Equilibrium freezing of leaf water and extracellular ice formation in Afroalpine ‘giant rosette’ plants

The water potentials of frozen leaves of Afroalpine plants were measured psychrometrically in the field. Comparison of these potentials with the osmotic potentials of an expressed cellular sap and the water potentials of ice indicated almost ideal freezing behaviour and suggested equilibrium freezing. On the basis of the osmotic potentials of expressed cellular sap, the fractions of frozen cellular water which correspond to the measured water potentials of the frozen leaves could be determined (e.g. 74% at -3.0° C). The freezing points of leaves were found to be in the range between 0° C and -0.5° C, rendering evidence for freezing of almost pure water and thus confirming the conclusions drawn from the water-potential measurements. The leaves proved to be frost resistant down to temperatures between -5° C and -15° C, as depending on the species. They tolerated short supercooling periods which were necessary in order to start ice nucleation. Extracellular ice caps and ice crystals in the intercellular space were observed when cross sections of frozen leaves were investigated microscopically at subfreezing temperatures.Symbols T temperature - water potential Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Effect of ‘in air’ freezing on post-thaw recovery of Callithrix jacchus mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds

Through enabling an efficient supply of cells and tissues in the health sector on demand, cryopreservation is increasingly becoming one of the mainstream technologies in rapid translation and commercialization of regenerative medicine research. Cryopreservation of tissue-engineered constructs (TECs) is an emerging trend that requires the development of practically competitive biobanking technologies. In our previous studies, we demonstrated that conventional slow-freezing using dimethyl sulfoxide (Me₂SO) does not provide sufficient protection of mesenchymal stromal cells (MSCs) frozen in 3D collagen-hydroxyapatite scaffolds. After simple modifications to a cryopreservation protocol, we report on significantly improved cryopreservation of TECs.Porous 3D scaffolds were fabricated using freeze-drying of a mineralized collagen suspension and following chemical crosslinking. Amnion-derived MSCs from common marmoset monkey Callithrix jacchus were seeded onto scaffolds in static conditions. Cell-seeded scaffolds were subjected to 24 h pre-treatment with 100 mM sucrose and slow freezing in 10% Me₂SO/20% FBS alone or supplemented with 300 mM sucrose. Scaffolds were frozen ‘in air’ and thawed using a two-step procedure. Diverse analytical methods were used for the interpretation of cryopreservation outcome for both cell-seeded and cell-free scaffolds. In both groups, cells exhibited their typical shape and well-preserved cell-cell and cell-matrix contacts after thawing. Moreover, viability test 24 h post-thaw demonstrated that application of sucrose in the cryoprotective solution preserves a significantly greater portion of sucrose-pretreated cells (more than 80%) in comparison to Me₂SO alone (60%).No differences in overall protein structure and porosity of frozen scaffolds were revealed whereas their compressive stress was lower than in the control group. In conclusion, this approach holds promise for the cryopreservation of ‘ready-to-use’ TECs.     

Effect of 2,2′-bipyridyl on porphyrin synthesis in etiolated and light-treated barley leaves

The effects of 2,2′-bipyridyl on porphyrin formation differed in illuminated and dark-treated barley leaves. In the dark, bipyridyl treatment increased photoconvertible protochlorophyllide (Pchlide, P650) and decreased the protohaem content. The increase in Pchlide could not be wholly accounted for by a diversion of ‘substrate’ from protohaem synthesis. The rate of Pchlide regeneration was slightly higher in chelator treated leaves which suggests increased δ-aminolaevulinic acid (ALA) synthesis. Only small quantities of Mg-protoporphyrinmonomethylester (Mg-protoME) were detected in etiolated leaves treated with bipyridyl in the dark. Protochlorophyll (P630) synthesis from exogenously supplied ALA was lower in the chelator treatments. The results suggest that only when substantial quantities of ALA are being utilized in dark-grown leaves does a ‘metal’ become limiting in the bipyridyl treated leaves. In the light, bipyridyl inhibited chlorophyll synthesis, again suggesting that when substantial amounts of ALA were being utilized a ‘metal’ becomes rate limiting. Bipyridyl treatment also inhibited ALA production in light-treated leaves. The incorporation of glycine-[¹⁴C] into ALA in the presence of bipyridyl was severely restricted compared to the incorporation of glutamate-[¹⁴C]. The data suggest two pathways for ALA synthesis; the classical ALA-synthetase which utilizes glycine and is operative in dark-grown leaves and a second enzyme system, which uses glutamate, and is of quantitative importance in the light.     

