N.W. Pammenter

Preservation of Recalcitrant Seeds

Cryogenic technologies help to preserve plant biodiversity in seed banks, particularly in the tropics. Concerns about the rapid erosion of plant diversity have spawned a host of seed-banking initiatives (1). These repositories provide critical germ plasm needed to understand, maintain, and manage natural variation within and among species (2). However, numerous plant species and much of the humid tropics are underserved in these endeavors because of the perceived problem of seed recalcitrance (3). About 75 to 80% of angiosperm species (4, 5) produce orthodox seeds that can survive drying and prolonged storage at −20°C. By contrast, 5 to 10% of angiosperm species produce recalcitrant seeds that do not survive desiccation (3) and are killed in the freezer when ice crystals form. How can their preservation be ensured?

The growth responses of coastal dune species are determined by nutrient limitation and sand burial

Past work suggests that burial and low nutrient availability limit the growth and zonal distribution of coastal dune plants. Given the importance of these two factors, there is a surprising lack of field investigations of the interactions between burial and nutrient availability. This study aims to address this issue by measuring the growth responses of four coastal dune plant species to these two factors and their interaction. Species that naturally experience either high or low rates of burial were selected and a factorial burial by nutrient addition experiment was conducted. Growth characteristics were measured in order to determine which characteristics allow a species to respond to burial. Species that naturally experience high rates of burial (Arctotheca populifolia and Scaevola plumieri) displayed increased growth when buried, and this response was nutrient-limited. Stable-dune species had either small (Myrica cordifolia, N-fixer) or negligible responses to burial (Metalasia muricata), and were not nutrient-limited. This interspecific difference in response to burial and/or fertiliser is consistent with the idea that burial maintains the observed zonation of species on coastal dunes. Species that are unable to respond to burial are prevented from occupying the mobile dunes. Species able to cope with high rates of burial had high nitrogen-use efficiencies and low dry mass costs of production, explaining their ability to respond to burial under nutrient limitation. The interaction between burial and nutrient limitation is understudied but vital to understanding the zonation of coastal dune plant species.

Implications of the lack of desiccation tolerance in recalcitrant seeds

A suite of interacting processes and mechanisms enables tolerance of desiccation and storage (conservation) of orthodox seeds in the dry state. While this is a long-term option under optimized conditions, dry orthodox seeds are not immortal, with life spans having been characterized as short, intermediate and long. Factors facilitating desiccation tolerance are metabolic “switch-off” and intracellular dedifferentiation. Recalcitrant seeds lack these mechanisms, contributing significantly to their desiccation sensitivity. Consequently, recalcitrant seeds, which are shed at high water contents, can be stored only in the short-term, under conditions not allowing dehydration. The periods of such hydrated storage are constrained by germination that occurs without the need for extraneous water, and the proliferation of seed-associated fungi. Cryopreservation is viewed as the only option for long-term conservation of the germplasm of recalcitrant-seeded species. This is not easily achieved, as each of the necessary procedures imposes oxidative damage. Intact recalcitrant seeds cannot be cryopreserved, the common practice being to use excised embryos or embryonic axes as explants. Dehydration is a necessary procedure prior to exposure to cryogenic temperatures, but this is associated with metabolism-linked injury mediated by uncontrolled reactive oxygen species generation and failing anti-oxidant systems. While the extent to which this occurs can be curtailed by maximizing drying rate (flash drying) it cannot be completely obviated. Explant cooling for, and rewarming after, cryostorage must necessarily be rapid, to avoid ice crystallization. The ramifications of desiccation sensitivity are discussed, as are problems involved in cryostorage, particularly those accompanying dehydration and damage consequent upon ice crystallization. While desiccation sensitivity is a “fact” of seed recalcitrance, resolutions of the difficulties involved germplasm conservation are possible as discussed.

An ultrastructural study using anhydrous fixation of Eragrostis nindensis, a resurrection grass with both desiccation-tolerant and -sensitive tissues

The ability of tissues to survive desiccation is common in seeds but rare in vegetative tissues. In this study the ultrastructure of hydrated and dehydrated tissues were examined at different stages of the life cycle of the resurrection grass, Eragrostis nindensis Ficalho & Hiern. Conventional fixation techniques are unsuitable for dry tissues as rehydration occurs during fixation in aqueous fixatives. Thus a cryofixation and freeze-substitution method was developed. As a result of the improved fixation methods, it was possible to identify the stage and nature of the damage in the desiccation-sensitive tissues. E. nindensis has desiccation-tolerant orthodox seeds, but the young seedlings are not tolerant to extreme water loss. However, like the seeds, most of the leaves of the adult plant are tolerant to desiccation (only the oldest outermost leaf on a tiller are not). Desiccation-induced damage in these outer leaves was observed in the later stage of dehydration, dominated by the appearance of abundant cell wall fractures (1 wall fracture per 50 μm2). Unlike the outer leaves, the leaves of seedlings appeared similar to those of the hydrated ones upon desiccation. Irreparable damage occurred on rehydration of these tissues possibly as a result of the absence of protection mechanisms observed during desiccation of the inner desiccation-tolerant leaves of the mature plants. The mesophyll tissues of these leaves become compact with extensive cell wall folding on drying. The bundle sheath cells maintained their shape with desiccation but became packed with small vacuoles.

