Analysis of CcDREB1D promoter region from drought-tolerant and susceptible clones of Coffea canephora by homologous genetic transformation of Coffea arabica

In several plant species, the DREB genes play a key role in responses to abiotic stress. Since the development of molecular markers is one of the major goals for accelerating breeding programs, a study was done to evaluate the sequence variability of the DREBID gene in several Coffee genotypes. The promoter and coding regions of DREBID gene were cloned and sequenced from 16 coffee plants (10 from C. arabica and 4 from C. canephora), most of them characterized by different phenotypes (tolerance vs. susceptibility) regarding to drought. This showed a high conservation of DREB1 D proteins among the homologous sequences due to the low level of diversity and the high number of synonymous mutations and neutral changes which represents the majority of sequence variations. However, several nucleic polymorphisms ("single nucleotide polymorphism" and insertion/deletion [InDels]) were found in the coffee DREBID promoters. A comparison of predicted cis-acting elements for all the promoter sequences signaled the loss of some regulatory DNA elements. The sequence variation and the loss of some regulatory DNA elements could explain the differences of DREBID gene expression previously observed in leaves of drought tolerant (clone 14) and susceptible (clone 22) clones of C. canephora. In fact, both clones 14 and 22, have one same CcDREBID allelic sequence (hp15), and diverge at a second allele. Thus, the CcDREBID allele in the tolerant 14 (hp16) was considered to be the favorable/tolerant allele and the allele in 22 (hp17) was inferior/sensitive. The capacity of CcDREBID promoter to control the expression of the uidA reporter gene is under evaluation in transgenic plants of Coffee arabica cv. caturra stably transformed by Agrobacterium tumefaciens mediated gene transfer procedure. Caturra transgenic embryos were placed on a clean bench and subjected to dehydration tests. Preliminary results of bioassays checking GUS (/3-glucuronidase) activities indicate that the observed sequence variations have a direct role in the regulation of CcDREBID expression. The proximal promoter of CcDREBID for the three alleles tested (hp15, hp16 and hp17) equally induced the uidA gene expression, however, expression of uidA under control of the complete CcDREBID promoter was significantly induced in the tolerant allele (hp16) in response to the osmotic stress, whereas, it was not significantly upregulated for the common (hp15) and sensitive alleles (hp17). These results also evidence that the sequence variation present at the first -700 by of CcDREBID promoter do not interfere the regulation activity of the promoter, probably due to the non-overlapping of SNPs and cis-regulatory elements. Though, the higher sequence variation and co-occurrence of SNPs and cis-regulatory elements observed between -700 and -1500 by seems to affect the regulation of CcDREBID promoter in response to drought stress.Support: CAPES COFECUB, INCT-Café, CNPq and ConsOrcio Pesquisa Café. (Texte intégral

Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures

<p>Abstract</p> <p>Background</p> <p>Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including <it>Coffea arabica</it>.</p> <p>Results</p> <p>We identified the conditions required for successful long-term proliferation of embryogenic cultures in <it>C. arabica </it>and designed a highly efficient and reliable <it>Agrobacterium tumefaciens</it>-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD₆₀₀= 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization.</p> <p>Conclusion</p> <p>Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make <it>Agrobacterium</it>-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes.</p