The world's largest potato cryobank at the International Potato Center (CIP) - Status quo, protocol improvement through large-scale experiments and long-term viability monitoring.

Long-term conservation of Plant Genetic Resources (PGR) is a key priority for guaranteeing food security and sustainability of agricultural systems for current and future generations. The need for the secure conservation of genetic resources collections ex situ is critical, due to rapid and extreme climatic changes which are threatening and reducing biodiversity in their natural environments. The International Potato Center (CIP) conserves one of the most complete and diverse genetic resources collections of potato, with more than 7500 accessions composed of 4900 cultivated potato and 2600 potato wild relative accessions. The clonal conservation of cultivated potato, principally landraces, through in vitro or field collections is indispensable to maintain fixed allelic states, yet it is costly and labor-intensive. Cryopreservation, the conservation of biological samples in liquid nitrogen (-196°C), is considered the most reliable and cost-efficient long-term ex-situ conservation method for clonal crops. Over the last decade, CIP has built one of the largest potato cryobanks worldwide, cyopreserving more than 4000 cultivated potato accessions which represents 84% of the total cultivated potato collection currently conserved at CIP. In approximately, four years the entire potato collection will be cryopreserved. The development of an applied, robust cryopreservation protocol for potato, serves as a model for other clonally maintained crop collections. The CIP cryobank designs experiments with a high number of genetically diverse genotypes (70-100 accessions, seven cultivated species), to obtain reliable results that can be extrapolated over the collection as genotypes can often respond variably to the same applied conditions. Unlike most published reports on cryopreservation of plants, these large-scale experiments on potato are unique as they examine the acclimatization process of in vitro plants prior to, as well as during cryopreservation on up to ten times the number of genotypes conventionally reported in the published literature. As a result, an operational cryopreservation protocol for potato has advanced that works well across diverse potato accessions, not only with reduced processing time and costs, but also with an increased average full-plant recovery rate from 58% to 73% (+LN) for routine cryopreservation. The present article describes the composition of CIP's cryobank, the cryopreservation protocol, methodology for the dynamic improvement of the operational protocol, as well as data collected on regeneration from long term cryopreserved potatoes.

Phased, chromosome-scale genome assemblies of tetraploid potato reveal a complex genome, transcriptome, and predicted proteome landscape underpinning genetic diversity.

Cultivated potato is a clonally propagated autotetraploid species with a highly heterogeneous genome. Phased assemblies of six cultivars including two chromosome-scale phased genome assemblies revealed extensive allelic diversity, including altered coding and transcript sequences, preferential allele expression, and structural variation that collectively result in a highly complex transcriptome and predicted proteome, which are distributed across the homologous chromosomes. Wild species contribute to the extensive allelic diversity in tetraploid cultivars, demonstrating ancestral introgressions predating modern breeding efforts. As a clonally propagated autotetraploid that undergoes limited meiosis, dysfunctional and deleterious alleles are not purged in tetraploid potato. Nearly a quarter of the loci bore mutations are predicted to have a high negative impact on protein function, complicating breeder's efforts to reduce genetic load. The StCDF1 locus controls maturity, and analysis of six tetraploid genomes revealed that 12 allelic variants of StCDF1 are correlated with maturity in a dosage-dependent manner. Knowledge of the complexity of the tetraploid potato genome with its rampant structural variation and embedded deleterious and dysfunctional alleles will be key not only to implementing precision breeding of tetraploid cultivars but also to the construction of homozygous, diploid potato germplasm containing favorable alleles to capitalize on heterosis in F1 hybrids.

Production and characterisation of a marine Halomonas surface-active exopolymer.

