Analysis of Thermal Characteristics of Potato and Hop Pollen for Their Cryopreservation and Cross-Breeding.

This study investigated the thermal properties of potato and hop pollen for cryopreservation and subsequent cross-breeding. Phase transitions and frozen water content in selected pollen samples were measured using a differential scanning calorimeter (DSC). Unlike hop pollen, potato pollen showed high variability in thermal properties and water content. Three specific types of pollen samples based on their thermal characteristics and water content were distinguished by DSC in potato: (1) 'glassy', with a water content lower than 0.21 g water per g dry matter; (2) 'transient', with a water content between 0.27 and 0.34 g of water per g of dry matter; (3) 'frozen', with a water content higher than 0.34 g of water per g of dry matter. Only the 'glassy' pollen samples with a low water content showed suitable properties for its long-term storage using cryopreservation in potato and hops. Cryopreservation of pollen did not significantly reduce its viability, and cryopreserved pollen was successfully used to produce both potato and hop hybrids. The results indicate that cryopreservation is a feasible technique for the preservation and utilization of pollen of these crops in the breeding process.

Vitrification Solutions for Plant Cryopreservation: Modification and Properties.

Many plants cannot vitrify themselves because they lack glassy state-inducing substances and/or have high water content. Therefore, cryoprotectants are used to induce vitrification. A cryoprotectant must have at least the following primary abilities: high glass-forming property, dehydration strength on a colligative basis to dehydrate plant cells to induce the vitrification state, and must not be toxic for plants. This review introduces the compounds used for vitrification solutions (VSs), their properties indicating a modification of different plant vitrification solutions, their modifications in the compounds, and/or their concentration. An experimental comparison is listed based on the survival or regeneration rate of one particular species after using more than three different VSs or their modifications. A brief overview of various cryopreservation methods using the Plant Vitrification Solution (PVS) is also included. This review can help in alert researchers to newly introduced PVSs for plant vitrification cryoprotocols, their properties, and the choice of their modifications in the compounds and/or their concentration.

Vitrification Ability of Combined and Single Cryoprotective Agents.

Cryoprotective agents (CPA) are an important part of many current vitrification methods. The vitrification ability of CPAs influences the probability of the glass transition and water crystallization occurrence. Thermal characteristics and the vitrification ability of two combined CPAs (PVS2 and PVS3), common plant vitrification solutions, and four single CPAs (ethylene glycol, DMSO, glycerol, and sucrose), the components of the mentioned PVSs, were evaluated utilizing a differential scanning calorimetry (DSC) during standard cooling/warming rates of 10 °C min-1. The effect of solute concentration on their vitrification ability was shown in the CPAs tested. Four typical concentration regions at which the glassy state and/or crystallization occurred were defined. We suggest the solute concentration of 0.7 g g-1 as the universal vitrification concentration, characterized by an actual Tg of CPA solution and limited water crystallization. Knowledge of the thermal properties of CPAs allows the design of new combined CPAs with the required vitrification ability respecting the cryopreservation method used and the characteristics of the cryopreserved sample.

The Survival of Pear Dormant Buds at Ultra-Low Temperatures.

Currently, there is a varietal diversity decline in pear orchards of the Czech Republic. Thus, the safe storage of their gene pool collections is becoming increasingly important. Therefore, the ultra-low temperature survival after two-step cryopreservation treatment of dormant buds was tested for a safe and rapid way to conserve pear germplasm in a broader range of varieties. The following varieties crucial for cultivation in the Czech Republic were tested; 'Amfora', 'Beurré Hardy', 'Bosc', 'Clapp's Favourite', 'Conference', 'Dicolor', 'Erika', 'Lucas', 'Williams' and 'Williams Red'. In 2011 and 2012, dormant pear buds were dehydrated to 40.1% and 36.0% water content, respectively, before cryopreservation. The average regeneration of the dormant pear buds after cryopreservation by the two-step cryoprotocol in 2011 and 2012 was 54.3% and 16.1%, respectively. The mentioned cryopreservation procedure is suitable for the safe storage of dormant buds in most tested pear varieties.

Cryotolerance of apple tree bud is independent of endodormancy.

Increasing interest in cryopreservation of dormant buds reveals the need for better understanding of the role of dormancy in cryotolerance. Dormancy stage and low-temperature survival of vegetative apple buds (Malus domestica Borkh.), cultivars 'Sampion' and 'Spartan', collected from orchard were evaluated during three seasons contrasting in temperature and precipitation throughout the arrested plant growth period. During each season, the cultivars differed either in the onset of the endodormancy or in the length of the endodormant period. A simple relation between endodormancy of the buds and their water content was not detected. The cryosurvival of vegetative apple buds of both cultivars correlated with their cold hardening without direct regard to their particular phase of dormancy. The period of the highest bud cryotolerance after low-temperature exposure overlapped with the endodormant period in some evaluated seasons. Both cultivars had the highest cryosurvival in December and January. The presented data were compared with our previous results from a dormancy study of in vitro apple culture. Endodormancy coincided with the period of successful cryosurvival of apple buds after liquid nitrogen exposure, but as such, it was not decisive for their survival and did not limit their successful cryopreservation.

The up-regulation of elongation factors in the barley leaf and the down-regulation of nucleosome assembly genes in the crown are both associated with the expression of frost tolerance.

