Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos for long-term germplasm storage

Cryopreservation using encapsulation-dehydration was developed for the long-term conservation of cocoa (Theobroma cacao L.) germplasm. Survival of individually encapsulated somatic embryos after desiccation and cryopreservation was achieved through optimization of cryoprotectants (abscisic acid (ABA) and sugar), duration of osmotic and evaporative dehydration, and embryo development stage. Up to 63% of the genotype SPA4 early-cotyledonary somatic embryos survived cryopreservation following 7 days preculture with 1 M sucrose and 4 h silica exposure (16% moisture content in bead). This optimized protocol was successfully applied to three other genotypes, e.g. EET272, IMC14 and AMAZ12, with recovery frequencies of 25, 40 and 72%, respectively (but the latter two genotypes using 0.75 M sucrose). Recovered SPA4 somatic embryos converted to plants at a rate of 33% and the regenerated plants were phenotypically comparable to non-cryopreserved somatic embryo-derived plants.

ICGD 2003: International Cocoa Germplasm Database Version 5.2. CD-ROM

The current version of this database on CD-ROM contains information on 14 127 cocoa (Theobroma cacao) clones and their 14 112 synonyms, the origin and history of the clones and the clone names, and accession lists for 48 of the major cocoa gene banks including quarantine stations. Also included are morphological data for leaves, fruits and seeds, disease reactions, quality and agronomic characters, and reference information on common abbreviations and acronyms, cocoa gene bank addresses and a full bibliography (with hyperlinked reference to data). New additions are 748 photographs and drawings of 428 individual clones in 11 different locations. Also included are 376 profiles for 15 simple sequence repeat primer pairs on 331 clones held in the University of Reading Intermediate Cocoa Quarantine Facility. Minimum system requirements are Windows 95 or later, a Pentium 166 with 32 MB RAM, CD-ROM drive and a minimum 20 MB hard disk space. A user guide is included in the package.

In situ analysis and structural elucidation of sainfoin (Onobrychis viciifolia) tannins for high-throughput germplasm screening

A rapid thiolytic degradation and cleanup procedure was developed for analyzing tannins directly in chlorophyll-containing sainfoin (Onobrychis viciifolia) plants. The technique proved suitable for complex tannin mixtures containing catechin, epicatechin, gallocatechin, and epigallocatechin flavan-3-ol units. The reaction time was standardized at 60 min to minimize the loss of structural information as a result of epimerization and degradation of terminal flavan-3-ol units. The results were evaluated by separate analysis of extractable and unextractable tannins, which accounted for 63.6−113.7% of the in situ plant tannins. It is of note that 70% aqueous acetone extracted tannins with a lower mean degree of polymerization (mDP) than was found for tannins analyzed in situ. Extractable tannins had between 4 and 29 lower mDP values. The method was validated by comparing results from individual and mixed sample sets. The tannin composition of different sainfoin accessions covered a range of mDP values from 16 to 83, procyanidin/prodelphinidin (PC/PD) ratios from 19.2/80.8 to 45.6/54.4, and cis/trans ratios from 74.1/25.9 to 88.0/12.0. This is the first high-throughput screening method that is suitable for analyzing condensed tannin contents and structural composition directly in green plant tissue.

Thiolytic screening method for exploring condensed tannin variation in a unique sainfoin germplasm bank

This EU funded 'HealthyHay'project stablished a sainfoin (Onobrychis vicifolia) germplasm bank at NIAB, Cambridge, with 306 accessions from around the world. A screening method was developed to characterise tannins by thiolytic degradation [1] directly in green plants for the first time. the method was validated by separate analysis of unextractable, extractable and purified tannins using thiolysis, HPLC-GPC and MALDI-TOF MS. Most tannins (58 to 73% of the total) could be recovered after Toyopearl HW50 fractionation with water, aqueous methanol and acetone. the greatest losses during purification occurred amongst larger molecular weight tannins with mean degree of polymerisation (mDP) > 18. The composition of water-,aqueous methanol- and acetone-soluble tannins differed considerably in their mDP and trans/cis ratios, but not in their prodelphinidin/orocyanidin (PD/PC) ratios.

