Physiological, cellular and molecular aspects of the desiccation tolerance in Anadenanthera colubrina seeds during germination / Aspectos fisiológicos, celulares e moleculares da tolerância à dessecação em sementes de Anadenanthera colubrina durante a germinação

Abstract During germination, orthodox seeds become gradually intolerant to desiccation, and for this reason, they are a good model for recalcitrance studies. In the present work, physiological, biochemical, and ultrastructural aspects of the desiccation tolerance were characterized during the germination process of Anadenanthera colubrina seeds. The seeds were imbibed during zero (control), 2, 8, 12 (no germinated seeds), and 18 hours (germinated seeds with 1 mm protruded radicle); then they were dried for 72 hours, rehydrated and evaluated for survivorship. Along the imbibition, cytometric and ultrastructural analysis were performed, besides the extraction of the heat-stable proteins. Posteriorly to imbibition and drying, the evaluation of ultrastructural damages was performed. Desiccation tolerance was fully lost after root protrusion. There was no increase in 4C DNA content after the loss of desiccation tolerance. Ultrastructural characteristics of cells from 1mm roots resembled those found in the recalcitrant seeds, in both hydrated and dehydrated states. The loss of desiccation tolerance coincided with the reduction of heat-stable proteins.

Resumo Durante a germinação, sementes ortodoxas tornam-se gradualmente intolerantes à dessecação, e por isso podem ser utilizadas como modelo para o estudo da recalcitrância. No presente trabalho realizou-se uma caracterização dos aspectos fisiológicos, bioquímicos e ultraestruturais da perda da tolerância à dessecação de sementes de Anadenanthera colubrina em processo germinativo. Para isso as sementes foram embebidas durante 0 (controle), 2,8,12 e aproximadamente 18 horas (sementes germinadas com 1 mm de radícula), secas por 72 horas, reidratadas e a sobrevivência avaliada. Ao longo da embebição foram realizadas análises citométricas, ultraestruturais e extração de proteínas resistentes ao calor e após embebição e secagem foram avaliados danos ultraestruturais. A tolerância à dessecação foi totalmente perdida após a protrusão radicular. Não houve aumento do conteúdo de DNA 4C quando a tolerância à dessecação foi perdida. Características ultraestruturais de células de radículas de 1 mm assemelharam-se às encontradas em sementes recalcitrantes tanto no estado hidratado quanto desidratado. A perda da tolerância à dessecação coincidiu com a redução do conteúdo de proteínas resistentes ao calor.

Characterization saprobic fungi on leaf litter of two species of trees in the Atlantic Forest, Brazil

Abstract We investigated the composition and structure of fungal communities associated with leaf litter generated by Clusia nemorosa and Vismia guianensis that belong to phylogenetically-related botanical families and exist together in a remnant of the Atlantic Forest in Bahia, Brazil. Samplings were conducted during wet (June 2011) and dry (January 2013) seasons in Serra da Jibóia. The fungi were isolated using particle filtration and the 1,832 isolates represented 92 taxa. The wet season yielded the largest number of isolates (1,141) and taxa (76) compared with the dry season (641 isolates and 37 taxa). The richness and diversity of fungal species associated with C. nemorosa (64 taxa, Simpson=0.95)were higher compared with those of V.guianensis (59 taxa, Simpson =0.90). Analysis of similarity (ANOSIM) revealed significant variations in the composition and community structure of fungi isolated from the two plants as a function of seasons. In contrast, nonmetric multidimensional scaling (NMDS) analysis show that the seasonality was an important influence on the distribution of fungal species. However, the populations of the saprobic fungal communities were dynamic, and several factors may influence such communities in the Atlantic Forest.

Propuesta de un método para evaluar resistencia genética en Schizolobium parahybum (Vell) Blake (Pachaco) frente a Ceratocystis spp: evidencias preliminares de resistencia en Ecuador / Proposal for a method to evaluate genetic resistance in Schizolobium parahybum (Vell) Blake (Pachaco) against ceratocystis spp: preliminary evidence of resistance in Ecuador

