The Effects of Moisture Content and Temperature on the Storability of an Orthodox, Intermediate and a Recalcitrant Forest Tree Seed

The effects of seed moisture and storage temperature on storability of an orthodox (Instia palembanica), an intermediate (Swietenia macrophylla) and a recalcitrant (Hopea odorata) forest tree seed were studied. The objectives were to determine the critical and optimum moisture and storage temperature for storage of each of the three species, and to evaluate the potential of these seeds for cryopreservation at ultra-low temperatures of liquid nitrogen. The study was divided into four parts of three experiments, each being for one of the three species. In Part I, the effects of desiccation on seed viability were evaluated. The orthodox seed, I. palembanica, has low shedding moisture of 10%. It was tolerant to desiccation as its critical and optimum moistures were low at 6% and 8-10% respectively. For the intermediate seed, S. macrophylla, it was more sensitive to desiccation. Its shedding moisture was high at 37% and it could not withstand excessive desiccation as its critical and optimum moistures were high at 1 5% and 25% respectively. The recalcitrant seed, H. odorata, has very high shedding moisture of 48% and was highly sensitive to even slight desiccation. Its critical and optimum moistures were very high at 29% and 32-35% respectively

Development of efficient experimental strategies for the cryopreservation of problematic tropical rain forest tree germplasm

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Seed Longevity - The Evolution of Knowledge and a Conceptual Framework

The lifespan or longevity of a seed is the time period over which it can remain viable. Seed longevity is a complex trait and varies greatly between species and even seed lots of the same species. Our scientific understanding of seed longevity has advanced from anecdotal 'Thumb Rules,' to empirically based models, biophysical explanations for why those models sometimes work or fail, and to the profound realisation that seeds are the model of the underexplored realm of biology when water is so limited that the cytoplasm solidifies. The environmental variables of moisture and temperature are essential factors that define survival or death, as well as the timescale to measure lifespan. There is an increasing understanding of how these factors induce cytoplasmic solidification and affect glassy properties. Cytoplasmic solidification slows down, but does not stop, the chemical reactions involved in ageing. Continued degradation of proteins, lipids and nucleic acids damage cell constituents and reduce the seed's metabolic capacity, eventually impairing the ability to germinate. This review captures the evolution of knowledge on seed longevity over the past five decades in relation to seed ageing mechanisms, technology development, including tools to predict seed storage behaviour and non-invasive techniques for seed longevity assessment. It is concluded that seed storage biology is a complex science covering seed physiology, biophysics, biochemistry and multi-omic technologies, and simultaneous knowledge advancement in these areas is necessary to improve seed storage efficacy for crops and wild species biodiversity conservation

Advances in cryopreservation of Syzygium maire (swamp maire, maire tawake) zygotic embryos, a critically endangered tree species endemic to New Zealand

IntroductionSyzygium maire is a threatened Myrtaceae tree species endemic to New Zealand. Due to its highly recalcitrant seed, cryopreservation is the only viable long-term ex situ conservation option for this species. Our previous attempts to cryopreserve the embryonic axis (EAs) of S. maire were unsuccessful but did provide a better understanding of desiccation behavior, biochemical composition, oxidative status, and ultrastructural changes associated with desiccation in EAs.MethodsWe incorporated this knowledge with biophysical information to investigate two advanced cryopreservation technologies: a droplet vacuum infiltration vitrification (DVIV) method and a novel metal-mesh vacuum infiltration vitrification (MVIV) method using Plant Vitrification Solution 2 (PVS2) for cryopreservation of the EAs.ResultsThe PVS2 treatment at room temperature (~20°C) proved phytotoxic with extended PVS2 incubation significantly reducing EA survival. No EAs survived cryopreservation using DVIV, however MVIV resulted in post-cryopreservation survival of up to 19% following PVS2 incubation for 20 min. Biophysical thermal analysis using Differential Scanning Calorimetry revealed a 15-fold reduction in ice crystallization following incubation in PVS2 for 20 min or more, with all freezable water removed after 60 min incubation.DiscussionThese results present a significant advance in being able to successfully cryopreserve S. maire EAs. The findings from this study will aid the development of cryopreservation protocols for other extremely recalcitrant seeded species, many of which are threatened with extinction due to climate change, plant pathogens, and habitat destruction

