Citogenética, quantidade de DNA e indução de poliploidia em Pfaffia glomerata Spreng

The genus Pfaffia (Amaranthaceae) comprises 33 species distributed in the neotropics. Thanks to some chemicals found in the genus that resemble those found in the genus Panax (Korean ginseng), the species P. glomerata is known as "Brazilian ginseng". P. glomerata is commonly used as tonic, phrodisiac, rejuvenator, anti-fatigue, anti-stress and against gastric disorders. Its medicinal properties are attributed to the compounds mainly found in their tuber roots, being the β-ecdysone the most explored by the pharmaceutical and cosmetic industries. Despite its economical and medicinal importance, few studies have been done to investigate the genetic aspects of the species. In this work, cytometric and chromosomal analyzes were made with diploid and induced polyploid of P. glomerata. The polyploidization occurred by in vitro chromosome doubling, using antimitotic agents (colchicine and oryzalin) at different concentrations and exposure times. Flow cytometry analysis showed 2.41 pg (2n = 34) and 4.79 pg (2n = 68) of DNA amounts. Both P. glomerata diploid and tetraploid were analyzed by conventional staining techniques and chromosomal differential staining by CMA3/DA/DAPI banding and rDNA FISH. In spite of the small sizes of chromosomes, it was possible to identify their primary constrictions and a pair of satellites chromosomes in the diploid and two pairs in the polyploid. Karyograms and idiograms were assembled. Karyotypic formulas were 16M + 1SM considering the 2n = 34 genome and 32M + 2SM considering 2n = 68. The interphase nuclei seem to reticulated. The fluorochrome CMA3/DA/DAPI banding revealed all chromosomes with centromeric DAPI+ bands indicating a heterochromatic region around the centromeres. A CMA+ band, with different extension between the homologous was observed on the short arm of one chromosome pair. The FISH technique with rDNA 45S and 5S sequences revealed two signals to each probe for the diploid genome and four signals in the polyploid genome. Homologous chromosomes are heteromorphic when considering the rDNA regions here investigated. The content of β ecdysone present in the methanol extract of the roots of P. glomerata with four months of cultivation was 0.32% for the diploid and 0.48% for the tetraploid suggesting an increase of 50% on the tetraploid comparing with the diploid. Pollen structure was determined by Erdtman acetolysis, and no differences were observed between diploid accessions. This is the first report of DNA amount in the genus Pfaffia and data obtained with FISH using 45S and 5S rDNA probes have not been published elsewhere. The β-ecdysone quantification suggests that chromosome duplication is an alternative to increase the economical value of the species.O gênero Pfaffia (Amaranthaceae) compreende 33 espécies distribuídas na Região Neotropical. Graças a algumas substâncias químicas encontradas no gênero que se assemelham àquelas encontradas no gênero Panax (ginseng coreano), a espécie Pfaffia glomerata é conhecida “ginseng brasileiro ”. Tal planta é utilizada como tônico, afrodisíaco, rejuvenescedor, anti-fadiga, antiestresse e contra distúrbios gástricos. Suas propriedades medicinais são atribuídas a compostos encontrados principalmente em suas raízes tuberosas, sendo a β-ecdisona a mais visada pela indústria farmacêutica e cosmética. Apesar de sua importância econômica e medicinal, são poucos os estudos que investigam aracterísticas genéticas da espécie. Neste trabalho foram feitas análises citométricas e cromossômicas de fáfias diploide e poliploide induzida. A poliploidização se deu por duplicação cromossômica in vitro com uso de agentes antimitóticos (colchicina e orizalina) em diferentes concentrações e tempo de exposição. A quantidade de DNA encontrada foi de 2,41 pg (2n=34) e 4,79 pg (2n=68). O genoma de P. glomerata diploide e tetraploide foi analisado por técnicas de coloração convencional e coloração diferencial dos cromossomos por bandamento com CMA3/DA/DAPI e FISH para DNAr. Embora os cromossomos da espécie sejam pequenos, foi possível identificar suas constrições primárias e encontrar um par de cromossomos com satélite no representante diploide e dois pares no poliploide. Foram montados cariogramas e idiogramas, sendo que as fórmulas cariotípicas foram: 16M + 1SM para o genoma 2n=34 e 32M + 2SM para o genoma 2n=68. Os núcleos interfásicos mostraram-se reticulados. Através do bandeamento com CMA3/DA/DAPI observou-se que todos os cromossomos possuem bandas centroméricas DAPI+, indicando uma região heterocromática ao redor dos centrômeros. Foi localizada uma banda CMA+ no braço curto de um dos cromossomos, sendo registrada diferença na extensão da banda entre os homólogos. O emprego da FISH com as sequências de DNAr 45S e 5S, gerou para cada tipo de sonda, dois sinais em um par de homólogos para o genoma diploide e quatro sinais no genoma poliploide. Os cromossomos homólogos apresentaram heteromorfismo para as regiões de DNAr invetigadas. O teor de β-ecdisona presente no extrato metanólico das raízes de fáfia com quatro meses de cultivo, foi de 0,32% para a planta diploide e 0,48% para a poliploide sugerindo um aumento de 50% na produção do composto no poliploide quando comparado com seu diploide. A estrutura do grão de pólen, determinada por acetólise Erdtman, mostrou-se estável entre acessos diploides da espécie. Este é o primeiro relato da quantidade de DNA no gênero Pfaffia. Também são inéditos os dados obtidos por FISH com sondas para DNAr 45S e 5S para a P. glomerata diploide e poliploide. Os resultados da quantificação do teor de β-ecdisona sugerem a duplicação cromossômica como alternativa para incremento do valor econômico da espécie.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorCNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerai

