Development, Management and Utilization of a Kiwifruit (Actinidia spp.) In Vitro Collection: A New Zealand Perspective.

The New Zealand Institute for Plant and Food Research Limited (PFR) supports a large kiwifruit breeding program that includes more than twenty Actinidia species. Almost all the kiwifruit accessions are held as field collections across a range of locations, though not all plants are at multiple locations. An in vitro collection of kiwifruit in New Zealand was established upon the arrival of Pseudomonas syringae pv. Actinadiae-biovar 3 in 2010. The value of an in vitro collection has been emphasized by restrictions on importation of new plants into New Zealand and increasing awareness of the array of biotic and abiotic threats to field collections. The PFR in vitro collection currently holds about 450 genotypes from various species, mostly A. chinensis var. chinensis and A. chinensis var. deliciosa. These collections and the in vitro facilities are used for germplasm conservation, identification of disease-free plants, reference collections and making plants available to users. Management of such a diverse collection requires appropriate protocols, excellent documentation, training, sample tracking and databasing and true-to-type testing, as well as specialized facilities and resources. This review also discusses the New Zealand biosecurity and compliance regime governing kiwifruit plant movement, and how protocols employed by the facility aid the movement of pathogen-free plants within and from New Zealand.

Seed Storage Physiology of Lophomyrtus and Neomyrtus, Two Threatened Myrtaceae Genera Endemic to New Zealand.

There is no published information on the seed germination or seed storage physiology of Lophomyrtus bullata, Lophomyrtus obcordata, and Neomyrtus pedunculata. This lack of information is hampering conservation efforts of these critically endangered species. This study investigated the seed morphology, seed germination requirements, and long-term seed storage methods for all three species. The impact of desiccation, desiccation and freezing, as well as desiccation plus storage at 5 °C, -18 °C, and -196 °C on seed viability (germination) and seedling vigour was assessed. Fatty acid profiles were compared between L. obcordata and L. bullata. Variability in storage behaviour between the three species was investigated through differential scanning calorimetry (DSC) by comparing thermal properties of lipids. L. obcordata seed were desiccation-tolerant and viability was retained when desiccated seed was stored for 24 months at 5 °C. L. bullata seed was both desiccation- and freezing-sensitive, while N. pedunculata was desiccation-sensitive. DSC analysis revealed that lipid crystallisation in L. bullata occurred between -18 °C and -49 °C and between -23 °C and -52 °C in L. obcordata and N. pedunculata. It is postulated that the metastable lipid phase, which coincides with the conventional seed banking temperature (i.e., storing seeds at -20 ± 4 °C and 15 ± 3% RH), could cause the seeds to age more rapidly through lipid peroxidation. Seeds of L. bullata, L. obcordata and N. pedunculata are best stored outside of their lipid metastable temperature ranges.

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.

Eradication of Potato Virus S, Potato Virus A, and Potato Virus M From Infected in vitro-Grown Potato Shoots Using in vitro Therapies.

Certain viruses dramatically affect yield and quality of potatoes and have proved difficult to eradicate with current approaches. Here, we describe a reliable and efficient virus eradication method that is high throughput and more efficacious at producing virus-free potato plants than current reported methods. Thermotherapy, chemotherapy, and cryotherapy treatments were tested alone and in combination for ability to eradicate single and mixed Potato virus S (PVS), Potato virus A (PVA), and Potato virus M (PVM) infections from three potato cultivars. Chemotherapy treatments were undertaken on in vitro shoot segments for four weeks in culture medium supplemented with 100 mg L-1 ribavirin. Thermotherapy on in vitro shoot segments was applied for two weeks at 40°C (day) and 28°C (night) with a 16 h photoperiod. Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture followed by exposure to PVS2 either without or with liquid nitrogen (LN, cryotherapy) treatment. The virus status of control and recovered plants following therapies was assessed in post-regeneration culture after 3 months and then retested in plants after they had been growing in a greenhouse for a further 3 months. Microtuber production was investigated using in vitro virus-free and virus-infected segments. We found that thermotherapy and cryotherapy (60 min PVS2 + LN) used alone were not effective in virus eradication, while chemotherapy was better but with variable efficacy (20-100%). The most effective result (70-100% virus eradication) was obtained by combining chemotherapy with cryotherapy, or by consecutive chemotherapy, combined chemotherapy and thermotherapy, then cryotherapy treatments irrespective of cultivar. Regrowth following the two best virus eradication treatments was similar ranging from 8.6 to 29% across the three cultivars. The importance of virus removal on yield was reflected in "Dunluce" free of PVS having higher numbers of microtubers and in "V500' free of PVS and PVA having a greater proportion of microtubers > 5 mm. Our improved procedure has potential for producing virus-free planting material for the potato industry. It could also underpin the global exchange of virus-free germplasm for conservation and breeding programs.