Cryopreservation of Recalcitrant (i.e. Desiccation-Sensitive) Seeds

cation and are often referred to as “recalcitrant” (Hong et al. 1998). Approximately 10–20% of angiosperm species produce seeds that acquire some, but not full, tolerance of desiccation during maturation (Dickie and Pritchard 2002). Incidence of recalcitrance does not distribute along phylogenetic clades, though some plant families include many species producing recalcitrant seeds (e.g., Fagaceae, Lauraceae, Sapindaceae, Meliaceae) while other families apparently lack species exhibiting this trait (e.g., Solanaceae, Asteraceae, Amaranthaceae). Life history traits of the plant, such as a long lived, perennial nature, and its habitat, such as aquatic or rainforest, are associated with seed recalcitrance, but not all plants with these characteristics produce recalcitrant seeds. The term “recalcitrant” is also used to describe seeds that are particularly difficult to germinate because they have deep dormancy or an unknown dormancy release mechanism. Though frustrating to work with, seeds with this dormancy physiology are amenable to

Cryoconservation of South African plant genetic diversity

South Africa has a rich flora which exhibits among the highest species density in the world, distributed across nine biomes that support an impressive diversity of animal life. However, a variety of human actions, invasion by alien species, natural disturbances and climate change collectively impact negatively on the great diversity of both plant and animal species. In situ conservation has long been practised, primarily in nature reserves, complemented by ex situ conservation in national botanic gardens, but in vitro plant conservation is not common. In the context of animal biodiversity conservation, the Wildlife Biological Resource Centre of the National Zoological Gardens utilises cryobanking as one of its major focuses and is now poised to expand as the repository for the cryoconservation of plant germplasm, particularly for indigenous recalcitrant-seeded and poor-seeding species. However, there are particular problems associated with successful germplasm cryostorage of such tropical and subtropical plants. As we see the science and application of cryobiology and cryoconservation as cross-cutting and transdisciplinary, we have entrained formal networking among scientists offering a range of specialisations aimed at a deeper understanding of common problems and practical outcomes to facilitate both plant and animal biobanking. The endeavours are aimed at elucidating the basis of both successes and failures in our efforts to attain optimal outcomes. With focus on best practices, standard operating procedures, validation and risk management for cryopreserved and cold-stored plant and animal material, our ultimate aim is to facilitate restoration by the safe reintroduction of indigenous species.

Differential drying rates of recalcitrant Trichilia dregeana embryonic axes: a study of survival and oxidative stress metabolism.

Studies to elucidate the biochemical basis of survival of excised embryonic axes (EAs) of recalcitrant seeds of Trichilia dregeana at different drying rates revealed significant differences between slow and rapid drying. Rapid drying allowed these EAs to survive dehydration to much lower water contents (WCs; ca. 0.31 g g⁻¹ dry mass basis with 73% germination) compared with slow drying, where 90% of the EAs lost viability at a WC of ca. 0.79 g g⁻¹. In EAs slowly dried within seeds, the levels of hydroxyl radical (three- to fivefold at WCs > 0.5 g g⁻¹) and lipid peroxidation (50% at similar WC) were significantly higher compared with those dried rapidly to comparable WCs. When EAs were dried slowly, enzymic antioxidant levels were not sustained and declined significantly with prolonged storage. In contrast, sustained activity of enzymic antioxidants was detected in rapidly dried EAs even at relatively low WCs. Furthermore, the greater decline in glutathione (GSH)/GSH disulphide ratio in EAs slowly dried within seeds compared with rapidly dried EAs and a shift in GSH redox potential to relatively more positive values in the EAs slowly dried within seeds was correlated with considerable viability loss. It is apparent from this study that greater retention of viability to lower WCs in rapidly dried EAs from recalcitrant seeds may at least be partly explained by the retention of functional antioxidant status. It is also suggested that the reduction of viability in rapidly dried EAs at very low WCs appears to be a non-oxidative process.

