Comportamento genômico de combinações híbridas entre capim-elefante e milheto

The objective of this work was to evaluate the genomic behavior of hybrid combinations between elephant grass (Pennisetum purpureum) and pearl millet (P. glaucum). Tetraploid (AAA'B) and pentaploid (AA'A'BB) chromosome races resulting from the backcross of the hexaploid hybrid to its parents elephant grass (A'A'BB) and pearl millet (AA) were analyzed as to chromosome number and DNA content. Genotypes of elephant grass, millet, and triploid and hexaploid induced hybrids were compared. Pentaploid and tetraploid genomic combinations showed high level of mixoploidy, in discordance with the expected somatic chromosome set. The pentaploid chromosome number ranged from 20 to 34, and the tetraploid chromosome number from 16 to 28. Chromosome number variation was higher in pentaploid genomic combinations than in tetraploid, and mixoploidy was observed among hexaploids. Genomic combinations 4x and 5x are mixoploid, and the variation of chromosome number within chromosomal race 5x is greater than in 4x.O objetivo deste trabalho foi avaliar o comportamento genômico de combinações híbridas resultantes do cruzamento entre capim-elefante (Pennisetum purpureum) e milheto (P. glaucum). Raças cromossômicas tetraploides (AAA'B) e pentaploides (AA'A'BB), resultantes do retrocruzamento do híbrido hexaploide com seus parentais capim-elefante (A'A'BB) e milheto (AA), foram avaliadas quanto ao número cromossômico e ao conteúdo de DNA. Foram comparados os genótipos de capim-elefante, milheto e de híbridos triploides e hexaploides induzidos. As combinações genômicas pentaploides e tetraploides mostraram elevado grau de mixoploidia, em desacordo com o complemento cromossômico somático esperado. O número cromossômico dos pentaploides variou de 20 a 34, e o dos tetraploides de 16 a 28. A variação do número cromossômico foi maior nas combinações genômicas pentaploides do que nas tetraploides, e a mixoploidia foi verificada entre hexaploides. As combinações genômicas 4x e 5x são mixoploides, e a variação do número de cromossomos na raça cromossômica 5x é maior do que na 4x

Comportamento genômico de combinações híbridas entre capim‑elefante e milheto

The objective of this work was to evaluate the genomic behavior of hybrid combinations between elephant grass (Pennisetum purpureum) and pearl millet (P. glaucum). Tetraploid (AAA’B) and pentaploid (AA’A’BB) chromosome races resulting from the backcross of the hexaploid hybrid to its parents elephant grass (A’A’BB) and pearl millet (AA) were analyzed as to chromosome number and DNA content. Genotypes of elephant grass, millet, and triploid and hexaploid induced hybrids were compared. Pentaploid and tetraploid genomic combinations showed high level of mixoploidy, in discordance with the expected somatic chromosome set. The pentaploid chromosome number ranged from 20 to 34, and the tetraploid chromosome number from 16 to 28. Chromosome number variation was higher in pentaploid genomic combinations than in tetraploid, and mixoploidy was observed among hexaploids. Genomic combinations 4x and 5x are mixoploid, and the variation of chromosome number within chromosomal race 5x is greater than in 4x.O objetivo deste trabalho foi avaliar o comportamento genômico de combinações híbridas resultantes do cruzamento entre capim-elefante (Pennisetum purpureum) e milheto (P. glaucum). Raças cromossômicas tetraploides (AAA’B) e pentaploides (AA’A’BB), resultantes do retrocruzamento do híbrido hexaploide com seus parentais capim-elefante (A’A’BB) e milheto (AA), foram avaliadas quanto ao número cromossômico e ao conteúdo de DNA. Foram comparados os genótipos de capim-elefante, milheto e de híbridos triploides e hexaploides induzidos. As combinações genômicas pentaploides e tetraploides mostraram elevado grau de mixoploidia, em desacordo com o complemento cromossômico somático esperado. O número cromossômico dos pentaploides variou de 20 a 34, e o dos tetraploides de 16 a 28. A variação do número cromossômico foi maior nas combinações genômicas pentaploides do que nas tetraploides, e a mixoploidia foi verificada entre hexaploides. As combinações genômicas 4x e 5x são mixoploides, e a variação do número de cromossomos na raça cromossômica 5x é maior do que na 4x