‘Hertwig Effect’ in plants: induced parthenogenesis through the use of irradiated pollen

Summary Studies of four combinations of Nicotiana involving four species, N. langsdorffii (N. l.), N. alata (N. a.), N. glutinosa (N. g.) and N. tabacum (N. t.), have shown that parthenogenetic haploid and diploid maternal individuals may arise with the use of male gametes (pollen) treated with high doses of ionising radiation in plants, similar to that found in animals (Hertwig Effect). At lower doses (10–20 Kr) rapidly diminishing numbers of seedlings were produced and many of these died soon after germination or before reaching maturity. In the intraspecific combination N. l. × N. l., viable seeds were produced only at the lower doses of 10 and 15 Kr. In the interspecific combination N. l. × N. a., at lower doses, all plants that came to bloom showed variable hybrid morphology. There were no plants resembling the female parent. In the combination N.t. × N.a., at lower doses there were rare surviving plants which were maternal dihaploid (1 plant out of 4 at 15 Kr) or tetraploid N. tabacum (all 5 plants at 20 Kr). All surviving plants at higher doses (50 and 100 Kr) were maternal tetraploids. In the combination N.t.×N.g., plants produced at lower than 20 Kr were almost all either aneuploid or triploid hybrids. Dihaploid, maternal N. tabacum plants appeared at 20 Kr and higher doses. After 50 Kr the large proportion of plants produced were maternal dihaploid or tetraploid N. tabacum.     

Effect of salt stress on photosystem Ⅱ heterogeneity in wheat leaves

In order to study the effects of salt stress on photosystem Ⅱ (PSⅡ) heterogeneity, signal from the fluorometer was digitized via a microcomputer interface to record PSⅡ fluorescence induction kinetics. Changes of parameters (Fm, Fp1 and F0) from the fast phase of fluorescence induction curve showed that the percentage of QB-nonreducing PSⅡ reaction centers dropped at first, and then rose with the increase of stress intensity and time. This indicated that the electron transfer from QA to QB is one of the sites where the photochemical and photophysical processes of PSⅡ are damaged by the salt stress.     

Effect of soil cadmium application and pH on growth and cadmium accumulation in roots, leaves and fruit of strawberry plants (Fragaria × ananassa Duch.)

Three strawberry (Fragaria × ananassa Duch.) cultivars Rainier, Totem and Selva were grown under greenhouse conditions in a Parkhill sandy loam soil with a background DTPA-extractable Cd concentration of 0.18 mg kg^-1 and a pH of 5.1. Experimental treatments included combinations of 4 Cd applications (0, 15, 30 and 60 mg Cd kg^-1 soil) applied as CdSO₄ and 2 soil pH values 5.1 and 6.8. Both the application of Cd and pH of the soil significantly affected plant growth, yield and Cd accumulation in plant tissue anf fruit. Although roots accumulated the highest concentrations of Cd of all plant parts investigated, increased soil Cd application reduced leaf weight more than root weight. In general, yield of strawberries was decreased by an increase in amount of soil-applied Cd, however the yield response varied among cultivars. At 60 mg Cd kg^-1 soil, yield of Rainier cultivar was reduced to 17.6% of control plants. Over 90% of total Cd taken up by plants grown in Cd-treated soil accumulated in roots, regardless of the Cd level in the soil. Root Cd concentrations ranged from 2.6 mg kg^-1 (control plants) to 505.7 mg kg^-1 (Totem plants grown in soil at highest Cd and a soil pH 5.1) and were directly related to soil Cd concentrations. Cd translocation from roots to leaves and fruit was very limited, resulting in a maximum Cd concentration in root leaf tissue of 10.2 mg kg^-1. Accumulation of Cd in fruit was found to correlate well with leaf Cd, although even at the highest amount of applied Cd, fruit Cd concentration did not exceed 700 g kg^-1 of fresh weight.Contribution no. 951     

