Rubens Onofre Nodari

Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses

The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species.

Genetic diversity and recruitment of the tropical palm, Euterpe edulis Mart., in a natural population from the Brazilian Atlantic Forest.

The genetic diversity and recruitment of plants of heart-of-palm tree (Euterpe edulis Mart.) were investigated in a natural population located in Southern Brazil. Five categories of plants, from seedlings to adults, were analysed using 16 allozymic loci. The results showed an average population level of genetic diversity (He=0.278) greater than the average of plant species already studied. The recruitment process of E. edulis is related to its genetic characteristics. A significant increase in the heterozygote frequency towards the adult stages was observed at three loci (Pgdh-2, G6pdh-1 and Mdh-1). This suggests the possible action of natural selection in promoting such heterozygote increase. In the same way, a linear increase in allele frequencies was observed at four loci (Prx-3, Prx-4, Pgdh-2 and G6pdh-1), indicating that recruitment is also related to a greater survival of individuals that are carriers of certain alleles. The maintenance of high diversity levels, as well as the increase in the heterozygote frequency, are positive aspects for in situ conservation. Furthermore, an increase in heterozygote frequency is favourable to the management of the species, since the maintenance of a stock of reproductive individuals with high heterozygosity levels favours the maintenance of the population dynamic and structure.

Estabelecimento de um protocolo regenerativo para a micropropagação do abacaxizeiro

Twenty-four treatment combinations among genotypes, physical medium composition and plant growth regulator levels of naphthaleneacetic acid (NAA) and 6-benzilaminopurine (BAP) were evaluated in order to identify the most efficient combination for the pineapple micropropagation in the laboratory of the Universidade Federal de Santa Catarina, Brazil, from 1993 to 1996. During seven weeks after the inoculation, the regeneration rate behaved as a quadratic function for most of the combinations. In one case (liquid medium, cultivar Primavera), the response followed a linear model. Among the treatments, the most efficient was the combination of MS (Murashige & Skoog, 1962) liquid medium with NAA (2.7 µM) and BAP (4.4 µM) which produced 19.7 shoot/explant, a significant deviation from the mean average (12 shoot/explant).The genotypic effect exhibited by two genotypes (Perolera and Primavera), expressed in terms of regeneration rate, was intermediary between the effect of the plant growth regulators levels and the physical medium constitution. The best treatment combination was tested in 17 other genotypes colected in the Santa Catarina State. In average, it was produced 15.3 shoot/explant, five weeks after the explant inoculation, 40% of which showing height near 3 cm. When submitted to acclimatization, 95.5% of the plantlets survived.

Somatic embryogenesis in goiabeira serrana: genotype response, auxinic shock and synthetic seeds

A embriogenese somatica in vitro e dependente de uma serie de fatores, dentre os quais o genotipo da planta matriz doadora de explantes e a fonte de auxina para conferir competencia embriogenica sao considerados como determinativos. Uma vez obtidos, os embrioes somaticos podem ser encapsulados em alginato para a obtencao de sementes sinteticas, permitindo assim o armazenamento em baixas temperaturas e o posterior cultivo de maneira semelhante a semente verdadeira. Os aspectos citados foram estudados no presente trabalho visando elucidar pontos de controle da embriogenese somatica. Assim, embrioes zigoticos excisados de frutos maduros de cinco acessos de Feijoa sellowiana Berg, foram avaliados quanto ao seu potencial embriogenetico. Choques auxinicos de 2,4-D (20 mM) em periodos de 1, 2, 4, 8 e 16 semanas foram utilizados para estabelecer o periodo minimo de aquisicao de competencia embriogenica dos explantes e o posterior efeito deste fitorregulador sobre o desenvolvimento e qualidade dos embrioes somaticos. Diferentes composicoes de capsulas de alginato foram empregadas para avaliar a polimerizacao das capsulas e a contaminacao das sementes sinteticas. A maior percentagem de inducao da embriogenese somatica e o maior numero de embrioes ocorreram nos acessos 101 e 50-4. Choques de 2,4-D de duas semanas foram suficientes para proporcionar valores similares de inducao e producao de embrioes somaticos, aqueles obtidos com choques de 4 e 8 semanas. Estudos histologicos demonstraram a origem direta da embriogenese somatica a partir da epiderme cotiledonar. A adicao dos sais de MS e sacarose ao alginato de sodio resultou em altos valores de contaminacao das sementes sinteticas, quando comparados as capsulas isentas destas substâncias. O emprego do KNO3 (100 mM) permitiu a despolimerizacao e a abertura de 81,2% das sementes sinteticas em comparacao aos valores de 0% para o tratamento com agua.

