Chromatin differentiation between Theobroma cacao L. and T. grandiflorum Schum

A comparative analysis of mitotic chromosomes of Theobroma cacao (cacao) and T. grandiflorum (cupuaçu) was performed aiming to identify cytological differences between the two most important species of this genus. Both species have symmetric karyotypes, with 2n = 20 metacentric chromosomes ranging in size from 2.00 to 1.19 μm (cacao) and from 2.21 to 1.15 μm (cupuaçu). The interphase nuclei of both species were of the arreticulate type, displaying up to 20 chromocentres, which were more regularly shaped in cacao than in cupuaçu. Prophase chromosomes of both species were more condensed in the proximal region, sometimes including the whole short arm. Both species exhibited only one pair of terminal heterochromatic bands, positively stained with chromomycin A ₃ , which co-localized with the single 45S rDNA site. Each karyotype displayed a single 5S rDNA site in the proximal region of another chromosome pair. Heterochromatic bands were also observed on the centromeric/pericentromeric regions of all 20 chromosomes of cacao after C-banding followed by Giemsa or DAPI staining, whereas in cupuaçu they were never detected. These data suggest that the chromosomes of both species have been largely conserved and their pericentromeric chromatin is the only citologically differentiated region.     

Serological relationships of some viruses from cocoa (Theobroma cacao L.) in Ghana

Strains A and Anibil of cocoa swollen-shoot virus are serologically closely related. They are more distantly related to strain M and cocoa mottle-leaf virus (Kpeve isolate), which are distantly related to one another. Strains A and Anibil protected against one another in cocoa seedlings, but no protection was found with any other combination between these four virus isolates. All mealybug-transmitted cocoa viruses in West Africa are probably best considered as strains of cocoa swollen-shoot virus.     

Microsatellite markers developed from Theobroma cacao L. expressed sequence tags

Theobroma cacao L. expressed sequence tags (ESTs) were converted into useful genetic markers for fingerprinting individuals and genetic linkage mapping. Primers were designed to microsatellite‐containing ESTs. Twenty‐two T. cacao accessions, parents of various mapping populations segregating for disease resistance and crop yield characteristics, were tested. Twenty‐seven informative loci were discovered with 26 primer pairs. The number of detected alleles ranged from two to 11 and averaged 4.4 per locus. All 27 markers could be mapped into at least one of the existing F₁ or F₂ populations segregating for agronomically important traits.     

A genetic linkage map of Theobroma cacao L.

A linkage map of the cocoa genome comprising 193 loci has been constructed. These loci consist of 5 isozymes, 101 cDNA/RFLPs, 4 loci from genes of known function, 55 genomic DNA/RFLPs and 28 RAPDs. A population of 100 individuals derived from a cross between two heterozygous genotypes was used. Segregation analyses were performed with the JoinMap program. Ten linkage groups, which putatively correspond to the ten gametic chromosomes of cocoa, were identified. The map covers a total length of 759 cM with a 3.9 cM average distance between 2 markers. A small fraction (9%) of the markers deviated significantly from the expected Mendelian ratios.     

The regulation of triacylglycerol biosynthesis in cocoa (Theobroma cacao) L.

Developing cocoa cotyledons accumulate initially an unsaturated oil which is particularly rich in oleate and linoleate. However, as maturation proceeds, the characteristic high stearate levels appear in the storage triacylglycerols. In the early stages of maturation, tissue slices of developing cotyledons (105 days post anthesis, dpa) readily accumulate radioactivity from [¹⁴C]acetate into the diacylglycerols and label predominantly palmitate and oleate. In older tissues (130 dpa), by contrast, the triacylglycerols are extensively labelled and, at the same time, there is an increase in the percentage labelling of stearate. Thus, the synthesis of triacylglycerol and the production of stearate are co-ordinated during development. The relative labelling of the phospholipids (particularly phosphatidylcholine) was rather low at both stages of development which contrasts with oil seeds that accumulate a polyunsaturated oil (e.g. safflower). Microsomal membrane preparations from the developing cotyledons readily utilised an equimolar [¹⁴C]acyl-CoA substrate (consisting of palmitate, stearate and oleate) and glycerol 3-phosphate to form phosphatidate, diacylglycerol and triacylglycerol. Analysis of the [¹⁴C]acyl constituents at the sn-1 and sn-2 positions of phosphatidate and diacylglycerol revealed that the first acylase enzyme (glycerol 3-phosphate acyltransferase) selectively utilised palmitate over stearate and excluded oleate, whereas the second acylase (lysophosphatidate acyltransferase) was highly selective for the unsaturated acyl-CoA. On the other hand, the third acylase (diacylglycerol acyltransferase) exhibited an almost equal selectivity for palmitate and stearate. Thus, stearate is preferentially enriched at position sn-3 of triacylglycerol at 120–130 dpa because of the relatively higher selectivity of the diacylglycerol acyltransferase for this fatty acid compared with those of the other two acylation enzymes.Abbreviation dpa days post anthesis We are grateful to Drs. G. Pettipher (Cadbury-Schweppes, Reading, UK), M. End and P. Hadley (Department of Horticulture, University of Reading) for the supply of cocoa pods and to the Agricultural and Food Research Council for financial support. We also wish to thank Dr. S. Stymne (Swedish University of Agricultural Sciences, Uppsala, Sweden) for a generous gift of acyl-CoA substrates.     

