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Modeste Kan Kouassi, Jane Kahia, Christophe N’guessan Kouame, Mathias Gnion Tahi and Edmond Kouablan Koffi

than 6 million people ( Conseil Café Cacao, 2014 ; ICCO, 2012 ). Theobroma cacao is generally a heterozygous plant with a high variability for agronomic and quality traits. Seed propagation, while efficient, is problematic, since many of the trees

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Jane Kahia, Siaka Kone, Lucien Diby, Georges Ngoran, Colombe Dadjo and Christophe Kouame

Cacao ( Theobroma cacao , Malvaceae) is a crop of major importance for the livelihoods of small-scale farmers and ecosystems in many tropical regions. About 72% of the world’s cocoa is produced in Africa, with Côte d’Ivoire being the top producer at

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Antonio Figueira, Jules Janick and Peter Goldsbrough

The size of the haploid genome of Theobroma cacao L. (cacao), estimated using laser flow cytometry, was 0.43 pg. An improved DNA extraction procedure was developed based on isolation of a crude nuclei preparation from leaf tissue that effectively eliminated contamination of the DNA by polysaccharides and produced DNA that was, on average, longer than 50 kb. DNA yields ranged from 2 to 10 μg·g-1 fresh weight of leaf tissue. DNA blot hybridization experiments with a flax (Linum usitatissimum L.) ribosomal DNA probe revealed restriction fragment length polymorphisms between three cacao genotypes. Differences between the DNA of these genotypes were also detected by polymerase chain reaction amplification of polymorphic DNA fragments, using random oligonucleotide primers.

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Catherine M. Ronning and Raymond J. Schneg

While Random Amplified Polymorphic DNA (KAPD) has been used successfully in genetic analysis of several crop plants, the method poses difficulties with a heterozyaous species such as Theobroma cacao due to the dominant phenotypic expression of bands. A backcross family of the cultivars `Catoneo' and `Pound 12' was analyzed to determine the efficacy of this technique in analyzing cacao populations. A primary screen of the parents and F1 was conducted with 180 KAPD primers; of these, 39.5% either-did not amplify or did so poorly or irreproducibly, while 60.5% amplified well. Phenotypes produced by 42 primers represented possible test crosses, which can be used in linkage mapping. Genomic DNA from 50 individuals of the backcross population were then amplified with these 42 primers, which in most cases resulted in 1:1 segregation of bands. Preliminary experiments show that the Stoffel fragment of Taq DNA polymerase may provide additional markers. These results indicate that it should be possible to use RAPD bands as molecular markers to study the cacao genome.

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R. Paul Baker, Karl H. Hasenstein and Michael S. Zavada

In order to characterize the self-incompatibility system in Theobroma cacao, the levels of ethylene, indole-3-acetic acid (IAA), and abscisic acid (ABA) were determined after pollination with compatible and incompatible pollen and in unpollinated flowers. Pollen tube growth rates after incompatible and compatible pollinations were identical, and the majority of the pollen tubes reached the ovules between 12 and 20 hours after pollination. ABA levels rose in incompatibly pollinated flowers, and fell in compatibly pollinated flowers, prior to pollen tube—ovule contact. Ethylene evolution remained stable in compatibly pollinated flowers and rose in incompatibly pollinated flowers. IAA concentrations increased in compatibly pollinated flowers, and remained stable in incompatibly pollinated flowers after pollination and subsequent to pollen tube—ovule contact.

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Antonio Figueira, Anna Whipkey and Jules Janick

Axillary shoots of cacao (Theobroma cacao L.), induced in vitro with cytokinins (BA or TDZ), elongated and produced leaves only in the presence of cotyledons and/or roots. Detached axillary shoots, which do not grow in `vitro under conventional tissue culture protocols, rooted with auxin and developed normally in vivo. Detached axillary shoots from cotyledonary nodes and single-node cuttings from mature plants were induced to elongate and produce normal leaves in the presence of 20,000 ppm CO2 and a photosynthetic photon flux density (PPFD) of 150 to 200 μmol·s-1·m-2. Subculture nodal cuttings continued to elongate and produce leaves under elevated CO2 and light levels, and some formed roots. Subculture of microcuttings under CO2 enrichment could be the basis for a rapid system of micropropagation for cacao. Chemical names used: N -(phenylmethyl) -1 H -purin-6-amine (BA); 1 H -indole-3-butyric `acid (IBA); α -naphthaleneacetic acid (NAA); thidiazuron (TDZ).