Fenologia reprodutiva de espécies arbóreas em uma formação secundária da floresta Atlântica

Studies on reproductive phenology of tree species provide the knowledge needed to define strategies for forest conservation and management. Thus, the objective of this work was to characterize the reproductive phenology of woody species in secondary forests of the Atlantic Rain Forest. From June/1998 to July/1999, flowering and fructification periods of five to 41 reproductive trees of 19 species were evaluated in an area displaying vegetation characteristic of an advanced stage of succession, located in Sao Pedro de Alcântara - Santa Catarina. Flowering and fructification observations were made every other week. The proportion of plants that produced flowers and fruits ranged from 5.5% to 80.0% (average 28.4%), depending on the species. The minimum DBH for a plant to produce fruits ranged from 5.6 to 21.5 cm. For most species, flowering occurred between October and January and fructification from January to March. In addition to these factors, the reproductive phenophases are also dependent on the ecosystem where they grow. This is why several species showed different reproductive periods compared to those observed in other regions. Thus, this work stresses the need of specific studies for the elaboration of a forest management plan for native timber species and seed collecting strategies.

An Improved Protocol for Intact Chloroplasts and cpDNA Isolation in Conifers

Background Performing chloroplast DNA (cpDNA) isolation is considered a major challenge among different plant groups, especially conifers. Isolating chloroplasts in conifers by such conventional methods as sucrose gradient and high salt has not been successful. So far, plastid genome sequencing protocols for conifer species have been based mainly on long-range PCR, which is known to be time-consuming and difficult to implement. Methodology/Principal Findings We developed a protocol for cpDNA isolation using three different conifer families: Araucaria angustifolia and Araucaria bidwilli (Araucariaceae), Podocarpus lambertii (Podocarpaceae) and Pinus patula (Pinaceae). The present protocol is based on high salt isolation buffer followed by saline Percoll gradient. Combining these two strategies allowed enhanced chloroplast isolation, along with decreased contamination caused by polysaccharides, polyphenols, proteins, and nuclear DNA in cpDNA. Microscopy images confirmed the presence of intact chloroplasts in high abundance. This method was applied to cpDNA isolation and subsequent sequencing by Illumina MiSeq (2×250 bp), using only 50 ng of cpDNA. Reference-guided chloroplast genome mapping showed that high average coverage was achieved for all evaluated species: 24.63 for A. angustifolia, 135.97 for A. bidwilli, 1196.10 for P. lambertii, and 64.68 for P. patula. Conclusion Results show that this improved protocol is suitable for enhanced quality and yield of chloroplasts and cpDNA isolation from conifers, providing a useful tool for studies that require isolated chloroplasts and/or whole cpDNA sequences.

Viabilidade do pólen e desenvolvimento do tubo polínico em macieira (Malus spp.)

In the present work it was studied features associated to the germination of pollen and the development of pollen tubes in six apple varieties as subsidies for the establishment of genetic improvement programs. Pollen grains were inoculated in culture medium with distilled water, agar (10 g. L-1), combined with sucrose at 0, 10, 20, 30, 40 and 50% and boric acid at 0 and 40 mg. L-1. For the study of pollen tubes development flowers were collected at 6, 12, 24 and 48 hours after antesis in M.9 x Marubakaido and in the self-pollinated M.9, being the pollen tubes analyzed in staining with blue aniline with acetic carmim and in fluorescence. Sucrose in concentrations ranging from 15% to 25% can successfully be used for the in vitro germination of pollen grains. Boric acid was not effective in promoting in vitro germination of pollen grains. In the absence of boric acid and in the presence of 15% of sucrose it was observed the largest germination rates: Fuji (51.1%), Imperatriz (31.7%), M.9 (20.8%), Catarina (19.2%), Gala (13.7%), and Marubakaido (6.1%). In the crossing M.9 x Marubakaido after 12 hours of pollination the pollen germination began in the stigma and after 24hrs it was observed 83% of germination. The techniques employed were efficient in the visualization and coloration of the pollen grains and of development of tube pollen.