Cell division study in ‘pollen mother cells’ and ‘pollen grains’ of in vivo and ex vitro plants in Drimiopsis botryoides

The various normal and abnormal stages of meiosis and pollen mitosis of Drimiopsis botryoides are described, and a comparison between naturally propagated in vivo and tissue culture derived ex vitro plants in respect to their cytological behaviour presented. We also describe the floral morphology and investigate the relationship between the floral developmental stages and the progression of microgametogenesis. In total, 33 bivalents are observed in diakinesis, which indicate the diploid number 2n = 66 and this number is cross-checked by a haploid set of n = 33 chromosomes in pollen mitosis. Only 6.8% and 4.9% meiotic abnormalities were recorded on in vivo and ex vitro plants, respectively, which led to the formation of non-viable pollen. Finally, the microspores have to develop into tri-cellular male gametophyte. Only 0.2% pollen grains are found with a micro-nucleus. Though the higher pollen viability was recorded on both in vivo (89.3 ± 4.1%) and ex vitro (92.1 ± 4.6%) plants, but surprisingly the pollen germination rate is extremely low with 13.6 ± 1.74% and 21.3 ± 1.55%, respectively. The present study obviously enriches the cytological database of D. botryoides and may help future research on androgenesis and genetic improvement.     

Genetic and biochemical studies on perchloroethylene ‘in vitro’ and ‘in vivo’

Perchloroethylene (PCE) was tested in a diploid strain (D7) of the yeast Saccharomyces cerevisiae in suspension tests with and without a mammalian microsomal activation system (S9) and ‘in vivo’ by the intrasanguineous host-mediated assay. In addition, enzyme alteration studies were performed in mice non-pretreated or pretreated with phenobarbital + β-naphthoflavone. PCE did not induce any genetic effect either ‘in vitro’ or ‘in vivo’. In the suspension test, PCE was more toxic without metabolic activation and less toxic with mammalian microsomal activation. The enzymatic determinations showed an increase of the aminopyrine demethylase activity and of the level of cytochrome P-450.     

Carboxydismutase activity in plants with and without β-carboxylation photosynthesis

Summary A comparison was made of the activity of carboxydismutase (ribulose-1,5-diphosphate carboxylase) between higher plant species which possess the -carboxylation (C₄-dicarboxylic acid) pathway for photosynthesis and species which lack this pathway. Contrary to earlier findings no marked difference in the level of this enzyme was found between the two groups of species. Chloroplast-containing vascular-bundle-sheath cells which seem to be present only in plants with -carboxylation apparently contain relatively high carboxydismutase activity.C.I.W.-D.P.B. Publ. No. 453.     

‘Fine’ and ‘coarse’ mycorrhizal fungi on red clover plants in acid soils: Root colonization and plant responses

