Genetic transformation of cocoa leaf cells using Agrobacterium tumefaciens

Leaf strips from cocoa tree (Theobroma cacao L.) clones ICS-16 and SIC-5 were cocultivated with the supervirulent Agrobacterium tumefaciens strain A281-Kan. A281-Kan contains a wild-type Ti plasmid and an additional plasmid, pGPTV-Kan, which confers kanamycin resistance to transformed plant cells after integration and expression of the neomycin phosphotransferase II (nptII) gene. Transformed cells were selected on callusing medium containing 100 g ml^-1 kanamycin. NptII assays confirmed that kanamycin-resistant cultures of ICS-16 and SIC-5 expressed the nptII gene, whereas control cultures did not. Genomic Southern blot analyses demonstrated single T-DNA insertions into ICS-16 and SIC-5. T-DNA/cocoa DNA border regions from transformed cultures were cloned and sequenced, revealing that in both transformed cell lines, the right T-DNA border was at the 5 end of the 25 bp right border repeat. Cocoa DNA probes from the T-DNA/cocoa DNA insertion sites were used in Southern blot analyses and showed that T-DNA from pGPTV-Kan had inserted into a unique region in ICS-16 and into a repetitive region in SIC-5. This study establishes that foreign genes can be inserted and expressed in cocoa using A. tumefaciens-mediated gene transfer.     

Effects of VA mycorrhiza on the growth of cacao seedlings under nursery conditions in Venezuela

The effects of vesicular-arbuscular mycorrhizae inoculation on the growth of cacao seedlings (Theobroma cacao var. Ocumare 60) grown for 5 months in a nursery were studied. The effects of introduced VAM fungi (Glomus occultum, Acaulospora appendicula, Glomus manihotis, Acaulospora morrowae and Scutellospora pellucida) in soils treated with copper oxychloride or methyl bromide, were compared with the indigenous VAM and with their respective non-inoculated controls.Cacao seedlings responded well to indigenous VAM fungi, which included Scutellospora calospora as the dominant species, inducing increases significant in height, dry weight and foliar uptake of P, Cu and Zn in relation to the sterile control. G. occultum and A. appendicula increased the height of cacao seedlings but to an extent not yet statistically significant through the duration of the experiment. S. pellucida and A. appendicula doubled the phosphorus uptake of cacao seedlings. The methyl bromide sterilization induced the lowest Cu and Zn uptake in the cacao seedlings but this effect was ameliorated with VAM inoculation. Manifest deficiency symptoms were not observed in the treatments.Copper oxychloride treatment depressed growth to the same level as the sterile control although its residual effects did not kill VAM. It could change the competitive relations among the VAM species and in this case seemed to affect adversely the more efficient native fungi.The influence of the presence and long permanence of large cotyledons in cacao on the results obtained is discussed.     

Effects of temperature on growth and water relations of cacao (Theobroma cacao var.Comum) seedlings

Growth of 55-day-oldTheobroma cacao var.Comum seedlings varied with temperature regimes, various plant parts, growth parameters, and time of harvesting. Over a 60-day period the optimal day-temperature regimes were near 33.3°C for dry weight increase and relative growth rates of seedlings and leaves; 30.5°C for increase in leaf area, height growth, and leaf abscission; 22.2°C for dry weight increase of stems or roots, stem diameter growth, and root-shoot ratio. The rates of increase in dry weights of stems or roots as well as root-shoot ratios declined progressively at temperatures above 22.2°C Partitioning of dry matter was affected by temperature regime, with proportionally more photosynthate retained by shoots and less translocated to roots at high temperatures. The progressive decrease in the root-shoot ratio at temperatures above 22.2°C may decrease drought tolerance of seedlings because roots will be less capable of absorbing endugh water to replace transpirational losses. This was shown by more negative shoot water potentials at high temperatures. Research supported by the College of Agricultural and Life Science. University of Wisconsin, Madison, Wi, USA and the Centro de Pesquisas do Cacau (CEPLAC/CEPEC), 45600 Itabuna, Ba, Brazil.     

Genetic diversity in cocoa revealed by cDNA probes

The variability of the cocoa (Theobroma cacao) nuclear genome was investigated. A total of 203 cocoa clones was surveyed for restriction fragment length polymorphisms (RFLPs) using four restriction endonuclease and 31 seed cDNA probes. A high level of polymorphism has been found. This study points to a structuring of the species that fits with the distinction between the Criollo and Forastero populations. These results combined with previously obtained nuclear rDNA and mtDNA data allow us to propose new hypotheses on the origin and evolution of the different cocoa populations.     

