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Au cours de la dernière décennie, les accessions du manioc de la République Centrafricaine ont fait l’objet de nombreux travaux entre autres les cultures in vitro et la micropagation. Cependant les plants issus de la culture in vitro nécessitent une période d'acclimatation avant leur transfert en champ, d’où l’intérêt de cette étude dont l’objectif était d’évaluer l’effet de la qualité du sol et des facteurs de croissance sur l’acclimatation. Des explants de jeunes pousses obtenus à partir de la culture des segments nodaux sous serre étaient transplantés dans des tubes à essai contenant le milieu MS et placés dans la salle de croissance. Les microboutures issues de la culture in vitro ont été traitées avec des phytohormones de croissances et repiquées dans des bacs de culture contenant le fumier et le sol et replacées sous serre. En outre, les plantules issues de la culture in vitro ont été transférées ex vitro en entier dans des pots de culture contenant le fumier et le sol pour suivie sous serre. La micropropagation ex vitro a montré le meilleur taux de survie dans le fumier que dans le mélange respectivement de 90% et 45%. Une meilleure croissance en hauteur a été révélée dans le fumier que dans le sol avec respectivement 2,68 cm et 1,05 cm. Ces observations étaient également confirmées par le plan d’expérience de transfert ex vitro des plantules entières dans les pots. Les données auxiniques sur le fumier ont révélé que l’Acide Indole-3-Acétique (ANA) semble être plus indiquée pour l’acclimatation que Acide Indole-3-Butyrique (AIB) et Acide α-Naphtalène acétique (ANA). Cette technique développée sur une accession de manioc pourrait être étendue à d'autres accessions et d’autres espèces végétales d’intérêt agronomique. Over the last decade, cassava accessions in the Central African Republic have been the subject of numerous studies, including in vitro culture and micropagation. However, the acclimatization of in vitro-grown plantlets to an ex vitro environment by gradually weaning them towards ambient relative humidity for the better survival of young and physiologically sensitive plantlets. This study therefore aimed to evaluate the effect of soil quality and growth factors on acclimatization. Sprouted shoots obtained from greenhouse nodal segment culture were transplanted into test tube containing MS medium were incubated in a controlled growth room. Microcuttings from in vitro culture were treated with growth phytohormones and transplanted in culture trays containing manure and soil and placed in the greenhouse. In addition, the seedlings from in vitro culture were transferred ex vitro whole into culture pots containing manure and soil for follow-up in the greenhouse. The ex vitro micropropagation showed the best survival rate in manure than in the mixture of 90% and 45% respectively. Better height growth was revealed in manure than in soil with 2.68 cm and 1.05 cm respectively. These observations were also confirmed by the experimental design of ex vitro transfer of whole seedlings in pots. The auxin data on manure revealed that Indole-3-Acetic Acid (IAA) seems to be more suitable for acclimatization than Indole-3-Butyric Acid (IBA) and α-Naphthalene Acetic Acid (NAA). This technique developed on a cassava accession could be extended to other accessions and other plant species of agronomic interest.

The quality of chocolate products proven by consumer acceptability mostly depends on cocoa volatile compounds. In fact, aroma profile of cocoa or chocolate is correlated to cocoa genotype, culture conditions, postharvest practices, and chocolate manufacturing. To the best of our knowledge, no study has been published on both sugars and aromas of controlled cocoa hybrids from Cameroon. The present study aimed at analyzing biochemical and volatile composition of six hand-pollinated cocoa hybrids belonging to different traditional groups. Specifically, the sugar profile was obtained by high performance ion chromatography-electrochemical detector (HPIC-ED) and aroma composition was analyzed using headspace solid phase micro-extraction coupled with gas chromatography-mass spectrometry (HS-SPME/GC-MS) on fermented dried beans and roasted nibs. The content of sugars was variable among hybrids and fructose showed the highest concentration in all hybrids in the two matrices. Roasting decreased sugar content. However, the decrease in sugar content seemed genotype dependent. A total of 48 volatile aromas were classified in six groups according to their odor description (fruity, floral, chocolate/nutty, buttery/creamy, undesirable, and unknown). Fruity flavors were not specific to a given chemical group. However, floral volatiles were specific to terpenes, chocolate/nutty to pyrazines, buttery/creamy to lactones and undesirable to phenols and acids. Principal component analysis (PCA) showed specificity of each hybrid and the hierarchical cluster analysis (HCA) Heatmap presented four distinct groups. SCA12 × ICS40 with the highest pyrazine concentration was associated with important content of sugars. Roasting of nibs reduced total volatiles and particularly fruity volatiles whereas other flavor groups moderately increased. In fact, some volatiles such as 2,3-butanediol, 2-nonanol, linalool, β-myrcene, cis/trans-β-ocimene, 2-nonanone, 2-heptanol, methyl acetate, acetophenone could be used as markers in separating traditional cocoa groups in Cameroon.