Three sterilized acid soils were inoculated with inocula of vesicular-arbuscular mycorrhizal fungi. Soils were limed and/or P fertilized to produce different fertility levels. Most inocula consisted of mixtures of fine + coarse type endophytes. Red clover (Trifolium pratense L.) was seeded in pots and grown in a glasshouse for 4 months. Root colonization by VAM fungi, the relative infection byGlomus tenue compared to that by coarse VAM fungi and the effect of inoculation on red clover growth and mineral nutrition (P, K, Ca and Mg) were studied. Spores were also checked and tentatively identified.Results showed that root colonization by VAM fungi was higher than 50% in most cases, the lower values being found in the soil with the highest P content. Inocula containingG. mosseae + G. tenue infected plant roots only in limed (pH>5.7) soils. A study of the relative colonization by fine and coarse endophytes showed that the competitive ability againstG. tenue followed the orderG. fasiculatum > G. mosseae > G. epigaeum > G. macrocarpum, although soil properties and fertility were crucial factors.Glomus lacteum was tentatively identified in two of the three experimental soils. The inoculum in whichGlommus tenue was most infective was also the most efficient in improving plant growth and nutrient uptake. The effect of inoculation on P and Mg uptake followed a similar pattern.     

Regeneration of plants from callus and embryos of ‘Royal’ apricot

Immature embryos of apricot (Prunus armeniaca L.) cv. Royal with a PF index of 25–100 were used to regenerate plants in vitro using two methods. In the first case, callus was initiated on MS medium with 4.5 M 2, 4-D plus 0.44 M BA and regeneration of shoots from the callus occurred on MS medium with 4.4 M BA plus 1.0 M 2, 4-D. In the second case, adventitious buds were directly regenerated from the cotyledons on MS medium with 4.4 M BA plus 1.0 M 2, 4-D.Abbreviations BA 6-benzyladenine - IBA dole-3-butyric acid - NAA -naphthylacetic acid - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PF (embryo length/seed length) x 100     

The ‘europium anomaly’ in plants: facts and fiction

Plant and Soil - Rare earth elements (REEs) and normalized REE patterns determined in plant and soil samples represent powerful tools to trace biogeochemical processes during weathering, soil...     

Effect of freezing–thawing process and quercetin on human sperm survival and DNA integrity

We aimed in the first part of our work to study the effect of cryopreservation on the human sperm DNA integrity and the activation of caspase 3, the main apoptosis indicator. In the second part, we were interested in testing the effect of quercetin, as an antioxidant, in preventing sperm damage during the freeze–thawing process. Seventeen semen samples were obtained from 17 men recruited for infertility investigations. Liquefied sperm was cryopreserved using spermfreeze®. Nine of the used samples were divided into two aliquots; the first one was cryopreserved with spermfreeze only (control) and the second one was cryopreserved with spermfreeze supplemented with quercetin to a final concentration of 50 μM. Sperm motility and viability were assessed according to WHO criteria. We used TUNEL assay and the Oxy DNA assay to assess sperm DNA integrity. Activated caspase 3 levels were measured in spermatozoa using fluorescein-labeled inhibitor of caspase (FLICA). Cryopreservation led to a significant increase in sperm DNA fragmentation, DNA oxidation and caspase 3 activation (p < 0.01). Supplementation of the cryopreservation medium with quercetrin induced a significant improvement in post thaw sperm parameters, compared to those of control, regarding sperm motility (p = 0.007), viability (p = 0.008) and DNA integrity (p = 0.02); however, it had no effect on caspase 3 activation (p = 0.3). We conclude that oxidative stress plays a major role in inducing sperm cryodamage but implication of apoptosis in this impairment requires further investigations. Quercetin could have protective effect during cryopreservation but further research is needed to confirm this effect.     

Effect of nickel concentration and soil pH on metal accumulation and growth in tropical agromining ‘metal crops’

Plant and Soil - Phosphorus (P) deficiency is a major constraint for rice production in the tropics. Field-specific P management is key for resource-limited farmers to increase yields with minimal...     