The roles of abscisic acid and ethylene in the abscission and senescence of cocoa flowers

Cocoa flowers have a limited period of longevity; more than 90% of unpollinated flowers abscised within 32 h after anthesis. Abscisic acid (ABA) levels increased significantly prior to abscission. By 21 h after anthesis, ABA levels had increased almost 10-fold, and by 32 h flowers had 20-fold higher levels of ABA than at anthesis. Fluridone completely inhibited both the increase in ABA, the formation of an abscission zone, and the abscission and senescence of flowers. In contrast, ethylene production increased only slightly 21 h after anthesis and was only 2-fold higher after 32 h. Aminoethoxyvinylglycine (AVG) delayed but did not prevent abscission. In cocoa flowers, ABA is the primary regulator of abscission; ethylene accelerates abscission but only in the presence of ABA. Naphthalene acetic acid (NAA) treatment of flowers at anthesis prevented abscission zone formation and flower abscission, but did not induce fruit set. All parts of the NAA-treated flower except the pedicel senesced after 6 days. NAA+AVG treatment only delayed, whereas fluridone treatment completely prevented flower senescence.     

Slow death of Atlantic forest trees in cocoa agroforestry in southeastern Brazil

Cocoa (Theobroma cacao) is cultivated in the states of Bahia and Espírito Santo in eastern Brazil under the so-called cabruca system, where the understorey of native Atlantic forest is cleared and the canopy is thinned out to provide adequate shading for the cocoa trees. Apart from its economic and social role, the cabruca system is said to be important for the conservation of Atlantic forest biodiversity. In this paper we studied tree species richness and forest structure of cabrucas to examine the demographic health of these forests and discuss their long-term survival. Data were collected in 20 farms located alongside a 30km track of the northern margin of the Rio Doce, in northern Espírito Santo. All trees 5cm DBH were identified and their diameter was measured in 80 plots (600m²), totalling 4.8ha of sampled area. Recorded trees were also allocated to four different regeneration phases (pioneers, early secondary, late secondary and climax). The inventory resulted in 507 trees belonging to 105 species in 39 families. This species richness is much lower than in less disturbed forests located in the region. Pioneers and early secondary species dominate the cabruca forest in terms of number of species (56.2%), density (71.0%) and basal area (72.3%). The distribution of diameter frequency showed an imbalance in tree regeneration. Most trees in the range of 5–30cm DBH were pioneers (40.7%), or early secondary species (32.6%), while late secondary and climax trees were less frequent (10.2 and 16.5% of the sampled trees, respectively). The dominance of species of early regeneration phases was also observed for trees >30cm DBH (69.0% of pioneers or early secondary and 31.0% of late secondary or climax species). The results indicated that the cabruca forests are not only less diverse and less dense than secondary or primary forests of the region, but also, and more importantly, their natural succession and gap dynamics are being severely impaired. As a consequence, cabrucas present a structure where tree species of late successional phases are becoming increasingly rare while pioneers and early secondary species are becoming dominant. If current management practices of thinning and clearing of native trees are not improved, the long-term survival of these forests is questionable and their role in maintaining biodiversity in the long run is limited.     

Morphological variation in Planococcoides njalensis occurring on cocoa in Ghana

A study was made to determine the extent of morphological variation in Planococcoides jalensis (Laing), the most important mealybug vector of cocoa swollen shoot virus (CSSV) in West Africa, and to resolve if more than one Planococcoides species is present. Fourteen populations of P. njalensis collected on cocoa from different localities in Ghana and one population each on Gliricidia sepium (Jacq.) Steud and Coffea canephora Pierre were compared by measured and meristic female characters using two types of morphometric analysis, Discriminant Functional Analysis and Principal Component Analysis. The analyses showed that P. njalensis exhibited a wide range of intraspecific morphological variation, particularly in meristic characters, but failed to identify new Planococcoides species. The results are discussed in relation to the control of the mealybug vectors of CSSV and to the suitability of Gliricidia as a shade tree for cocoa.     

