Studies on factors influencing viability after cryopreservation of excised zygotic embryos from recalcitrant seeds of two amaryllid species.

Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2010.Recalcitrant unlike orthodox seeds do not show a sharp border between maturation and germination and remain highly hydrated and desiccation-sensitive at all developmental and post-harvest stages. In contrast with recalcitrant seeds, orthodox types retain viability for predictably long periods in the dry state and hence can be stored under low relative humidity and temperature conditions. Storage of recalcitrant seeds under conditions allowing little to no water loss, at moderate temperatures, allows for short- to medium-term storage but only facilitates viability retention for a matter of a few weeks to months, at best, because the seeds are metabolically active and initiate germination while stored. Cryopreservation, i.e. storage at ultra-low temperatures (usually in liquid nitrogen [LN] at -196°C), is a promising option for the long-term germplasm conservation of recalcitrant-seeded species but their seeds present some unavoidable difficulties in terms of the amenability of their germplasm to cryopreservation. Pre-conditioning treatments can reduce the amount of ‘free’ water available for freezing and may increase the chances of cells or tissues surviving exposure to cryogenic temperatures. Such conditioning may be imposed by physical dehydration or cryoprotection, i.e. exposure to compounds that depress the kinetic freezing point of water and so reduce the likelihood of lethal ice-crystal formation during cooling (i.e. exposure to LN at -196°C or sub-cooled LN at -210°C) and subsequent thawing. Partial dehydration is presently a standard pre-treatment for the cryopreservation of recalcitrant zygotic germplasm and explant cryoprotection has been shown to improve postthaw survival in some recalcitrant-seeded species. However, there is a paucity of information on the physiological and biochemical basis of post-thaw survival or death in recalcitrant seeds, and this is the major focus of the current contribution. Additionally, in light of the lack of understanding on how cryo-related stresses imposed at the embryonic stage are translated or manifested during subsequent seedling growth, this study also investigated the effects of partial dehydration and the combination of partial dehydration and cooling of recalcitrant zygotic embryos on subsequent in and ex vitro seedling vigour. All studies were undertaken on the zygotic embryos of two recalcitrant-seeded members of the Amaryllidaceae, viz. Amaryllis belladonna (L.) and Haemanthus montanus (Baker); both of which are indigenous to South Africa. Studies described in Chapter 2 aimed to interpret the interactive effects of partial dehydration (rapidly to water contents > and <0.4 g g-1), cryoprotection (with sucrose [Suc; nonpenetrative] or glycerol [Gly; penetrative]) and cooling rate (rapid and slow) on subsequent zygotic embryo vigour and viability, using three stress markers: electrolyte leakage (an indicator of membrane integrity); spectrophotometric assessment of tetrazolium chloride-reduction (an indicator of respiratory competence); and rate of protein synthesis (an indicator of biochemical competence). These studies showed that in recalcitrant A. belladonna and H. montanus zygotic embryos, stresses and lesions, metabolic and physical, induced at each stage of the cryopreservation protocol appear to be compounded, thus pre-disposing the tissues to further damage and/or viability loss with the progression of each step. Maximum post-thaw viability retention in both species appeared to be based on the balance between desiccation damage and freezing stress, and the mitigation of both of these via Gly cryoprotection. Post-thaw viabilities in both species were best when Gly cryoprotected + partially dried zygotic embryos were rapidly, as opposed to slowly, cooled. However, the rate at which water could be removed during rapid drying was higher in A. belladonna and this may explain why the optimum water content range for post-thaw survival was 0.40 g g-¹ for H. montanus. These results suggest that to optimise cryopreservation protocols for recalcitrant zygotic germplasm, attention must be paid to pre-cooling dehydration stress, which appears to be the product of both the ‘intensity’ and ‘duration’ of the stress. Cryoprotection and