Control of cocoa swollen shoot disease by eradicating infected trees in Ghana: A survey of treated and replanted areas

Cocoa farms that had been treated and replanted in Ghana during the most recent phase of the cocoa swollen shoot virus (CSSV) eradication campaign were surveyed. Farms that were replanted close to adjoining old cocoa farms or which contained old trees were common in most (38) of the 41 cocoa farms surveyed. CSSV infections were apparent in 20 (53%) out of these 38 farms and they pose a serious risk of causing early infections of the re-planted farms. Control strategies that isolate the newly planted farms by a boundary of immune crops as barriers to reduce CSSV re-infection are discussed. (c) 2005 Elsevier Ltd. All rights reserved.

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.

Texture development in ice cream - effects of adding cocoa solids

Dynamic rheological techniques can aid the understanding of the factors contributing to ice cream structure, though the data obtained differs from that deduced from destructive techniques. Studies have shown that ice cream systems are both strain- and frequency-dependent. Chocolate ice cream is normally more viscous than the equivalent vanilla ice cream during mix preparation and has more body on freezing. Ice creams were prepared with and without cocoa solids and frequency sweeps were made from 0.1 to 100 Hz at 0.1% strain. With rapidly frozen ice creams, both G' and G" increased in the presence of cocoa solids. Comparison of mixes made with and without low-fat cocoa powder or non-gelatinizing starch demonstrated a similar relationship, with higher apparent viscosities in those mixes containing either cocoa powder or the starch. The results were consistent with the cocoa particles adding to the effect of the fat globules in increasing viscosity.

Detection of somaclonal variation during cocoa somatic embryogenesis characterised using cleaved amplified polymorphic sequence and the new freeware Artbio

The scarcity and stochastic nature of genetic mutations presents a significant challenge for scientists seeking to characterise de novo mutation frequency at specific loci. Such mutations can be particularly numerous during regeneration of plants from in vitro culture and can undermine the value of germplasm conservation efforts. We used cleaved amplified polymorphic sequence (CAPS) analysis to characterise new mutations amongst a clonal population of cocoa plants regenerated via a somatic embryogenesis protocol used previously for cocoa cryopreservation. Efficacy of the CAPS system for mutation detection was greatly improved after an ‘a priori’ in silico screen of reference target sequences for actual and potential restriction enzyme recognition sites using a new freely available software called Artbio. Artbio surveys known sequences for existing restriction enzyme recognition sites but also identifies all single nucleotide polymorphism (SNP) deviations from such motifs. Using this software, we performed an in silico screen of seven loci for restriction sites and their potential mutant SNP variants that were possible from 21 restriction enzymes. The four most informative locus-enzyme combinations were then used to survey the regenerant populations for de novo mutants. We characterised the pattern of point mutations and, using the outputs of Artbio, calculated the ratio of base substitution in 114 somatic embryo-derived cocoa regenerants originating from two explant genotypes. We found 49 polymorphisms, comprising 26.3% of the samples screened, with an inferred rate of 2.8 × 10−3 substitutions/screened base. This elevated rate is of a similar order of magnitude to previous reports of de novo microsatellite length mutations arising in the crop and suggests caution should be exercised when applying somatic embryogenesis for the conservation of plant germplasm.

Progressive erosion of genetic and epigenetic variation in callus-derived cocoa (Theobroma cacao) plants

Relatively little is known about the timing of genetic and epigenetic forms of somaclonal variation arising from callus growth. We surveyed for both types of change in cocoa (Theobroma cacao) plants regenerated from calli of various ages, and also between tissues from the source trees. For genetic change, we used 15 single sequence repeat (SSR) markers from four source trees and from 233 regenerated plants. For epigenetic change, we used 386 methylation-sensitive amplified polymorphism (MSAP) markers on leaf and explant (staminode) DNA from two source trees and on leaf DNA from 114 regenerants. Genetic variation within source trees was limited to one slippage mutation in one leaf. Regenerants were far more variable, with 35% exhibiting at least one mutation. Genetic variation initially accumulated with culture age but subsequently declined. MSAP (epigenetic) profiles diverged between leaf and staminode samples from source trees. Multivariate analysis revealed that leaves from regenerants occupied intermediate eigenspace between leaves and staminodes of source plants but became progressively more similar to source tree leaves with culture age. Statistical analysis confirmed this rather counterintuitive finding that leaves of ‘late regenerants’ exhibited significantly less genetic and epigenetic divergence from source leaves than those exposed to short periods of callus growth.