Effect of metabolites ofAspergillus niger andTrichoderma viride on development and structure of radicle of cocoa (Theobroma cacao) seedlings

Both the volume and concentration of filtrate ofAspergillus niger andTrichoderma viride influenced the development of the radicles of cocoa seedlings. Radicles placed in Petri dishes containing filter paper moistened with 20 and 30 ml of undiluted filtrate and filtrate dilutions of 1∶1 and 1∶2 failed to develop lateral roots and eventually died. The culture filtrate ofA. niger was more repressive. Radicles in the same volume of filtrate (20 and 30 ml) of higher dilutions (1∶5 and 1∶10) developed lateral roots and survived. Radicles placed in less volume (≤10 ml) survived and produced lateral roots irrespective of concentration of filtrates. Development of the radicles and roots in the control was consistently better (P=0.05) than in filtrate solutions of eitherA. niger orT. viride. The hypocotyls of seedlings under the influence of metabolites ofA niger showed greater cambial activity and formed xylem vessels and tracheids with larger lumina.     

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.     

Recovery of species richness and conservation of native Atlantic forest trees in the cacao plantations of southern Bahia in Brazil

The Atlantic forests of southern Bahia in Brazil present great species richness and a high degree of endemism. A large part of these native forests were transformed into cacao plantations in an agroforestry system known locally as cabrucas, where native trees were culled and cacao was planted under the shade of remaining trees. The present study analyzed the influence of time of implantation (age) and time of abandonment of management practices on tree species diversity of cabruca plantations to evaluate the capacity for conservation and recovery of species richness of native Atlantic Forest trees in cabrucas. Phytosociological surveys were conducted in five cabrucas with different conditions of age and state of abandonment. All trees, including hemiepiphytes and excluding the cacao plants, with a minimum stem diameter of 10 cm at breast height, were surveyed within a 3-ha sampling area in each plantation. A total of 2514 individual trees belonging to 293 species and 52 families were recorded in the five cabrucas. The Shannon diversity index varied from 3.31 to 4.22 among the cabrucas and was positively correlated with the time of abandonment (r = 0.97). The new cabrucas showed the highest values of estimated total richness (Chao) and the highest proportion of late successional species than the old ones. All areas preserved a very high proportion of native forest species while the three old cabrucas showed a higher proportion of exotic species than the two new ones. Thus the exotic species seem to replace more of the native species in the long run because of management practices and local preferences. The cabrucas presented also a high capacity for the regeneration of tree species richness after abandonment. Simple alterations in management practices could improve the recruitment of late successional species in these areas. Economic incentives may be necessary for the farmers to adopt management practices to retain native species which bring no economic returns.     

A genomewide admixture mapping study for yield factors and morphological traits in a cultivated cocoa (Theobroma cacao L.) population

The selection of productive varieties of modern Criollo cocoa, showing fine aromatic qualities in their beans, is of major interest for some producing countries, such as Venezuela. Cultivated populations of Modern Criollo or Trinitario varieties may be suitable for admixture mapping analysis, as large blocks of alleles derived from two identified divergent ancestors, recently admixed, are still preserved, after a few generations of recombination, similar to experimental mapping progenies. Two hundred and fifty-seven individuals from a cultivated population of Modern Criollo were selected and analysed with 92 microsatellite markers distributed along the genome. This population exhibited a wide range of variability for yield factors and morphological features. Population structure analysis identified two main subgroups corresponding to the admixture from the two ancestors Criollo and Forastero. Several significant associations between markers and phenotypic data (yield factors and morphological traits) were identified by a least squares general linear model (GLM) taking into account the population structure and the percentage of admixture of each individual. Results were compared with classical QTL analyses previously reported for other cacao populations. Most markers associated to quantitative traits were very close to QTLs detected formerly for the same traits. Associations were also identified between markers and several qualitative traits including the red pigmentation observed in different organs, mainly associated to common markers in linkage group 4.     

Landscape and farm scale management to enhance biodiversity conservation in the cocoa producing region of southern Bahia, Brazil

In southern Bahia, Brazil, large land areas are used for the production of cocoa (Theobroma cacao), which is predominantly grown under the shade of native trees in an agroforestry system locally known as cabruca. As a dominant forest-like landscape element of the cocoa region, the cabrucas play an important role in the conservation of the region’s biodiversity. The purpose of this review is to provide the scientific basis for an action plan to reconcile cocoa production and biodiversity conservation in southern Bahia. The available research collectively highlights the diversity of responses of different species and biological groups to both the habitat quality of the cabrucas themselves and to the general characteristics of the landscape, such as the relative extent and spatial configuration of different vegetation types within the landscape mosaic. We identify factors that influence directly or indirectly the occurrence of native species in the cabrucas and the wider landscape of the cocoa region and develop recommendations for their conservation management. We show that the current scientific knowledge already provides a good basis for a biodiversity friendly management of the cocoa region of southern Bahia, although more work is needed to refine some management recommendations, especially on shade canopy composition and density, and verify their economic viability. The implementation of our recommendations should be accompanied by appropriate biological and socioeconomic monitoring and the findings should inform a broad program of adaptive management of the cabrucas and the wider cocoa landscape.     