Gene discovery and microarray analysis of cacao (Theobroma cacao L.) varieties

The cacao bean harvest from the relatively under developed tropical tree cacao (Theobroma cacao L.) is subject to high losses in potential production due to pests and diseases. To discover and understand the stability of putative natural resistance mechanisms in this commodity crop, essential for chocolate production, we undertook a gene-discovery program and demonstrated its use in gene-expression arrays. Sequencing and assembling bean and leaf cDNA library inserts produced a unique contig set of 1,380 members. High-quality annotation of this gene set using Blast and MetaFam produced annotation for 75% of the contigs and allowed us to identify the types of gene expressed in cacao beans and leaves. Microarrays were constructed using amplified inserts of the uni-gene set and challenged with bean and leaf RNA from five cacao varieties. The microarray performed well across the five randomly chosen cacao genotypes and did not show a bias towards either leaf or bean tissues. This demonstrates that the gene sequences are useful for microarray analysis across cacao genotypes and tissue types. The array results, when compared with real-time PCR results for selected genes, showed a correlation with differential gene-expression patterns.We intend that the resultant DNA sequences and molecular microarray platform will help the cacao community to understand the basis, likely stability and pathotype resistance range of candidate cacao plants.     

The relationship between lignin and morphological characteristics of the tracheary elements from cacao(Theobroma cacao L.) Hulls

Tracheary elements (TEs) were physically separated from the hulls of cacao pods(Theobroma cacao L). Their morphological features were extensively investigated with scanning electron microscopy and chemical characterization. Spiral TEs were covered with a thin layer of primary wall that had a web-like structure on its outer surface. These TEs had a spiral circularity diameter of 8.2 ± 0.6 μm and an estimated secondary wall thickness of about 2.1 ± 0.2 μm. Polarized microscopy analysis revealed that the cellulose microfibrils were aligned parallel to that thickening. Lignin content was 36.1%, with a 0.13:1.00 molar ratio of syringyl to guaiacyl units and a 1.09:1.00 molar ratio of erythronic acid and threonic acid. Total yields of the alkaline nitrobenzene oxidation and ozonation products were 324.5 and 148.8 μmol g^-1 of extract-free TEs, respectively. Based on these morphological and lignin characteristics, we conclude that fully ripened cacao hulls exhibit the same features of secondary wall thickening as those seen at an earlier stage.     

Somatic embryogenesis and plant regeneration from floral explants of cacao (Theobroma cacao L.) using thidiazuron

Summary A procedure for the regeneration of cacao (Theobroma cacao) plants from staminode explants via somatic embryogenesis was developed. Rapidly growing calli were induced by culturing staminode explants on a DKW salts-based primary callus growth (PCG) medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and thidiazuron (TDZ) at various concentrations. Calli were subcultured onto a WPM salts-based secondary callus growth medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and 1.4 nM kinetin. Somatic embryos were formed from embryogenic calli following transfer to a hormone-free DKW salts-based embryo development medium containing sucrose. The concentration of TDZ used in PCG medium significantly affected the rate of callus growth, the frequency of embryogenesis, and the number of somatic embryos produced from each responsive explant. A TDZ concentration of 22.7 nM was found to be the optimal concentration for effective induction of somatic embryos from various cacao genotypes. Using this procedure, we recovered somatic embryos from all 19 tested cacao genotypes, representing three major genetic group types. However, among these genotypes, a wide range of variation was observed in both the frequency of embryogenesis, which ranged from 1 to 100%, and the average number of somatic embryos produced from each responsive explant, which ranged from 2 to 46. Two types of somatic embryos were identified on the basis of their visual appearance and growth behavior. A large number of cacao plants have been regenerated from somatic embryos and established in soil in a greenhouse. Plants showed morphological and growth characteristics similar to those of seed-derived plants. The described procedure may allow for the practical use of somatic embryogenesis for clonal propagation of elite cacao clones and other applications that require the production of a large number of plants from limited source materials.     