dehydration increased the chances of post-thaw survival in A. belladonna and H. montanus zygotic embryos. However, transmission electron microscopy studies on the root meristematic cells from the radicals of these embryos (described in Chapter 3) suggest that their practical benefits appear to have been realised only when damage to the sub-cellular matrix was minimised: when (a) pre-conditioning involved the combination of cryoprotection and partial dehydration; (b) the cryoprotectant was penetrating (Gly) as opposed to non-penetrating (Suc); and (c) embryos were rapidly cooled at water contents that minimised both dehydration and freezing damage. The ability of A. belladonna and H. montanus embryos to tolerate the various components of cryopreservation in relation to changes in extracellular superoxide (.O2 -) production and lipid peroxidation (a popular ‘marker’ for oxidative stress) was investigated in studies featured in Chapter 4. Pre-conditioning and freeze-thawing led to an increase in oxidative stress and the accompanying decline in viability suggests that oxidative stress was a major component of cryoinjury in the embryos presently investigated. Post-thaw viability retention in Gly cryoprotected + partially dried embryos was significantly higher than noncryoprotected + partially dried embryos, possibly due to the relatively lower post-drying lipid peroxidation levels and relatively higher post-drying and post-thawing enzymic antioxidant activities in the former. Exposure of certain plant tissues to low levels of oxidative or osmotic stress can improve their tolerance to a wide range of stresses. In contrast, exposure of H. montanus zygotic embryos to low levels of oxidative stress provoked by exogenously applied hydrogen peroxide (H2O2) or exposure of A. belladonna embryos to low levels of osmotic stress provoked by low water potential mannitol and polyethylene glycol solutions (in studies featured in Chapter 5) increased their sensitivity to subsequent dehydration and freeze-thaw stresses. Exposure of Gly cryoprotected and non-cryoprotected amaryllid embryos to such stress acclimation treatments may pre-dispose A. belladonna and H. montanus embryos to greater post-drying and post-thaw total antioxidant and viability loss than untreated embryos. To assess the vigour of seedlings recovered from partially dried H. montanus embryos, seedlings recovered from fresh (F) and partially dried (D) embryos in vitro were hardened-off ex vitro, and subsequently subjected to either 42 days of watering or 42 days of water deficit (in studies described in Chapter 6). In a subsequent study (described in Chapter 7), seedlings recovered from fresh (F), partially dried (D) and cryopreserved (C) A. belladonna embryos were regenerated in vitro, hardened-off ex vitro and then exposed to 12 days of watering (W) or 8 days of water stress (S) followed by 3 days of re-watering. Results of these studies suggest that the metabolic and ultrastructural lesions inflicted on A. belladonna and H. montanus zygotic embryos during cryopreservation may compromise the vigour (e.g. development of persistent low leaf water and pressure potentials and reduced photosynthetic rates) and drought tolerance of recovered seedlings, compared with seedlings recovered from fresh embryos. While the adverse effects of freeze-thawing were carried through to the early ex vitro stage, certain adverse effects of partial drying were reversed during ex vitro growth (e.g. the increased relative growth rate of seedlings from partially dried embryos). The reduced vigour and drought tolerance of seedlings recovered from partially dried and cryopreserved embryos in the present work may therefore disappear with an extension in the period afforded to them for hardening-off under green-house conditions, and in the field. The results presented in this thesis reinforce the notion that each successive manipulation involved in the cryopreservation of recalcitrant zygotic germplasm has the potential to inflict damage on tissues and post-thaw survival in such germplasm relies on the minimisation of structural and metabolic damage at each of the procedural steps involved in their cryopreservation. The results also highlight the need to design research programmes aimed not only at developing protocols for cryopreservation of plant genetic resources, but also at elucidating and understanding the fundamental basis of both successes and failures