Somatic embryogenesis in hybrids of Pennisetum sp. and genomic stability evaluation by cytometry

The objectives of this study were to establish an efficient protocol for somatic embryogenesis in triploid hybrids between napiergrass (Pennisetum purpureum Schumach.) and pearl millet (P. glaucum (L.) R. Br.), and to assess the genomic stability by flow cytometry of the plants obtained in vitro. Somatic embryogenesis and plant regeneration were successfully established from mature zygotic embryos of napiergrass and pearl millet hybrids. Four treatments with 2,4 dichlorophenoxyacetic acid (2,4-D) at 0, 1, 2 e 3 mg L-1 were tested for embryogenic calli induction and two treatments with inositol at 1 e 2 g L-1 were tested for plant regeneration. The treatments were arranged in a completely randomized design. The optimum hormone combinations were 2 mg L-1 of 2,4 D for embryogenic callus induction, and 1 g L-1 of inositol for embryos conversion and plant regeneration. The analysis of DNA content by flow cytometry of the regenerated plantlets indicated that no ploidy changes had been induced during somatic embryogenesis and plant regeneration. The nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous in comparison to those of the control plants. There was no occurrence of karyological instability in the regeneration system utilized for Pennisetum hybrid.Os objetivos deste trabalho foram estabelecer um protocolo eficiente de embriogênese somática, em híbridos triploides entre capim elefante (Pennisetum purpureum Schumach.) e milheto (P. glaucum (L.) R. Br.), e avaliar por citometria de fluxo a estabilidade genômica das plantas obtidas in vitro. A embriogênese somática e a regeneração das plantas foram estabelecidas a partir de embriões zigóticos maduros de híbridos entre capim elefante e milheto. Foram testados quatro tratamentos com 2,4 ácido diclorofenoxiacético (2,4 D), nas concentrações 0, 1, 2 e 3 mg L-1, para indução de calos embriogênicos, e dois tratamentos com inositol a 1 e 2 g L-1, para regeneração das plantas. Os tratamentos foram dispostos em delineamento inteiramente ao acaso. A combinação ótima de hormônios foi de 2 mg L-1 de 2,4 D, para indução de calos embriogênicos, e de 1 g L-1 de inositol, para conversão de embriões e regeneração de plantas. A análise de quantidade de DNA, por citometria de fluxo das plantas regeneradas, indicou a não ocorrência de alterações em ploidia durante a embriogênese somática e a regeneração das plantas. A quantidade de DNA nuclear e a ploidia das plantas regeneradas foram estáveis e homogêneas em comparação às das plantas controle. Não ocorreu instabilidade cariotípica no sistema de regeneração usado para híbridos de Pennisetum

Embriogênese somática em híbridos de Pennisetum sp. e avaliação de estabilidade genômica por citometria

