Genetic transformation of tobacco plants with a cacao pathogenesis-related protein 4 for tolerance to water stress study : S03P06

Drought is an important environmental factor limiting the productivity of various crops worldwide. The development of crop cultivars with improved adaptation to drought is a major goal in many crop breeding programs. In addition to classical breeding approaches, genetic transformation to introduce candidate genes into plants for better tolerance to water deficit has been successfully developed. Pathogenesis-related proteins (PR proteins) are defined as plant proteins induced in response to pathogen attack. However, it is known that these proteins may also be involved in response to abiotic stresses. The objective of this study was to transform tobacco plants (as plant model for subsequent analysis of cultivated plant such as citrus) with a PR-4b protein from Theobroma cacao (TcPR-4b) and to select and test the tolerance of such transformed plants to water/osmotic stress. First, an in silico analysis of the TcPR-4b using the BLAST, Pfam,InterProScan, ORF-Finder programs, as well as a search on Cocoa GenDB databank were performed. The TcPR-4b belongs to a small family of PR-4 proteins whose members were mainly located on the chromosomes 5 (five genes) and 10 (one gene). The complete TcPR-4b sequence is 802 bp in length and contains two exons (171 and 258 bp), and one intron (82 bp); the corresponding protein is 142 amino acids in length. For plant transformation experiment, the TcPR-4b cDNA (from cacao-M. perniciosa interaction library) was cloned into the pGem- T Easy vector then subcloned on the pCambia binary vector 1390. Then, Agrobacterium tumefaciens strain EHA 105 was transformed with the35S::TcPR-4b::pCambia construction, and the transformed A. tumefaciens used for Nicotiana tabacum cv.Havana transformation by co-cultivation of leaf segments in selection medium. Transformed shoots from three transformation events are under selection for subsequent in vitro water/osmotic stress, using, among others, mannitol and NaCl. Funding Agency: FAPESB, CNPq, CAPES, EMBRAPA, FINEP/RENORBIO and CIRAD. (Texte intégral

A protocol for large scale genomic DNA isolation for cacao genetics analysis

Advances in DNA technology, such as marker assisted selection, detection of quantitative trait loci and genomic selection also require the isolation of DNA from a large number of samples and the preservation of tissue samples for future use in cacao genome studies. The present study proposes a method for the preservation of sample tissues for DNA extraction and for manual extraction of large number of samples using spheres. The integrity and concentration of the DNA by these methods were assessed and compared with conventional method using mortar. The best parameters in order to obtain a fine powder using spheres was the use of 4 lyophilized leaf disks (50 mg), a single steel ball of 6 mm in diameter, followed by 30 s of manual maceration. The quantity of DNA obtained was four times higher than the conventional method. The purity of the DNA obtained was satisfactory and proved to be amplifiable by PCR using SSR primers. The present approach is a reliable, rapid, simple and consistent DNA isolation method for cacao, compared to the conventional methods. The protocol greatly increases the efficiency of extraction and suggests an inexpensive and practical way of DNA isolation of cacao for large scale.Keywords: DNA extraction, cacao, spheres, lyophilized

Encrypted antimicrobial peptides from plant proteins.

Abstract Examples of bioactive peptides derived from internal sequences of proteins are known for decades. The great majority of these findings appear to be fortuitous rather than the result of a deliberate and methodological-based enterprise. In the present work, we describe the identification and the biological activities of novel antimicrobial peptides unveiled as internal fragments of various plant proteins founded on our hypothesis-driven search strategy. All putative encrypted antimicrobial peptides were selected based upon their physicochemical properties that were iteratively selected by an in-house computer program named Kamal. The selected peptides were chemically synthesized and evaluated for their interaction with model membranes. Sixteen of these peptides showed antimicrobial activity against human and/or plant pathogens, some with a wide spectrum of activity presenting similar or superior inhibition efficacy when compared to classical antimicrobial peptides (AMPs). These original and previously unforeseen molecules constitute a broader and undisputable set of evidences produced by our group that illustrate how the intragenic concept is a workable reality and should be carefully explored not only for microbicidal agents but also for many other biological functions

Genome size, cytogenetic data and transferability of EST-SSRs markers in wild and cultivated species of the genus Theobroma L. (Byttnerioideae, Malvaceae).

The genus Theobroma comprises several trees species native to the Amazon. Theobroma cacao L. plays a key economic role mainly in the chocolate industry. Both cultivated and wild forms are described within the genus. Variations in genome size and chromosome number have been used for prediction purposes including the frequency of interspecific hybridization or inference about evolutionary relationships. In this study, the nuclear DNA content, karyotype and genetic diversity using functional microsatellites (EST-SSR) of seven Theobroma species were characterized. The nuclear content of DNA for all analyzed Theobroma species was 1C = ~ 0.46 pg. These species presented 2n = 20 with small chromosomes and only one pair of terminal heterochromatic bands positively stained (CMA+/DAPI− bands). The small size of Theobroma ssp. genomes was equivalent to other Byttnerioideae species, suggesting that the basal lineage of Malvaceae have smaller genomes and that there was an expansion of 2C values in the more specialized family clades. A set of 20 EST-SSR primers were characterized for related species of Theobroma, in which 12 loci were polymorphic. The polymorphism information content (PIC) ranged from 0.23 to 0.65, indicating a high level of information per locus. Combined results of flow cytometry, cytogenetic data and EST-SSRs markers will contribute to better describe the species and infer about the evolutionary relationships among Theobroma species. In addition, the importance of a core collection for conservation purposes is highlighted

Honey and propolis production, hygiene and defense behaviors of two generations of Africanized honey bees

Phenotypic characters of honeybees, relevant to beekeepers, can be evaluated by studying correlations between them, and the correlated characteristics can be evaluated in the short term to assist in monitoring of annual genetic progress. This work therefore aims to evaluate the production of honey and propolis, the hygiene and defensive behaviours of two generations of Africanized Apis mellifera (Hymenoptera, Apidae), to estimate the correlations between them and their heritability. We used 30 Langstroth beehives in apiaries in Marechal Cândido Rondon, Paraná State, Brazil. We used a method of drilling pupae to evaluate hygiene behaviour and the velveteen ball method to test defensive behaviour. We selected ten colonies which had the best honey and propolis production, and which produced F1 queens that were then transferred to beehives at an experimental farm, in order to observe honey and propolis production, hygiene and defence behaviours of their female offspring. The estimated differences for each characteristic between the generations, the correlations between them within each generation and their heritability suggest that selection of colonies based on propolis production was more efficient at maintaining this high production than was selection based on honey production according to the performance of the colonies for this characteristic. The selected behavioural characteristics can be used to enhance performance, but not for improving yield characteristics evaluated