Proanthocyanidin diversity in the EU 'HealthyHay' sainfoin (onobrychis viciifolia) germplasm collection

This study investigated 37 diverse sainfoin (Onobrychis viciifolia Scop.) accessions from the EU ‘HealthyHay’ germplasm collection for proanthocyanidin (PA) content and composition. Accessions displayed a wide range of differences: PA contents varied from 0.57 to 2.80 g/100 g sainfoin; the mean degree of polymerisation from 12 to 84; the proportion of prodelphinidin tannins from 53% to 95%, and the proportion of trans-flavanol units from 12% to 34%. A positive correlation was found between PA contents (thiolytic versus acid–butanol degradation; P < 0.001; R2 = 0.49). A negative correlation existed between PA content (thiolysis) and mDP (P < 0.05; R2 = −0.30), which suggested that accessions with high PA contents had smaller PA polymers. Cluster analysis revealed that European accessions clustered into two main groups: Western Europe and Eastern Europe/Asia. In addition, accessions from USA, Canada and Armenia tended to cluster together. Overall, there was broad agreement between tannin clusters and clusters that were based on morphological and agronomic characteristics.

The role of the international cocoa germplasm database and the international cocoa quarantine centre in information management and distribution of cocoa genetic resources

A range of physiological parameters (canopy light transmission, canopy shape, leaf size, flowering and flushing intensity) were measured from the International Clone Trial, typically over the course of two years. Data were collected from six locations, these being: Brazil, Ecuador, Trinidad, Venezuela, Côte d’Ivoire and Ghana. Canopy shape varied significantly between clones, although it showed little variation between locations. Genotypic variation in leaf size was differentially affected by the growth location; such differences appeared to underlie a genotype by environment interaction in relation to canopy light transmission. Flushing data were recorded at monthly intervals over the course of a year. Within each location, a significant interaction was observed between genotype and time of year, suggesting that some genotypes respond to a greater extent than others to environmental stimuli. A similar interaction was observed for flowering data, where significant correlations were found between flowering intensity and temperature in Brazil and flowering intensity and rainfall in Côte d’Ivoire. The results demonstrate the need for local evaluation of cocoa clones and also suggest that the management practices for particular planting material may need to be fine-tuned to the location in which they are cultivated.

Haplotype analysis of vernalization loci in European barley germplasm reveals novel VRN-H1 alleles and a predominant winter VRN-H1/VRN-H2 multi-locus haplotype

In barley, variation in the requirement for vernalization (an extended period of low temperature before flowering can occur) is determined by the VRN-H1, -H2 and -H3 loci. In European cultivated germplasm, most variation in vernalization requirement is accounted for by alleles at VRN-H1 and VRN-H2 only, but the range of allelic variation is largely unexplored. Here we characterise VRN-H1 and VRN-H2 haplotypes in 429 varieties representing a large portion of the acreage sown to barley in Western Europe over the last 60 years. Analysis of genotype, intron I sequencing data and growth habit tests identified three novel VRN-H1 alleles and determined the most frequent VRN-H1 intron I rearrangements. Combined analysis of VRN-H1 and VRN-H2 alleles resulted in the classification of seventeen VRN-H1/VRN-H2 multi-locus haplotypes, three of which account for 79% of varieties. The molecular markers employed here represent powerful diagnostic tools for prediction of growth habit and assessment of varietal purity. These markers will also allow development of germplasm to test the behaviour of individual alleles with the aim of understanding the relationship between allelic variation and adaptation to specific agri-environments.