We report a series of microarray-based leaf and crown transcriptome comparisons involving three barley cultivars (cvs. Luxor, Igri and Atlas 68) which express differing degrees of frost tolerance. The transcripts were obtained following the exposure of seedlings to low (above and below zero) temperatures, aiming to identify those genes and signalling/metabolic pathways which are associated with frost tolerance. Both the leaves and the crowns responded to low temperature by the up-regulation of a suite of abscisic acid (ABA)-responsive genes, most of which have already been recognized as components of the plant low temperature response. The inter-cultivar comparison indicated that genes involved in maintaining the leaf's capacity to synthesize protein and to retain chloroplast activity were important for the expression of frost tolerance. In the crown, the repression of genes associated with nucleosome assembly and transposon regulation were the most relevant transcriptional changes associated with frost tolerance, highlighting the role of gene repression in the cold acclimation response.

The choice of reference gene set for assessing gene expression in barley (Hordeum vulgare L.) under low temperature and drought stress.

Drought and low temperature are the two most significant causes of abiotic stress in agricultural crops and, therefore, they pose considerable challenges in plant science. Hence, it is crucial to study response mechanisms and to select genes for identification signaling pathways that lead from stimulus to response. The assessment of gene expression is often attempted using real-time RT-PCR (qRT-PCR), a technique which requires a careful choice of reference gene(s) for normalization purpose. Here, we report a comparison of 13 potential reference genes for studying gene expression in the leaf and crown of barley seedlings subjected to low temperature or drought stress. All three currently available software packages designed to identify reference genes from qRT-PCR data (GeNorm, NormFinder and BestKeeper) were used to identify informative sets of up to three reference genes. Interestingly, the data obtained from the separate treatment of leaf and crown have led to the recommendations that HSP70 and S-AMD (and possibly HSP90) to be used as the reference genes for low-temperature stressed leaves, HSP90 and EF1α for low-temperature stressed crowns, cyclophilin and ADP-RF (and possibly ACT) for drought-stressed leaves, and EF1α and S-AMD for drought-stressed crowns. Our results have demonstrated that the gene expression can be highly tissue- or organ-specific in barley and have confirmed that reference gene choice is essential in qRT-PCR. The findings can also serve as guidelines for the selection of reference genes under different stress conditions and lay foundation for more accurate and widespread use of qRT-PCR in barley gene analysis.

Transcriptional responses of winter barley to cold indicate nucleosome remodelling as a specific feature of crown tissues.

We report a series of microarray-based comparisons of gene expression in the leaf and crown of the winter barley cultivar Luxor, following the exposure of young plants to various periods of low (above and below zero) temperatures. A transcriptomic analysis identified genes which were either expressed in both the leaf and crown, or specifically in one or the other. Among the former were genes responsible for calcium and abscisic acid signalling, polyamine synthesis, late embryogenesis abundant proteins and dehydrins. In the crown, the key organ for cereal overwintering, cold treatment induced transient changes in the transcription of nucleosome assembly genes, and especially H2A and HTA11, which have been implicated in cold sensing in Arabidopsis thaliana. In the leaf, various heat-shock proteins were induced. Differences in expression pattern between the crown and leaf were frequent for genes involved in certain pathways responsible for osmolyte production (sucrose and starch, raffinose, γ-aminobutyric acid metabolism), sugar signalling (trehalose metabolism) and secondary metabolism (lignin synthesis). The action of proteins with antifreeze activity, which were markedly induced during hardening, was demonstrated by a depression in the ice nucleation temperature.

Hodgkin's lymphoma in the elderly: the results of 10 years of follow-up.

Elderly patients with Hodgkin's lymphoma carry a worse prognosis than younger patients because of a higher incidence of advanced stages, a worse performance status and the intolerance of full-dose curative treatment. A retrospective analysis of patients treated at our institution was performed. Our retrospective study summarizes the treatment results for 52 Hodgkin's lymphoma patients aged older than 60 years between 1973 and 1993. These patients were treated with combination of less toxic chemotherapy schedule (cyclophosphamide, vincristine, procarbazine and prednisone) and/or involved-field radiotherapy. The aim was to maintain an acceptable quality of life in spite of lower remission rate. The 5- and 10-year overall survival rates were 48% and 33%, respectively. We found two independent prognostic factors for overall survival: (i) stage of the disease and (ii) accomplishment of the treatment. Combined modality treatment yielded better results than chemotherapy. Tolerance of the treatment was acceptable. The present study demonstrates that a combination of mild chemotherapy with limited radiotherapy is a feasible way of treating elderly patients with Hodgkin's lymphoma.

Impact of lowering the screening and confirmation cutoff values for urine drug testing based on dilution indicators.

Many clinical and forensic toxicology laboratories establish criteria for identifying a random urine specimen submitted for drug screening as being "normally concentrated" or "dilute" by incorporating creatinine analysis and/or specific gravity measurement into their testing protocols. The objective of this study is to describe the importance of urine creatinine analysis and specific gravity measurements in the Correctional Service of Canada (CSC) drug-testing program. The CSC program uses the Substance Abuse and Mental Health Services Administration (SAMHSA) creatinine cutoff value (20 mg/dL) mandated for workplace drug testing in the United States. In the CSC program, urine specimens must have a creatinine concentration <20 mg/dL and specific gravity value

Substance abuse monitoring by the Correctional Service of Canada.

The Correctional Service of Canada implemented a urine drug-testing program over a decade ago. Offenders residing in federal correctional institutions and living in the community on conditional release were subject to urine drug testing. The objective of this study is to describe this testing program and the extent of drug use by conditional release offenders in 2000. Urine specimens were tested for drugs of abuse and prescription drugs including amphetamines, cannabinoids, cocaine metabolite, opiates, phencyclidine, benzodiazepines, methyl phenidate, meperidine, pentazocine and fluoxetine by immunoassay screening followed by GC-MS confirmation. Ethyl alcohol was analyzed when specifically requested. Alternative screening and confirmation methods with lower cut-off values were used whenever urine specimens were dilute (creatinine <20 mg/dL and specific gravity