Flavonoids are phenolic compounds involved in defense mechanisms against black pod disease (BPD). Crossing between Forastero and Trinitario-resistant cocoa genotypes usually produce resistant progenies with high contents of bioactive components. This study aims at analyzing flavan-3-ol and flavonol content in the defense against P. megakarya in T. cacao genotypes for their potential selection as markers of partial resistance to BPD. Assessment of necrosis development and biochemical markers of stress (total polyphenols (TPP), total flavonoids (TF), and condensed tannins (CT)) through spectrophotometric methods of 19 genotypes identified eight hybrids that contained higher amounts (P˂0.05) of bioactive components than the better parent T79/467. The necrosis length revealed to be negatively correlated with metabolite concentrations (P

Black Sigatoka disease (BSD) is a foliar disease caused by Mycosphaerella fijiensis, responsible of reduction of the photosynthetic area of banana plant and yield at harvest since it has an influence on fruit physiology. The control of BSD relies on the use of chemicals which are not affordable for the small holder farmers and increase the cost of production. Moreover, this chemical control is ineffective, negatively impacting the environment and human health, and is at the origin of strain resistance. Tithonia diversifolia is known as rich in many compounds such as mineral elements, defense metabolites, some phytochemicals; and it is increasingly used in agriculture. Recently, the protective effect of Tithonia diversifolia liquid extract against BSD development on plantain vivoplants in the nursery was highlighted. The aim of our study was to evaluate the efficacy of a biopesticide base on Tithonia diversifolia on the BSD development in a plantain field under high disease pressure. The effect of Tithonia diversifolia biopesticide on Mycosphaerella fijiensis mycelial growth in vitro was evaluated. An experimental field at the flowering stage was selected and treated with the biopesticide base on Tithonia diversifolia at three different concentrations: undiluted (100%), diluted at 1/2 (50%) and diluted at 1/4 (25%) for 17 weeks. The disease severity, the number of functional leaves, the youngest spotted leaf (YSL) and the youngest necrotic leaf (YNL) were evaluated in course of time. The biopesticide treatments significantly (P Tithonia diversifolia could be a hopeful ecoresponsible solution for the plantain sector in general and in particular for poor small farmers.

Prunus africana is an endangered medicinal species and has been classified as a priority for domestication in Cameroon. However, the seeds rapidly lose their viability during storage at room temperature after 2–3 months. This study aimed to improve seed germination of P. africana by germinating at different temperatures (4 °C, 28 °C) using different concentrations of growth stimulators (sodium nitrate and gibberellic acid) and different concentrations of salts. P. africana seeds had 91.7% germination at 4 °C after 1 month of storage. Growth regulators considerably influenced germination after 6 months and reached 66.0% with 10 mM gibberellic acid and 100% with 10 mM sodium nitrate. Approximately three shoots per seed were developed, an indication of polyembryony. Histochemical analyses revealed the presence of protein-like bodies close to the embryo axis and accumulations of starch after 7 days of germination. After 14 days, amyloplasts and dark protein bodies of various sizes were observed. The outcome of this study will contribute to improve the germination of P. africana for better domestication and conservation.

The spatial location and timing of plant developmental events are largely regulated by the well balanced effects of auxin and cytokinin phytohormone interplay. Together with transport, localized metabolism regulates the concentration gradients of their bioactive forms, ultimately eliciting growth responses. In order to explore the dynamics of auxin and cytokinin metabolism during early seedling growth in Theobroma cacao (cacao), we have performed auxin and cytokinin metabolite profiling in hypocotyls and root developmental sections at different times by using ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS). Our work provides quantitative characterization of auxin and cytokinin metabolites throughout early root and hypocotyl development and identifies common and distinctive features of auxin and cytokinin metabolism during cacao seedling development.

Genetic diversity of species is an important baseline for the domestication process. In Cameroon, Prunus africana, an important and threatened medicinal tree, is among the priority species for domestication. The bark extract has been used to treat various diseases, mainly benign prostatic hyperplasia which affects men above the age of 50. As little is known about the genetic diversity of P. africana in Cameroon, we aimed to determine the genetic diversity and differentiation of several P. africana populations in the western provinces, using sets of chloroplast DNA markers and nuclear microsatellites previously developed for Prunus species. Genetic diversity in the observed populations was considerable and genetic differentiation between populations proved substantial with 21% of the total observed variation detected among populations, revealing a distinct genetic structure among certain populations. However, the lack of correlation between genetic and geographic distances does not support isolation by distance (IBD). The analysis of chloroplast DNA haplotypes revealed no strong phylogeographic component in the genetic structure observed in the western populations of P. africana in Cameroon. The outcome of this study will contribute to improve the genetic characterization of P. africana for its better domestication and conservation in the Cameroon agroforestry system.