A baseline study of vicine–convicine levels in faba bean (Vicia faba L.) germplasm

Vicine and convicine are anti-nutritional compounds that accumulate in the cotyledons of faba beans. When humans consume beans with high levels of these compounds, it can cause a condition called favism in individuals harbouring a deficiency in the activity of their glucose-6-phosphate dehydrogenase. When faba beans are used in animal feeds, there can be effects on performance. These concerns have resulted in increasing interest within plant breeding in developing low vicine and convicine faba bean germplasm. In order to facilitate this objective, we developed a rapid and robust screening method for vicine and convicine, capable of distinguishing between faba beans that are either high (wild type) or low in vicine and convicine. In the absence of reliable commercial reference materials, we report an adaptation of a previously published method where a biochemical assay and spectral data were used to confirm the identity of our analytes, vicine and convicine. This method could be readily adopted in other facilities and open the way to the efficient exploitation of diverse germplasm in regions where faba beans play a significant role in human nutrition. We screened a collection of germplasm of interest to a collaborative plant breeding programme developing between the National Institute for Agricultural Botany in the UK and L'Institut Nationale d'Agronomie de Tunisie in Tunisia. We report the results obtained and discuss the prospects for developing molecular markers for the low vicine and convicine trait.

Analysis of the genetic diversity and structure across a wide range of germplasm reveals prominent gene flow in apple at the European level

Abstract Background: The amount and structure of genetic diversity in dessert apple germplasm conserved at a European level is mostly unknown, since all diversity studies conducted in Europe until now have been performed on regional or national collections. Here, we applied a common set of 16 SSR markers to genotype more than 2,400 accessions across 14 collections representing three broad European geographic regions (North+East, West and South) with the aim to analyze the extent, distribution and structure of variation in the apple genetic resources in Europe. Results: A Bayesian model-based clustering approach showed that diversity was organized in three groups, although these were only moderately differentiated (FST=0.031). A nested Bayesian clustering approach allowed identification of subgroups which revealed internal patterns of substructure within the groups, allowing a finer delineation of the variation into eight subgroups (FST=0.044). The first level of stratification revealed an asymmetric division of the germplasm among the three groups, and a clear association was found with the geographical regions of origin of the cultivars. The substructure revealed clear partitioning of genetic groups among countries, but also interesting associations between subgroups and breeding purposes of recent cultivars or particular usage such as cider production. Additional parentage analyses allowed us to identify both putative parents of more than 40 old and/or local cultivars giving interesting insights in the pedigree of some emblematic cultivars. Conclusions: The variation found at group and sub-group levels may reflect a combination of historical processes of migration/selection and adaptive factors to diverse agricultural environments that, together with genetic drift, have resulted in extensive genetic variation but limited population structure. The European dessert apple germplasm represents an important source of genetic diversity with a strong historical and patrimonial value. The present work thus constitutes a decisive step in the field of conservation genetics. Moreover, the obtained data can be used for defining a European apple core collection useful for further identification of genomic regions associated with commercially important horticultural traits in apple through genome-wide association studies.

Ensemble yield simulations: crop and climate uncertainties, sensitivity to temperature and genotypic adaptation to climate change

Estimates of the response of crops to climate change rarely quantify the uncertainty inherent in the simulation of both climate and crops. We present a crop simulation ensemble for a location in India, perturbing the response of both crop and climate under both baseline (12 720 simulations) and doubled-CO2 (171720 simulations) climates. Some simulations used parameter values representing genotypic adaptation to mean temperature change. Firstly, observed and simulated yields in the baseline climate were compared. Secondly, the response of yield to changes in mean temperature was examined and compared to that found in the literature. No consistent response to temperature change was found across studies. Thirdly, the relative contribution of uncertainty in crop and climate simulation to the total uncertainty in projected yield changes was examined. In simulations without genotypic adaptation, most of the uncertainty came from the climate model parameters. Comparison with the simulations with genotypic adaptation and with a previous study suggested that the relatively low crop parameter uncertainty derives from the observational constraints on the crop parameters used in this study. Fourthly, the simulations were used, together with an observed dataset and a simple analysis of crop cardinal temperatures and thermal time, to estimate the potential for adaptation using existing cultivars. The results suggest that the germplasm for complete adaptation of groundnut cultivation in western India to a doubled-CO2 environment may not exist. In conjunction with analyses of germplasm and local management

