Effects of some cryopreservation procedures on recalcitrant zygotic embryos of Ammocharis coranica.

BACKGROUND: Cryopreservation, the most promising method for the long-term conservation of recalcitrant (desiccation-sensitive) seed germplasm, is often associated with high viability losses. Cryo-procedures involve a sequence of steps which must be optimised to reduce the impact of the stresses. OBJECTIVE: This study reports on the effects of some of the steps of cryopreservation on the recalcitrant zygotic embryos of the amaryllid, Ammocharis coranica. MATERIALS AND METHODS: Embryos were subjected to cryoprotection with glycerol and/or DMSO, rapid (flash) drying, and rapid (>100 degree C s(-1)) or slow (1 degree C s(-1)) cooling. RESULTS: Rapid dehydration (from c. 2.7 to 0.9 g g(-1) over 60 min) and cooling had a detrimental effect on the viability of the embryos, which was exacerbated when these steps were applied sequentially. After cooling, seedling production (30%) was obtained only from embryos that had been cryoprotected with glycerol prior to drying and rapid cooling, while 30% of non-treated embryos and 70% of those that had undergone cathodic protection during flash drying produced callus. CONCLUSION: Noting that no post-cryo survival of A. coranica embryos had previously been obtained, this study identified cryoprotection with glycerol and the incorporation of cathodic protection during flash drying as promising intervention points for future studies.

A novel means for cryopreservation of germplasm of the recalcitrant-seeded species, Ekebergia capensis.

Cryopreserved zygotic embryonic axes offer the best means of genetic diversity conservation of recalcitrant-seeded species, but frequently shoots fail to develop following processing for, and after, cryostorage. The present work offers a means to overcome this, by generating adventitious shoots from seedling roots produced after axis cryopreservation. Embryonic axes of Ekebergia capensis were exposed to cryoprotectants, flash dried, and rapidly cooled in nitrogen slush. Cryoprotection was an essential step, with both glycerol and DMSO permitting survival after cryogen exposure, but sucrose alone, or in combination with glycerol, was deleterious. Adventitious shoots were formed from seedling roots developed by axes germinated after cryogen exposure, after being subjected to intermittent flushing with a BAP-containing medium for 24 h in a temporary immersion system and subsequent culture on a semi-solid BAP-containing medium. After excision, a high proportion of the adventitious shoots produced roots in vitro, with most of these rooted plantlets being subsequently successfully acclimated.

Exploring the use of DMSO and ascorbic acid to promote shoot development by excised embryonic axes of recalcitrant seeds.

Seeds of Trichilia dregeana, T. emetica and Protorhus longifolia are recalcitrant (desiccation-sensitive), hence cryopreservation is the only ex situ means feasible for long-term conservation of these germplasm. For cryopreservation of these species, the excised embryonic axis is the explant of choice due to their small size and higher tolerance to desiccation. However, for many species with seeds having fleshy cotyledons, shoot development fails to occur after excision, which has been attributed to a reactive oxygen species (ROS) burst during excision wounding. This is a critical limiting step in developing cryopreservation protocols for such species. In embryos of T. dregeana, T. emetica and P. longifolia, the cotyledonary insertions are in close proximity to the shoot apical meristem and oxidative stress upon excision of the axis from cotyledons has been consistently associated with shoot tip necrosis, which precludes shoot development. This study tested the effects of dimethyl sulphoxide (DMSO) pre-culture prior to complete removal of the cotyledons, and post-excision soaking in DMSO or in the antioxidant, ascorbic acid, on shoot development by axes of T. dregeana and P. longifolia. These treatments had a significant (P < 0.05) positive effect on shoot production with a 6 h DMSO pre-culture combined with a DMSO post-excision soak being optimal for promoting shoot production in 70 percent of the axes of T. dregeana and 60 percent of those of P. longifolia. Embryonic axes of T. emetica responded best to a 6 h DMSO pre-culture alone, with 55 percent of axes producing shoots. It was further shown that two different post-harvest developmental stages of T. dregeana axes differed significantly initially (P < 0.05) in their response to DMSO and ascorbic acid treatments.

Viability of stored seed: extension by cathodic protection.

Placing seeds on a negatively charged conductor extended their viability during artificial aging. Such cathodic protection may reduce free radical attack by providing a source of electrons. The results stupport the hypothesis of free radical damage to cellular components and are consistent with such damage being important in deteriorative senescence changes.

Cryopreservation of shoot tips of Trichilia emetica, a tropical recalcitrant-seeded species.