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C.M. Ronning, R.J. Schnell and D.N. Kuhn

RAPD markers have been used successfully in genetic analysis of several crop plants. This method poses difficulties with a highly heterozygous species such as Theobroma cacao because of the dominant phenotypic expression of bands. A backcross family derived from ctultivars Catongo and Pound 12 was analyzed to determine the efficacy of RAPD markers in analyzing cacao populations. A preliminary screen of the parents and the F1 plant used as the backcross parent was conducted with 180 RAPD primers; of these, 26% were polymorphic and reproducible and produced 104 storable loci. Genomic DNA from 54 individuals of the backcross population was then amplified with these primers; 68.3 % of the loci segregated as expected in a Mendelian fashion. Separation of RAPD fragments on acrylamide revealed an additional polymorphic locus from one primer that was indistinguishable on agarose. The results demonstrated that RAPD markers can be used to study the cacao genome.

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Antonio Figueira and Jules Janick

In vitro culture of axillary cotyledonary shoots of Theobroma cacao L. (cacao) under increasing CO2 concentration from ambient to 24,000 ppm (culture tube levels) significantly increased total shoot elongation, number of leaves, leaf area per explant, and shoot dry and fresh weight. Although light was necessary for the CO2 response, the effect of various photon fluxes was not significant for the measured growth parameters. Net photosynthesis estimated on the basis of CO2 depletion in culture tubes increased 3.5 times from 463 to 2639 ppm CO2, and increased 1.5 times from 2639 to 14,849 ppm CO2, but declined from 14,849 to 24,015 ppm CO2. Ethylene concentration in culture vessels increased under enriched CO2 conditions. Depletion of nutrients (fructose, K, Ca, Mg, and P) from the medium was increased under enriched CO2 conditions.

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Antonio Figueira, Anna Whipkey and Jules Janick

Cacao (Theobroma cacao) has long been considered a recalcitrant species in regard to microproagation. Although axillary shoots from cotyledonary nodes will proliferate and grow in vitro provided either cotyledons or roots are attached, excised shoots fail to grow in spite of conventional medium and hormonal modifications. Charcoal supplemented medium and rapid medium change are only marginally effective in inducing shoot elongation. The recalcitrance of cacao appear to be due to the presence of gums which are produced from stem tissues in response to wounding. However, growth of axillary cotyledonary shoots as well as mature shoots was obtained under conditions of high CO2 (20,000 ppm) and high light (quantum flux of 200 μmol m-2s-1) with or without sugar. Under these conditions leaves develop and shoots elongate which can be subdivided and subcultured. Shoots root under these conditions in vitro. We hypothesize that growth of cacao shoots in response to high CO2 is due to translocation of metabolizes from photosynthesizing leaves and stems.

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A.J. Daymond, P. Hadley, R.C.R. Machado and E. Ng

Biomass partitioning of cacao (Theobroma cacao L.) was studied in seven clones and five hybrids in a replicated experiment in Bahia, Brazil. Over an 18-month period, a 7-fold difference in dry bean yield was demonstrated between genotypes, ranging from the equivalent of 200 to 1389 kg·ha-1. During the same interval, the increase in trunk cross-sectional area ranged from 11.1 cm2 for clone EEG-29 to 27.6 cm2 for hybrid PA-150 × MA-15. Yield efficiency increment (the ratio of cumulative yield to the increase in trunk circumference), which indicated partitioning between the vegetative and reproductive components, ranged from 0.008 kg·cm-2 for clone CP-82 to 0.08 kg·cm-2 for clone EEG-29. An examination of biomass partitioning within the pod of the seven clones revealed that the beans accounted for between 32.0% (CP-82) and 44.5% (ICS-9) of the pod biomass. The study demonstrated the potential for yield improvement in cacao by selectively breeding for more efficient partitioning to the yield component.