Traditional Knowledge and Management of Feijoa (Acca sellowiana) in Southern Brazil

Traditional Knowledge and Management of Feijoa (Acca sellowiana) in Southern Brazil. This paper investigates traditional knowledge of the use and management of Acca sellowiana in southern Brazil. Fifty-six informants from three rural communities were assigned to one of four subgroups (“maintainers,” “managers,” “cultivators,” or “users”) based on their responses regarding management and use of A. sellowiana. Traditional knowledge related to use of this species is widespread among rural residents, but traditional knowledge related to management is fragmented depending on whether one uses, manages, or cultivates the species. Knowledge held in rural communities suggests that A. sellowiana could play an expanded role in local economies as well as biodiversity conservation. We suggest that participatory research could stimulate greater local use as well as on-farm conservation of A. sellowiana.

Somatic Embryogenesis in Araucaria angustifolia (Bert) O. Ktze

Brazilian pine tree (Araucaria angustifolia), the only native conifer of economic importance in Brazil (Figure 1A), representing the most exploited timber source up to the 70’s (Mattos, 1994; Shimizu and Oliveira, 1981). This tree can provide seed, wood, fiber and resin. The seeds are of high nutritious value and are generally consumed by humans and also by the wild fauna on the end of the fall and beginning of the winter season. Young trees are typically used as Christmas trees in the region of natural occurrence, and the wood of adult trees is used for furniture, structural timber, and almost all other kinds of wood applications. The tonality of its wood varies from yellow to brownish with a light and soft brilliant surface (Longhi, 1993). The wood of A. angustifolia contains 58.3% cellulose and 28.5% lignin with long fibres, resulting in the production of high quality paper. The resin serves as a base for the production of varnish, acetone and other chemical products (Carvalho, 1994).

The complete chloroplast genome sequence of Podocarpus lambertii: genome structure, evolutionary aspects, gene content and SSR detection.

Background Podocarpus lambertii (Podocarpaceae) is a native conifer from the Brazilian Atlantic Forest Biome, which is considered one of the 25 biodiversity hotspots in the world. The advancement of next-generation sequencing technologies has enabled the rapid acquisition of whole chloroplast (cp) genome sequences at low cost. Several studies have proven the potential of cp genomes as tools to understand enigmatic and basal phylogenetic relationships at different taxonomic levels, as well as further probe the structural and functional evolution of plants. In this work, we present the complete cp genome sequence of P. lambertii. Methodology/Principal Findings The P. lambertii cp genome is 133,734 bp in length, and similar to other sequenced cupressophytes, it lacks one of the large inverted repeat regions (IR). It contains 118 unique genes and one duplicated tRNA (trnN-GUU), which occurs as an inverted repeat sequence. The rps16 gene was not found, which was previously reported for the plastid genome of another Podocarpaceae (Nageia nagi) and Araucariaceae (Agathis dammara). Structurally, P. lambertii shows 4 inversions of a large DNA fragment ∼20,000 bp compared to the Podocarpus totara cp genome. These unexpected characteristics may be attributed to geographical distance and different adaptive needs. The P. lambertii cp genome presents a total of 28 tandem repeats and 156 SSRs, with homo- and dipolymers being the most common and tri-, tetra-, penta-, and hexapolymers occurring with less frequency. Conclusion The complete cp genome sequence of P. lambertii revealed significant structural changes, even in species from the same genus. These results reinforce the apparently loss of rps16 gene in Podocarpaceae cp genome. In addition, several SSRs in the P. lambertii cp genome are likely intraspecific polymorphism sites, which may allow highly sensitive phylogeographic and population structure studies, as well as phylogenetic studies of species of this genus.