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The acquisition of embryogenic cell suspension (ECS) cultures has been one of the main objectives to maximize clonal propagation of the coffee plant. However, the majority of somatic embryogenesis induction requirements are genotype-dependent. Therefore, molecular markers linked to the embryogenic transition events may be useful. The BABY BOOM (BBM) gene can be considered as one of those markers, as it is related to the embryogenic process and to cell proliferation. BBM homologous sequences were obtained from Expressed Sequence Tags (ESTs) in a databank generated by the Brazilian Coffee Genome Project. We selected ESTcontigs that showed similarities with BBM sequence from different species. Two EST-contigs (C2 and C9) were expressed in silico in cellular suspension libraries and embryogenic calli of coffee. Contig C9, defined as BBM-like (CaBBM), presented similarity with BBM genes and showed 2-fold change in expression in ECS relative to embryogenic calli (EC). Contig C2, on the other hand, was related to the ERF-like family. It showed basal expression in nonembryogenic calli (NEC) and approximately 66-and 311-fold less in ECS and EC compared with CaBBM in the same samples, respectively. These data suggest that CaBBM is likely to be a BBM ortholog in Coffea arabica, which has potential for use as a molecular marker to further increase the methodological efficiency of in vitro culture of coffee.

ABSTRACT. 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 1699©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 12 (2): 1698-1709 (2013) Putative sequences in somatic embryogenesis of coffee 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.

The analysis of the developmental stages during somatic embryogenesis has been essential to elucidate the in vitro plants embryogenesis process and can validate the somatic embryogenesis induction and progression. This work aimed to characterize the embryogenic calluses and cell suspension of Banana cv. Prata-Anã, anatomically and ultrastructurally, respectively. For the calluses induction and anatomical analyses, the male flower were disinfested and inoculated on MA1 solid medium. At intervals of 30 days up to 11 months, calluses were collected, fixed in FAA and prepared for analysis in optical microscopy. For the cell suspension and ultrastructural analyses, calluses4 months old calluses, obtained on the first experiment, were transferred to different liquids cultures mediums: MRT, MGM, CNPMF or MA2 and maintained in aggitation. At intervals of 30 days calluses were collected and prepared for analysis in scanning electron microscope. The male flowers showed, after three months on culture medium, heterogeneous calluses formed by clusters of small isodiametric cells that gave rise to globular structure with meristems tissues, clearly differentiated from other cells around the calluses. The globular structure, after ten months on the culture medium, developed to torpedo stage showing the meristematic tissues protoderm, ground meristem and procambium. The cell suspension cultivated on MRT culture medium showed embryos on globular and torpedo stage. RESUMO:A análise dos estágios de desenvolvimento durante a embriogênese somática tem sido essencial para elucidar os processos de embriogênese in vitro de muitas plantas e pode ser utilizada para validar a indução e a progressão da embriogênese somática. O objetivo deste trabalho foi caracterizar anatomicamente os calos embriogênicos e ultraestruturalmente as suspensões celulares de Banana cv. Prata-Anã. Para a indução dos calos e análises anatômicas, flores masculinas foram desinfestadas e inoculados em meio de cultura sólido MA1. Em intervalos de 30 dias, até 11 meses, os calos foram coletados, fixados e preparados para análise em microscópio óptico. Para o estabelecimento das suspensões celulares e análise ultraestrutural, calos obtidos no experimento anterior, com 4 meses de cultivo, foram transferidos para diferentes meios de cultura líquidos -MRT, MGM, CNPMF ou MA2, mantidos sob agitação após 30 dias foram coletados e preparados para análise em microscópio eletrônico de varredura. As flores masculinas apresentaram, após 3 meses em meio de cultura, calos heterogêneos formados por um grupo organizado células pequenas de isodiamétricas que deram origem a estruturas globulares com tecidos meristemáticos diferentes das outras células do calo. Depois de 10 meses no meio de cultura, as estruturas globulares evoluíram para o estágio torpedo, no qual foi observada a formação dos tecidos meristemáticos primários, protoderme, procâmbio e meristema fundamental. As suspensões celulares cultivadas em meio de cultura MRT também apresentaram embriões nos estágios globular e torped...

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