Effects of greywater irrigation on germination, growth and photosynthetic characteristics in selected African leafy vegetables

The reuse of greywater, wastewater from sources other than toilets, could enable low-income  households to save potable water for drinking and cooking. Greywater irrigation of food crops is widely practised but its effects on African leafy vegetables (ALVs), which hold potential for cultivation to improve food security, are unknown. This study investigated the effects of synthetic greywater  irrigation on germination in three ALVs, viz., Amaranthus dubius, Cleome gynandra and Solanum nigrum, and subsequent seedling growth in A. dubius and S. nigrum. Seeds and seedlings were treated with chlorinated and dechlorinated greywater and tap water, supplemented with nutrients. Greywater application decreased germination capacity (by 23–25%) when assessed in Petri dishes in A. dubius only. However, greywater application was less harmful to A. dubius seeds sown in soil. Vigour was compromised in greywater-treated seeds of all three species but greywater can be used to irrigate freshly-sown seeds of A. dubius without reducing percentage seedling production. However, greywater irrigation reduced capacity (by 21–23%) and rate of shoot emergence in S. nigrum, and growth and chlorophyll content in both species. These negative effects were accompanied by increased soil  electrical conductivity (after 21 d) and pH (after 14 d). The reduced growth under greywater irrigation was most likely based on a reduction in light-harvesting capacity and/or nutrient availability. Overall, S. nigrum seedlings were significantly more sensitive to the negative effects of greywater, possibly due to increased transpirational water loss under greywater irrigation. The effects of greywater were largely independent of chlorine content. Applying greywater in excess of plant requirements and/or alternating greywater irrigation events with freshwater watering events could promote leaching of salts found in greywater. The effects of greywater irrigation on soil water and nutrient availability demand further investigation for ALVs.Keywords: African leafy vegetables, greywater irrigation, germination, seedling growt

Impacts of plastic debris on biota and implications for human health: A South African perspective

Entanglement and ingestion of plastics are the main ecological impacts of marine plastic debris on marine biota, but indirect effects such as the transport of alien species and benthic smothering are also important to note. Entanglement of invertebrates, sharks, turtles, birds and marine mammals is mainly caused by macroplastics (>5 mm), and leads to reduced mobility, ineffective foraging and subsequent mortality. The main plastic types associated with entanglement are improperly discarded fishing nets, lines, ropes and straps

A leadership model validation: Dimensions influential to innovation

Background: Current pressures for globalisation force organisations to explore, create and implement new ideas in order to remain competitive. This necessitates the need to utilise innovation to diversify products and services, introduce new technology, establish new managerial and administrative practices, and initiate transformation in other areas of the organisation. Objectives: This article explored the relationship between the latent variables, namely, rewards, resources, leadership vision and innovation, as postulated by De Jong and Den Hartog’s leadership model for stimulating innovation. Method: The research approach chosen to investigate the research questions was an ex post facto, cross-sectional field survey. Secondary data from a reputable financial institution with extensive business in three African countries (Botswana, Lesotho and Namibia) were used as the data were collected by the institution and made available to the researcher for further analysis. A complete sample size of N = 584 was obtained across the three countries. Confirmatory factor analysis was initially used to provide a confirmatory test of the measurement theory followed by structural equation modelling which allowed to test for regression amongst the latent variables (rewards, resources, leadership vision and innovation). Results: Structural equation modelling revealed that only leadership vision and resources were found to be statistically significant; rewards showed a negative relationship (r = −0.02) with innovation. Resources made the greater contribution (r = 0.75) to innovation, compared to leadership vision (r = 0.28). Conclusion: The study empirically validate and support the assertion of De Jong and Den Hartog (2007) that the latent variables resources and leadership vision positively correlate with innovation in the context of the financial services industry

Quantification and characterisation of microplastics ingested by selected juvenile fish species associated with mangroves in KwaZulu- Natal, South Africa

Though the number studies on microplastic ingestion by fish is growing, data on fish species charac- teristic of the South African coastline are scarce. This study quantified and characterised (physically and chemically) microplastics ingested by four species of juvenile fish (viz. Oreochromis mossambicus [Peters, 1852], Terapon jarbua [Forsskål, 1775], Ambassis dussumieri [Cuvier, 1828] and Mugil sp.), within four mangroves along the east coast of South Africa. Microplastics were isolated from whole fish using a proteinase K digestion method, and then quantified and characterised in terms of shape, chemical nature (plastic type), colour and length. Fibres (68%) and fragments (21%) were the dominant shapes found. Of the 174 fish sampled, 52% contained microplastic particles, with 0.79 ± 1.00 particles per fish. The average number of particles per fish did not differ significantly across species within sites and across sites but was higher than in juvenile fish of other species sampled in oceanic habitats. The main plastic types collected using 10 mm filters and identified with Fourier Transform Infrared Spectroscopy (FTIR), were rayon (70.4%), polyester (10.4%), nylon (5.2%) and polyvinylchloride (3.0%). Particle length ranged from 0.1 to 4.8 mm, averaging 0.89 ± 0.77 mm, but irrespective of length, particles were mostly blue in colour. This study provides evidence that juvenile fish inhabiting mangroves are consuming significant quan- tities of microplastics. Importantly, it should be noted that rayon, though the most abundant plastic type found, is a semi-synthetic fibre made from regenerated cellulose that is commonly reported in studies of this nature. The habitats studied serve as nurseries for numerous fish species; however, more detailed studies are needed to assess whether microplastic ingestion could compromise the health of these fish or whether these effects are dependent on species, feeding habit and/or plastic type

