Tracing the native ancestors of the modern Theobroma cacao L. population in Ecuador

The native Theobroma cacao L. population from Ecuador, known as Nacional, is famous for its fine cocoa flavour. From the beginning of the twentieth century, however, it has been subjected to genetic erosion due principally to successive introductions of foreign germplasm whose hybrid descendants gradually replaced the native plantations, implying a decrease in cocoa quality. We attempted to trace this native cacao within a wide pool of modern Ecuadorian cacao population. Three hundred and twenty-two cacao accessions collected from different geographical areas along the pacific coast of Ecuador and maintained in two living collections were analysed using 40 simple-sequence repeat markers. Most of Ecuadorian cacao accessions displayed a high diversity and heterozygosity level. A factorial analysis of correspondence (FAC) showed a continuous variation among them, with a few ones, grouped at an extreme side of the FAC cloud, showing higher levels of homozygosity and lower introgression level by foreign cacaos. A paternity analysis revealed that these highly homozygous individuals are the most probable ancestors of the modern Nacional hybrid pool. These particular accessions studied could represent the native Nacional cacao present in Ecuador before the foreign introductions. Their identification will help to conserve valuable genetic material and to improve cocoa quality in new cacao varieties.     

Strcture of the Mature Embryo of Theobroma cacao L.

The mature embryo of Theobroma cacao L. consists of an embryonicaxis and two cotyledons. The cotyledonary tissue comprises thebulk of the embryo and contains mature and maturing vasculartissue surrounded by a bundle sheath. The cells of the groundtissue are filled with starch grains, anthocyanin pigments,or mucilage. The embryonic axis consists of epicotyl, hypocotyl,and root meristem. Maturation of the vascular tissue has begunin the procambium, where xylem maturation is ahead of phloemmaturation. Mucilage cells are found throughout the tissue of the axis.Most of the cells of the embryonic tissue contain starch grains.     

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.     

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.     

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.     

Phenol content, acidic peroxidase and IAA-oxidase during somatic embryogenesis in Theobroma cacao L.

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. This revised version was published online in July 2006 with corrections to the Cover Date.     

Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides

Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.     

Gene structure,phylogeny and expression profile of the sucrose synthase gene family in cacao (Theobroma cacao L.)

Journal of Genetics - In higher plants, sucrose synthase (Sus, EC 2.4.1.13) is widely considered as a key enzyme involved in sucrose metabolism. Although, several paralogous genes encoding...     

Molecular,Physiological and Biochemical Responses of Theobroma cacao L. Genotypes to Soil Water Deficit

Six months-old seminal plants of 36 cacao genotypes grown under greenhouse conditions were subjected to two soil water regimes (control and drought) to assess, the effects of water deficit on growth, chemical composition and oxidative stress. In the control, soil moisture was maintained near field capacity with leaf water potentials (ΨWL) ranging from −0.1 to −0.5 MPa. In the drought treatment, the soil moisture was reduced gradually by withholding additional water until ΨWL reached values of between −2.0 to −2.5 MPa. The tolerant genotypes PS-1319, MO-20 and MA-15 recorded significant increases in guaiacol peroxidase activity reflecting a more efficient antioxidant metabolism. In relation to drought tolerance, the most important variables in the distinguishing contrasting groups were: total leaf area per plant; leaf, stem and total dry biomass; relative growth rate; plant shoot biomass and leaf content of N, Ca, and Mg. From the results of these analyses, six genotypes were selected with contrasting characteristics for tolerance to soil water deficit [CC-40, C. SUL-4 and SIC-2 (non-tolerant) and MA-15, MO-20, and PA-13 (tolerant)] for further assessment of the expression of genes NCED5, PP2C, psbA and psbO to water deficit. Increased expression of NCED5, PP2C, psbA and psbO genes were found for non-tolerant genotypes, while in the majority of tolerant genotypes there was repression of these genes, with the exception of PA-13 that showed an increased expression of psbA. Mutivariate analysis showed that growth variables, leaf and total dry biomass, relative growth rate as well as Mg content of the leaves were the most important factor in the classification of the genotypes as tolerant, moderately tolerant and sensitive to water deficit. Therefore these variables are reliable plant traits in the selection of plants tolerant to drought.     

