ABSTRACT. One of the main objectives of sugarcane plantations is to increase their longevity without decreasing agricultural productivity. In the present study, we analyzed the proteome of the axillary buds of ‘RB966928’ to investigate possible changes in the number of proteins at different cutting stages. Using tryptic digestion followed by ultra-performance liquid chromatography coupled with high-resolution time-of-flight mass spectrometry, 122 proteins were identified from the proteome of the axillary buds of ‘RB966928’. Of the 122, respectively 97 and 95 proteins were detected at the first and fifth cutting stages, of which 27 and 25 proteins were unique to the respective stage. Proteins that prevent the misfolding of polypeptides generated under stress were exclusively detected at the first cutting stage. Meanwhile, proteins associated with stress responses and disease resistance were exclusively detected at the fifth cutting stage. The present proteomic analysis in the regrowth cycles and axillary bud development of ‘RB966928’ significantly advanced our understanding of the biological processes linked to the reduction of agricultural productivity of sugarcane with the advancement of cutting age. Absence of proteins to tolerate adverse growth conditions at the fifth cutting stage may be related to reduced agricultural productivity, in addition to environmental stress, soil compaction, nutrient availability, cultural practices, and pests or pathogen attacks at different phenological stages of crops.

ABSTRACT. The genetic differentiation of grapevine rootstock varieties was inferred by the Artificial Neural Network approach based on the Self-Organizing Map algorithm. A combination of RAPD and SSR molecular markers, yielding polymorphic informative loci, was used to determine the genetic characterization among the rootstock varieties 420-A, Schwarzmann, IAC-766 Campinas, Traviú, Kober 5BB, and IAC-572 Jales. A neural network algorithm, based on allelic frequency, showed that the individual grapevine rootstocks (n = 64) were grouped into three genetically differentiated clusters. Cluster 1 included only the Kober 5BB rootstock, Cluster 2 included rootstocks of the varieties Traviú and IAC-572, and Cluster 3 included 420-A, Schwarzmann and IAC-766 plants. Evidence from the current study indicates that, despite the morphological similarities of the 420-A and Kober 5BB varieties, which share the same genetic origin, two new varieties were generated that are genetically divergent and show differences in performance.

RESUMO Somaclones de cactos mandacaru (Cereus peruvianus Mill.) com caules típicos e atípicos foram regenerados a partir do cultivo de calos. Uma vez que a relação genética entre estes somaclones ainda não foi investigada, o presente estudo empregou primers de microssatélites heterólogos para avaliar a diversidade genética dentro e entre os somaclones com fenótipos típicos e atípicos com alta divergência morfológica. O valor de identidade genética entre os somaclones foi alto (I = 0,929) e AMOVA mostrou maior variação genética dentro (96%) do que entre (4%) as amostras de somaclones. O polimorfismo nos locos microssatélites indicou altos níveis de heterozigosidades observada e esperada nos somaclones atípicos, presumivelmente com potencial alto de adaptação e como fonte de genótipos para a geração de novas variedades de cactos ornamentais. Por outro lado, a divergência genética baixa entre os somaclones típicos e atípicos é uma indicação promissora para o uso dos somaclones atípicos como fonte de compostos químicos de interesse comercial e industrial. As variações somaclonais que ocorreram no cultivo in vitro dos calos geraram subpopulações fenotipicamente diferenciadas com baixa divergência genética, porém com alta variabilidade genética, suficiente para recomendar os somaclones atípicos como fonte de genótipos para gerar novas variedades de cactos ornamentais e de plantas com novas características, necessários para a implementação de programas de melhoramento.

ABSTRACT Somaclones of mandacaru cactus (Cereus peruvianus Mill.) with typical and atypical shoots were regenerated from a callus. Since the genetic relationship among regenerated somaclones has not been investigated, current study employed heterologous microsatellite primers to examine the molecular diversity within and among somaclones, showing typical and atypical phenotypes with high morphological divergence. Nei identity value calculated between the somaclones was high (I = 0.929) and AMOVA showed higher genetic variation within (96%) than among (4%) the samples of somaclones. The polymorphism in the microsatellite loci indicated high levels of mean observed and expected heterozygosity in atypical somaclones, presumably with high adaptive potential and as source of genotypes for generation of new varieties of ornamental cacti. On the other hand, low molecular divergence between typical and atypical morphologies of somaclones is a promising perspective for use of the atypical somaclones as source of chemical compounds of commercial and industrial interest. The somaclonal variations occurring in vitro callus culture has generated phenotypically differentiated subpopulations with low molecular divergence, however with high genetic variability, enough to be recommended as a source of genotypes to generate new varieties of ornamental cacti and of plants with new traits, necessary for breeding programs.