Effects of inorganic salt solutions on vigour, viability, oxidative metabolism and germination enzymes in aged cabbage and lettuce seeds

This study assessed the potential of pre-hydration treatment with aqueous solutions (electrolysed [cathodic water; CW] and non-electrolysed) prepared from four different inorganic ion combinations: 1 mM CaCl2, 1 m CaCl2 and 1 mM MgCl2 (CaMg, hereafter), 1 mM MgCl2 and 1 mM NaCl to invigorate controlled deteriorated (CDd) Brassica oleracea (cabbage) and Lactuca sativa (lettuce) seeds by assessing germination, vigour and biochemical markers (electrolyte leakage, lipid peroxidation products, protein carbonylation, and defence and germination associated enzymes) of oxidative stress. Additionally, the possible e ects of pH of electrolysed CaMg and NaCl solutions were assessed. The inorganic salt solutions were applied to fresh seeds and seeds deteriorated to 75% viability (P75), 50% viability (P50) and 25% viability (P25); deionised water served as control. The pre-hydration treatment did not enhance normal seedling production in cabbage. However, Ca-containing and CW hydration treatments (CaCl2 CW, CaMg and CaMg CW [6.5], MgCl2 CW, NaCl CW and NaCl CW [6.5]) promoted normal seedling production of CDd lettuce seeds, while seedling vigour was enhanced by CaMg, CaMg CW (6.5), NaCl CW and NaCl CW (6.5) in CDd cabbage seeds, and CaCl2, CaCl2 CW, CaMg, CaMgCW(6.5), MgCl2 CW, NaClCWand NaClCW(6.5) in CDd lettuce seeds. The supplementation of Ca, a component of the ionised solutes, and/or the reducing potential of CW contributed to increased normal seedling production in lettuce seeds irrespective of the pH of treatment solutions or degree of deterioration. Overall, the pre-hydration treatments enhanced endogenous antioxidants leading to reduced levels of electrolyte leakage, lipid peroxidation, protein carbonylation, and enhanced germination enzyme activities in lettuce seeds. The study concluded that pre-hydration with selected inorganic salt solutions can invigorate debilitated lettuce seeds

Marine seismic surveys for hydrocarbon exploration: What’s at stake?

With a coastline extending approximately 3900 km, South Africa exercises jurisdiction over a vast Exclusive Economic Zone (EEZ) that exceeds 1.5 million km2 . 1 South Africa is located at an ecologically important crossroad for inter-ocean exchange of heat, salt and biota2 involving the warm, fast-flowing Agulhas current of the Indian Ocean and the cold, nutrient-rich Benguela upwellings of the Atlantic Ocean, and within the range of influence of the world’s most biologically productive ocean3 , the Southern Ocean4-6. South Africa’s marine territory is also characterised by spectacular topography, including dramatic canyons, slopes, plateaus, and seamounts.7 Unsurprisingly, South Africa’s complex oceanographic influences, coastal topography, and geology boasts 179 marine ecosystem types, with 150 around South Africa and 29 in the country’s sub-Antarctic territory.8 South Africa may also be richly endowed with hydrocarbon deposits.9,10 Seismic surveys are a routine and key upstream component of the hydrocarbon sector and crucial to understanding where recoverable oil and gas resources likely exist. Hydrocarbon extraction is largely dependent on seismic data acquisition and processing technology, with exploration companies relying on seismic survey results to decide whether or where to extract hydrocarbon deposits. While seismic surveys pose an immediate threat to South Africa’s exceptionally rich marine life, the downstream implications of such surveys – the extraction and use of non-renewable energy sources – are more profound