During screening for novel emulsifiers and surfactants, a marine gammaproteobacterium, Halomonas sp. MCTG39a, was isolated and selected for its production of an extracellular emulsifying agent, P39a. This polymer was produced by the new isolate during growth in a modified Zobell's 2216 medium amended with 1% glucose, and was extractable by cold ethanol precipitation. Chemical, chromatographic and nuclear magnetic resonance spectroscopic analysis confirmed P39a to be a high-molecular-weight (~ 261,000 g/mol) glycoprotein composed of carbohydrate (17.2%) and protein (36.4%). The polymer exhibited high emulsifying activities against a range of oil substrates that included straight-chain aliphatics, mono- and alkyl- aromatics and cycloparaffins. In general, higher emulsification values were measured under low (0.1 M PBS) compared to high (synthetic seawater) ionic strength conditions, indicating that low ionic strength is more favourable for emulsification by the P39a polymer. However, as observed with other bacterial emulsifying agents, the polymer emulsified some aromatic hydrocarbon species, as well as refined and crude oils, more effectively under high ionic strength conditions, which we posit could be due to steric adsorption to these substrates as may be conferred by the protein fraction of the polymer. Furthermore, the polymer effected a positive influence on the degradation of phenanthrene by other marine bacteria, such as the specialist PAH-degrader Polycyclovorans algicola. Collectively, based on the ability of this Halomonas high-molecular-weight glycoprotein to emulsify a range of pure hydrocarbon species, as well as refined and crude oils, it shows promise for the bioremediation of contaminated sites.

Using Genomic Sequence Information to Increase Conservation and Sustainable Use of Crop Diversity and Benefit-Sharing.

This article describes how CGIAR centers and partners are using genomic sequence information to promote the conservation and sustainable use of crop genetic diversity, and to generate and share benefits derived from those uses. The article highlights combined institutional, and benefit-sharing-related challenges that need to be addressed to support expanded use of digital sequence information in agricultural research and development.

A Case of Need: Linking Traits to Genebank Accessions.

Genebanks are responsible for collecting, maintaining, characterizing, documenting, and distributing plant genetic resources for research, education, and breeding purposes. The rationale for requests of plant materials varies highly from areas of anthropology, social science, small-holder farmers, the commercial sector, rehabilitation of degraded systems, all the way to crop improvement and basic research. Matching "the right" accessions to a particular request is not always a straightforward process especially when genetic resource collections are large and the user does not already know which accession or even which species they want to study. Some requestors have limited knowledge of the crop; therefore, they do not know where to begin and thus, initiate the search by consultation with crop curators to help direct their request to the most suitable germplasm. One way to enhance the use of genebank material and aid in the selection of genetic resources is to have thoroughly cataloged agronomic, biochemical, genomic, and other traits linked to genebank accessions. In general, traits of importance to most users include genotypes that thrive under various biotic and abiotic stresses, morphological traits (color, shape, size of fruits), plant architecture, disease resistance, nutrient content, yield, and crop specific quality traits. In this review, we discuss methods for linking traits to genebank accessions, examples of linked traits, and some of the complexities involved, while reinforcing why it is critical to have well characterized accessions with clear trait data publicly available.

Hydrocarbon-degradation and MOS-formation capabilities of the dominant bacteria enriched in sea surface oil slicks during the Deepwater Horizon oil spill.

A distinctive feature of the Deepwater Horizon (DwH) oil spill was the formation of significant quantities of marine oil snow (MOS), for which the mechanism(s) underlying its formation remain unresolved. Here, we show that Alteromonas strain TK-46(2), Pseudoalteromonas strain TK-105 and Cycloclasticus TK-8 - organisms that became enriched in sea surface oil slicks during the spill - contributed to the formation of MOS and/or dispersion of the oil. In roller-bottle incubations, Alteromonas cells and their produced EPS yielded MOS, whereas Pseudoalteromonas and Cycloclasticus did not. Interestingly, the Cycloclasticus strain was able to degrade n-alkanes concomitantly with aromatics within the complex oil mixture, which is atypical for members of this genus. Our findings, for the first time, provide direct evidence on the hydrocarbon-degrading capabilities for these bacteria enriched during the DwH spill, and that bacterial cells of certain species and their produced EPS played a direct role in MOS formation.

A randomised, double blind comparison of tecarfarin, a novel vitamin K antagonist, with warfarin. The EmbraceAC Trial.