Se condujo una investigación en el Trópico Húmedo Ecuatoriano (THE), dirigida a encontrar un método eficiente para evaluar la resistencia genética en árboles de Schizolobium parahybum (pachaco) frente al complejo Ceratocystis: C. paradoxa, C. moniliformis, y C. fimbriata. Se estudiaron dos métodos basados en el empleo de dos tipos de tejidos vegetales: a) tejidos de ramas laterales, y 2) tejidos de corteza fustal. Se emplearon cinco rodales de pachaco, tres de los cuales son considerados de introducción original de la especie forestal al THE desde la amazonía, y dos que son descendientes de los primeros. Los resultados permitieron definir que el método basado en tejidos de corteza fustal, fue el más eficiente y logísticamente viable. La metodología final aplicada, consistió en extraer corteza desde árboles adultos, reducirla a secciones pequeñas de 1,5 cm x 4 cm (6 cm2) y mantenerlas en una cámara húmeda durante 96 horas. Una vez distribuidas las secciones de corteza, se inocularon con 0,45mL-1 de una suspensión calibrada a razón de 30.000 unidades de infección (ascosporas, conidias y micelio). Para la evaluación, se empleó una escala arbitraria de 0 a 4 que permitió estimar el crecimiento de micelio y número de peritecios para cada uno de los hongos. Esta metodología permitió discriminar entre árboles: resistentes (0,0 a 1,0), moderadamente resistentes (1,1 a 2,0), susceptibles (2,1 a 3,0), y muy susceptibles (3,1 a 4,0), lo cual la hace viable para futuros trabajos de selección de individuos y mejoramiento genético de la especie.

A research was conducted in the Humid Tropics of Ecuador (THE), aimed at finding an efficient method to evaluate genetic resistance in Schizolobium parahybum (Pachaco) trees against Ceratocystis complex: C. paradoxa, C. moniliformis and C. fimbriata. We studied two methods based on the use of two types of plant tissues: a) tissue of lateral branches, and 2) stem bark tissues. Five forest of pachaco were used, three of which are considered original introduction of forestry specie to THE from the Amazon, and two who are descendants of the former. The results allowed to define the method based on stem bark tissue was the most efficient and logistically feasible. The final methodology applied, consisted in to remove bark from mature trees, reducing it to small sections of 1.5 cm x 4 cm (6cm2) and maintained in a moist chamber for 96 hours. Once distributed the sections of bark, were inoculated with 0.45mL-1 of a suspension calibrated at a rate of 30.000 units of infection (ascospores, conidia and mycelium). For evaluation, we used an arbitrary scale from 0 to 4, which allowed to estimate the growth of mycelium and perithecia number for each of the fungi. This methodology allows us to discriminate between trees: resistant (0.0 to 1.0), moderately resistant (1.1 to 2.0), susceptible (2.1 to 3.0), and very susceptible (3.1 to 4 , 0), which makes it viable for future selection of individuals and breeding of the forest species.

Genetic diversity and structure of wild populations of the tropical dry forest tree Jacaratia Mexicana (Brassicales: Caricaceae) at a local scale in Mexico

The tropical dry forest is a greatly endangered ecosystem, from which Jacaratia mexicana is a native tree. With the aim to assess the levels of genetic variation and population structure, four wild populations of J. mexicana were studied in the Sierra de Huautla Biosphere Reserve, Morelos, Mexico. For this, DNA was extracted from 159 individuals and were amplified with six random primers using the Random Amplified Polymorphic DNA (RAPD). A total of 54 bands were obtained, of which 50 (92.6%) were polymorphic. The total genetic diversity found within the four populations was 0.451 when estimated by Shannon’s index. An AMOVA analysis showed that 84% of the total genetic variation was found within populations and 16% was among populations. The UPGMA dendrogram showed that all individuals from one of the populations (Huaxtla) formed one distinct genetic group, while the rest of the individuals did not cluster according to population. A Mantel test did not show an association between genetic and geographical distances among populations (r=0.893, p=0.20). A Bayesian cluster analysis performed with STRUCTURE, showed that the most probable number of genetic groups in the data was four (K=4), and confirmed the distinctness of Huaxtla population. Our results showed that important genetic differentiation among populations can occur even at this small geographic scale and this has to be considered in conservation actions for this genetic resource.