This paper reports the successful cryopreservation of shoot tips of Trichilia emetica, a tropical tree species producing recalcitrant seeds. Preculture of shoot tips on MS medium with 0.7 M sucrose or with 0.3 M sucrose + 0.5 M glycerol followed by cryoprotection with a mixture of glycerol and DMSO or with PVS2 was crucial for successful recovery following cryostorage. Three cooling rates were applied to assess the effects on post-thaw regrowth of shoot tips. Slow cooling of the shoot tips (WC 1.24 g/g DW) precultured on medium with 0.3 M sucrose + 0.5 M glycerol and cryoprotected with PVS2 resulted in high shoot production (71 percent). Subsequent to relatively faster cooling, only 38 percent of the shoot tips developed shoots. Ultra-rapid cooling with PVS2 resulted in callus formation with 55 percent regrowth. We report one of the very few successful attempts to cryopreserve explants alternative to zygotic axes of tropical tree species producing recalcitrant seeds.

Sucrose treatment and explant water content: critical factors to consider in development of successful cryopreservation protocols for shoot tip explants of the tropical species Dioscorea rotundata (YAM).

A study was conducted to determine the optimum methods for conditioning explants to be used in the development of a simple protocol for long-term conservation of the germplasm of Dioscorea rotundata via cryopreservation. Shoot tips from cultures maintained in vitro were exposed to high concentrations of sucrose prior to silica gel-based dehydration and vitrification solution-based cryopreservation protocols. Explant water contents were determined, and ultrastructural studies were also carried out. Initially, culturing explants on medium supplemented with 0.3 M sucrose for 3-5 d considerably reduced tissue water content from about 12.2 g/g dry mass to between 4.8 and 5.5 g/g dry mass before cryoprotection with modified PVS2 (MPVS2) or silica gel dehydration. Ultrastructural studies indicated that cells had deposits of starch in plastids following sucrose treatments. Survival for D. rotundata shoot tips treated with MPVS2 vitrification solution, unloaded with 1.0 M sucrose medium and cooled to -7 degree C, was 16 percent for 15 min treatment and 44 percent for 40 min. After the 40 min MPVS2 treatment the TTZ test indicated 88 percent viability retention of explants cooled to -70 degree C, and 44 percent at -196 degree C. Plantlet development was obtained for -70 degree C-cooled shoot tips, whereas only callus development occurred from tissues exposed to liquid nitrogen. Explant regeneration was not obtained with air-dehydration techniques. It was concluded that vitrification-solution based cryopreservation presently offers the best option for conservation of this Dioscorea species.

Aflatoxin, kwashiorkor, and morbidity.

Children suffering from kwashiorkor could be grouped as those in whom aflatoxin was detectable in both serum and urine, and those in whom this mycotoxin was undetectable. Examination of the clinical records of the aflatoxin-positive and -negative children (58% and 42% of the sample, respectively) showed several other differences between the two groups. Compared with the aflatoxin-negative group, the children scored as aflatoxin-positive showed a significantly lower haemoglobin level (P = 0.02), a longer duration of oedema (P = 0.057), an increased number of infections (P = 0.037), and a longer duration of hospital stay (P = 0.008). The present findings suggest that the consumption of a staple food such as maize that is contaminated with the fungus Aspergillus flavus exposes susceptible kwashiorkor children to the metabolic hazards of aflatoxins, resulting in a greater risk of frequent infections.

In vitro studies on the potential for biological control of Aspergillus flavus and Fusarium moniliforme by Trichoderma species. A study of the production of extracellular metabolites by Trichoderma species.

Culture filtrates of Trichoderma viride and Trichoderma harzianum were inhibitory of Fusarium moniliforme and, to a lesser extent, Aspergillus flavus. The degree of inhibition was, however, dependent on the carbon or nitrogen source incorporated into the medium. Scanning electron microscopy revealed the development of abnormal fruiting structures on exposure to some Trichoderma culture filtrate, while macroscopically, growth restriction and, in the case of A. flavus, altered colony colouration were observed. Based on the results of inverted colony culture, it would appear that some isolates of Trichoderma produce inhibitory volatile compounds. The production of possible antibiotics was also demonstrated. The aggressive behaviour (towards A. flavus and F. moniliforme) demonstrated by Trichoderma spp. may be partly explained by the liberation of extracellular enzymes by these fungi. An isolate of T. viride exhibited amylolytic, pectinolytic, proteolytic and cellulolytic activity. Based on the results of the present investigation, Trichoderma spp. are potential candidates for biocontrol of some mycotoxin-producing fungi, but there exists some doubt as to their osmotolerance within the air-dry seed.

In vitro studies on the potential for biological control of Aspergillus flavus and Fusarium moniliforme by Trichoderma species. 1. Macroscopical and microscopical observations of fungal interactions.