Information on genetic diversity is essential to the characterization and utilization of germplasm. The genetic diversity of twenty-two sweet corn cultivars (seventeen open-pollinated varieties, OPV, and five hybrids, H) was investigated by applying simple sequence repeat markers. A total of 257 primers were tested, of which 160 were found to be usable in terms of high reproducibility for all the samples tested; 45 were polymorphic loci, of which 30 were used to assess the genetic diversity of sweet corn cultivars. We detected a total of 86 alleles using 30 microsatellite primers. The mean polymorphism was 82 %. The highest heterozygosity values (Ho = 0.20) were found in the PR030-Doce Flor da Serra and BR427 III OPVs, whereas the lowest values (0.14) were recorded in the MG161-Branco Doce and Doce Cubano OPVs. The polymorphism information content ranged from 0.19 (Umc2319) to 0.71 (Umc2205). The analysis of molecular variance revealed that most of the genetic variability was concentrated within the cultivars of sweet corn (75 %), with less variability between them (25 %). The consensus tree derived from the neighbor-joining (NJ) algorithm using 1,000 bootstrapping replicates revealed seven genetically different groups. Nei’s diversity values varied between 0.103 (Doce do Hawai × CNPH-1 cultivars) and 0.645 (Amarelo Doce × Lili cultivars), indicating a narrow genetic basis. The Lili hybrid was the most distant cultivar, as revealed by Principal Coordinates Analysis and the NJ tree. This study on genetic diversity will be useful for planning future studies on sweet corn genetic resources and can complement the breeding programs for this crop.

No presente estudo foram adicionados ao meio de cultura Knudson-C (KC), suplementos específicos (micronutriente, peptona, carvão ativado) e complexos (água de coco e polpa de banana) para estimular a propagação in vitro das espécies de orquídeas Hadrolaelia purpurata (=Laelia purpurata) e Encyclia randii. O número maior de plântulas de H. purpurata foi observado no meio KC contendo 90 g L-1 de polpa de banana com iluminação contínua (24h). O efeito positivo da adição de polpa de banana foi descrito para a formação de plântulas e folhas de E. randii. O número de plântulas de E. randii mantidas com iluminação contínua também foi alto em meio contendo água de coco. Um efeito positivo da adição de peptona foi descrito somente na formação de plântulas de E. randii mantidas sob iluminação contínua. O maior número de plântulas de H. purpurata e folhas de E. randii ocorreu com iluminação contínua. O sucesso na germinação assimbiótica de sementes com três anos de idade, das duas espécies de orquídeas nativas do Brasil, de regiões diferentes, com custo reduzido, é uma técnica oportuna para ser usada em viveiros comerciais, e para a conservação ex-situ destas espécies nativas e exploradas de orquídeas.

In the current study, complex (coconut water and ripe-banana pulp) and specific (micronutrients, peptone and activated charcoal) supplements were added to Knudson-C medium (KC) medium to stimulate the in vitro propagation of Hadrolaelia purpurata (=Laelia purpurata) and Encyclia randii orchid species. The highest number of H. purpurata seedlings occurred in KC medium containing 90 g L-1 banana pulp maintained with 24-hour illumination. Positive effects of the banana pulp addition in KC medium were reported for the formation of seedlings and leaves from E. randii seeds. The seedling number from seeds of E. randii maintained with continuous illumination was also high in KC medium containing coconut water. Positive effects due to the addition of peptone were reported only in the case of seedling formation from E. randii seeds maintained under continuous illumination. The highest number of H. purpurata seedlings and leaves of E. randii occurred in cultures maintained in continuous illumination. The successful asymbiotic germination of the three-year-old seeds of two Brazilian native species from different regions at reduced costs offers an opportunity for the use of the techniques in commercial nurseries and for ex situ conservation of the native and explored orchid species.

Nenhum estudo prévio tem investigado o uso de grafite como substituto do carvão ativado em orquídeas. No presente trabalho, concentrações diferentes de carvão ativado ou de grafite foram adicionadas ao meio KC para escurecer o meio de cultura e estimular a propagação in vitro de Cattleya bicolor e de um duplo híbrido de orquídea ('BLC Pastoral Innocence'). As plântulas foram inoculadas em meio KC sem reguladores de crescimento; os efeitos de concentrações de carvão ativado (0,0; 1,5; 3,0; 4,5; 6,0 and 7,5 g L-1) e de grafite (0,0; 1,5; 3,0; 4,5; 6,0 and 7,5 g L-1) foram testados separadamente. Foram inoculadas quinze plântulas em cada frasco e os experimentos foram conduzidos sob esquema de planejamento fatorial com dois fatores: meios de cultura e luminosidade; o meio de cultura com onze níveis (diferentes concentrações de grafite ou de carvão ativado) e luminosidade com dois níveis (fotoperíodo de 14h e iluminação contínua). Para cada combinação do nível fatorial foram feitas três repetições. Após seis meses de cultivo, foram registrados os números médios de brotos e raízes induzidos em cada concentração de carvão ativado e de grafite. O maior número de brotos nas plântulas de C. bicolor foi observado no meio contendo 6,0 e 7,5 g L-1 de grafite, e o maior número de raízes foi verificado no meio contendo 6,0 g L-1 de carvão ativado. No duplo híbrido o maior número de brotos e raízes foi observado no meio contendo 4,5 g L-1 de carvão ativado. O grafite não pode ser recomendado para substituir o carvão ativado.