Impacts of Rapid Desiccation on Oxidative Status, Ultrastructure and Physiological Functions of Syzygium maire (Myrtaceae) Zygotic Embryos in Preparation for Cryopreservation.

Syzygium maire is a highly threatened Myrtaceae tree species endemic to New Zealand. Due to its recalcitrant seed storage behaviour, cryopreservation is the only viable long-term ex situ conservation option for this species. This study investigated viability, oxidative stress, thermal properties, and ultrastructure of zygotic embryo axes (EAs) desiccated to various moisture contents (MC). Fresh EAs had a MC of c. 1.9 g/g with 100% viability but rapid desiccation to MC < 0.3 g/g significantly reduced viability and decreased the activities of the enzymatic antioxidants superoxide dismutase, catalase and glutathione peroxidase, with a sevenfold increase in the production of protein carbonyls and lipid peroxides. Differential Scanning Calorimetry analysis showed no thermal events in EAs desiccated to a MC of <0.2 g/g, indicating that all freezable water had been removed, but this was lethal to both EAs and enzymatic antioxidants. The ultrastructure of desiccated EAs showed signs of plasmolysis, while fully hydrated EAs exposed to cryogenic temperature had ultrastructural disintegration and membrane damage. The decline in enzymatic antioxidant activities and the increase in lipid peroxidation suggest that S. maire EA viability loss is due to oxidative stress rather than structural impacts.

Volatile signature indicates viability of dormant orthodox seeds.

All seeds eventually die even under optimal storage conditions. The moment of viability loss is difficult to predict and detect. In order to differentiate between dead and viable dormant orthodox seeds, GC-MS analysis was used to non-invasively evaluate the volatile signature of seeds of Pyrus communis L. and Sorbus aucuparia L. Dormant seeds are capable of extended metabolic depression. However, their low metabolic rate remains largely unquantified, and there are no measurements of metabolites, i.e. volatile organic compounds (VOC) for physiologically dormant seeds during storage. Therefore, to address this issue, seeds were stored at a broad range of moisture content (MC) ranging from 2 to 30% under cryogenic (-196°C), cool (5°C) and elevated (40°C) temperatures. Volatile emission was highly dependent on seed MC and storage temperature and was higher under conditions associated with seed viability loss. However, changes in the emission of volatiles entrapped in seeds and released during 24 h after storage were detected for all conditions, providing insight into the processes occurring in dry dormant seeds. Among the 36 volatiles identified, three (acetaldehyde, ethanol, ethyl acetate) were highly correlated with seed germinability and show potential for the non-invasive screening of viability. Significantly, all three VOC are derived mostly from glycolysis and peroxidation and were detected even under very low moisture and temperature storage conditions. This is the first study to report on VOC accumulation and emission from physiologically dormant seeds and provide a broader view into their viability.

Resistance of New Zealand Provenance Leptospermum scoparium, Kunzea robusta, Kunzea linearis, and Metrosideros excelsa to Austropuccinia psidii.

Resistance to the pandemic strain of Austropuccinia psidii was identified in New Zealand provenance Leptospermum scoparium, Kunzea robusta, and K. linearis plants. Only 1 Metrosideros excelsa-resistant plant was found (of the 570 tested) and no resistant plants of either Lophomyrtus bullata or L. obcordata were found. Three types of resistance were identified in Leptospermum scoparium. The first two, a putative immune response and a hypersensitive response, are leaf resistance mechanisms found in other myrtaceous species while on the lateral and main stems a putative immune stem resistance was also observed. Both leaf and stem infection were found on K. robusta and K. linearis plants as well as branch tip dieback that developed on almost 50% of the plants. L. scoparium, K. robusta, and K. linearis are the first myrtaceous species where consistent infection of stems has been observed in artificial inoculation trials. This new finding and the first observation of significant branch tip dieback of plants of the two Kunzea spp. resulted in the development of two new myrtle rust disease severity assessment scales. Significant seed family and provenance effects were found in L. scoparium, K. robusta, and K. linearis: some families produced significantly more plants with leaf, stem, and (in Kunzea spp.) branch tip dieback resistance, and provenances provided different percentages of resistant families and plants. The distribution of the disease symptoms on plants from the same seed family, and between plants from different seed families, suggested that the leaf, stem, and branch tip dieback resistances were the result of independent disease resistance mechanisms.