Cacao fermentation induces biochemical changes in the beans that lead to different cacao grades such as slaty (unfermented), violet (under fermented) and brown (fully fermented) beans. This grade heterogeneity in a sample set can impact the quality of the end-products. In the present study, amino acids and polyphenol contents of slaty, violet and brown beans were evaluated. Free amino acids were derivatized from o–phthalaldehyde and detected with a fluorescence spectrophotometer coupled with a reverse phase HPLC system. Polyphenols were analysed at 280 nm by HPLC using a Photodiode Array Detector. Amino acids content in the violet beans were significantly higher (11,165 ± 4281 mg kg(−1) fat free dry material, ffdm) than that of the slaty beans (4304.5 ± 1927.6 mg kg(−1) ffdm), meanwhile there was no significant difference between violet and brown beans. Epicatechin, catechin, cyanidin-3-arabinoside and cyanidin-3-galactoside contents were significantly lower in violet and brown beans when compared to slaty beans. Our results have shown that a mixture of violet and brown beans is suitable to obtain polyphenols and amino acids in the development of cacao products. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13197-019-03749-y) contains supplementary material, which is available to authorized users.

Fresh, ripe cocoa beans from Cameroon (German cocoa/Amelonado group and ICS 40/Trinitario group) were subjected to fermentation-like incubations in acetic acid, lactic acid, or both and to natural fermentation. Two naturally fermented samples from Cuba (UF 654/Trinitario group and C 411/Criollo group) were also investigated. Both cyanidin-3-galactoside and cyanidin-3-arabinoside (found as major anthocyanins in colored beans only) were drastically degraded through fermentation, especially in small beans and in the presence of acetic acid. On the other hand, emergence of a cyanidin-rhamnose isomer was evidenced, even in Criollo beans. In addition to the recently described structures F1 and F2 [m/z = 575 in ESI(-)], three additional polyphenolic structures [F3, F4, and F5; m/z = 557 in ESI(+)] were found after fermentation, the two former ones resulting from epicatechin oxidation. Synthesis of F5 requires an interclass reaction between cyani(di)n and epicatechin, which explains its absence in fermented Criollo beans.

The biochemical reactions occurring inside cocoa beans during fermentation are mostly due to penetration of lactic and acetic acids issued from microbial activities. In the present study, fresh, ripe cocoa beans were subjected to three fermentation-like incubation schemes: incubation for 2 days at pH 4 in a solution of acetic acid, lactic acid, or both, followed by a 3-days incubation at pH 5 in acetic acid solution. After each treatment applied, the fermentation index was above 1 and a cut test revealed a brown color, characteristic of well-fermented beans. As shown by RP-HPLC-ESI(−)-MS/MS analysis, the main flavan-3-ols found in German Cocoa (Amelonado group) and ICS 40 (Trinitario group) ranked as follows: epicatechin > C1 > B2 > catechin > B5 > dehydrodiepicatechin A. In both natural fermentations and fermentation-like incubations, these compounds showed a sharp decrease, this effect being strongest when acetic acid was present from the start. Lactic acid exhibited a somewhat polyphenol-protective effect. Two procyanidins with a molecular weight of 576, undetected before fermentation, were evidenced here for the first time in both incubated and naturally fermented cocoa beans.

This study investigates the heritability of polyphenolic, anthocyanin and antioxidant capacity of beans derived from four cocoa clones and their offsprings. These compounds were analyzed at 280 nm (polyphenols) and 520 nm (anthocyanins) by reversed-phase high-performance liquid chromatography (RP-HPLC) method using a photodiode array detector (PDA). The antioxidant capacity of methanolic extracts prepared from cocoa beans was measured by the DPPH and ABTS assays. Samples displayed catechin and epicatechin as the two main polyphenols. Epicatechin, which represents 1 to 5% of defatted cocoa seed powder, was hundred-fold higher than catechin while cyanidin-3-arabinoside was three-fold higher than cyanidin-3-galactoside. The two main anthocyanins found in our samples represent about 0.05% of defatted cocoa seed powder. All these compounds were genotype-dependent. Unidentified substances called A, B and C were also found in cocoa seeds. Substance A is discussed as a derivative of caffeic acid and an ester-bound compound. Substances B and C are oligomers of proanthocyanidins. Antioxidant capacity of cocoa beans obtained by 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay was higher than those obtained by DPPH assay. According to correlation tests, flavanols contributed better in the antioxidant capacity than anthocyanin. No maternal effect was detected in the transmission of polyphenol compounds suggesting a nuclear heritability. Key words: Theobroma cacao, cocoa beans, polyphenolic compounds, anthocyanins, heritability.