The objectives of this study were to establish an efficient protocol for somatic embryogenesis in triploid hybrids between napiergrass (Pennisetum purpureum Schumach.) and pearl millet (P. glaucum (L.) R. Br.), and to assess the genomic stability by flow cytometry of the plants obtained in vitro. Somatic embryogenesis and plant regeneration were successfully established from mature zygotic embryos of napiergrass and pearl millet hybrids. Four treatments with 2,4-dichlorophenoxyacetic acid (2,4-D) at 0, 1, 2 e 3 mg L-1 were tested for embryogenic calli induction and two treatments with inositol at 1 e 2 g L-1 were tested for plant regeneration. The treatments were arranged in a completely randomized design. The optimum hormone combinations were 2 mg L-1 of 2,4-D for embryogenic callus induction, and 1 g L-1 of inositol for embryos conversion and plant regeneration. The analysis of DNA content by flow cytometry of the regenerated plantlets indicated that no ploidy changes had been induced during somatic embryogenesis and plant regeneration. The nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous in comparison to those of the control plants. There was no occurrence of karyological instability in the regeneration system utilized for Pennisetum hybrid.Os objetivos deste trabalho foram estabelecer um protocolo eficiente de embriogênese somática, em híbridos triploides entre capim-elefante (Pennisetum purpureum Schumach.) e milheto (P. glaucum (L.) R. Br.), e avaliar por citometria de fluxo a estabilidade genômica das plantas obtidas in vitro. A embriogênese somática e a regeneração das plantas foram estabelecidas a partir de embriões zigóticos maduros de híbridos entre capim-elefante e milheto. Foram testados quatro tratamentos com 2,4-ácido diclorofenoxiacético (2,4-D), nas concentrações 0, 1, 2 e 3 mg L-1, para indução de calos embriogênicos, e dois tratamentos com inositol a 1 e 2 g L-1, para regeneração das plantas. Os tratamentos foram dispostos em delineamento inteiramente ao acaso. A combinação ótima de hormônios foi de 2 mg L-1 de 2,4-D, para indução de calos embriogênicos, e de 1 g L-1 de inositol, para conversão de embriões e regeneração de plantas. A análise de quantidade de DNA, por citometria de fluxo das plantas regeneradas, indicou a não ocorrência de alterações em ploidia durante a embriogênese somática e a regeneração das plantas. A quantidade de DNA nuclear e a ploidia das plantas regeneradas foram estáveis e homogêneas em comparação às das plantas controle. Não ocorreu instabilidade cariotípica no sistema de regeneração usado para híbridos de Pennisetum

Genetic diversity and population structure of Musa accessions in ex situ conservation

Abstract\ud \ud \ud \ud Background\ud \ud Banana cultivars are mostly derived from hybridization between wild diploid subspecies of Musa acuminata (A genome) and M. balbisiana (B genome), and they exhibit various levels of ploidy and genomic constitution. The Embrapa ex situ Musa collection contains over 220 accessions, of which only a few have been genetically characterized. Knowledge regarding the genetic relationships and diversity between modern cultivars and wild relatives would assist in conservation and breeding strategies. Our objectives were to determine the genomic constitution based on Internal Transcribed Spacer (ITS) regions polymorphism and the ploidy of all accessions by flow cytometry and to investigate the population structure of the collection using Simple Sequence Repeat (SSR) loci as co-dominant markers based on Structure software, not previously performed in Musa.\ud \ud \ud \ud Results\ud \ud From the 221 accessions analyzed by flow cytometry, the correct ploidy was confirmed or established for 212 (95.9%), whereas digestion of the ITS region confirmed the genomic constitution of 209 (94.6%). Neighbor-joining clustering analysis derived from SSR binary data allowed the detection of two major groups, essentially distinguished by the presence or absence of the B genome, while subgroups were formed according to the genomic composition and commercial classification. The co-dominant nature of SSR was explored to analyze the structure of the population based on a Bayesian approach, detecting 21 subpopulations. Most of the subpopulations were in agreement with the clustering analysis.\ud \ud \ud \ud Conclusions\ud \ud The data generated by flow cytometry, ITS and SSR supported the hypothesis about the occurrence of homeologue recombination between A and B genomes, leading to discrepancies in the number of sets or portions from each parental genome. These phenomenons have been largely disregarded in the evolution of banana, as the “single-step domestication” hypothesis had long predominated. These findings will have an impact in future breeding approaches. Structure analysis enabled the efficient detection of ancestry of recently developed tetraploid hybrids by breeding programs, and for some triploids. However, for the main commercial subgroups, Structure appeared to be less efficient to detect the ancestry in diploid groups, possibly due to sampling restrictions. The possibility of inferring the membership among accessions to correct the effects of genetic structure opens possibilities for its use in marker-assisted selection by association mapping.This work was funded by FAPESP [2008/03470-0] and CNPq. Technical assistance by Luis Eduardo Fonseca was greatly appreciated. The authors (ONJ, SSO, EP, AF) are grateful for the fellowships provided by CNPq and GGS to FAPESP [2010/01398-0]

