Microsatellite loci transferability from Theobroma cacao to Theobroma grandiflorum

Theobroma grandiflorum (cupuassu) is an important fruit tree native to the Brazilian Amazon. Forty‐eight microsatellite loci developed for the congener Theobroma cacao were tested in cupuassu, and 29 (60.4%) produced robust alleles. The analyses of 216 cupuassu accessions using the 21 polymorphic microsatellite loci revealed a total of 113 alleles. The number of alleles per polymorphic locus ranged from two to 11, with an average of 5.38 alleles per locus. The average observed heterozygosity was 0.343, while the mean expected heterozygosity was 0.614. The successful transferability of T. cacao microsatellite primers to cupuassu was consistent with currently accepted phylogeny.     

Mineral nutrition of cacao (Theobroma cacao L.)

Summary Two experiments on cacao seedlings grown in sand culture are described, the first of which was concerned with variations in the levels of nitrogen, phosphorus, potassium, calcium, magnesium and sulfur (the macronutrient experiment) and the second dealing with variations in the levels of iron, copper, zinc, boron, manganese, and molybdenum (the micronutrient experiment).Many of the deficiency symptoms obtained were similar to those reported in the literature and they have not been described again. However, additional information is provided for symptoms of phosphorus, potassium, calcium, iron, manganese, copper, boron, and molybdenum deficiencies.The effects of all treatments on the dry weights of leaves, stems, and roots are presented. The effects of the macronutrient treatments on the levels of nitrogen, phosphorus, potassium, calcium, and magnesium in the leaves of eight month old plants and the effects of micronutrient treatments on the levels of nitrogen, phosphorus, potassium, calcium, magnesium, iron, manganese, copper, zinc, boron, molybdenum, sodium, and aluminium in the leaves of eleven-month-old plants are presented and discussed.     

A new glycosylated dihydrophaseic acid from cacao germs (Theobroma cacao L.)

Cacao beans are composed of cacao nibs and germs. Although numerous chemical and physiological studies on cacao nib compounds have been reported, there is little information on cacao germ compounds. We therefore analyzed an extract from the cacao germ, and found two compounds that were specific to the germ. One of these two compounds was identified as the new glycosylated abscisic acid metabolite, dihydrophaseic acid-4'-O-6″-(β-ribofuranosyl)-β-glucopyranoside, and the other as the known compound, dihydrophaseic acid-4'-O-β-D-glucopyranoside.     

Cryopreservation of immature embryos of Theobroma cacao

Immature, white zygotic embryos of Theobroma cacao L. (cacao) retained the ability to produce callus and to undergo somatic embryogenesis after slow hydrated freezing and desiccated fast freezing in liquid nitrogen. The highest rate of somatic embryogenesis occurred in embryos which were precultured on a medium containing 3% sucrose, frozen slowly with cryoprotectants before exposure to liquid nitrogen, and recovered on a medium containing 3 mg/liter NAA. Embryos precultured on media containing sucrose increasing to 21% had a higher rate of survival but were less embryogenic after freezing. These results suggest that immature embryos might be used for long-term germplasm storage of T. cacao germplasm.     

Culture of Shoot Apices of Theobroma cacao

Surface sterilized buds of young cocoa plants (Theobroma cacao L.) taken at particular stages of the flush cycle were placed in Linsmaier and Skoog agar medium supplemented with a range of growth regulators. Only buds taken at the 1–2 (dormant) stage of the flush cycle and treated with gibberellic acid (GA₃) alone and GA₃plus kinetin (KN) supplement showed bud opening. In liquid Linsmaier and Skoog medium buds isolated at the 1–2 stage also responded to GA₃ and KN. In this case addition of KN caused bud opening, while GA₃ either initiated bud opening only or opening followed by leaf expansion depending on the concentration of GA₃ supplied. Bud development was inhibited when ABA was included in the medium hut this was overcome by the presence of GA₃ but not KN. Since a hormonal supplement was required for any response from the excised buds, it is suggested that the intermittent growth of the shoot apex in the intact plant may be determined by hormonal stimuli derived from other parts of the plant. The findings also indicate that the bud apices could be maintained in culture for long periods which may provide a basis for the development of a micropropagation procedure for cocoa.     

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.     

The status of cacao (Theobroma cacao,sterculiaceae) in the western hemisphere

There are, at the present time, effectively no long-range, ongoing programs in any tropical country of the western hemisphere dedicated to the improvement of cacao (Theobroma cacao, Sterculiaceae). While some effort is currently made to obtain new acquisitions of cacao cultivars exhibiting desirable characteristics and to maintain genepools of these trees, there are few data from field trials to prove and substantiate these qualities. In addition, there is a growing concern regarding the disparities between predicted yields of cacao trees through the use of “hybrid” seed and from actual production under field conditions. This has stimulated an awareness of the current inadequate understanding of the genetics of cacao and the lack of comprehension as to which cultivars, under distinct ecological conditions, are precocious, resistant to disease, or heavy bearing, or indeed demonstrate those traits vital to the success of farming programs adapted to today’s market conditions. This paper examines the events that have led to the current status of selection, development, and breeding of cacao. Alternative approaches are suggested.     