Comparative Seed Morphology of Tropical and Temperate Orchid Species with Different Growth Habits.

Seed morphology underpins many critical biological and ecological processes, such as seed dormancy and germination, dispersal, and persistence. It is also a valuable taxonomic trait that can provide information about plant evolution and adaptations to different ecological niches. This study characterised and compared various seed morphological traits, i.e., seed and pod shape, seed colour and size, embryo size, and air volume for six orchid species; and explored whether taxonomy, biogeographical origin, or growth habit are important determinants of seed morphology. We investigated this on two tropical epiphytic orchid species from Indonesia (Dendrobium strebloceras and D. lineale), and four temperate species from New Zealand, terrestrial Gastrodia cunnninghamii, Pterostylis banksii and Thelymitra nervosa, and epiphytic D. cunninghamii. Our results show some similarities among related species in their pod shape and colour, and seed colouration. All the species studied have scobiform or fusiform seeds and prolate-spheroid embryos. Specifically, D. strebloceras, G. cunninghamii, and P. banksii have an elongated seed shape, while T. nervosa has truncated seeds. Interestingly, we observed high variability in the micro-morphological seed characteristics of these orchid species, unrelated to their taxonomy, biogeographical origin, or growth habit, suggesting different ecological adaptations possibly reflecting their modes of dispersal.

Lipid Thermal Fingerprints of Long-term Stored Seeds of Brassicaceae.

Thermal fingerprints for seeds of 20 crop wild relatives of Brassicaceae stored for 8 to 44 years at the Plant Germplasm Bank-Universidad Politécnica de Madrid and the Royal Botanic Gardens, Kew's Millennium Seed Bank-were generated using differential scanning calorimetry (DSC) and analyzed in relation to storage stability. Relatively poor storing oily seeds at -20 °C tended to have lipids with crystallization and melting transitions spread over a wide temperature range (c. 40 °C) that spanned the storage temperature, plus a melting end temperature of around 15 °C. We postulated that in dry storage, the variable longevity in Brassicaceae seeds could be associated with the presence of a metastable lipid phase at the temperature at which they are being stored. Consistent with that, when high-quality seed samples of various species were assessed after banking at -5 to -10 °C for c. 40 years, melting end temperatures were observed to be much lower (c. 0 to -30 °C) and multiple lipid phases did not occur at the storage temperature. We conclude that multiple features of the seed lipid thermal fingerprint could be used as biophysical markers to predict potential poor performance of oily seeds during long-term, decadal storage.

Plant species with extremely small populations (PSESP) in China: A seed and spore biology perspective.

Approximately one fifth of the world's plants are at risk of extinction. Of these, a significant number exist as populations of few individuals, with limited distribution ranges and under enormous pressure due to habitat destruction. In China, these most-at-risk species are described as 'plant species with extremely small populations' (PSESP). Implementing conservation action for such listed species is urgent. Storing seeds is one of the main means of ex situ conservation for flowering plants. Spore storage could provide a simple and economical method for fern ex situ conservation. Seed and spore germination in nature is a critical step in species regeneration and thus in situ conservation. But what is known about the seed and spore biology (storage and germination) of at-risk species? We have used China's PSESP (the first group listing) as a case study to understand the gaps in knowledge on propagule biology of threatened plant species. We found that whilst germination information is available for 28 species (23% of PSESP), storage characteristics are only known for 8% of PSESP (10 species). Moreover, we estimate that 60% of the listed species may require cryopreservation for long-term storage. We conclude that comparative biology studies are urgently needed on the world's most threatened taxa so that conservation action can progress beyond species listing.

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.

Post desiccation germination of mature seeds of tea (Camellia sinensis L.) can be enhanced by pro-oxidant treatment, but partial desiccation tolerance does not ensure survival at -20°C.