Characterisation and genetic mapping of resistance and defence gene analogs in cocoa (Theobroma cacao L.)

Disease resistance and defence gene analog (RGA/DGA) sequences were isolated in cocoa using a PCR approach with degenerate primers designed from conserved domains of plant resistance and defence genes: the NBS (nucleotide binding site) motif present in a number of resistance genes such as the tobacco N, sub-domains of plant serine/threonine kinases such as the Pto tomato gene, and conserved domains of two defence gene families: pathogenesis-related proteins (PR) of classes 2 and 5. Nucleotide identity between thirty six sequences isolated from cocoa and known resistance or defence genes varied from 58 to 80%. Amino acid sequences translated from corresponding coding sequences produced sequences without stop codons, except for one NBS –like sequence. Most of the RGAs could be mapped on the cocoa genome and three clusters of genes could be observed : NBS-like sequences clustered in two regions located on chromosomes 7 and 10, Pto-like sequences mapped in five genome regions of which one, located on chromosome 4, corresponded to a cluster of five different sequences. PR2-like sequences mapped in two regions located on chromosome 5 and 9 respectively. An enrichment of the genetic map with microsatellite markers allowed us to identify several co-localisations of RGAs, DGAs and QTL for resistance to Phytophthora detected in several progenies, particularly on chromosome 4 where a cluster of Pto-like sequences and 4 QTL for resistance to Phytophthora were observed. Many other serious diseases affect cocoa and the candidate genes, isolated in this study, could be of broader interest in cocoa disease management.     

Early growth and nutritional response to resource competition in cocoa-shade intercropped systems

Intercropping is often promoted for effective mutualism between species, thus compensating for external inputs. However, for optimal farm design resulting in superior production and nutrition, an accurate assessment of plant inter- and intra-specific competition is required. In predominant shade tree-cocoa (Theobroma cacao) systems, inconclusive evidence remains on species interactions, limitations to resource availability and subsequent growth and nutritional response, particularly in early growth. We examined cocoa biomass and foliar nutrition as well as nutrient supply through rates of decomposition and N mineralization after 1-year growth. Our approach employed fertilization and mixed planting treatments in an additive design of cocoa in monoculture (control), under artificial shade, and intercropped under two separate shade species (Terminalia superba and Newbouldia laevis). Intercropping had no effect on cocoa biomass production in comparison to monoculture cocoa. However, artificial shading stimulated foliage and root production both with and without fertilization, suggesting strong effects of light regulation on growth in the absence of belowground competition. Nutritionally, intercropping suppressed K uptake in cocoa foliage as K concentration was reduced by 20–25%, signifying dilution of this nutrient, presumably due to interspecific competition for mobile elements. Foliar N content under N. laevis was raised, where N concentration kept up with growth under this intercropped species. Intercropping also delayed decomposition rates, suggesting slower but sustained release of available nutrients into the topsoil. Cocoa under artificial shade, both with and without fertilization, exhibited the greatest nutrient responses as compared to unfertilized monoculture cocoa, where P uptake was stimulated most (175 and 112%), followed by K (69 and 71%), and then N (54 and 42%). Intercropping with shade trees failed to increase cocoa biomass, however, nutrient uptake was sustained for N and P, suggesting low interspecific competition. When fertilizers are undesirable or unavailable, intercropping of appropriately selected shade trees will not competitively suppress early growth of cocoa but will improve light regulation and nutritional status of cocoa saplings.     

Isolation of DNA from plants high in polyphenolics

DNA suitable for digestion with restriction endonucleases has been isolated fromTheobroma cacao, a plant high in polyphenolic compounds, and several other plant taxa using the method presented in this paper. The method relies on concentrating nuclei away from most of the cytoplasmic components and inhibiting the formation of oxidized polyphenolic compounds and their interaction with the DNA fraction in the remaining steps.     

