhibits new insights into a potentially ancestral microsporogenesis.

Impact of spatial constraints during seed germination on the evolution of angiosperm cotyledons: A case study from tropical Hydatellaceae (Nymphaeales)

UNLABELLED PREMISE OF THE STUDY A bipolar embryo with cotyledons is a characteristic feature that appeared early in the evolution of seed plants. Cotyledon number is an important character in angiosperm classification. We explore the links between functional aspects of seed germination and the number and location of the cotyledons, using as a model the early-divergent angiosperm family Hydatellaceae, in which seedlings are superficially monocot-like. • METHODS Seedlings of two species of tropical Hydatellaceae were studied using light and scanning electron microscopy. • KEY RESULTS Seedlings of Trithuria cowieana bear two free cotyledons. Each cotyledon possesses a green, filiform, vascularized blade that resembles subsequent leaves, and a basal, nonvascularized, haustorial outgrowth that remains in close contact with the endosperm. Seedlings of Trithuria konkanensis have two free cotyledonary haustoria inserted close to each other and a leaf blade probably belonging to one of the cotyledons. The cotyledonary node elongates between the haustoria and the leaf blade to form a mesocotyl. • CONCLUSIONS To date, the absence or presence of a cotyledonary tube represents the only known qualitative morphological difference between the two major clades of Hydatellaceae. Cotyledons with a haustorium and leaf blade are unusual at the scale of seed plants and probably evolved due to homeosis. The mesocotyl of T. konkanensis resembles that of grasses and sedges. Seedling diversity in Hydatellaceae and other seed plants is linked with the principal physical and spatial constraint of their embryo structure, with the primary root and shoot apical meristems located at opposite poles, and haustorial cotyledon tips.

The Role of Botanic Gardens in the Science and Practice of Ecological Restoration: Role of Botanic Gardens in Restoration

Many of the skills and resources associated with botanic gardens and arboreta, including plant taxonomy, horticulture, and seed bank management, are fundamental to ecological restoration efforts, yet few of the worlds botanic gardens are involved in the science or practice of restoration. Thus, we examined the potential role of botanic gardens in these emerging fields. We believe a reorientation of certain existing institutional strengths, such as plant-based research and knowledge transfer, would enable many more botanic gardens worldwide to provide effective science-based support to restoration efforts. We recommend botanic gardens widen research to include ecosystems as well as species, increase involvement in practical restoration projects and training practitioners, and serve as information hubs for data archiving and exchange.

Large Scale Propagation and In vitro Weaning for the Restoration Viola palustris to Support Assisted Colonisation of a Threatened Butterfly

The distribution and abundance of Boloria selene (small pearl-bordered fritillary butterfly SPBF) declined over the decades in many parts of the UK. Availability of food plants, especially marsh violet (Viola palustris), for the caterpillars of the SPBF has been identified as one of the major limiting factors for this decline. To achieve augmentation of existing colonies and develop new populations of SPBF large numbers of marsh violet propagules were required specifically to feed the larvae. The main objectives of the study were to produce thousands of good quality marsh violet propagules to restore selected habitats in the Heart of Durham, Northern England, using in vitro methods. Preliminary trials showed that in vitro multiplication of seedlings from wild collected seeds through conventional agar-based cultures was lengthy, expensive and turned out to be a non-viable route to achieve the objectives. This study explored the potential of bioreactor-based cloning and cost-effective one step rooting and weaning. Robust propagules, ready for transplantation following rapid propagation and one step rooting and weaning in vitro, were raised in a plug system for transplantation and establishment under field conditions. This was achieved by using simple and cost-effective methods to support the large-scale restoration exercise using 14,000 propagules. Application of high throughput micropropagation and low cost one step weaning systems for time-bound conservation and restoration projects are discussed in detail. This research highlights the important role of in vitro methods to support integrated biodiversity conservation of threatened native plant and butterfly.