Distribution of assimilate during the flush cycle of growth in Theobroma cacao L.

The growth of the shoot and roots of seedling plants of cocoa (Theobroma cacao L.) under constant glasshouse conditions showed a rhythmic cycle, with the maximum growth stages of each alternating in a regular sequence. When the growth cycle of the shoot was upset by removing all new leaves immediately after unfolding, the roots showed a high constant growth rate during this period, suggesting that normally the rapidly expanding leaves exert an inhibitory influence on the roots. Conversely removal of portions of the root delayed the production of new leaves in the shoot. The level of soluble and starch carbohydrate in the mature leaves, roots and stem declined during the period of expansion of the flush leaves, but accumulated again at the end of the leaf expansion stage. It is likely that this reserve carbohydrate was remobilised and translocated to the flush leaves during their period of expansion. A large proportion of newly formed photoassimilate, as shown by the distribution of ¹⁴C radioactivity from different source leaves, was also translocated to the young leaves during expansion. The large sink created by these leaves may cause photoassimilate and reserve carbohydrate to be diverted from the roots, thereby inhibiting root growth during the stage of leaf expansion. It is suggested that the rhythmic leaf production at the apex may control the growth cycle of the roots.     

Genetic diversity assessment of sub-samples of cacao, Theobroma cacao L. collections in West Africa using simple sequence repeats marker

Knowledge of genebank and on-farm genetic diversity, particularly in an introduced crop species, is crucial to the management and utilization of the genetic resources available. Microsatellite markers were used to determine genetic diversity in 574 accessions of cacao, Theobroma cacao L., representing eight groups covering parental populations in West Africa, genebank, and farmers’ populations in Nigeria. From the 12 microsatellite markers used, a total of 144 alleles were detected with a mean allelic richness of 4.39 alleles/locus. The largest genetic diversity was found in the Upper Amazon parent population (H _nb  = 0.730), followed by the 1944 Posnette’s Introduction (H _nb  = 0.704), and was lowest in the Local parent population (H _nb  = 0.471). Gene diversity was appreciably high in the farmers’ populations (H _nb  = 0.563–0.624); however, the effective number of alleles was lower than that found in the genebank’s Posnette’s population. Fixation index estimates indicated deficiency of heterozygotes in the Upper Amazon and the Local parent populations (F _is  = 0.209 and 0.160, respectively), and excess of heterozygotes in the Trinitario parent population (F _is  = −0.341). The presence of inbreeding in the Local parent populations and substructure (Wahlund effect) in the Upper Amazon were suggested for the deficiency of heterozygotes observed. Non-significant genetic differentiation observed between the genebank’s and farmers’ populations indicated significant impact of national breeding programs on varieties grown in farmers’ plantations. From this study, we showed that appreciable genetic diversity was present in on-farm and field genebank collections of cacao that can be exploited for crop improvement in West Africa. Suggestions for future conservation of on-farm genetic diversity and local landraces are further discussed.     

Isolation and characterisation of cell wall polysaccharides from cocoa (Theobroma cacao L.) beans

 Cell wall material (CWM) was prepared from sun-dried cocoa (Theobroma cacao L.) bean cotyledons before and after fermentation. The monosaccharide composition of the CWM was identical for unfermented and fermented beans. Polysaccharides of the CWM were solubilised by sequential extraction with 0.05 M trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), 0.05 M Na₂CO₃, and 1 M, 4 M and 8 M KOH. The non-cellulosic sugar composition for each fraction was similar for unfermented and fermented samples, indicating that fermentation caused no significant modification of the structural features of individual cell wall polysaccharides. Pectic polysaccharides accounted for 60% of the cell wall polysaccharides but only small amounts could be solubilised in solutions of CDTA, Na₂CO₃, and 1 M and 4 M KOH. The bulk of the pectic polysaccharides were solubilised in 8 M KOH and were characterised by a rhamnogalacturonan backbone heavily substituted with side-chains of 5-linked arabinose and 4-linked galactose. Linkage analysis indicated the presence of additional acidic polysaccharides, including a xylogalacturonan and a glucuronoxylan. Cellulose, xyloglucan and a galactoglucomannan accounted for 28%, 8% and 3% of the cell wall polysaccharides, respectively. It is concluded that the types and structural features of cell wall polysaccharides in cocoa beans resemble those found in the parenchymatous tissue of many fruits and vegetables rather than those reported for many seed storage polysaccharides. Received: 29 May 1999 / Accepted: 19 October 1999     