Jacaratia mexicana es un árbol nativo del bosque tropical seco, que es considerado el tipo de vegetación en mayor riesgo de desaparecer completamente. Se utilizaron polimorfismos de ADN amplificados al azar (RAPD, Random Amplified Polymorphic DNA), para evaluar los niveles de variación y estructura genética en cuatro poblaciones silvestres de J. mexicana en la Reserva de la Biósfera Sierra de Huautla (Morelos, México). Se amplificó el ADN de 159 individuos utilizando seis oligonucleótidos (“primers”) aleatorios. Se obtuvieron en total 54 bandas, de las cuales 50 (92.6%) fueron polimórficas. La diversidad genética total que se encontró en las cuatro poblaciones de J. mexicana fue de 0.451 de acuerdo con el índice de Shannon. Un análisis de varianza molecular (AMOVA) mostró que el 84% de la variación genética total se encuentra dentro de las poblaciones y el 16% entre las poblaciones. Un dendrograma construido mediante el algoritmo UPGMA mostró que los individuos de una población (Huaxtla) formaron un grupo, mientras que el resto de los individuos no se agruparon de acuerdo a su población de origen. Una prueba de Mantel no mostró una asociación entre las distancias genéticas y geográficas entre las poblaciones (r=0.893, p=0.20). Un análisis de agrupamiento Bayesiano realizado mediante STRUCTURE mostró que el número más probable de grupos genéticos es cuatro (K=4) y confirmó la diferenciación de la población Huaxtla. Nuestros resultados muestran que una considerable diferenciación genética entre poblaciones puede existir incluso a esta escala geográfica, lo cual es de interés para la conservación de este recurso genético.

Genetic diversity in natural populations of Jacaranda decurrens Cham. determined using RAPD and AFLP markers

Jacaranda decurrens (Bignoniaceae) is an endemic species of the Cerrado with validated antitumoral activity. The genetic diversity of six populations of J. decurrens located in the State of São Paulo was determined in this study by using molecular markers for randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Following optimization of the amplification reaction, 10 selected primers generated 78 reproducible RAPD fragments that were mostly (69.2 percent) polymorphic. Two hundred and five reproducible AFLP fragments were generated by using four selected primer combinations; 46.3 percent of these fragments were polymorphic, indicating a considerable level of genetic diversity. Analysis of molecular variance (AMOVA) using these two groups of markers indicated that variability was strongly structured amongst populations. The unweighted pair group method with arithmatic mean (UPGMA) and Pearson's correlation coefficient (RAPD -0.16, p = 0.2082; AFLP 0.37, p = 0.1006) between genetic matrices and geographic distances suggested that the population structure followed an island model in which a single population of infinite size gave rise to the current populations of J. decurrens, independently of their spatial position. The results of this study indicate that RAPD and AFLP markers were similarly efficient in measuring the genetic variability amongst natural populations of J. decurrens. These data may be useful for developing strategies for the preservation of this medicinal species in the Cerrado.

Molecular phylogenetic relationships among four species of the mangrove tree genus Bruguiera (Rhizophoraceae), as revealed by chromosome and RAPD markers

Analysis of karyotype, nuclear DNA content and RAPD markers were performed in four species of Bruguiera (Rhizophoraceae) of Bhitarkanika mangrove forests, Orissa, India. Detailed karyotype analysis revealing 2n=34 in B. cylindrica and 2n=36 in B. gymnorrhiza was reported for the first time and 2n=34 in B. parviflora and B. sexangula was confirmed. On the basis of the common types of chromosomes present among Bruguiera, two distinct groups were found; one consists of B. cylindrica and B. parviflora and the other of B. gymnorrhiza and B. sexangula. The symmetrical karyotype with same chromosome types grouped B. cylindrica and B. parviflora together and presence of Type E chromosomes placed B. gymnorrhiza and B. sexangula in a separate group, suggesting their closer affinity in their respective group. Analysis of chromosome length, volume, INV and 4C DNA content confirmed this division. Nuclear DNA content was two-fold higher (~17.0 pg) in the second group than in the first (~8.0 pg). The amplification products generated through RAPD revealed 1-9 amplicons with size variations from 600 bp to 2 500 bp with 49.31% genetic similarity between B. gymnorrhiza and B. sexangula and 47.10% in between B. cylindrica and B. parviflora. The high copy number marker band (~ 1 100 bp) yielded in OPN-15 primer in B. parviflora the characteristic DNA marker, which was cloned and used as probes for assessment of genetic diversity, and demonstrated its close genetic affinity to B. cylindrica. B. gymnorrhiza and B. sexangula also produced similar marker bands of ~600 bp and ~2 200 bp in the same primer. All of the cytological, 4C DNA content and RAPD data confirmed the existence of two taxonomically distinct groups of Bruguiera: one consisting of B. cylindrica and B. parviflora and the other of B. gymnorrhiza and B. sexangula as placed earlier (1862) in the tribe Rhizophoreae by Bentham and Hooker, on the basis of the flowering habits of Bruguiera. Genetically, the B. sexangula and B. gymnorrhiza group was found to be very closely, rather than distantly, related to B. parviflora and B. cylindrica. Our results demonstrate that molecular markers together with cytological evidence provide an effective tool to access the existing interspecific genetic polymorphism in mangrove species, to solve the taxonomic problems and to design their conservation strategy. Rev. Biol. Trop. 55 (2): 437-448. Epub 2007 June, 29.