Prunus africana (Kanda stick) is a valuable medicinal tree classified as priority species for domestication in Cameroon. To measure the ability for developing axillary buds and multiple shoots in relation with the origin and state of explants, some biochemical parameters were assessed in eight types of single-node cuttings. These cuttings were treated with different concentrations of kinetin (kin) indole butyric acid (IBA) and thidiazuron (TDZ) and cultured in Murashige and Skoog (MS) medium. The glutelin content (1.13 ± 0.201 µg/mg FW) was higher compared to those of total albumin, globulin and prolamin and most representative at mature vegetative apical stage (MVA). The highest (15.60 ± 1.966 µg/mg FW) and lower (2.82 ± 0.072 µg/mg FW) amount of phenol compound was observed in the mature dormant stages and immature vegetative distal stage (IVD), respectively. Regarding the peroxidases, highest activity (0.453 ± 0.055 OD/mg/ FW.5 min -1 ) was obtained in mature dormant apical stage. Apart from IVA state, there was no reactive explant cultured in vitro. The high percentage of budding and proliferation of budding (100%) and rooting (100%) were achieved with 12 μM Kin and 24 μM IBA, respectively. In the presence of 24 μM TDZ, multiple shoot was induced with a maximum of 5 shoots/explants. Micropropagation success of P. africana is influenced by cytokinin, physiological and biochemical status of single node cutting. Key words : Biochemical profile, Prunus africana, budding, multiple shoots, rooting.

The cacao tree (Theobroma cacao L.) is the only source for the key raw material in chocolate manufacturing. However, fresh cocoa seeds do not develop any chocolate flavour during chocolate manufacturing because they do not contain the precursors necessary. The latter are formed only during the fermentation process that the seeds are subjected to in the countries of origin. During this in most cases spontaneous fermentation of the fruit pulp surrounding the seeds, the pulp is degraded by yeasts and bacteria. This degradation results in heat and organic acid formation. Heat effect and tissue acidification are the key parameters guiding flavour precursor formation. Accordingly, not microorganisms themselves but exclusively their metabolites are necessary for successful fermentation. In the present study we subjected fresh cocoa seeds to three types of fermentation-like incubation with defined amounts of acetic acid, controlled temperature regime and in the absence of microorganisms. All raw cocoa samples were of high reproducible quality as determined by biochemical quality parameter analysis. We conclude that control of the heat effect and the acidity is the reason for these quality characteristics and that application of this technique may permit standardization and mechanization of cocoa fermentation.

Two dimensional electrophoresis and nano-LC–MS were performed in order to identify alterations in protein abundance that correlate with maturation of cacao zygotic and somatic embryos. The cacao pod proteome was also characterized during development. The recently published cacao genome sequence was used to create a predicted proteolytic fragment database. Several hundred protein spots were resolved on each tissue analysis, of which 72 variable spots were subjected to MS analysis, resulting in 49 identifications. The identified proteins represent an array of functional categories, including seed storage, stress response, photosynthesis and translation factors. The seed storage protein was strongly accumulated in cacao zygotic embryos compared to their somatic counterpart. However, sucrose treatment (60 g L −1 ) allows up-regulation of storage protein in SE. A high similarity in the profiles of acidic proteins was observed in mature zygotic and somatic embryos. Differential expression in both tissues was observed in proteins having high p I . Several proteins were detected exclusively in fruit tissues, including a chitinase and a 14-3-3 protein. We also identified a novel cacao protein related to known mabinlin type sweet storage proteins. Moreover, the specific presence of thaumatin-like protein, another sweet protein, was also detected in fruit tissue. We discuss our observed correlations between protein expression profiles, developmental stage and stress responses.

This study aims at establishing a temporary immersion technique for large-scale propagation of cocoyam (Xanthosoma sagittifolium). Sucrose was experimented with as a determinant factor for shoot multiplication in this culture system. The highest proliferation rate (68 ± 7) occurred with 20 g l−1 sucrose in the culture medium. This concentration appeared to be the optimal amount due to its promoting effect on plantlet development. The acclimatized plantlets showed a continuous effect of sucrose treatment during ex vitro growth, especially in low sucrose concentration. This fact is evidenced by the low survival rate (0.13 ± 0.12) and the poor chlorophyll content (1.180 ± 0.076 mg g−1) recorded on plantlets derived from 15 g l−1 of sucrose. The treatment with 60 g l−1 of sucrose prior to acclimatization was efficient for roots induction and elongation. The analysis of pH revealed a fluctuation from one subculture to another, with an overall pH decrease under all treatments tested and, thus, indicates that plants release proton during growth. This feature had an impact on in vitro plantlets growth. The potentiality of the temporary immersion technique to foster the production of Xanthosoma sagittifolium is discussed.