Physiological and biochemical responses of Theobroma cacao L. genotypes to flooding

Flooding is common in lowlands and areas with high rainfall or excessive irrigation. A major effect of flooding is the deprivation of O₂ in the root zone, which affects several biochemical and morphophysiological plant processes. The objective of this study was to elucidate biochemical and physiological characteristics associated with tolerance to O₂ deficiency in two clonal cacao genotypes. The experiment was conducted in a greenhouse with two contrasting clones differing in flood tolerance: TSA-792 (tolerant) and TSH-774 (susceptible). Leaf gas exchange, chlorophyll (Chl) fluorescence, chemical composition and oxidative stress were assessed during 40 d for control and flooded plants. Flooding induced a decrease in net photosynthesis, stomatal conductance and transpiration of both genotypes. In flood conditions, the flood-susceptible clone showed changes in chlorophyll fluorescence, reductions in chlorophyll content and increased activity of peroxidase and polyphenol oxidase. Flooding also caused changes in macro- and micronutrients, total soluble sugars and starch concentrations in different plant organs of both genotypes. Response curves for the relationship between photosynthetically active radiation (PAR) and net photosynthetic rate (P _N) for flooded plants were similar for both genotypes. In flood conditions, the flood-susceptible clone exhibited (1) nonstomatal limitations to photosynthesis since decreased in maximum potential quantum yield of PSII (F_v/F_m) values indicated possible damage to the PSII light-harvesting complex; (2) oxidative stress; (3) increased leaf chlorosis; and (4) a reduction in root carbohydrate levels. These stresses resulted in death of several plants after 30 d of flooding.     

Genetic diversity and relationships of cacao (Theobroma cacao L.) in southern Mexico

 Neotropical tree crops are affected by a combination of biological and human factors that complicate the study of genetic diversity and crop evolution. Genetic diversity and relationships among southern Mexican populations and horticultural collections of Theobroma cacao (chocolate, cocoa, cacao) are examined in light of the agricultural practices of the Maya. Collections of cacao were obtained from the extremes of its geographic range including archeological sites in southern Mexico where cacao was first domesticated. Genetic diversity was assayed by 57 informative random amplified polymorphic DNA (RAPD) marker loci. A unique sample of the total diversity found in this study exists in the southern Mexican populations. These populations are significantly different from all other cacao with regards to their profile of RAPD bands, including the ‘criollo’ variety, their morphological and geographical group. A population of cacao found in a sinkhole (cenote) in northern Yucatan with genetic affinities to populations in Chiapas suggests the Maya maintained plants far away from their native habitat. This finding concurs with known agroforestry practices of the Maya. Modern efforts to increase germplasm of tropical tree crops such as cacao should carefully examine archeological sites where genetic diversity, either deliberately or by chance, was collected and maintained by ancient cultures. Received: 21 May 1997 / Accepted: 9 October 1997     

A comparison between Theobroma cacao L. zygotic embryogenesis and somatic embryogenesis from floral explants

Summary In order to improve the late phases of Theobroma cacao L. embryogenesis from tissues of maternal origin, zygotic embryogenesis and somatic embryogenesis were compared, with respect to morphological, histological, and physiological parameters. Zygotic embryogenesis could be divided into three steps: (a) embryogenesis sensu stricto, (b) a growth period in which cotyledonary embryos reached their final dimensions, and (c) a maturation period in which embryos accumulated protein and starch reserves, dehydrated to a water content equal to 30%, and underwent a modification in soluble sugar composition. Monosaccharides and sucrose contents decreased to the benefit of the oligosaccharides raffinose and stachyose. The formation of somatic embryos by use of basic protocols was studied to define the limiting factors that could lie behind their poor development. Morphological abnormalities of somatic embryos, which represented 80% of the total population, were described. A histological study showed that somatic embryos lacked starch and protein reserves; moreover, their water content was much higher than that of their zygotic counterparts. Introducing a growth period into the culture protocol made for better embryo development. Adding sucrose and abscisic acid to the maturation medium was effective in increasing reserve synthesis and resulted in higher germination, conversion, and acclimatization rates.     

Localization and identification of phenolic compounds in Theobroma cacao L. somatic embryogenesis

Cocoa breeders and growers continue to face the problem of high heterogeneity between individuals derived from one progeny. Vegetative propagation by somatic embryogenesis could be a way to increase genetic gains in the field. Somatic embryogenesis in cocoa is difficult and this species is considered as recalcitrant. This study was conducted to investigate the phenolic composition of cocoa flowers (the explants used to achieve somatic embryogenesis) and how it changes during the process, by means of histochemistry and conventional chemical techniques. In flowers, all parts contained polyphenolics but their locations were specific to the organ considered. After placing floral explants in vitro, the polyphenolic content was qualitatively modified and maintained in the calli throughout the culture process. Among the new polyphenolics, the three most abundant were isolated and characterized by 1H- and 13C-NMR. They were hydroxycinnamic acid amides: N-trans-caffeoyl-l-DOPA or clovamide, N-trans-p-coumaroyl-l-tyrosine or deoxiclovamide, and N-trans-caffeoyl-l-tyrosine. The same compounds were found also in fresh, unfermented cocoa beans. The synthesis kinetics for these compounds in calli, under different somatic embryogenesis conditions, revealed a higher concentration under non-embryogenic conditions. Given the antioxidant nature of these compounds, they could reflect the stress status of the tissues.     