No previous studies have investigated the use of graphite instead of activated charcoal in orchids. In this work, different concentrations of activated charcoal or graphite were added to KC medium to darken the culture medium and stimulate the in vitro propagation of Cattleya bicolor and of a double hybrid orchid ('BLC Pastoral Innocence'). The seedlings were inoculated on growth regulator-free KC medium; the effects of activated charcoal (0.0, 1.5, 3.0, 4.5, 6.0, and 7.5 g L-1) and graphite (0.0, 1.5, 3.0, 4.5, 6.0, and 7.5 g L-1) concentrations were tested separately. Each flask contained fifteen seedlings, and the experiments had a randomised, two-way factorial design. The two tested variables were culture medium and illumination levels; eleven culture levels (different concentrations of graphite or activate charcoal) and two illumination levels (14-hr photoperiod or continuous illumination) were evaluated. Three replicates of each combination were utilised. After six months of culture, the average numbers of induced buds and roots per seedling were recorded for each concentration of activated charcoal or graphite. Whereas the largest number of buds in C. bicolor seedlings occurred on media containing 6.0 or 7.5 g L-1 graphite, the largest number of roots occurred on media containing 6.0 g L-1 activated charcoal. In the hybrid 'BLC Pastoral Innocence', the largest number of buds and roots was reported in medium with 4.5 g L-1 activated charcoal. When using graphite in place of activated charcoal, we obtained disparate the results in root formation that suggest that graphite is not a recommended substitute for activated charcoal.

A técnica de propagação in vitro é considerada efetiva para fins comerciais e de conservação de orquídeas. A metodologia de modelos lineares generalizados (MLG) foi usada para analisar um experimento de germinação de Cattleya bicolor. O propósito do experimento foi avaliar os efeitos da temperatura de armazenamento e dos meios de cultivo sobre a germinação, cuja resposta foi considerada binária. Análise convencional com ou sem transformação de dados foram também realizados. Todas as abordagens estatísticas indicaram a importância da temperatura sobre a viabilidade das sementes. Entretanto, os efeitos de meios de cultivo e interação foram significativos apenas para MLG. As sementes armazenadas a 10°C incrementaram sua viabilidade, onde o meio a base de coco atingiu o melhor desempenho. Os resultados enfatizam a importância de adotar MLG, para melhorar a confiabilidade em situações onde a variável resposta segue uma distribuição distinta à normal.

This study aimed at applying the generalized linear models (GLM) for the analysis of a germination experiment of Cattleya bicolor in which the response variable was binary. The purpose of this experiment was to assess the effects of the storage temperatures and culture mediums on the seed viability. The analyses of variance was also carried out either with or without the data transformation. All the statistical approaches indicated the importance of the storage temperature on the seed viability. But, the culture media and interaction effects were significant only by the GLM. Based on the GLM, the seeds stored at 10°C increased viability, in which the coconut medium achieved the best performance. The results emphasized the importance of adopting the GLM to improve the reliability in many situations where the response variable followed a non-normal distribution.

Isozyme biochemical marker may be useful tool for genomic analysis of maize populations undergoing recurrent selection. Thus, isozymes markers was utilized for assess the changes in the genetic variability and distance in a Brazilian composite population of popcorn following four cycles of recurrent selection for yield. One hundred and ninety-six half-sib families were evaluated from each cycle and the ten highest-yielding families (5.2%) were recombined to produce the next cycle. Isozyme analysis considered 80 seedlings per cycle. Simple linear regression equations were estimated among the allele frequencies in each locus in function of the selection cycles, the genetic distances among the cycles and the average heterozygosity per locus for each cycle. Regression analysis did not reveal any common trend for changes in allele frequencies presumably due to selection. The estimates of the number of polymorphic locus, of the mean of allele per locus and the mean heterozygosity did not reveal any reduction in variability. It was concluded that four selection cycles did not cause relevant changes in the variability or genetic distance among the selection cycles of CMS-43 popcorn population. Isozymes markers analysis showed that the number of recombined half-sib families in recurrent selection was suitable.