Carolus Linnaeus a ete le pere des reseaux phenologiques modernes. Dans son ouvrage " Philosophia Botanica ", il a decrit des methodes permettant d'elaborer des calendriers annuels des plantes precisant l'ouverture des feuilles, la floraison, la fructification et la chute des feuilles, le tout associe a des observations climatologiques " de facon a montrer les differences selon les regions " (Schnelle, 1955). Le Programme GLOBE (Global Learning and Observations to Benefit the Environment, www.globe.gov) fonde en 1998, a reconnu la valeur des observations phenologiques dans l'education, et encourage les etudiants a prendre des mesures valides sur le plan scientifique dans les domaines de l'atmosphere, de l'hydrologie, des sols et de la couverture vegetale/phenologie, associant la recherche scientifique et l'education. La description detaillee des etapes de croissance de plantes utiles, suivie d'une codification adequate, facilite la communication entre les scientifiques et les chercheurs, lorsque par ex. de nouvelles decouvertes scientifiques doivent etre transferees sous forme de procedures de gestion, ou lorsque des experiences menees sur un site de culture doivent etre adaptees a un autre site. Nous decrivons les etapes de la croissance d'especes de cacaoyers (Theobroma sp.) dans le monde entier afin de preparer les informations qui serviront de base a la gestion de la production, a des comparaisons d'etudes epidemiologiques de maladies, de schemas de croissance suivant differents facteurs environnementaux et de parametres genetiques specifiques a certains clones. La codification suit l'echelle " BBCH etendue " (BBCH = Biologische Bundesantalt, Bundessortenamt and CHemische Industrie, Allemagne), un systeme numerique qui distingue les etapes de croissance principale, secondaire et tertiaire. Chaque etape de croissance presentee, de la germination des semences jusqu'au developpement de la cime et a la recolte, est mise en correlation avec les pratiques de gestion generales. Cette echelle sera tres utile aux cacaoculteurs et aux chercheurs du monde entier pour permettre une meilleure communication, une planification plus efficace des pratiques de gestion et des experimentations. (Resume d'auteur)

Accessions of Cola acuminata , Cola nitida and Cola anomala were analysed based on the variation of polyphenol and alkaloid contents in order to gain insight on the genetic relationships within and between the taxonomic entities. These compounds were analysed at 280 nm by HPLC using a Photodiode Array Detector (PDA). Catechin, epicatechin, theobromine and caffeine were detected in Cola seeds. Among the three species, C. nitida is found to be highest in flavonoid and caffeine content while C. anomala possessed high amount of theobromine. Catechin was the dominant flavonoid. Caffeine was the major alkaloid in Cola seeds and was considered as one of the signature compounds due to its concentration range. The average concentrations in C. acuminata , C. nitida and C. anomala accessions were 11066 ± 3166, 13761 ± 2728 and 7013 ± 2369 mg/kg FW respectively. Garcinia kola seeds displayed higher levels of total phenolic compounds compared to Cola sp. and lacked purine alkaloids and catechin. The former displayed many unidentified polyphenolic compounds most of which were apolar. Principal component and cluster analyses of Cola sp. categorized accessions into two groups. The first chemotype encompassed C. anomala accessions and exhibited high theobromine content. The second cluster enclosed C. nitida and C. acuminata accessions and displayed high caffeine and catechin contents. Genetic distances between the accessions varied from 0.0 to 1.26. The importance of Cola phenolic compounds and alkaloids in food chemistry and chemotaxonomy is discussed.

Somatic embryogenesis in cacao is difficult and this species is considered as recalcitrant. Therefore, reformulation of culture media might be a breakthrough to improve its somatic embryogenesis. In cacao, acquisition of somatic embryogenesis competence involves three main stages: induction of primary callus, induction of secondary callus and embryo development. Screening for MgSO4 and K2SO4 concentrations for somatic embryo differentiation was conducted on three genotypes (Sca6, IMC67 and C151-61) at the three stages. The effect of these two salts in culture media appears to be most efficient at the embryo development stage. At this stage, high MgSO4 (24 mM) and K2SO4 (71.568 mM) in the culture media induced direct somatic embryos on staminodes and petals of the Sca6 and IMC67 genotypes. Media supplemented with 6.0 mM and 12.0 mM MgSO4 enabled high responsive of explants and produced high proportion of embryos. The positive effect of MgSO4 and K2SO4 on the acquisition of embryogenesis competence was further tested on seven cacao genotypes reputed as non embryogenic: SNK12, ICS40, POR, IMC67, PA121, SNK64 and SNK10. All these genotypes were able to produce somatic embryos depending on the MgSO4 concentration. Thus, our results showed that the recalcitrance of cacao to somatic embryo differentiation can be overcome by screening for the suitable MgSO4 or K2SO4 concentration. Studies of the influence of different K+/Mg2+ ratios (at normal sulphate concentration) on somatic embryo differentiation revealed that sulphate supply was the main factor promoting responsive explants and the proportion of embryos. Cysteine synthase isoforms showed patterns related to morphogenetic structures sustaining that sulphur supply and its assimilation improve somatic embryogenesis in cacao.

Little is known of the mechanisms that induce the dedifferentiation of a single somatic cell into a totipotent embryogenic cell that can either be regenerated or develop into an embryo and subsequently an entire plant. In this Opinion article, we examine the cellular, physiological and molecular similarities and differences between different plant stem cell types. We propose to extend the plant stem cell concept to include single embryogenic cells as a totipotent stem cell based on their capacity to regenerate or develop into an embryo under certain conditions. Our survey suggests that differences in chromatin structure might ensure that meristem-localized stem cells have supervised freedom and are pluripotent, and that embryogenic stem cells are unsupervised, autonomous and, hence, freely totipotent.