Estudiamos cuatro especies del mangle Bruguiera (Rhizophoraceae) en Orissa, India. Los cromosomas indican queB. cylindrica y B. parviflora son un grupo taxonómico, y que B. gymnorrhiza y B. sexangula son otro. Genéticamente, el par B. sexangula y B. gymnorrhiza está cercanamente emparentado con B. parviflora and B. cylindrica. Nuestros datos indican que el uso combinado de marcadores genéticos y evidencia citológica permiten discernir el polimorfismo genético interespecífico en los mangles, tanto para resolver problemas taxonómicos como para diseña estrategias eficaces de conservación.

A micromethod for high throughput RNA extraction in forest trees

A large quantity of high quality RNA is often required in the analysis of gene expression. However, RNA extraction from samples taken from woody plants is generally complex, and represents the main limitation to study gene expression, particularly in refractory species like conifers. Standard RNA extraction protocols are available but they are highly time consuming, and not adapted to large scale extraction. Here we present a high-throughput RNA extraction protocol. This protocol was adapted to a micro-scale by modifying the classical cetyltrimethylammonium (CTAB) protocol developed for pine: (i) quantity of material used (100-200 mg of sample), (ii) disruption of samples in microtube using a mechanical tissue disrupter, and (iii) the use of SSTE buffer. One hundred samples of woody plant tissues/organs can be easily treated in two working days. An average of 15 /ig of high quality RNA per sample was obtained. The RNA extracted is suitable for applications such as real time reverse transcription polymerase chain reaction, cDNA library construction or synthesis of complex targets for microarray analysis.

Contributions of weather variables for specific adaptation of rubber tree (Hevea brasiliensis Muell.- Arg) clones

The specific adaptation of 15 rubber tree (Hevea brasiliensis) clones was assessed by analyzing yield during a normal year (1997-98) and a year (1998-99) in which the yield was exceptional. Differences in yield in response to changes in weather conditions over the years were evident with clones RRII 203, RRIM 703, PB 5/51 and PB 235 which all exhibited a negative trend with increasing wind velocity during 1997-98, these clones also exhibited a negative correlation with minimum temperature during 1998-99. The prominent yield differences across the years made selection based on both yield and stability inevitable through computing weather variables and environmental index as covariant. To determine the contribution of variable(s) to genotype-environment (GE) interactions, the GE interaction was partitioned into heterogeneity and residual GE interaction. Heterogeneity only for environmental index was highly significant (p = 0.01), meaning that stability or instability of clones was due to a linear effect of the environmental index. The non-significant values of heterogeneity for the weather variables revealed that none of these factors individually was sufficient to explain heterogeneity. A QBASIC computer program called STABLE was used to select simultaneously for yield and stability. Clones PB 235, RRII 118, RRII 203, RRIM 703 and RRIM 600 were stable over the years investigated.

Genotype-environment interaction and phenotypic stability for girth growth and rubber yield of Hevea clones in São Paulo State, Brazil

The best-yielding, best vigour and most stable Hevea clones are identified by growing clones in different environments. However, research on the stability in Hevea brasiliensis (Willd. Adr. ex Juss.) Muell.-Arg. is scarce. The objectives of this work were to assess genotype-environment interaction and determine stable genotypes. Stability analysis were performed on results for girth growth and rubber yield of seven clones from five comparative trials conducted over 10 years (girth growth) and four years (rubber yield) in São Paulo State, Brazil. Stability was estimated using the Eberhart and Russell (1966) method. Year by location and location variability were the dominant sources of interactions. The stability analysis identified GT 1 and IAN 873 as the most stable clones for girth growth and rubber yield respectively since their regression coefficients were almost the unity (beta = 1) and they had one of the lowest deviations from regressions (S2di). Their coefficient of determination (Rý) was as high as 89.5 percent and 89.8 percent confirming their stability. In contrast, clones such as PB 235, PR 261, and RRIM 701 for girth growth and clones such as GT 1 for rubber yield with regression coefficients greater than one were regarded as sensitive to environment changes.

