Observing the devastating coffee berry borer (Hypothenemus hampei) inside the coffee berry using micro-computed tomography.

The coffee berry borer is the most devastating insect pest of coffee throughout the world. The insect spends most of its life cycle inside the coffee berry, which makes it quite difficult to observe its behaviour. Micro-computed tomography (micro-CT) was used to observe all developmental stages of the coffee berry borer inside coffee berries (Coffea canephora). An interesting oviposition pattern involving a sequential placement of eggs starting in the periphery of the seed and moving inwards was observed. Micro-CT should be useful in elucidating unknown life history aspects of other seed-feeding bark beetles as well as of bark and ambrosia beetles in general.

Identification of expressed sequences in the coffee genome potentially associated with somatic embryogenesis.

Brazil possesses the most modern and productive coffee growing farms in the world, but technological development is desired to cope with the increasing world demand. One way to increase Brazilian coffee growing productivity is wide scale production of clones with superior genotypes, which can be obtained with in vitro propagation technique, or from tissue culture. These procedures can generate thousands of clones. However, the methodologies for in vitro cultivation are genotype-dependent, which leads to an almost empirical development of specific protocols for each species. Therefore, molecular markers linked to the biochemical events of somatic embryogenesis would greatly facilitate the development of such protocols. In this context, sequences potentially involved in embryogenesis processes in the coffee plant were identified in silico from libraries generated by the Brazilian Coffee Genome Project. Through these in silico analyses, we identified 15 EST-contigs related to the embryogenesis process. Among these, 5 EST-contigs (3605, 9850, 13686, 17240, and 17265) could readily be associated with plant embryogenesis. Sequence analysis of EST-contig 3605, 9850, and 17265 revealed similarity to a polygalacturonase, to a cysteine-proteinase, and to an allergenine, respectively. Results also show that EST-contig 17265 sequences presented similarity to an expansin. Finally, analysis of EST-contig 17240 revealed similarity to a protein of unknown function, but it grouped in the similarity dendrogram with the WUSCHEL transcription factor. The data suggest that these EST-contigs are related to the embryogenic process and have potential as molecular markers to increase methodological efficiency in obtaining coffee plant embryogenic materials.

Purification of legumin-like proteins from Coffea arabica and Coffea racemosa seeds and their insecticidal properties toward cowpea weevil ( Callosobruchus maculatus ) (Coleoptera: Bruchidae).

Legumin-like proteins from seeds of Coffea arabica (CaL-1 and CaL-2) and Coffea racemosa (CrL-1 and CrL-2) were characterized and isolated by gel filtration and reverse-phase chromatography. The insecticidal properties of the purified proteins were tested against Callosobruchus maculatus using artificial diets. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses indicated that CaL-1 is composed of two subunits of 33 and 24 kDa, while CaL-2, CrL-1, and CrL-2 were monomeric with a single band of 14 kDa. The LD(50) values were 0.5% (w/w) for CaL-1 and 0.3% (w/w) for CaL-2, CrL-1, and CrL-2. ED(50) at 0.3% was assessed for all protein concentrations. The legumin-like proteins were not digested by midgut homogenates of C. maculatus until 8 h of incubation. CaL-1 and CaL-2 ( C. arabica ) and CrL-1 and CrL-2 ( C. racemosa ) are chitin-binding proteins, and their insecticidal properties toward C. maculatus larvae might be related to their capacity to bind chitin present in the larval gut and their associated low digestibility.

[Massive multiplication of coffee (Coffee arabica L. cv. Catimor) through embryogenic cell suspension culture]. / Multiplicacion masiva del cafe (Goffea arabica L. cv. Catimor) mediante cultivo de suspensiones celulares embriogenicas.

Cell suspensions offer several advantages as a system for massive propagation because of the high rates of multiplication, the higher homogeneity in the culture conditions and the possibility of automatization. In this study, different experimental conditions were analyzed to establish embryogenic cell suspension cultures of coffee. The best conditions to establish the embryogenic cell suspension cultures of coffee were as follows: coffee leaf sections were cultivated during 12 weeks (Stage I) in a solid medium with the Murashige and Skoog salts, 2 mg/l kinetin and 0.5 mg/l 2,4-dichlorophenoxiacetic acid (medium 1). Under these conditions the explants formed a callus tissue that was transferred to a liquid medium containing 5 mg/l of 6-benzylamlno-purine (medium 2). After 12 days in a shaking liquid medium (Stage II), the cultures were sieved and were maintained In the same media, which was renewed every eight days (Stage III). This method yielded 1884 embryos in 50 ml; placing the embryos under conditions for germination yielded plantlets of normal appearance.

Multiplicacion masiva del cafe (coffea arabica L. cv. Catimor) mediante cultivo de suspensiones celulares embriogenicas / Massive multiplication of coffee (Coffea arabica L. cv. Catimor) through embryogenic cell suspension culture

Las suspensiones celulares ofrecen ventajas como sistema de propagación masiva de plantas por las altas tasas de multiplicación que presentan, una mayor homogeneidad en las condiciones de cultivo y la posibilidad de automatización. En el presente estudio se analizaron distintas condiciones experimentales para el establecimiento de suspensiones celulares embriogénicas de café (Coffea arabica L. cv. Catimor). Las condiciones óptimas para el establecimiento de suspensiones celulares embriogénicas de café consistieron en cultivar secciones de hoja durante 12 semanas (Etapa I) en un medio sólido con las sales de Murashige y Skoog (MS), suplementado con 2 mg/l de cinetina y 0,5 mg/l de ácido 2,4-diclorofenoacético (medio 1). Bajo estas condiciones de cultivo se formó un tejido de callo que fue transferido a medio líquido con 5 mg/l de 6-bencilaminopurina (medio 2). Después de permanecer 12 días en medio líquido con agitación (Etapa II), los cultivos fueron tamizados y se mantuvieron en el mismo medio, renovándolo cada 8 días (Etapa III). Con el método utilizado se logró la diferenciación de 1884 embriones en 50 ml, los cuales, una vez colocados en las condiciones para su germinación, formaron plántulas de apariencia normal. Palabras clave: Café, embriogénesis somática, propagación clonal, suspensión celular.