Polyphenols were analysed at 280 nm by HPLC device using a Photodiode Array Detector (PDA). Anthocyanins were separated with the SEP-PAK Vac 6cc 1000 mg (Waters) column and measured at 520 nm with a PDA. Nineteen cacao clones from Cameroon genebank were analysed. Fresh and fermented-like seeds were used. Two main polyphenols were present in our samples: catechin and epicatechin. Epicatechin represents 2–4% DM of defatted cocoa seed powder. Undefined substances called A, B and C were also found in cocoa seeds. Substance A is discussed as a derivative of caffeic acid and an ester-bound compound. Substances B and C are oligomeres of proanthocyanidins. Protocatechiuc acid and quercetin were not detected. Two anthocyanins were found in cocoa seeds: cyanidin-3-galactoside and cyanidin-3-arabinoside. They represent 0.02–0.4% DM of defatted cocoa seed powder. Total phenols, catechin, epicatechin and anthocyanin in fresh and fermented-like beans were genotype-dependent. Polyphenols from seeds of two different pods from the same clone showed a quantitative significant difference. Spearman's correlation test showed that there is no correlation between the number of seeds per pod, weight of pod and content of polyphenolic compounds. Nevertheless, a negative correlation was found between the number of seeds per pod and the catechin content ( r = - 0.4 6 3 , P 0.0 1 ). Groupings of samples were observed using PCA and hierarchical cluster analysis. The separation between groups is related to their polyphenol and anthocyanin contents.

Amino acids and soluble carbohydrates were analyzed in cacao (Theobroma cacao L.) pods to demonstrate whether or not there is a relationship between these compounds and the susceptibility of the different cacao clones to Phytophthora megakarya. Nine cacao clones were used: SNK10, UPA134, SNK13, SNK213, SNK64, ICS95, SNK416, ICS84 and SNK413. Analyses were carried out 5 days after pod infection. The clones SNK213 and SNK416, documented as mildly susceptible and less susceptible respectively, were found to be highly susceptible in our experiments. Seven amino acids (asparagine, cysteine, glycine, isoleucine, proline, serine and tyrosine) were identified but the occurrence of each in the pods varied with the genotype and with the treatment. Total amino acid content was 745% higher in the less susceptible clone SNK413 than in the highly susceptible clone SNK10. A significant negative correlation (rp=−0.646,P

Introduction. La plupart des travaux sur la multiplication in vitro de l'ananas ont utilise des explants de diverses origines, mais peu font etat de l'utilisation de couronnes pour l'obtention de plantules in vitro. Par ailleurs, la regeneration de plants effectuee via la formation de cals est susceptible d'induire des variations somaclonales. De ce fait, une methode de regeneration directe in vitro a partir des couronnes a ete testee. Materiel et methodes. Apres desinfection, des couronnes d'ananas de la variete cayenne ont ete cultivees sur un milieu liquide de Murashige et Skoog dilue de moitie, contenant (2 a 10) mg BAP × L-1 ou (2 a 12) mg kinetine × L-1. L'induction des bourgeons axillaires a ete observee apres 14 j de culture et le nombre de plantules developpees a ete compte apres 65 j. Certaines plantules ont alors ete acclimatees sur un melange de terre et vermiculite avant d'etre transferees au champ. Resultats et discussion. Les meilleurs taux d'induction et de developpement de plantules ont ete obtenus avec 4 mg BAP × L-1 (en moyenne 6,7 plantules par couronne en 65 j) et 6 mg kinetine × L-1 (11,9 plantules par couronne). Les plantules acclimatees et transferees au champ ont survecu a 100 %. Conclusion. Le demarrage direct d'un nombre important de bourgeons axillaires de couronne grâce a l'utilisation d'une cytokinine constitue une technique intermediaire entre les pratiques d'horticulture et la culture in vitro. Toutefois, la productivite des plants issus des plantules ainsi regenerees devra etre evaluee comparativement a celle des plants obtenus de facon traditionnelle afin d'en tester les performances.