A análise do polimorfismo de isozimas esterases foi usada para reportar a estrutura genética de duas populações remanecentes da espécie de árvore em extinção Aspidosperma polyneuron Müll.Arg. (Apocynaceae). Variação alélica foi detectada em três locos de isoesterases (Est-3, Est-9, e Est-10). A proporção de locos polimórficos de ambas as populações foi de 30%, sendo observado um desvio do equilíbrio de Hardy-Weinberg no loco Est-3 na população da região norte do Estado do Paraná. Uma distorção na segregação e um mais baixo nível de heterozigosidade observada e esperada nesta população foram atribuídos ao efeito do genótipo fundador. Os valores altos de identidade genética das populações do norte e noroeste do Estado estão de acordo com o baixo nível de divergência genética interpopulacional demonstrado pelo valor de F(ST) (0,03). O valor de F(IS) (0,23) indicou moderado nível de endocruzamentos. A. polyneuron pode ser indicada como um exemplo de espécie em extinção apresentando variação genética alta, contrastando com a variação genética baixa reportada sobre espécies em extinção. As isozimas esterases podem ser um bom marcador genético em estudos de populações naturais de A. polyneuron.

The population genetic structure of the endangered tree species Aspidosperma polyneuron Mull.Arg. (Apocynaceae) was reported based on analysis of esterase polymorphism in two remanant populations. Allelic variation was detected at three isoesterase loci (Est-3, Est-9, and Est-10). The proportion of polymorphic loci for both populations was 30% and deviation from Hardy-Weinberg equilibrium was observed for the Est-3 locus observed in the northern population. Segregation distortion and the lower level of observed and expected heterozygosity in this population were attributed to founder genotype. The high genetic identity values for northern and northwestern populations are in accordance with the low levels of interpopulation genetic divergence demonstrated by the F(ST) (0.03) value. The F(IS) value (0.23) indicated moderate levels of inbreeding. A. polyneuron can be indicated as an example of endangered species suggesting high genetic variation in contrast to the low genetic variation reported for endangered species. The esterase isozymes may be a good genetic marker for studies of natural A. polyneuron populations.

O presente estudo relata um método simples e promissor para multiplicação in vitro de Tabernaemontana fuchsiaefolia, uma espécie abundantemente encontrada no sul do Brasil, utilizada com propósitos medicinais e como fonte de compostos que podem ser usados para desenvolver novas drogas sintéticas. Para estimular o crescimento e a proliferação de caules, os explantes apicais e os hipocótilos foram cultivados em meio MS contendo diferentes concentrações das citocininas 6-benzil-amino-purina (BA) e cinetina (KIN) e suplementado com fluoroglucinol (1, 3, 5-hidroxibenzeno). A micropropagação de T. fuchsiaefolia foi positivamente influenciada por citocininas e a cinetina induziu à formação de mais caules por explantes do que a citoquinina BA. O efeito favorável de fluoroglucinol na indução de gemas apicais e laterais a partir dos hipocótilos foi verificado na ausência de cinetina ou em meio com todas as concentrações de cinetina testadas. Os caules recém-formados foram cortados dos explantes e o enraizamento satisfatório destes caules foi obtido após um tratamento com pulsos breves em 5,0 mg/l da auxina ácido 3-indolbutírico (IBA). A adição de fluoroglucinol no meio MS induziu ao enraizamento em 29% dos segmentos nodais transferidos para o meio MS na ausência de IBA, em 50% dos segmentos nodais transferidos para o meio MS contendo 0,5 mg/l de IBA e em segmentos nodais previamente submetidos aos breves pulsos de IBA.

This study describes a simple and promising for in vitro multiplication of Tabernaemontana fuchsiaefolia, a species abundantly found in southern Brazil utilized for medicinal purposes and as a source of compounds that may be used to develop new synthetic drugs. Apical and hypocotyl explants were cultured in MS medium containing different concentrations of the cytokinins benzylaminopurine (BA) and 6-furfurylaminopurine (kinetin), supplemented with phloroglucinol (1, 3, 5-hydroxybenzene) to stimulate growth and shoot proliferation. Cytokinin added to the culture media positively influenced the micropropagation of T. fuchsiaefolia.and kinetin induced more shoots per explant than BA cytokinin. A favorable effect of phloroglucinol on apical and lateral buds from hypocotyls was also achieved in medium containing no kinetin or in all kinetin concentrations tested. Short pulses of auxin 3-indolebutyric acid (IBA) 5.0 mg/l resulted in satisfactory rooting in apical microcuttings. The addition of phloroglucinol to MS medium induced rhizogenesis in 29% of the nodal segments transferred to MS medium in the absence of IBA and in 50% of the nodal segments transferred to MS medium containing 0.5 mg/l IBA and in nodal segments previously submitted to short pulses of IBA.