Structural Changes in the Embryonic Axis of Theobroma cacao L., during Germination and Early Seedling Establishment

The changes occurring in the axis of the developing seedlingof Theobroma cacao L. were observed over a 96-h period. It wasfound that during the first 24-h period, greatest change hadoccurred in the hypocotyl; the epicotyl and the root meristemhad apparently remained dormant. In the hypocotyl, vascularizationwhich had begun in the mature embryo continued: starch grainshad disappeared from the cells of the cortex and pith, the latterof which were seen to be binucleate. During the second 24-hperiod, greatest change was seen to have taken place in theroot meristem, resulting in the production of a root which consistedof a central pith, a poly-arch stele, and cortical tissue. Betweenthe root and the hypocotyl an apparently undifferentiated regionwas observed. This region was seen to have differentiated duringthe third 24-h period, leading to the production of a completering of xylem, external to which were groups of phloem, theformer being separated from the latter by parenchymatous tissue.This region is interpreted as being transitional between rootand shoot. During the fourth 24-h period, the adventitious andlateral root primordia initiated earlier, were seen to havedifferentiated to the extent that the disposition of their tissueswas evident.     

New unsaturated fatty acid and other chemical constituents from the roots of Cola rostrata K. Schum. (Malvaceae)

Phytochemical investigation of the roots of Cola rostrata K. Schum. led to the isolation of a new unsaturated fatty acid, named rostratanic acid (1), together with fourteen known compounds, lignoceric acid, friedelan (7), friedelanone (8), bauerenol (3), lupeol (4), herranone (9), acotatarone A (11), betulinic acid (6), betulin (5), nonanedioc acid (2), arjunolic acid (10) stigmasterol, β−sitosterol, and β−sitosterol-3-O-β-D-glucopyranoside. The structure of the new compound as well as those of the known compounds were established by means of spectroscopic methods: NMR analysis (¹H and ¹³C NMR, ¹H–¹H–COSY, HSQC and HMBC) and high-resolution mass spectrometry (HR-ESI-MS), and by comparison with previously reported data. Two of those known compounds were modified chemically to afford three new derivatives. All those compounds were tested for their cytotoxic activity against the human cervix carcinoma KB-3-1 cells and their antibacterial activity against Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa and Staphylococcus aureus. Although the crude extract gave weak antibacterial activity, none of the isolated compounds showed antibacterial activity, and, only the prenylated derivative showed weak cytotoxicity. In addition, the chemotaxonomic significance of the species Cola rostrata is discussed.     

Initiation and Growth of Callus and Cell Suspensions of Theobroma cacao L

Callus and suspension cultures of Theobroma cacao L., initiatedfrom immature cotyledons of beans from pods harvested 120–130days after pollination were established. A modified B-5 or Murashige—Skoogagar medium sustained growth of callus without loss of vigourafter each sub-culture. A 15-fold weight increase occurred duringthe 4 week culture periods at 30 ± 1 °C. Coconutwater improved callus growth substantially. The optimum hormonalconcentrations for growth of suspensions were 0.5 mg 1^–1of 2, 4-dichlorophenoxyacetic acid and 0.1 mg I^–1 of kinetinin a Murashige—Skoog basal medium liquid medium. The optimumtemperature for growth of suspensions was 25–30 °C.The cell number and cell mass of suspensions increased 20-foldin 14 days. No organogenesis or embryogenesis was observed. Theobroma cacao L., acao, cell culture, suspension culture, tissue culture.     

Effect of pollination intensity on fruit and seed set in cacao (Theobroma cacao L.)

We studied the functional relationship between pollination intensity and fruit survival as well as the number of seeds per pod in the tropical tree Theobroma cacao L. on a Forastero Upper-Amazon clone (UPA 409) in Ivory Coast. Cutting the style 24 h after pollination allowed for counting the number of pollen grains deposited on a stigma without affecting fruit set and seed development. Forty-three pollen grains were necessary to reach 50% of maximum fruit set 28 days after pollination. Above 115 pollen grains, the proportion of developing ovaries reached a maximum of 88% 28 days after pollination and 75% at maturity. With fewer than 238 pollen grains per stigma, there was a close relationship between pollination intensity and number of seeds per pod; the pollenseed ratio increased from 1.61 to 3.81 for PI increasing from 30 to 238 pollen grains. For higher pollination intensities, the average number of seeds per pod reached a maximum of 58. The relationship between pollination intensity and seed content was modelled. Results are consistent with the hypothesis that ovules attracted pollen tubes in a similar way regardless of whether or not they had already been reached by another pollen tube.