Background Theobroma cacao L. is a tropical fruit tree, the seeds of which are used to create chocolate. In vitro somatic embryogenesis (SE) of cacao is a propagation system useful for rapid mass-multiplication to accelerate breeding programs and to provide plants directly to farmers. Two major limitations of cacao SE remain: the efficiency of embryo production is highly genotype dependent and the lack of full cotyledon development results in low embryo to plant conversion rates. With the goal to better understand SE development and to improve the efficiency of SE conversion we examined gene expression differences between zygotic and somatic embryos using a whole genome microarray. Results The expression of 28,752 genes was determined at 4 developmental time points during zygotic embryogenesis (ZE) and 2 time points during cacao somatic embryogenesis (SE). Within the ZE time course, 10,288 differentially expressed genes were enriched for functions related to responses to abiotic and biotic stimulus, metabolic and cellular processes. A comparison ZE and SE expression profiles identified 10,175 differentially expressed genes. Many TF genes, putatively involved in ethylene metabolism and response, were more strongly expressed in SEs as compared to ZEs. Expression levels of genes involved in fatty acid metabolism, flavonoid biosynthesis and seed storage protein genes were also differentially expressed in the two types of embryos. Conclusions Large numbers of genes were differentially regulated during various stages of both ZE and SE development in cacao. The relatively higher expression of ethylene and flavonoid related genes during SE suggests that the developing tissues may be experiencing high levels of stress during SE maturation caused by the in vitro environment. The expression of genes involved in the synthesis of auxin, polyunsaturated fatty acids and secondary metabolites was higher in SEs relative to ZEs despite lack of lipid and metabolite accumulation. These differences in gene transcript levels associated with critical processes during seed development are consistent with the fact that somatic embryos do not fully develop the large storage cotyledons found in zygotic embryos. These results provide insight towards design of improved protocols for cacao somatic embryogenesis.

Calli were induced in cacao cotyledon explants on a half-strength Murashige and Skoog medium containing 6 × 10-2 g m-3 saccharose and various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) with kinetin (kin), benzylaminopurine (BAP) or 2-isopentenylphosphate (2-iP). Experiments were carried out on two clones of cacao differing in their susceptibility to black pod disease. The highest percentage of explants forming callus and the most rapid callus development were obtained with 10-6 g m-3 2,4-D and 0.5× 10-6 g m-3 kin. Somatic embryogenesis and rhizogenesis were induced by transferring 3-week-old callus in a half strength Murashige and Skoog medium containing 3 × 10-2 g m-3 saccharose and NAA or IBA in the 0 to 5 × 10-6 g m-3 concentration range. No differentiation could be observed when the medium was supplemented with kin or BAP. The conversion of callus into somatic embryos and roots was accompanied by a drop in phenol content and an increase in peroxidase and IAA-oxidase activities. Moreover, cell differentiation was characterized by the persistence in the callus of one acidic soluble isoperoxidase which was not detected in nondifferentiating callus. Although some differences were noticed between the clones, alterations responsible for cell differentiation were the same in both genotypes.

Somatic embryogenesis is a useful tool for Theobroma cacao improvement and propagation. Depending on culture medium composition, different morphogenetic structures (including somatic embryo) occur in response to alteration of genes expression patterns and biochemical changes. The effect of SO42- ion deficiency in culture media on somatic embryogenesis was studied through sequential replacement of MgSO4 and K2SO4 by MgCl2 and KCl, respectively, at different steps of somatic embryogenesis. It appears that explants gradually lost their embryogenic competence as the period of exposition to sulphur free medium increases. These results suggest that, sulphur availability and the duration to sulphur exposition might modulate the expression of genes involved in somatic embryo differentiation in T. cacao. Cysteine, glutathione, reducing sugars, cysteine synthase and cysteine desulfurase activities were analysed in different morphogenetic structures obtained in vitro. Cysteine and reducing sugars contents appeared to be higher in embryogenic calli than their nonembryogenic homologues, whereas glutathione content appears to be lower in embryogenic calli. Cysteine synthase activities also discriminate the embryogenic calli from non embryogenic calli. In the embryogenic calli, the ratio cysteine synthase/cysteine desulfurase activities were above unit. The assimilation of exogenous sulphur (sulphate) for the synthesis of cysteine might hence be crucial for somatic embryogenesis in T. cacao. This explains the reduction and the absence of somatic embryo response observed during sulphur depletion in culture media. Sulphur nutrition is therefore critical in cacao somatic embryogenesis.Keyswords: Cacao, embryo, sulphate, cysteine synthase, glutathione, deficiencyAfrican Journal of Biotechnology Vol. 9(35), pp. 5665-5675, 30 August, 2010

Cocoa ( Theobroma cacao L.) is an important allogamous tropical tree crop, whose centre of diversity is considered to be in Central America. Dry cocoa beans from five cocoa clones, and their intercrossed hybrids were analysed based on the variation of alkaloids and polyphenolic compounds contents, in order to gain insights on the heterosis and broad-sense heritability. Polyphenols and alkaloids were analysed at 280 nm by HPLC, using a Photodiode Array Detector (PDA); while anthocyanins were separated with the SEP-PAK Vac 6cc 1000 mg (waters) column and measured at 520 nm with a PDA. Dry cocoa beans displayed high content of purine alkaloids (2.1 and 8.8 mg g-1 for caffein and theobromine, respectively), and polyphenols (25 and 2978 μg g-1 for catechin and epicatechin, respectively). Among the five cocoa clones, SNK16 was the highest in purine alkaloid (caffein and theobromin) and flavanol (catechin and epicatechin); while T79/467 possessed the greatest quantity of cyanidin-3-galactoside and cyanidin-3-arabinoside. From all the parameters studied, anthocyanins (Cyanidin- 3-galactoside and cyanidin-3-arabinoside) exhibited the highest level of heterosis. Parental genotypes SNK16 and T79/467 showed good aptitudes for the combination of characters because their reciprocal hybrids F5 and F9, distinguished themselves by better levels of mid-parent heterosis values. Besides, the heritability value in strict sense of this Cyanidin-3-galactoside was very high. Absence of significant difference between genotypes, coming from reciprocal crossbreeding for Cyanidin-3-galactoside, suggests that this character in cocoa would be nuclear contrary to purine alkaloids and flavan-3-ols, where their transmission to offsprings can be stated as cytoplasmic. Key Words : Caffein, catechin, Theobroma cacao