Antimicrobial properties of two novel peptides derived from Theobroma cacao osmotin

The osmotin proteins of several plants display antifungal activity, which can play an important role in plant defense against diseases. Thus, this protein can be useful as a source for biotechnological strategies aiming to combat fungal diseases. In this work, we analyzed the antifungal activity of a cacao osmotin-like protein (TcOsm1) and of two osmotin-derived synthetic peptides with antimicrobial features, differing by five amino acids residues at the N-terminus. Antimicrobial tests showed that TcOsm1 expressed in Escherichia coli inhibits the growth of Moniliophthora perniciosa mycelium and Pichia pastoris X-33 in vitro. The TcOsm1-derived peptides, named Osm-pepA (H-RRLDRGGVWNLNVNPGTTGARVWARTK-NH₂), located at R23-K49, and Osm-pepB (H-GGVWNLNVNPGTTGARVWARTK-NH₂), located at G28-K49, inhibited growth of yeasts (Saccharomyces cerevisiae S288C and Pichia pastoris X-33) and spore germination of the phytopathogenic fungi Fusarium f. sp. glycines and Colletotrichum gossypi. Osm-pepA was more efficient than Osm-pepB for S. cerevisiae (MIC = 40 μM and MIC = 127 μM, respectively), as well as for P. pastoris (MIC = 20 μM and MIC = 127 μM, respectively). Furthermore, the peptides presented a biphasic performance, promoting S. cerevisiae growth in doses around 5 μM and inhibiting it at higher doses. The structural model for these peptides showed that the five amino acids residues, RRLDR at Osm-pepA N-terminus, significantly affect the tertiary structure, indicating that this structure is important for the peptide antimicrobial potency. This is the first report of development of antimicrobial peptides from T. cacao. Taken together, the results indicate that the cacao osmotin and its derived peptides, herein studied, are good candidates for developing biotechnological tools aiming to control phytopathogenic fungi.     

Auxin modification of the incompatibility response in Theobroma cacao

The time course and control of floral abscission and fruit set in Theobroma cacao were studied after spray application of growth regulators. 1-Naphthaleneacetic acid (NAA) prevented flower abscission in a concentration dependent manner and induced the early stages of fruit development. The cytokinin benzylaminopurine (BAP) counteracted NAA but resulted in longer fruit retention. Measurements of endogenous levels of indole-3-acetic acid showed an inverse correlation between the number of flowers per plant and auxin content. The results suggest that the genetic control of self-incompatibility in T. cacao may be modulated by the hormonal content of the flower.     

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.     

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.     

Initiation and Growth of Tissue Cultures of Theobroma cacao

Difficulties were experienced in obtaining sterile explant materialfrom which to initiate the callus cultures. The material whichprovided the most effective source in terms of numbers of explants,levels of contamination and ease of sterilization was from seedlingtissue, grown from seeds which had been transported in the intactpod. The seeds were removed asceptically and germinated in sterileflasks on White's medium solidified with agar, then at 3–4weeks old the seedling was sectioned into root, stem, cotyledon,hypocotyl and apical bud explants. The medium which promotedthe most rapid and sustained callus growth was a modified Murashigeand Skoog medium. Callus transferred to a liquid medium formeda cell suspension which by selecting for a low level of aggregationand a fast growth rate eventually developed these characteristics.There was evidence for clonal differences in the callus basedon growth rate data and appearance and related to the originof the initial explant tissue. Although the callus was exposedto considerable variation in the nutrient medium, except forthe occasional appearance of root-like structures, no regularorganogenesis was found.     

Out of the Amazon: Theobroma cacao enters the genomic era

Theobroma cacao has long been a crop of near-mystical human importance among indigenous Mesoamerican cultures and its importance in modern culture is growing with new realizations of the potential health benefits of cocoa polyphenolic compounds. However, cultivated T. cacao is vulnerable to emerging disease pressures because it has a narrow genetic base, and systematic genetic improvement of the crop is imperative. A wide range of genomic tools and resources has been developed and are providing the basis for genome-based breeding and gene discovery.     

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.     

Evaluation of the extent of genetic variability among Theobroma cacao accessions using RAPD and RFLP markers

 Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were used to evaluate genetic relationships within the Theobroma cacao species and to assess the organization of its genetic diversity. Genetic variability was estimated with 18 primers and 43 RFLP probes on 155 cocoa trees belonging to different morphological groups and coming from various geographic origins. The majority of the RFLP probes issued from low-copy DNA sequences. On the basis of on the genetic distance matrices, the two molecular methods gave related estimates of the genetic relationship between genotypes. Although an influence of cocoa morphological groups and geographical origins of trees was observed, a lack of gene differentiation characterized the T. cacao accessions studied. The continuous RFLP variability observed within the species may reflect the hybridization and introgressions between trees of different origins. Nevertheless, the Nacional type was detected to be genetically specific and different from well-known types such as Forastero, Criollo and Trinitario. Some of those genotypes were characterized by a low heterozygosity rate and may constitute the original Nacional pool. These results also provide information for the constitution of a cocoa tree core collection. Received: 10 June 1996/Accepted: 11 October 1996     

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