Tecarfarin is a novel vitamin K antagonist that is metabolised by carboxyl estererase, thereby eliminating the variability associated with cytochrome-mediated metabolism. EmbraceAC was designed to compare the quality of anticoagulation with tecarfarin and warfarin as determined by time in therapeutic range (TTR). In this phase 2/3 randomised and blinded trial, 607 patients with indications for chronic anticoagulation were assigned to warfarin (n=304) or tecarfarin (n=303). Dosing of study drugs was managed by a centralised dose control centre, which had access to genotyping. The primary analysis tested superiority of tecarfarin over warfarin for TTR. Patients were recruited between May 12, 2008 and May 12, 2009. TTR with tecarfarin and warfarin were similar (72.3 % and 71.5 %, respectively; p=0.51). In those taking CYP2C9 interacting drugs, the TTR on tecarfarin (n=92) was similar to that on warfarin (n=87, 72.2 % and 69.9 %, respectively; p=0.15). In patients with mechanical heart valves, the TTR of tecarfarin (n=42) was similar to that of warfarin (n=42, 68.4 % and 66.3 %, respectively; p=0.51). The same was true for the TTR in patients with any CYP2C9 variant allele and on CYP2C9-interacting drugs (tecarfarin, n=24, 76.5 % vs warfarin, n=31, 69.5 %; p=0.09). There was no difference in thromboembolic or bleeding events. In conclusion, superiority of tecarfarin over warfarin for TTR was not demonstrated. The TTR with tecarfarin was similar to that with well-controlled warfarin and tecarfarin appeared to be safe and well tolerated with few major bleeding and no thrombotic events. Favourable trends in certain subpopulations make tecarfarin a promising oral anticoagulant that deserves further study.

Metabolism and pharmacokinetics of naronapride (ATI-7505), a serotonin 5-HT(4) receptor agonist for gastrointestinal motility disorders.

The absorption and disposition of the serotonin 5-HT(4) receptor agonist, naronapride (6-[(3S,4R)-4-(4-amino-5-chloro-2-methoxy-benzoylamino)-3-methoxy-piperidin-1-yl]-hexanoic acid 1-aza-bicyclo[2,2,2]oct-(R)-3-yl ester dihydrochloride; ATI-7505), were evaluated in healthy males given a single 120-mg oral dose of (14)C-labeled compound. Serial blood samples and complete urine and feces were collected up to 552 h postdose. Naronapride was extensively metabolized, undergoing rapid hydrolysis to 6-[(3S,4R)-4-(4-amino-5-chloro-2-methoxy-benzoylamino)-3-methoxy-piperidin-1-yl]-hexanoic acid (ATI-7500) with stoichiometric loss of quinuclidinol. ATI-7500 was either N-glucuronidated on the phenyl ring or its hexanoic acid side chain underwent two-carbon cleavage, probably through a ß-oxidation metabolic pathway, to form 4-[(3S,4R)-4-(4-amino-5-chloro-2-methoxy-benzoylamino)-3-methoxy-piperidin-1-yl]-butanoic acid (ATI-7400). ATI-7400 underwent further side-chain oxidation to form 2-[(3S,4R)-4-(4-amino-5-chloro-2-methoxy-benzoylamino)-3-methoxy-piperidin-1-yl]-acetic acid (ATI-7100). Quinuclidinol, ATI-7500, ATI-7400, and ATI-7100 were the major metabolites, with plasma area under the curve values approximately 72-, 17-, 8-, and 2.6-fold that of naronapride. Naronapride, ATI-7500, ATI-7400, and ATI-7100 accounted for 32.32, 36.56, 16.28, and 1.58%, respectively, of the dose recovered in urine and feces. ATI-7400 was the most abundant radioactive urinary metabolite (7.77%), and ATI-7500 was the most abundant metabolite in feces (35.62%). Fecal excretion was the major route of elimination. Approximately 32% of the dose was excreted unchanged in feces. Naronapride, ATI-7500, and quinuclidinol reached peak plasma levels within 1 h postdose. Peak ATI-7400 and ATI-7100 concentrations were reached within 1.7 h, suggesting rapid ATI-7500 metabolism. Naronapride plasma terminal half-life was 5.36 h, and half-lives of the major metabolites ranged from 17.69 to 33.03 h. Naronapride plasma protein binding was 30 to 40%. The mean blood/plasma radioactivity ratio indicated minimal partitioning of (14)C into red blood cells.

Identification of amide bioisosteres of triazole oxytocin antagonists.

A series of amides were investigated as potential bioisosteres of previously reported triazole oxytocin antagonists. A range of potent analogues were identified, although SAR for potency and selectivity over the related V(1A) and V(2) receptors was found to be somewhat divergent from that observed for the corresponding triazole series. The high synthetic accessibility of this new amide series also facilitated the identification of a range of alternative left hand side (biaryl replacement) substituents which gave good levels of oxytocin antagonism.