The maximal potential desiccation tolerance (MPDT) of tea (Camellia sinensis) seeds has been a matter of debate for decades. Here we assessed the ability of tea seeds from three sites in China to germinate after desiccation. Desiccation tolerance was greatest in Kunming, followed by Puer and Lincang, with Kunming seeds tolerating drying to 8% moisture content (MC), or ∼0.5 water activity (a(w)). Such tolerance was observed in Lincang seeds only when hydrogen peroxide (H2O2) at 0.5 or 1M was applied to seeds, indicating a stimulatory role for H2O2 in post-desiccation germination. Puer seeds exhibited MPDT of 16% MC (∼0.7 a(w)). Therefore, seeds from all three sites were not recalcitrant. The length of the dry season after dispersal and the high ratio of seed coat to seed mass (>0.3) support the observation of non-recalcitrant behaviour. The seeds were not immature, as the lipid signal in embryonic axes mirrored that of the cotyledons (30% oil). Even after high survival [>60% total germination (TG)] on drying to 10-13% MC, no Kunming seeds tolerated 1 month storage at -20 °C coinciding with lipid transitional changes at this temperature. The results indicate that tea seeds from China are neither recalcitrant nor storable at -20 °C.

Applications of differential scanning calorimetry in developing cryopreservation strategies for Parkia speciosa, a tropical tree producing recalcitrant seeds.

Shoot-tips of Parkia speciosa, a recalcitrant seed producing tropical leguminous tree withstood cryopreservation using encapsulation-vitrification in combination with trehalose preculture. Differential scanning calorimetry (DSC) revealed that trehalose moderated the thermal characteristics of the shoot-tips. A 30 min PVS2 treatment had the lowest glass transition temperature (Tg) (-50.2 +/- 1.1 degree C) when applied in combination with 5% (w/v) trehalose. The Tg increased to -40.2 +/- 1.0 degree C as the sugar concentration was decreased to 2.5 percent (w/v). Tg heat capacity for shoot-tips treated with 2.5 percent and 5 percent (w/v) trehalose and exposed to PVS2 for 30 min increased from 0.17 +/ 0.05 to 0.23 +/- 0.01 J per gram, respectively. Enthalpies of the melt-endotherm varied in proportion to trehalose concentration, for the 30 min PVS2 treatment, whereas the melt enthalpy for control shoots was greater than 150 J per gram and decreased to ca. 60 J per gram with 2.5 percent (w/v) trehalose. For 5 percent and 10 percent (w/v) trehalose treatments, enthalpy declined to ca. 24 and 12 J per gram respectively and freezing points were depressed to -75 degree C and -85 degree C with 2.5 percent and 5 percent trehalose (w/v), respectively. DSC elucidated the critical points at which vitrification occurred in germplasm exposed to trehalose and PVS2. A 60 min PVS2 treatment supporting ca. 70 percent survival was found optimal for stable glass formation during cooling and on rewarming.

Optimisation of the azinobis-3-ethyl-benzothiazoline-6-sulphonic acid radical scavenging assay for physiological studies of total antioxidant activity in woody plant germplasm.

A robust spectroscopic method for determining total antioxidant activity in aqueous extractions has been applied to tissues from diverse woody plant species, including seeds of Coffea arabica and in vitro shoots from Ribes nigrum, Picea sitchensis and Shorea leprosula. The assay involves scavenging of an ABTS [2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulphonic acid)] radical generated by the reaction of potassium persulphate with ABTS to produce an ABTS*(+) chromophore (lambda=734 nm). Antioxidants reduce ABTS*(+) back to ABTS with a concomitant decrease in absorbance. Aqueous extractions from C. arabica and S. leprosula had considerably higher (110-205 micromol Trolox eq. g(-1) FW) total antioxidant activities than P. sitchensis and R. nigrum (6-11 micromol Trolox eq. g(-1) FW). Further studies in two of these species showed that the inclusion of water-insoluble polyvinylpyrrolidone during aqueous tissue extraction enabled the combined phenolic and alkaloid antioxidant activity to be determined. These fractions accounted for 85% and 60% of total antioxidant activity for C. arabica seeds and R. nigrum shoots, respectively. The ABTS radical scavenging assay is presented herein as a robust method for determining total antioxidant activity in germplasm from diverse woody plant tissues and species. Its applicability to study oxidative stress in tissue cultures and germplasm employed in plant biotechnology, breeding and stress physiology programmes is discussed.