Prebiotic evaluation of cocoa-derived flavanols in healthy humans by using a randomized, controlled, double-blind, crossover intervention study

BACKGROUND: The absorption of cocoa flavanols in the small intestine is limited, and the majority of the flavanols reach the large intestine where they may be metabolized by resident microbiota. OBJECTIVE: We assessed the prebiotic potential of cocoa flavanols in a randomized, double-blind, crossover, controlled intervention study. DESIGN: Twenty-two healthy human volunteers were randomly assigned to either a high-cocoa flavanol (HCF) group (494 mg cocoa flavanols/d) or a low-cocoa flavanol (LCF) group (23 mg cocoa flavanols/d) for 4 wk. This was followed by a 4-wk washout period before volunteers crossed to the alternant arm. Fecal samples were recovered before and after each intervention, and bacterial numbers were measured by fluorescence in situ hybridization. A number of other biochemical and physiologic markers were measured. RESULTS: Compared with the consumption of the LCF drink, the daily consumption of the HCF drink for 4 wk significantly increased the bifidobacterial (P < 0.01) and lactobacilli (P < 0.001) populations but significantly decreased clostridia counts (P < 0.001). These microbial changes were paralleled by significant reductions in plasma triacylglycerol (P < 0.05) and C-reactive protein (P < 0.05) concentrations. Furthermore, changes in C-reactive protein concentrations were linked to changes in lactobacilli counts (P < 0.05, R(2) = -0.33 for the model). These in vivo changes were closely paralleled by cocoa flavanol-induced bacterial changes in mixed-batch culture experiments. CONCLUSION: This study shows, for the first time to our knowledge, that consumption of cocoa flavanols can significantly affect the growth of select gut microflora in humans, which suggests the potential prebiotic benefits associated with the dietary inclusion of flavanol-rich foods. This trial was registered at clinicaltrials.gov as NCT01091922.

In vitro bioaccessibility and gut biotransformation of polyphenols present in the water-insoluble cocoa fraction

Scope: Cocoa, especially the water-insoluble cocoa fraction (WICF), is a rich source of polyphenols. In this study, sequential in vitro digestion of the WICF with gastrointestinal enzymes as well as its bacterial fermentation in a human colonic model system were carried out to investigate bioaccessibility and biotransformation of WICF polyphenols, respectively. Methods and results: The yield of each enzymatic digestion step and the total antioxidant capacity (TAC) were measured and solubilized phenols were characterized by MS/MS. Fermentation of WICF and the effect on the gut microbiota, SCFA production and metabolism of polyphenols was analyzed. In vitro digestion solubilized 38.6% of WICF with pronase and Viscozyme L treatments releasing 51% of the total phenols from the insoluble material. This release of phenols does not determine a reduction in the total antioxidant capacity of the digestion-resistant material. In the colonic model WICF significantly increased of bifidobacteria and lactobacilli as well as butyrate production. Flavanols were converted into phenolic acids by the microbiota following a concentration gradient resulting in high concentrations of 3-hydroxyphenylpropionic acid (3-HPP) in the last gut compartment. Conclusion: Data showed that WICF may exert antioxidant action through the gastrointestinal tract despite its polyphenols being still bound to macromolecules and having prebiotic activity.

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.

Genotypic variation in photosynthetic and leaf traits in cocoa

The photosynthetic characteristics of eight contrasting cocoa genotypes were studied with the aim of examining genotypic variation in maximum (light-saturated) photosynthetic rates, light-response curve parameters and water use efficiency. Photosynthetic traits were derived from single leaf gas exchange measurements using a portable infra-red gas analyser. All measurements were conducted in a common greenhouse environment. Significant variation was observed in light-saturated photosynthesis ranging from 3.4 to 5.7 µmol CO2 m-2 s-1 for the clones IMC 47 and SCA 6, respectively. Furthermore, analyses of photosynthetic light response curves indicated genotypic differences in light saturation point and quantum efficiency (i.e. the efficiency of light use). Stomatal conductance was a significant factor underlying genotypic differences in assimilation. Genotypic variation was also observed in a number of leaf traits, including specific leaf area (the ratio of leaf area to leaf weight), chlorophyll concentration and nitrogen content. There was a positive correlation between leaf nitrogen per unit area and light-saturated photosynthesis. Water use efficiency, defined as the ratio of photosynthetic rate to transpiration rate, also varied significantly between clones (ranging from 3.1 mmol mol-1 H2O for the clone IMC 47 to 4.2 mmol mol-1 H2O for the clone ICS 1). Water use efficiency was a negative function of specific leaf area, suggesting that low specific leaf area might be a useful criterion for selection for increased water use efficiency. It is concluded that both variation in water use efficiency and the photosynthetic response to light have the potential to be exploited in breeding programmes.