Innovative approaches to the preservation of forest trees

AbstractThe recent acceleration of actions to conserve plant species using ex situ and in situ strategies has revealed the need to understand how these two approaches might be better developed and integrated in their application to tree species. Here we review some of the recent successes relating mainly to tree seed biology that have resulted in the development and application of innovative actions across five areas: (i) the expansion of living collections to conserve threatened tree species in sufficient numbers to ensure a broad genetic diversity in their progeny; (ii) the generation of viability constants to enable estimates to be made of storage longevity of tree seeds in the dry state; (iii) improvement in the diagnosis of tree seed storage behaviour through the development of predictive models, reliable prognoses of desiccation tolerance and use of botanical information systems, such as GIS, to correlate information on species distribution and their physiological characteristics; (iv) advances in storage preservation biotechnology to enhance the future application of cryopreservation procedures to recalcitrant species in biodiversity hotspots where many are under threat of extinction; and (v) integration of ex situ and in situ conservation approaches to ensure that best practice in horticultural and forestry are combined to maintain or enhance genetic diversity, especially in high value species and those with small and vulnerable populations. These actions can lead to greater impact if supported by greater efforts to create seed banks and to collate databases world-wide so that data, knowledge and collections are more available to the scientific, forestry and NGO communities. Throughout this review we have used examples from the mega-biodiversity countries of Brazil and China, as a way of illustrating wider principles that can be applied in many countries. Future development of current research approaches, the adherence to conservation policy and the expanding needs for education are also considered briefly

Biophysical characteristics of successful oilseed embryo cryoprotection and cryopreservation using vacuum infiltration vitrification: an innovation in plant cell preservation.

Heterogeneity in morphology, physiology and cellular chemistry of plant tissues can compromise successful cryoprotection and cryopreservation. Cryoprotection is a function of exposure time × temperature × permeability for the chosen protectant and diffusion pathway length, as determined by specimen geometry, to provide sufficient dehydration whilst avoiding excessive chemical toxicity. We have developed an innovative method of vacuum infiltration vitrification (VIV) at 381 mm (15 in) Hg (50 kPa) that ensures the rapid (5 min), uniform permeation of Plant Vitrification Solution 2 (PVS2) cryoprotectant into plant embryos and their successful cryopreservation, as judged by regrowth in vitro. This method was validated on zygotic embryos/embryonic axes of three species (Carica papaya, Passiflora edulis and Laurus nobilis) up to 1.6 mg dry mass and 5.6 mm in length, with varying physiology (desiccation tolerances) and 80 °C variation in lipid thermal profiles, i.e., visco-elasticity properties, as determined by differential scanning calorimetry. Comparisons between the melting features of cryoprotected embryos and embryo regrowth indicated an optimal internal PVS2 concentration of about 60% of full strength. The physiological vigour of surviving embryos was directly related to the proportion of survivors. Compared with conventional vitrification, VIV-cryopreservation offered a ∼ 10-fold reduction in PVS2 exposure times, higher embryo viability and regrowth and greater effectiveness at two pre-treatment temperatures (0 °C and 25 °C). VIV-cryopreservation may form the basis of a generic, high throughput technology for the ex situ conservation of plant genetic resources, aiding food security and protection of species from diverse habitats and at risk of extinction

Embryos internal concentration of PVS2 and their corresponding regrowth.

<p>Legend: Regrowth following cryopreservation compared to internal concentration of PVS2 (defined by melt enthalpy using a DSC) of <i>C. papaya</i> (A), <i>P. edulis</i> (B) and <i>L. nobilis</i> (C) embryos/embryonic axes with a polynomial fit. (<b>○</b> = CV at 0°C; • = CV at 25°C; Δ = VIV at 0°C; ▴ = VIV at 25°C).</p

DSC thermograms for dry seeds and embryos.

<p>Legend: DSC warming thermograms for <i>C. papaya</i> (A), <i>P. edulis</i> (B) and <i>L. nobilis</i> (C) dry seeds and embryos/embryonic axes showing lipid melt endotherms. Samples were cooled from 25°C to −100°C and rewarmed to 25°C (50°C for <i>L. nobilis</i>) at ±10°C min⁻¹. Numbers on each peak indicating different lipid melt peaks corresponding to peaks in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096169#pone-0096169-t001" target="_blank">Table 1</a>.</p

DSC warming thermograms for <i>C. papaya</i> embryos following cryoprotection.

<p>Legend: Representative DSC warming thermograms for <i>C. papaya</i> embryos untreated and treated with PVS2 under CV for 30, 60 and 90 min at 0°C showing ice melt (I), lipid melt (L), PVS2 melt (P) with melt enthalpies in brackets and PVS2 de-vitrification (P_dv). Samples were warmed from −100 to 25°C at 10°C min⁻¹. Inset: Warming thermal profile of PVS2 solution with melt enthalpy in bracket (warming program: from −150 to 0°C at 10°C min⁻¹).</p