Headspace volatile markers for sensitivity of cocoa (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) somatic embryos to cryopreservation

The mechanisms that reduce the viability of plant somatic embryos following cryopreservation are not known. The objective of the present study was to evaluate the sensitivity of cocoa (Theobroma cacao L.) somatic embryos at different stages of an encapsulation–dehydration protocol using stress-related volatile hydrocarbons as markers of injury and recovery. The plant stress hormone ethylene and volatile hydrocarbons derived from hydroxyl radicals (methane) and lipid peroxidation (ethane) were determined using gas chromatography headspace analysis. Ethylene and methane were the only volatiles detected, with both being produced after each step of the cryogenic protocol. Ethylene production was significantly reduced following exposure to liquid nitrogen, but then increased in parallel with embryo recovery. In contrast, the production of methane was cyclic during recovery, with the first cycle occurring earlier for embryos recovered from liquid nitrogen and desiccation than those recovered from earlier steps in the protocol. These results suggest that loss of somatic embryo viability during cryopreservation may be related to the oxidative status of the tissue, and its capacity to produce ethylene. This study has demonstrated that headspace volatile analysis provides a robust non-destructive analytical approach for assessing the survival and recovery of plant somatic embryos following cryopreservation.     

The vesicular-arbuscular (VA) mycorrhiza and its effects on growth of vegetatively propagated Theobroma cacao L.

A greenhouse experiment was conducted to study the effects of mycorrhizal inoculation on growth and nutrient uptake of vegetatively propagated cocoa plants from the Sabah hybrid (NA 33 × PA 7) raised on unsterilized Munchong soils. Inoculation with mixed species of Scutellospora and Glomus, resulted in higher dry matter yield and stem diameter of mycorrhizal plants obtained through budding, air layering or marcotting (a form of asexual plant propagation using vegetative parts and the selection of a healthy branch and scraping off the bark at the lower end of the branch, forcing it to root by covering it with a ball of soil) and stem cutting. Budded and marcotted mycorrhizal plants gave a significant increase in P content of shoots. In contrast, only budded mycorrhizal plants (on both young and old rootstocks) gave a significantly higher Ca concentration in tops, compared to uninoculated plants.     

Substrate specificity of N-methyltransferase involved in purine alkaloids synthesis is dependent upon one amino acid residue of the enzyme

Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are the major purine alkaloids in plants. To investigate the diversity of N-methyltransferases involved in purine alkaloid biosynthesis, we isolated the genes homologous for caffeine synthase from theobromine-accumulating plants. The predicted amino acid sequences of N-methyltransferases in theobromine-accumulating species in Camellia were more than 80% identical to caffeine synthase in C. sinensis. However, there was a little homology among the N-methyltransferases between Camellia and Theobroma. The recombinant enzymes derived from theobromine-accumulating plants had only 3-N-methyltransferase activity. The accumulation of purine alkaloids was, therefore, dependent on the substrate specificity of N-methyltransferase determined by one amino acid residue in the central part of the protein.     

Mapping of Quantitative Trait Loci for Butter Content and Hardness in Cocoa Beans (Theobroma cacao L.)

Cocoa butter is an important raw material for the chocolate, pharmaceutical, and cosmetic industries. The butter content and quality in cocoa beans are genetically controlled characteristics, and affect its commercial value and industrial applicability. In the present work, an F₂ population derived from the cross between the ICS-1 and Scavina-6 cocoa clones was used for molecular mapping. A linkage map was constructed based on amplified fragment length polymorphism, random amplified polymorphic DNA, and simple sequence repeat markers, resulting in a total of 273 markers, distributed in 14 linkage groups (LGs). Phenotyping of butter content was performed after ether extraction and butter hardness was determined by sweeping differential calorimetry. One quantitative trait locus (QTL) associated to butter content was mapped at linkage group 9 (LG9) and two QTLs for butter hardness were identified at linkage groups 9 and 7 (LG9 and LG7). The two QTLs mapped at the LG9 explained 51.0% and 28.8% of the phenotypic variation for butter content and hardness, respectively. These QTLs were concentrated in the same map region, suggesting a close genetic linkage or pleiotropic effect. The QTLs identified may be useful in further marker-assisted selection breeding programs aimed at cocoa butter quality improvement.     

Trichoderma species form endophytic associations within Theobroma cacao trichomes

Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species (Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized stems from cacao seedlings that had been inoculated with each of the four Trichoderma strains. Fungal hyphae were observed under the microscope emerging from the trichomes as soon as 6 h after their isolation from surface sterilized cacao seedling stems. Hyphae were also observed, in some cases, emerging from stalk cells opposite the trichome head. Repeated single trichome/hyphae isolations verified that the emerging hyphae were the Trichoderma strains with which the cacao seedlings had been inoculated. Strains of four Trichoderma species were able to enter glandular trichomes during the colonization of cacao stems where they survived surface sterilization and could be re-isolated. The penetration of cacao trichomes may provide the entry point for Trichoderma species into the cacao stem allowing systemic colonization of this tissue.