The possible biological control of the seed-associated fungi, Aspergillus flavus and Fusarium moniliforme by Trichoderma species was investigated. A study of the fungal growth in dual cultures revealed that four of nine isolates of two Trichoderma spp. (T. harzianum and T. viride) inhibited fungal growth. A scanning electron microscopical investigation of fungal interactions demonstrated no obvious hyphae penetration of A. flavus or F. moniliforme by Trichoderma spp. Morphological alteration of Fusarium hyphae with pronounced collapse and the production of aberrant conidial heads and microheads by A. flavus were observed. The results suggest that mycoparasitism is not the mechanism involved in the inhibitory interaction of either A. flavus or F. moniliforme with Trichoderma spp.

In defence of aldehyde-osmium fixation and critical-point drying for characterization of seed-storage fungi by scanning electron microscopy.

Low-temperature scanning electron microscopy cannot be used as a general approach when resolution of definitive surface features is required for fungal characterization, because of the production of a pervading exudate by some of the species, strains or varieties. As the resolution of the light microscope is obviously limiting, scanning electron microscopy remains the best microscopical mode. Results are presented for four species within the Aspergillus flavus group which indicate that minimal (if any) distortion or collapse can be achieved with concomitant preservation of surface morphology, when particular precautions are taken during fixation and critical-point drying. Details of the steps pertaining to preparation for scanning electron microscopy following critical-point drying that are considered collectively to contribute to the success of the approach are discussed.

Germination-associated events and the desiccation sensitivity of recalcitrant seeds - a study on three unrelated species.

The storage behaviour of recalcitrant seeds was assessed using three diverse species: a gymnosperm, Araucaria angustifolia (Bert.) O. Kuntze; a herbaceous monocotyledon, Scadoxus membranaceus (Bak.) Friis Nordal; and a woody dicotyledon, Landolphia kirkii Dyer. Seeds were stored under conditions of high relative humidities that maintained seed moisture content and under low relative humidities that caused drying. At regular intervals moisture content was determined, germinability assessed and the ultrastructure of radicle meristem cells examined. Under storage at high relative humidity, seed moisture content was maintained at the original level and subcellular germination events were initiated in the short-term. Such seeds showed enhanced rates of germination when removed from storage and planted. Long-term storage under these conditions resulted in the initiation of subcellular damage which intensified with time and ultimately resulted in the loss of viability. The rate at which germination events proceeded varied among the three species, and could be directly correlated with the period of viability retention under humid storage conditions. Storage under desiccating conditions resulted in subcellular damage and rapid loss of viability. The rate at which the seeds dried varied among the three species. The proportion of water loss tolerated by the different species before loss of viability, correlated with the rate of drying. The storage behaviour of the seeds of these three species is discussed in terms of a previously described model.

Homoiohydrous (recalcitrant) seeds: Developmental status, desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer.

The desiccation sensitivity in relation to the stage of development was investigated in embryonic axes from the homoiohydrous (recalcitrant) seeds of Landolphia kirkii. Electrolyte leakage, used to assess membrane damage after flash (very rapid) drying, indicated that axes from immature (non-germinable) seeds were the most desiccation-tolerant, followed by those from mature seeds, while axes from seeds germinated for increasing times were progressively more desiccation-sensitive. Differential scanning calorimetry was used to study the relationship between desiccation sensitivity and the properties of water in the tissues. Axes from immature seeds had a lower content of non-freezable water than that of any other developmental stage and a higher enthalpy of melting of freezable water. For mature and immature axes electrolyte leakage increased at the point of loss of freezable water. At other developmental stages the water content at which electrolyte leakage increased markedly correlated with the other properties of the water, such as the change in the shape of the melting endotherm and the onset temperature. Ultrastructural studies of axes at the various developmental stages showed changes in the degree and pattern of vacuolation, the presence and quantities of lipid and starch, and the degree of endomembrane development. The results are discussed in relation to current hypotheses on the basis of desiccation tolerance.

Homeohydrous (Recalcitrant) Seeds: Dehydration, the State of Water and Viability Characteristics in Landolphia kirkii.

Differential scanning calorimetry was used to study the relationships among drying rate, desiccation sensitivity, and the properties of water in homeohydrous (recalcitrant) seeds of Landolphia kirkii. Slow drying of intact seeds to axis moisture contents of approximately 0.9 to 0.7 gram/gram caused lethal damage, whereas very rapid (flash) drying of excised embryonic axes permitted removal of water to approximately 0.3 gram/gram. The amount of nonfreezable water in embryonic axes (0.28 gram H(2)O/gram dry mass) did not change with drying rate and was similar to that of desiccation-tolerant seeds. These results suggest that the amount of nonfreezable water per se is not an important factor in desiccation sensitivity. However, flash drying that removed all freezable water damaged embryonic axes. Differences between desiccation-sensitive and -tolerant seeds occur at two levels: (a) tolerant seeds naturally lose freezable water, and sensitive seeds can lose this water without obvious damage only if it is removed very rapidly; (b) tolerant seeds can withstand the loss of a substantial proportion of nonfreezable water, whereas sensitive seeds are damaged if nonfreezable water is removed.