Variação genética no complexo poliploide Zygopetalum maculatum (Orchidaceae)

The orchids of the "Zygopetalum maculatum" (Kunth) Garay complex are typical of the “campos rupestres” of the east side of Brazil. Currently, six species are recognized for this group which, due to their morphological similarity, is recognized here as a species complex. In order to understand the phenotypic pattern observed among the populations and contribute to clarify the formation of this complex, this study characterized 22 populations of “Z. maculatum”, being 21 populations from Brazil and one population from Bolivia. The study included DNA amount estimation, determination of the chromosome number, meiotic behavior and genetic diversity estimation. The average of DNA content revealed three 2C values, 7.36 pg, 10.52 pg and 14.09 pg of DNA, which were associated to 2n = 48; 2n = 72 and 2n = 96 chromosomes, respectively. The variation was observed within and among populations. Karyotypic data (morphometry, 5S and 45S rDNA) showed similarity among all ploidy levels. Meiosis behavior was not regular being observed chromosome delay and loss, bridges, micronuclei, chromosomal stickiness and asynchrony. The dendrogram based on ISSR revealed similarity regarding the ploidy level and the geographical distribution of the individuals. The data also suggested low genetic diversity within populations being most of the diversity observed among populations. In general, the results indicate that polyploidization events are probably related to the Zygopetalum diversification and speciation, being an important process to explain the morphological variations observed within the complex.As orquídeas do complexo "Zygopetalum maculatum" (Kunth) Garay são típicas dos campos rupestres e de altitude do leste do Brasil. Atualmente são reconhecidas seis espécies para este grupo que, por compartilharem características morfológicas diagnósticas, são reconhecidas aqui como um complexo de espécies. De forma a contribuir para entender o padrão de variação fenotípica observado entre as populações e com isso tentar esclarecer a formação deste complexo, este trabalho caracterizou do ponto de vista genético ar 22 populações de "Z. maculatum", sendo 21 provenientes do Brasil e uma da Bolívia. As análises compreenderam estimativa da quantidade de DNA nuclear, determinação do número cromossômico, comportamento meiótico e diversidade genética identificada por marcadores do tipo ISSR. O conteúdo médio de DNA revelou três níveis de ploidia no complexo, 7,36 pg, 10,52 pg e 14,09 pg de DNA, sendo estes relacionados a números cromossômicos de 2n=48; 2n=72; 2n=96, respectivamente. Tal variação ocorreu entre e dentro das populações. Dados cariotípicos (morfometria, DNAr 5S e 45S) evidenciaram similaridade entre os três níveis de ploidia. A meiose mostrou-se irregular, com ocorrência de atraso e perda cromossômica, pontes, “cromossomos aderentes”, micronúcleos e assincronia em todos os níveis de ploidia. O dendrograma baseado em marcadores ISSR revelou tendência de agrupamento de indivíduos com o mesmo nível de ploidia e originados das mesmas regiões geográficas. Os resultados sugerem que as populações possuem baixos índices de diversidade genética entre indivíduos e que a maior diversidade encontra-se entre as populações. De forma geral, os resultados indicam que eventos de poliploidização estejam relacionados à diversificação e especiação de “Z. maculatum”, sendo um processo importante para explicar as variações morfológicas observadas dentro do complexo