Mapping QTL for yield components, vigor, and resistance to Phytophthora palmivora in Theobroma cacao L.

Quantitative trait loci (QTL) mapping for agronomic traits was carried out in cocoa (Theobroma cacao L.). Regions of the genome involved in yield, vigor, and resistance to Phytophthora palmivora were identified. Three heterozygous clones, one upper Amazon Forastero (IMC78) and two Trinitario (DR1 and S52), were crossed with the same male parent, a lower Amazon Forastero (Catongo), known to be highly homozygous. Observations were made on progeny over nine consecutive years. One to three QTL related to yield were detected in each of the three populations, located on chromosomes 1, 2, 4, 5, 9, and 10. They explained between 8.1 and 19.3% of the phenotypic variation and showed various levels of repeatability. In IMC78, the QTL detected on chromosome 5 was the most repeatable over years. The QTL for the average individual pod weight on chromosome 4 was the most significant with an LOD of 17.3 and an R2 of 43.7. QTL related to these traits were identified in the same region of the genome in clones of different genetic groups. This suggests that molecular markers can be used to improve cocoa varieties.     

The host range and vector species of viruses from Cola chlamydantha K. Schum., Adansonia digitata L. and Theobroma cacao L

Seventeen species in the Tiliaceae, Sterculiaceae and Bombacaceae were tested for susceptibility to three virus isolates from naturally infected Cola chlamydantha trees and three from Adansonia digitata trees in Ghana. Seven species of Pseudococcidae were tested as vectors of the six isolates. These studies indicate that the Cola isolates should be classified with cocoa swollen shoot virus and those from Adansonia with cocoa mottle leaf virus.     

Comparison of two cacao (Theobroma cacao L.) clones for the effect of pollination intensity on fruit set and seed content

We compared the influence of pollination intensity (PI) on fruit set and seed number per pod in two cacao (Theobroma cacao L.) clones, IFC5 (Forastero Lower-Amazon Amelonado), which is self-compatible and known to produce a high number of seeds per pod under open pollination, and SCA6 (Forastero Upper-Amazon), which is self-incompatible and known to produce a low number of seeds per pod under open pollination. With both clones, PI had a positive effect on fruit set, with the maximum rate requiring more than 150 pollen grains per pod. One-half of the maximum rate of fruit set was reached with 39 pollen grains per pod for SCA6, and 78 for IFC5. With SCA6, a significant positive effect of PI on seed number per pod was also observed, with maximum seed number requiring more than 200 pollen grains per pod. In contrast, seed numbers with IFC5 were approximately equal over the PI range 100–800 pollen grains per pod. Patterns of seed number per pod were compared after: (1) open insect pollination, (2) low-intensity hand pollination and (3) high-intensity hand pollination. The patterns obtained with IFC5 under open pollination showed a peak of 40–50 seeds per pod, whereas the distribution patterns were more even with SCA6. The pattern of seed number under open pollination was similar to that obtained with high-intensity hand pollination for IFC5, and with low-intensity hand pollination for SCA6. We concluded that the high number of seeds per pod observed with IFC5 under natural insect pollination may be explained by a high number of compatible self-pollen grains on the stigma and by a severe drop of low-pollinated flowers eliminating potential lowfilled fruits. With SCA6, however, the number of compatible pollen grains deposited on the stigma was probably low under open pollination, and the flowers required lower pollen quantities to set fruit, which resulted in a high frequency of low seed numbers per fruit. This difference in the capacity to set low-seeded fruits might be considered as an adaptive trait related to the mode of reproduction.