The effects of various parameters during processing for cryopreservation on the ultrastructure and viability of recalcitrant zygotic embryos of Amaryllis belladonna

Cryostorage (usually in, or above liquid nitrogen) is presently the only option for long-term germplasm conservation of species producing recalcitrant (desiccation-sensitive) seeds. The present study investigated the ultrastructural responses of zygotic embryos excised from recalcitrant Amaryllis belladonna seeds to the sequential steps involved in cryopreservation. Flash-dried embryos, with and without prior sucrose (non-penetrating) or glycerol (penetrating) cryoprotection, were cooled rapidly or slowly, recovered in vitro and then assessed for ultrastructural and viability responses. Untreated embryos were 100% viable, the ultrastructure being indicative of their actively metabolic condition. Although nuclear morphology changed, viability was unaffected after exposure to either glycerol or sucrose, but mitochondrial ultrastructure suggested enhancement of metabolic activity particularly after sucrose treatment. When flash dried after sucrose cryoprotection, a significant increase in the degree of vacuolation, abnormal plastid ultrastructure and some wall abnormality accompanied a decline in survival to 70% and 60% at water contents > and <0.4xa0gxa0g−1, respectively. In contrast, glycerol cryoprotection, which promoted retention of generally normal ultrastructure and also counteracted any increase in the degree of vacuolation, was associated with 100% and 90% survival of embryos at the higher and lower water contents. After exposure to liquid nitrogen (LN), ultrastructural irregularities were minimal in rapidly cooled glycerol-cryoprotected embryos, at water content <0.4xa0gxa0g−1, which showed 70% survival after retrieval from cryogenic conditions. At the other extreme, no embryos survived LN exposure when sucrose cryoprotected. The study relates the cumulative effects of subcellular abnormality and declining viability, in relation to experimental parameters for cryopreservation.

Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures

BACKGROUND AND AIMSnCryopreservation is the only long-term conservation strategy available for germplasm of recalcitrant-seeded species. Efforts to cryopreserve this form of germplasm are hampered by potentially lethal intracellular freezing events; thus, it is important to understand the relationships among cryo-exposure techniques, water content, structure and survival.nnnMETHODSnUndried embryonic axes of Acer saccharinum and those rapidly dried to two different water contents were cooled at three rates and re-warmed at two rates. Ultrastructural observations were carried out on radicle and shoot tips prepared by freeze-fracture and freeze-substitution to assess immediate (i.e. pre-thaw) responses to cooling treatments. Survival of axes was assessed in vitro.nnnKEY RESULTSnIntracellular ice formation was not necessarily lethal. Embryo cells survived when crystal diameter was between 0·2 and 0·4 µm and fewer than 20 crystals were distributed per μm(2) in the cytoplasm. Ice was not uniformly distributed within the cells. In fully hydrated axes cooled at an intermediate rate, the interiors of many organelles were apparently ice-free; this may have prevented the disruption of vital intracellular machinery. Intracytoplasmic ice formation did not apparently impact the integrity of the plasmalemma. The maximum number of ice crystals was far greater in shoot apices, which were more sensitive than radicles to cryo-exposure.nnnCONCLUSIONSnThe findings challenge the accepted paradigm that intracellular ice formation is always lethal, as the results show that cells can survive intracellular ice if crystals are small and localized in the cytoplasm. Further understanding of the interactions among water content, cooling rate, cell structure and ice structure is required to optimize cryopreservation treatments without undue reliance on empirical approaches.

Effects of simulated acid rain on germination, seedling growth and oxidative metabolism of recalcitrant‐seeded Trichilia dregeana grown in its natural seed bank

Increased air pollution in a number of developing African countries, together with the reports of vegetation damage typically associated with acid precipitation in commercial forests in South Africa, has raised concerns over the potential impacts of acid rain on natural vegetation in these countries. Recalcitrant (i.e. desiccation sensitive) seeds of many indigenous African species, e.g. must germinate shortly after shedding and hence, may not be able to avoid exposure to acid rain in polluted areas. This study investigated the effects of simulated acid rain (rainwater with pH adjusted to pH 3.0 and 4.5 with 70:30, H2 SO4 :HNO3 ) on germination, seedling growth and oxidative metabolism in a recalcitrant-seeded African tree species Trichilia dregeana Sond., growing in its natural seed bank. The results suggest that acid rain did not compromise T. dregeana seed germination and seedling establishment significantly, relative to the control (non-acidified rainwater). However, pH 3.0 treated seedlings exhibited signs of stress typically associated with acid rain: leaf tip necrosis, abnormal bilobed leaf tips, leaf necrotic spots and chlorosis, reduced leaf chlorophyll concentration, increased stomatal density and indications of oxidative stress. This may explain why total and root biomass of pH 3.0 treated seedlings were significantly lower than the control. Acid rain also induced changes in the species composition and relative abundance of the different life forms emerging from T. dregeanas natural seed bank and in this way could indirectly impact on T. dregeana seedling establishment success.