Identification of a urea bioisostere of a triazole oxytocin antagonist.

A series of azetidine ureas were investigated as potential bioisosteres of previously reported azetidinyltriazole oxytocin antagonists. Although potency was somewhat reduced in several close-in analogues, one compound, 9, was both a potent oxytocin antagonist and demonstrated significant selectivity over the closely related vasopressin V(1A) receptor.

Triazole oxytocin antagonists: Identification of an aryloxyazetidine replacement for a biaryl substituent.

A series of aryloxyazetidines, aryloxypyrrolidines and aryloxypiperidines were designed based on structural overlap with previously reported arylpyrazine Oxytocin antagonists. Similarly high levels of Oxytocin antagonism were achievable in these new series. Several aryloxyazetidines also showed high levels of selectivity, with one compound, 25, displaying promising in vivo pharmacokinetics and significantly improved aqueous solubility over related compounds containing a biaryl substituent.

Aryloxypyrazines as highly selective antagonists of Oxytocin.

A series of aryloxypyrazines were designed based on structural overlap with previously reported arylpyrazine Oxytocin antagonists. Similarly high levels of Oxytocin antagonism were achievable in this new series. In addition, significant improvements in selectivity over the related vasopressin V(1A) receptor were also possible.

Triazole oxytocin antagonists: identification of aryl ether replacements for a biaryl substituent.

Several potent aryl ether/triazole oxytocin antagonists are described. The lead compound in this series had significantly improved aqueous solubility over related systems containing a biaryl substituent.

Design and optimization of potent, selective antagonists of Oxytocin.

A novel series of Oxytocin antagonists are described. This series was identified through pharmacophoric overlap of in-house and literature antagonists. Subsequent optimization led to a series of potent, selective antagonists. Several analogues displayed oral bioavailability in vivo in the rat.

Up-regulation of sucrose synthase and UDP-glucose pyrophosphorylase impacts plant growth and metabolism.

The effects of the overexpression of sucrose synthase (SuSy) and UDP-glucose pyrophosphorylase (UGPase) on plant growth and metabolism were evaluated in tobacco (Nicotiana tabacum cv. Xanthi). T(1) transgenic plants expressing either gene under the control of a tandem repeat cauliflower mosaic virus 35S promoter (2x35S) or a xylem-localized 4CL promoter (4-coumarate:CoA ligase; 4CL) were generated, and reciprocally crossed to generate plants expressing both genes. Transcript levels, enzyme activity, growth parameters, fibre properties and carbohydrate content of stem tissue were quantified. The expression profiles of both genes confirmed the expression pattern of the promoters: 2x35S expressed more strongly in leaves, while 4CL expression was highest in stem tissue. In-depth plant characterization revealed that the single-transgene lines showed significant increases in the height growth compared with corresponding control lines. The double-transgene plants demonstrated an additive effect, proving to be even taller than the single-transgene parents. Several of these lines had associated increases in soluble sugar content. Although partitioning of storage carbohydrates into starch or cellulose was not observed, the increased height growth and increases in soluble carbohydrates suggest a role for SuSy as a marker in sink strength and lend credit to the function of UGPase in a similar role. The up-regulation of these two genes, although not increasing the percentage cellulose content, was effective in increasing the total biomass, and thus the overall cellulose yield, from a given plant.

Implementation of garlic cryopreservation techniques in the national plant germplasm system.

The USDA-ARS National Plant Germplasm System (NPGS) maintains more than 200 Allium sativum (garlic) accessions at the Western Regional Plant Introduction Station in Pullman, WA. All accessions must be grown out in the field annually since garlic plants from these accessions do not reliably produce seeds and bulbs do not store well. Shoot tips excised from garlic cloves can be successfully cryopreserved using either Plant Vitrification Solution 2 (PVS2; 15 percent v/v DMSO, 15 percent v/v ethylene glycol, 30 percent v/w glycerol, 0.4 M sucrose) or Plant Vitrification Solution 3 (PVS3; 50 percent v/w sucrose, 50 percent v/w glycerol). We compared regrowth of shoot tips representing diverse garlic germplasm after exposure to either PVS2 or PVS3 during the cryopreservation procedure. At the USDA-ARS National Center for Genetic Resources Preservation, a component of the NPGS, we consider accessions successfully preserved if a minimum of 40 percent of explants exhibit regrowth after liquid nitrogen exposure and at least 60 viable shoot tips remain in long-term storage. Ten of twelve diverse garlic accessions were successfully cryopreserved using either PVS2 or PVS3 as cryoprotectants. Five genotypes had the best post liquid nitrogen regrowth after exposure to PVS2, four genotypes had the best regrowth after exposure to PVS3, and three genotypes performed equally well using either cryoprotectant solution. This project is part of an ongoing program to cryopreserve accessions of NPGS clonal crop collections.