The present study aimed at developing temporary immersion bioreactor techniques for multiplication of cacao somatic embryos. Temporary Immersion System (TIS), i.e. flooding of plant tissue at regular time intervals provides an efficient way to propagate plants. Somatic embryos were regenerated in twin flask bioreactors. The TIS proved to be suitable for mass regeneration of somatic embryos and for their subsequent direct sowing. The number of embryos after 3 months of culture was significantly higher in TIS cultures than in the solid medium variant. TIS also improved embryo development regarding the conversion to torpedo shaped forms. Matured embryos derived from TIS and pre-treated with 6% sucrose were converted into plants after direct sowing. Additionally to the influence of culture conditions on the development of somatic embryogenesis the content and composition of free amino acids were analysed. The content of free amino acids in somatic embryos rose as immersion frequency increased. The endogenous free GABA content in embryogenic callus was significantly higher than in non-embryogenic callus.

Theobroma cacao L., an economically important crop for developing countries, can be experimentally propagated by somatic embryogenesis. Because of their potential roles in embryogenesis, a gene candidate strategy was initiated to find gene homologues of the members of the leafy cotyledon family of transcription factors. A homologue of the leafy cotyledon1-like gene, that encodes the HAP 3 subunit of the CCAAT box-binding factor, was found in the cocoa genome (TcL1L). The translated peptide shared a high amino acid sequence identity with the homologous genes of Arabidopsis thaliana, Phaseolus coccineus and Helianthus annuus. TcL1L transcripts mainly accumulated in young and immature zygotic embryos, and, to a lesser extent, in young and immature somatic embryos. In situ hybridization specified the localization of the transcripts as being mainly in embryonic cells of young embryos, the meristematic cells of the shoot and root apex of immature embryos, and in the protoderm and epidermis of young and immature embryos, either zygotic or somatic. Non-embryogenic explants did not show TcL1L expression. Ectopic expression of the TcL1L gene could partially rescue the Arabidopsis lec1 mutant phenotype, suggesting a similarity of function in zygotic embryogenesis.

Cacao ( Theobroma cacao L.) clones that differ in susceptibility to black pod disease were analysed for response to stress induced by pod inoculation with the fungus Phytophthora megakarya Braz. Et Griff. Fungal inoculation significantly stimulated β-1,3-glucanase activity in both soluble and ionically-bound fractions of the less susceptible clones. No significant correlation was found in the other clones. Chitinase activity in both soluble and ionically-bound fractions was generally reduced upon pod inoculation with the fungus, but this pattern did not correlate with disease severity. Polyphenoloxidase activity, following pod inoculation, was genotype dependent in the soluble fractions, whereas in the ionically-bound fractions, stimulation of this activity was recorded in the less susceptible clones. Moreover, pod inoculation was characterised by the appearance of a new polyphenoloxidase isoform A\'2 in the less susceptible clones. Key Words: Absorbance, cortex, ionically-bound, Theobroma cacao, wonding Resume Les reponses aux stress induits par innoculation de gousses de cacao (Theobroma cacao L.), aux susceptibilites differentes a la maladie de Blackpod, par le Phytophthora megakarya Braz etaient analysees. L\'inoculation de fungi a significativement stimule les activites de « glucanase » β-1,3 dans les deux fractions solubles et a liaison ionique des clones moins susceptibles. Pas des correlations significatives etaient observees dans d\'autres clones. L\'activite de la chitanase dans les deux milieux etait generalement reduite juste apres l\'inoculation par le fongi, mais cette tendance n\'a pas correlee avec la severite de la maladie. L\'activite du polyphenoloxidase, apres inoculation de gousses, etait generalement dependante du genotype dans la fraction soluble, alors que la stimulation de cette activite etait observee dans les clones moins susceptibles. En plus, l\'inoculation des gousses etait caracterisee par l\'apparition de nouveau polyphenolosidase isoformes A\'2 dans les clones les moins susceptibles. Mots Cles: Absorbance, cortex, ionically-bound, Theobroma cacao, wonding (Af Crop Sci J 2003 Vol 11 No 2 pp.97-106)