Growth and inorganic composition of ‘Nova’ mandarin plants grafted on two commercial rootstocks in response to salinity and silicon

The effects of salinity and its combination with silicon (Si) were studied in ‘Nova’ mandarin plants grafted on Citrus aurantium L. or Swingle Citrumelo to determine: (1) which combination is more tolerant to salt stress and (2) the impact of Si in limiting the harmful effects of salinity. Six groups of plants were grown in a greenhouse for 120 days and irrigated with: (1) 50 % Hoagland’s solution (Control), (2) 50 % Hoagland’s solution plus 80 mM NaCl (NaCl), and (3) 50 % Hoagland’s solution plus 80 mM NaCl plus 0.5 mM Si (NaCl + Si). Grafted plants exhibited accumulation of Na and Cl in their tissues following exposure to salinity. The ability of S. Citrumelo to retain the toxic ions in the roots in corroboration with the observation that the dry weights (DWs) of S. Citrumelo tissues were not influenced by NaCl treatment indicates that this rootstock is more tolerant to salinity. Silicon supplementation into the saline medium promoted the accumulation of toxic ions, whereas, when compared to NaCl treatment, it increased the DW of S. Citrumelo roots. Mineral concentrations were significantly affected by rootstock, treatment, and their interaction with S. Citrumelo, which presented better nutrient status than Sour Orange; and Si which differed depending on citrus tissue. It appears that S. Citrumelo rootstock is the most tolerant for ‘Nova’ mandarin plants under salinity, whereas salt tolerance in grafted citrus plants is not improved by Si application, indicating that the beneficial role of Si depends on the cultivar or rootstock–scion combinations.     

The role of ‘Norin 10’ dwarfing genes in photosynthetic and respiratory activity of wheat leaves

Summary A comparative analysis of eight cultivars of spring wheat (Triticum aestivum) classified by height as tall (T), semi-dwarf (D₁), dwarf (D₂) and very dwarf (D₃) was conducted to study their efficiency of oxygen exchange during photosynthesis and dark respiration. Two cultivars were included in each height group.Cultivars carrying Norin 10 dwarfing genes (D₁, D₂ and D₃) were found to have a significantly higher photosynthetic rate per unit leaf area than talls (T) that lack these genes. Among the Norin gene carriers, dwarf group (D₂) was most efficient, followed by very dwarf (D₃) and semi-dwarf (D₁).Photosynthetic rate and respiratory rate were found to have a positive relationship.     

Changes in chlorophyll,polyamines and chloroplast ultrastructure of Puccinia striiformis induced ‘green islands’ on detached leaves of Triticum aestivum

Biochemical and ultrastructure features of ‘green islands’ were investigated using detached wheat leaves infected with the yellow rust Puccinia striiformis. Chlorophylls appear to culminate 10 d after inoculation at which point ‘green islands’ were fully developed. These changes were paralleled by an increase in spermidine and spermine content which play an important role in formation of ‘green islands’. Retention of chlorophyll was demonstrated in leaf tissues of wheat plants supplied with exogenous putrescine, spermidine and spermine. Putrescine was least and spermidine and spermine most effective in retarding loss of chlorophylls. Ultrastructural observation revealed that chloroplasts were regenerated in ‘green islands’ where many proplastids were detected. The regeneration of chloroplasts coincided with the high concentration of chlorophylls and polyamines particularly spermidine and spermine. The ultrastructural changes of chloroplasts in leaf cell containing infection structures were parallel to physiological changes.     

The ‘Obligation’ to Screen and its Effect on Autonomy

In the United States, disease screening is offered to the public as a consumer service. It has been proposed that the act of “consumption” is a manifestation of agency and that the decision to consume is an exercise of autonomy. The enthusiasm of the American public for disease screening and the expansion in the demand for all sorts of disease screening in recent years can be viewed as an expression of such autonomy. Here, we argue that the enthusiasm for disease screening witnessed in the American public today may be more a reflection of the constraint on autonomy than its facilitation. It is our opinion that the articulation of socio-historical processes has contributed to a moral imperative which is reflected in the decision making of individuals around disease screening. We suggest medical and health professionals have a responsibility to facilitate the exercise of individual autonomy in health care decision making as an integral component of professional obligation. These professionals need to problematise healthcare activities that constrain individual autonomy.