The potential of metabolite profiling as a selection tool for genotype discrimination in Populus.

Differences between wild-type Populus tremulaxalba and two transgenic lines with modified lignin monomer composition, were interrogated using metabolic profiling. Analysis of metabolite abundance data by GC-MS, coupled with principal components analysis (PCA), successfully differentiated between lines that had distinct phenotypes, whether samples were taken from the cambial zone or non-lignifying suspension tissue cultures. Interestingly, the GC-MS analysis detected relatively few phenolic metabolites in cambial extracts, although a single metabolite associated with the differentiation between lines was directly related to the phenylpropanoid pathway or other down-stream aspects of lignin biosynthesis. In fact, carbohydrates, which have only an indirect relationship with the modified lignin monomer composition, featured strongly in the line-differentiating aspects of the statistical analysis. Traditional HPLC analysis was employed to verify the GC-MS data. These findings demonstrate that metabolic traits can be dissected reliably and accurately by metabolomic analyses, enabling the discrimination of individual genotypes of the same tree species that exhibit marked differences in industrially relevant wood traits. Furthermore, this validates the potential of using metabolite profiling techniques for marker generation in the context of plant/tree breeding for industrial applications.

Proteome analysis of early somatic embryogenesis in Picea glauca.

Forestry is a valuable natural resource for many countries. Rapid production of large quantities of genetically improved and uniform seedlings for restocking harvested lands is a key component of sustainable forest management programs. Clonal propagation through somatic embryogenesis has the potential to meet this need in conifers and can offer the added benefit of ensuring consistent seedling quality. Although in commercial use, mass production of conifers through somatic embryogenesis is relatively new and there are numerous biological unknowns regarding this complex developmental pathway. To aid in unravelling the embryo developmental process, two-dimensional electrophoresis was employed to quantitatively assess the expression levels of proteins across four stages of somatic embryo maturation in white spruce (0, 7, 21 and 35 days post abscisic acid treatment). Forty-eight differentially expressed proteins have been identified, which display a significant change in abundance as early as day 7 of embryo development. These proteins are involved in a variety of cellular processes, many of which have not previously been associated with embryo development. The identification of these proteins was greatly assisted by the availability of a substantial expressed sequence tag (EST) resource developed for white, sitka and interior spruce. The combined use of these spruce ESTs in conjunction with GenBank accessions for other plants improved the rate of protein identification from 38% to 62% when compared with GenBank alone using automated, high-throughput techniques. This underscores the utility of EST resources in a proteomic study of any species for which a genome sequence is unavailable.

Significant increases in pulping efficiency in C4H-F5H-transformed poplars: improved chemical savings and reduced environmental toxins.

The gene encoding ferulate 5-hydroxylase (F5H) was overexpressed in poplar (Populus tremula x Populus alba) using the cinnamate-4-hydroxylase (C4H) promoter to drive expression specifically in cells involved in the lignin biosynthetic pathway and was shown to significantly alter the mole percentage of syringyl subunits in the lignin, as determined by thioacidolysis. Analysis of poplar transformed with a C4H-F5H construct demonstrated significant increases in chemical (kraft) pulping efficiency from greenhouse-grown trees. Compared to wild-type wood, decreases of 23 kappa units and increases of >20 ISO brightness units were observed in trees exhibiting high syringyl monomer concentrations. These changes were associated with no significant modification in total lignin content and no observed phenotypic differences. C4H-F5H-transformed trees could increase pulp throughputs at mills by >60% while concurrently decreasing chemicals employed during processing (chemical pulping and bleaching) and, consequently, the amount of deleterious byproducts released into the environment.