Heat tolerance in groundnut

Tolerance to high soil and air temperature during the reproductive phase is an important component of adaptation to and and semi-arid cropping environments in groundnut. Between 10 and 22 genotypes were screened for tolerance to high air and soil temperature in controlled environments. To assess tolerance to high soil temperature, 10 genotypes were grown from start of podding to harvest at ambient (28 degrees) and high (38 degreesC) soil temperatures, and crop growth rate (CGR), pod growth rate (PGR) and partitioning (ratio PGR:CGR) measured. To assess tolerance to high air temperature during two key stages-microsporogenesis (3-6 days before flowering, DBF) and flowering, fruit-set was measured in two experiments. In the first experiment, 12 genotypes were exposed to short (3-6 days) episodes of high (38 degreesC) day air temperature at 6 DBF and at flowering. In the second experiment, 22 genotypes were exposed to 40 degreesC day air temperature for I day at 6 DBF, 3 DBF or at flowering. Cellular membrane thermostability (relative injury, RI) was also measured in these 22 genotypes. There was considerable variation among genotypes in response to high temperature, whether assessed by growth rates, fruit-set or RI. Pod weight at high soil temperature was associated with variation in CGR rather than partitioning. Flowering was more sensitive to high air temperature than microsporogenesis. Genotypes tolerant to high air temperature at microsporogenesis were not necessarily tolerant at flowering, and nor was tolerance correlated with RI. Six genotypes (796, 55-437, ICG 1236, ICGV 86021, lCGV 87281 and ICGV 92121) were identified as heat tolerant based on their performance in all tests. These experiments have shown that groundnut genotypes can be easily screened for reproductive tolerance to high air and soil temperature and that several sources of heat tolerance are available in groundnut germplasm. (C) 2003 Elsevier Science B.V. All rights reserved.

Receptivity to social research by the CGIAR

This paper analyses the way the CGIAR system has incorporated social research in its agenda. Since 1995, the social science staff capacity in the CGIAR has decreased by 24%, and the overall balance of social science research is still significantly tilted away from the core germplasm enhancement, production systems/natural resources management, and technology adoption work - the 'bread and butter' of technology generation and development effort - toward ex-ante and ex-post activities, Further, the bulk of the social science research has low social research content despite the significant expansion of the CGIAR initial goal of increasing the proverbial pile of rice' to poverty alleviation and sustainable food security. The paper concludes that a concerted effort is now required to mainstream social research in the CGIAR system, and this cannot occur without the full support of the CGIAR donors, the CGIAR senior managers, and the centre boards and executive staff.

Use of secondary somatic embryos promotes genetic fidelity in cryopreservation of cocoa (Theobroma cacao L.)

The inability to conserve cocoa (Theobroma cacao L.) germplasm via sced storage and the vulnerability of field collections make the establishment of cryopreserved genebanks for the crop a priority. An effective encapsulation-dehydration based cryopreservation system has been developed for cocoa but because the somatic embryos used for freezing arise after a protracted period of callus culture there is concern about maintenance of genetic fidelity during the process. Microsatellite markers for seven of the 10 cocoa linkage groups were used to screen a population of 189 primary somatic embryo-derived emblings and the 43 secondary somatic embryos they gave rise to. Of the primary somatic embryos, 38.1% exhibited polymorphic microsatellite profiles while for secondary somatic embryos the frequency was 23.3%. The same microsatellite markers used to screen another population of 44 secondary somatic embryos cryopreserved through encapsulation-dehydration revealed no polymorphisms. Scanning electron microscopy showed the secondary somatic embryos were derived from cotyledonary epidermal cells rather than callus. The influence of embryo ontogeny on somaclonal variation is discussed.