MICROPROPAGATION AND PLOIDY STABILITY OF Lippia lacunosa Mart. & Schauer: AN ENDANGERED BRAZILIAN MEDICINAL PLANT

Lippia lacunosa is a Brazilian savanna plant that belongs to the Verbenaceae family. It has been used in folk medicine as a treatment for different diseases. This species represents an endangered Brazilian medicinal plant, and this is the first report documenting a reliable protocol for the in vitro propagation and regeneration of L. lacunosa. Axenic explants were cultivated in MS medium containing different concentrations of naphthalene acetic acid (NAA) to induce root growth. The mean shoot length and the number of roots were highest with 0.06 mg·L-1 NAA. The highest number of buds in shoot regeneration was induced with 2 mg·L-1 6-benzylaminopurine (BA). To obtain a long-term culture, the dwarf shoots were elongated on MS media containing 0.5 mg·L-1 BA alternated with MS containing 2 mg·L-1 BA every 40 days. In the present protocol, the long-term shoots retained the ability to root even after long periods of BA treatment. In addition, we evaluated the nuclear DNA content and ploidy levels, including the occurrence of endopolyploidy, in long-term micropropagated plant leaves using flow cytometry analysis. The plants propagated in vitro over several years possessed nuclear DNA contents ranging from 2.940 to 3.095 pg, and no differences in DNA content were found among in vitro plants or between these plants and the control (L. lacunosa from a greenhouse with a DNA content of 3.08 pg). The flow cytometry analysis also demonstrated that there was no polyploidization. The present study will be useful for biotechnological approaches and provides the first estimate of the nuclear DNA content of this species using flow cytometry

Mitodepressive and clastogenic effects of aqueous extracts of the lichens Myelochroa lindmanii and Canoparmelia texana (Lecanorales, Parmeliaceae) on meristematic cells in plant bioassays

The cytotoxicity effect of aqueous extracts of the lichens species Myelochroa lindmanii and Canoparmelia texana (Lecanorales, Parmeliaceae) were evaluated using meristematic cells of lettuce (Lactuca sativa) and maize (Zea mays). The seasonal effect was also evaluated. Extracts of M. lindmanii and C. texana inhibited root growth and/or percentage germination, possibly due to alterations in the cell cycle. The M. lindmanii extract showed anti-mitotic effects and blocked the cell cycle in metaphase so that c-mitosis and cells with chromosome duplication were produced. The C. texana extract appeared to hinder cell division, increasing the number of interphase cells. In addition, both extracts caused an increase in percentage of cell death. Clastogenic effects were also observed, such as sticky chromosomes, bridges, fragments and later segregation. Both lichen species are thus potential sources of biologically active substances with possible applications in biology, medicine and agronomy

In vitro plant regeneration of Passiflora setacea D.C. (Passifloraceae): the influence of explant type, growth regulators, and incubation conditions

The present study aimed to establish a protocol for in vitro organogenesis of Passiflora setacea and to determine the genetic stability of regenerated plants. Three types of explants (leaf, hypocotyl, and root), four growth regulator combinations [Murashige and Skoog (MS) salts, MS + 6-benzyladenine (BA), MS + thidiazuron (TDZ), and MS + BA + TDZ], and two light regimes (16-h photoperiod and continuous darkness) were tested. After 30 d on induction medium, the percentage of explants forming shoots was evaluated. Direct and indirect organogenesis was evident from hypocotyl- and root-derived explants, whereas only indirect organogenesis was observed from leaf explants. The presence of BA was essential for shoot formation from leaf explants and improved the response of hypocotyl segments under a 16-h photoperiod compared to the cytokinin-free control. However, after transfer to shoot elongation medium, the greatest number of elongated shoots was obtained from hypocotyl segments that had been induced on BA + TDZ medium under a 16-h photoperiod, as was also observed for root explants. Flow cytometry analysis confirmed the genetic stability of the regenerants based on DNA quantity (2C = 2.57 pg) in comparison with seed-derived plantlets (2C = 2.60 pg). This is the first report on the in vitro regeneration of P. setacea