Aflatoxins and kwashiorkor in Durban, South Africa.

The present investigation has indicated that maize seeds stored under various simulated seasonal conditions show a spectrum of fungi that appear as a succession. The aflatoxin-producing fungus, Aspergillus flavus, is favoured by storage conditions of high temperature and humidity (summer and autumn seasons). This coincides with the more frequent admission of children suffering from kwashiorkor at King Edward VIII Hospital in Durban. Aflatoxin analysis was undertaken on 74 children diagnosed at King Edward VIII Hospital in Durban as cases of kwashiorkor, marasmus or underweight (Wellcome classification). The control group consisted of 35 age-matched patients with no symptoms of protein energy malnutrition. Aflatoxins were detected in serum and/or urine from all groups, including the controls. The serum/urine ratio was significantly higher in the kwashiorkor group than in the other groups. The control group, however, had a higher proportion of urine aflatoxins than the kwashiorkor group. These findings were interpreted in terms of impaired liver function in kwashiorkor. Aflatoxins may have a rôle in the pathogenesis of kwashiorkor, although the present findings do not indicate that they are a causal factor.

Ultrastructure of the salt glands of the mangrove, Avicennia marina (Forssk.) Vierh., as indicated by the use of selective membrane staining.

Each salt-excreting gland of the mangrove Avicennia marina (Forsskål) Vierh. consists of two to four collecting cells, one stalk cell, and eight to twelve excretory cells. Differential membrane staining by zinc iodide-osmium tetroxide (as a post-fixative) or phosphotungstic acid (as a section-stain) was used to characterise the ultrastructure of the glands. A large amount of tubular endoplasmic reticulum was found in the stalk and excretory cells of the gland, but not in the collecting cells. The ultrastructural arrangement of the endoplasmic reticulum indicates that salt is loaded from the apoplasm into the endoplasmic reticulum of the symplasm at the base of the stalk cell, traverses both cell types in the endoplasmic reticulum, and is excreted at the outer edge of the gland by an eccrine-type mechanism. Increasing development of the tubular endoplasmic reticulum accompanied differentiation of the gland cells.

Interactions among water content, rapid (nonequilibrium) cooling to -196 degrees C, and survival of embryonic axes of Aesculus hippocastanum L. seeds.

This study investigated the interactions among water content, rapid (nonequilibrium) cooling to -196 degrees C using isopentane or subcooled nitrogen, and survival of embryonic axes of Aesculus hippocastanum. Average cooling rates in either cryogen did not exceed 60 degrees C s(-1) for axes containing more than 1.0 g H(2)O g(-1)dw (g g(-1)). Partial dehydration below 0.5 g gg(-1) facilitated faster cooling, averaging about 200 and 580 degrees C s(-1) in subcooled nitrogen and isopentane, respectively. The combination of partial drying and rapid cooling led to increased survival and reduced cellular damage in axes. Electrolyte leakage was 10-fold higher from fully hydrated axes cooled in either cryogen than from control axes that were not cooled. Drying of axes to 0.5 g g(-1), reduced electrolyte leakage of cryopreserved axes to levels similar to those of control material. Axis survival was assayed by germination in vitro. Axes with water contents greater than 1.0 g g(-1), did not survive cryogenic cooling. Between 1.0 and 0.75 g g(-1), axes survived cryogenic exposure but developed abnormally. The proportion of axes developing normally after being cooled in isopentane increased with increasing dehydration below 0.75 g g(-1), reaching a maximum between 0.5 and 0.25 g g(-1) after being cooled at > or =300 degrees C s(-1). Cooling rates attained in subcooled nitrogen did not exceed 250 degrees C s(-1), and normal development of axes was observed only at < or =0.4 g g(-1). These results support the hypothesis that rapid cooling enhances the feasibility of cryopreservation of desiccation-sensitive embryonic axes by increasing the upper limit of allowable water contents and overall survival.

Aflatoxin B1--its effects on an in vitro plant system.

The phytotoxic effects of aflatoxin B1 (AFB1) on in vitro cultures of differentiating calli and regenerating plantlets of Nicotiana tabacum were assessed. Callus appeared more sensitive to the effects of AFB1, with fresh mass accumulation and callus chlorophyll levels affected at low (approximately 0.5 micrograms/ml) aflatoxin concentrations. Transmission electron microscopy revealed early deteriorative alterations in chloroplast morphology. Inhibitory effects of the toxin (up to and including 10 micrograms/ml) on callus fresh mass accumulation were reversed following a 3 week toxin-free recovery period. In tobacco plantlets, root and leaf development, and root and leaf mass were significantly inhibited in a dose-dependent fashion with increasing AFB1 concentration above 0.5 micrograms/ml. Inhibitory effects on plantlet root development were more pronounced that on leaf development.