Genetic structure and gene flow in Eugenia dysenterica DC in the Brazilian Cerrado utilizing SSR markers

The ''cagaita tree'' (Eugenia dysenterica) is a plant found widespread in the Brazilian Cerrado. Its fruit is used for popular consumption and for industrial purposes. This study opens a new perspective for the generation of population genetic data and parameters estimates for devising sound collection and conservation procedures for Eugenia dysenterica. A battery of 356 primer pairs developed for Eucalyptus spp. was tested on the ''cagaita tree''. Only 10 primer pairs were found to be transferable between the two species. Using a polyacrilamide gel, an average of 10.4 alleles per locus was detected, in a sample of 116 individuals from 10 natural ''cagaita tree'' populations. Seven polymorphic loci allowed estimation of genetic parameters, including expected average heterozygosity He = 0,442, among population diversity, R ST = 0,268 and gene flow Nm = 0,680. Results indicated a potential of SSR locus transferability developed for Eucalyptus to other species of different genera, such as in the case of the ''cagaita tree''. The high genetic diversity among populations detected with SSR markers indicated that these markers are highly sensitive to detect population structure. Estimated Nm values and the existence of private alleles indicated reduced gene flow and consequently possible damage to the metapopulation structure.

Clonal stability of latex yield in eleven clones of Hevea brasiliensis Muell. Arg

Eleven Hevea brasiliensis clones were evaluated for clonal stability of latex yield. A randomized complete block design was used with four replicates, two locations, seven years and three periods per year. Stability analysis was based on clone x year and clone x year x location interactions. Five stability parameters viz environmental variance, shukla's stability variance, regression of clonal latex yield on environmental index, variance due to regression and variance due to deviation from regression were applied. There was significant clone x environment effect at the two levels of interaction. Among the eleven clones, C 162 was outstanding for clonal stability and it can serve as donor parent for stability alleles. Three clones (C 76, C 150 and C 154) were also stable. The four stable clones (C 76, C 150, C 154 and C 162) are suitable for broad-spectrum recommendation for latex yield. Five clones (C 83, C 143, C 163, C 202 and RRIM 600) will require environment-specific recommendation because of their unstable phenotype. The stability feature of two clones (C 145 and C 159) was not clear and this will be investigated in subsequent studies

Diurnal regulation of plastid genes in Populus deltoides.

Light regulates leaf and chloroplast development, together with overall chloroplast gene expression at various levels. Plants respond to diurnal and seasonal changes in light by changing expression of photosynthesis genes and metabolism. In Populus deltoides, a deciduous tree species, leaf development begins in the month of March and leaf maturation is attained by summer, which is subsequently followed by autumnal senescence and fall. In the present study, diurnal changes in the steady state transcript levels of plastid genes were examined in the fully developed leaves during summer season. Our results show that steady state level of the psaA/B, psbA, psbEFLJ and petA transcripts showed differential accumulation during diurnal cycle in summer. However, there was no significant change in the pigment composition during the day/night cycle. Our studies suggest that the diurnal regulation of steady state mRNA accumulation may play a crucial role during daily adjustments in plants life with rapidly changing light irradiance and temperature.

Hermaphroditism in the rubber tree Hevea brasiliensis (Willd. ex Adr. de Juss.) Müell. Arg

The aim of this work was to obtain infomiation about the reproductive biology of Hevea brasiliensis (Willd. ex Adr. de Juss.) Müell. Arg. The intemal organization of the flowers of 13 clones of the species (GT 711, PR 107, Fx 25, AVROS 1328, RRIM 526, RRIM 600, Tjir 1, Tjir 16, IAC 1, IAC 2, IAN 2652, IAN 2813 and PB 86) was studied by analysing the Mstological sections of flower buds. Among female flowers 25.8 per cent were hermaphodites (in clones GT 711, Tjir 16, PR 107 and AVROS 1328). There was evidence of protandry and cleistogamy in H. brasiliensis.