Bromelain crude extract to improve the emergence and growth of pepper seedlings

Pepper (Capsicum annuum L.) is one of the most consumed horticultural products worldwide. However, pepper seeds have slow and asynchronous germination. Organic agriculture encourages using natural substances for the pre-germinative treatment of seeds. The objective of the present work was to evaluate the effect of priming sweet pepper seeds with bromelain crude extract (obtained from pineapple stem residues) on subsequent seedling emergence and growth. The effects of the proteolytic activity of the extract (0, 3.16, 6.25, 12.25, and 25.0 totalU), imbibition time (0, 3, 6, 9, 12, and 15 h), and imbibition temperature (15, 25, 35 and 45 ℃) on seedling emergence and growth were assessed. The significantly best emergence, based on a range of indicators (emergence percentage: E, time to 50% emergence: TE50, emergence index: EI, mean emergence rate: MER, mean emergence time: MET, and synchronization index: SI), was obtained when the seeds were treated for three h at 35°C with a 6.25 totalU bromelain crude extract. Seeds subjected to this treatment combination also yielded the most vigorous seedlings (number of true leaves, length and thickness of the stems, fresh mass, dry mass, leaf area) . In conclusion, pre-germinative treatment with bromelain crude extract of 6.25 totalU proteolytic activity for three h at 35°C improved seedling emergence and growth in sweet pepper seedlings relative to untreated seed. This result offers an organic priming alternative for seeds of sweet pepper

A critical comparative review of evidence on alternative instruments for supporting research and innovation in agri-food systems in the Global South

Financial support is a critical enabling factor for healthy agri-food innovation systems, particularly within resource-limited settings, though additional forms of support are also necessary. This motivated a critical comparative review of evidence in peer-reviewed and gray literature on the range of instruments that support innovation in agri-food systems in the Global South, toward achieving sustainable agriculture intensification. The main aim is to provide recommendations to innovation managers on the choice of different instruments for supporting innovation. The key guiding questions for the comparative analysis were whether the instrument fosters uptake of innovation and whether it promotes inclusive development. A review of the literature was supplemented with a scan of websites for sources of peer-reviewed and gray literature documenting the application of the 12 selected instruments. The study revealed three categories of instruments: (Type A) those that support entrepreneurship; (Type B) those that primarily finance innovation; and (Type C) those that support innovation in real-life contexts. Our analysis indicates that innovation managers and funders need to select instruments that are likely to fit the specific context as well as to address the mandates of their organizations, and in so doing, they must consider how to ensure the sustainability of their investments and meet the needs of their beneficiaries. This review represents one of a handful that have compared the use of multiple instruments across multiple continents in the Global South, and can serve as an important decision-making tool for investors and funders looking to invest in agri-food innovation systems

Yeast supplementation alleviates the negative effects of greywater irrigation on lettuce and maize

Water scarcity has led to increased use of wastewater, particularly greywater, for crop irrigation. This study investigated whether the addition of yeast can alleviate the potential negative effects of greywater use on lettuce (Lactuca sativa L.) and maize (Zea mays L.). Seeds and seedlings were treated with 4 concentrations (0.005; 0.01; 0.015 and 0.020 g‧mL−1) of yeast-treated tapwater (YTW) and greywater (YGW). Tapwater (TW) and greywater (GW) without yeast served as controls. In general, an increase in yeast concentration compromised seed germination in Petri dishes, but improved germination in soil. Tapwater was more effective than GW in promoting germination and growth in both species. Lower concentrations of yeast generally increased germination capacity in both species compared to the controls. Total biomass, number of leaves, chlorophyll content, leaf area, photosynthetic rate and maximum quantum yield of photosystem II (Fv/Fm) were significantly higher in yeast treatments in both species, compared with the controls. Biomass accumulation, total leaf area, chlorophyll content and photosynthesis were higher in YGW than controls and YTW. Differences in biomass allocation between treatments may be due to changes in soil moisture, pH and electrical conductivity of the soil caused by yeast supplementation. This study showed that plants treated with YGW performed better than those treated with YTW and without yeast. Yeast supplementation of greywater could increase water recycling and provide a cheap bio-fertilizer to home growers, whilst significantly improving yield in both species. This innovative approach may enhance water and food security of subsistence farmers in rural areas