Karyotype, genome size, and in vitro chromosome doubling of Pfaffia glomerata (Spreng.) Pedersen

Pfaffia glomerata (Spreng.) Pedersen, known worldwide as Brazilian ginseng, has an important commercial value due to its pharmaceutical properties. In addition to the newly described karyological traits and the first estimation of DNA content, this study reports a protocol for the successful induction of tetraploidy. Natural diploid individuals (2n = 34) showed a symmetric karyotype, centromeric DAPI+ bands, one chromosome pair with a CMA+ band and 45S rDNA site and another with one 5S rDNA site. To induce chromosome duplication, small nodal buds were cultured in semi-solid MS-based medium with 2.22 μM BA, 2.69 μM NAA, and colchicine or oryzalin at 10, 15, 20, 25, and 30 μM for 1 or 2 weeks before being transferred to MS basal medium. The results showed that colchicine induced tetraploid plants, mainly after 1 week of exposure, whereas oryzalin treatment induced only mixoploid plants. The tetraploid plants exhibited twice the chromosome number and DNA content and twice the number of chromosome markers observed for the diploids. Chromosome duplication reduced the dry mass of the stems and roots of the polyploid plants compared to the diploids, and the stomatal density was also reduced on the abaxial and adaxial leaf surfaces of the polyploids. Additionally, the production of β-ecdysone was 50 % higher in the tetraploids than in the diploids. Thus, chromosome doubling showed that is possible to increase the content of β-ecdysone, highlighting the considerable potential of this technique to produce new cultivars with high commercial value

The phytoecdysteroid β-ecdysone is genotoxic in Rodent Bone Marrow Micronuclei and Allium cepa L. Assays

Ethnaopharmacologial relevance In South America, the β-ecdysone ecdysteroid has been found in species of the genus Pfaffia Mart. Due to the similar morphology of its roots to the Panax ginseng C. A. Mey. (Korean ginseng), some species of this genus has been known as Brazilian ginseng and have been used as tonic and aphrodisiac, as well as for the treatment of diabetes and rheumatism. Here we report a cytogenotoxic evaluation of β-ecdysone (a natural ecdysteroid found in plants) in Rodent Bone Marrow Micronuclei and Allium cepa Assays. Three β-ecdysone (pure) concentrations (based in human therapeutic dosage) were used in the Micronucleus Assay. The animals were treated during two consecutive days. Micronucleated cells were counted in 2000 polychromatic erythrocytes per animal. For A. cepa L. Assay, one β-ecdysone concentration was analyzed. The onions bulbs were exposed for 24 h. The Micronucleus Assay showed genotoxic effects for all treatments, expressed by an increase of micronucleated cells. In A. cepa L. Assay, cell abnormalities associated to the malfunction/non-formation of mitotic spindle (aneugenic effect) and chromosomal bridges (clastogenic effect) were observed. The results indicate a cytogenotoxic activity of β-ecdysone. Therefore, the popular use of Pfaffia and others species containing β-ecdysone should be considered with caution