RESUMO

Asepsis, defined as the absence of microbial contamination, is one of the most important requirements of plant micropropagation. In long-term micropropagated cultures, there may occasionally occur scattered microorganism growth in the culture medium. These microorganisms are common plant components and are known as latent endophytes. Thus, the aim of this research was to investigate the presence of endophytic bacteria in asymptomatic pineapple and orchid microplants, which were cultivated in three laboratories for 1 year. Isolation and characterization of bacterial isolates, PCR-DGGE from total genomic DNA of microplants and ultrastructural analysis of leaves were performed. In the culture-dependent technique, it was only possible to obtain bacterial isolates from pineapple microplants. In this case, the bacteria genera identified in the isolation technique were Bacillus, Acinetobacter, and Methylobacterium. The scanning electron microscopy and transmission electron microscopy (SEM and TEM) analyses revealed the presence of endophytic bacteria in intracellular spaces in the leaves of pineapple and orchid microplants, independent of the laboratory or cultivation protocol. Our results strongly indicate that there are endophytic bacterial communities inhabiting the microplants before initiation of the in vitro culture and that some of these endophytes persist in their latent form and can also grow in the culture medium even after long-term micropropagation, thus discarding the concept of "truly axenic plants."

Assuntos

RESUMO

ABSTRACT: This research aimed to evaluate the antibiotic therapy effects in the growing of micropropagated pineapple plants as well as in the community of the endophytic bacterial of the species. It was performed molecular analyzes and verified the presence of endophytes within microplants even after 30 days of culture on MS medium supplemented with different antibiotics and analyzed the development growth of shoots and roots of microplants. After the period of 30 days of cultivation, even being checked the presence of endophytic bacteria, it was verified that the microplants showed reduction of the growing of shoots and roots, thereby demonstrating the influence of in vitro endophytic microorganisms in the development of microplants, and the importance of maintaining it in equilibrium with endophytes/ microplants.

RESUMO: Com o intuito de avaliar os efeitos da antibioticoterapia no crescimento de plantas micropropagadas de abacaxizeiros, bem como na comunidade bacteriana endofítica da espécie, realizaram-se análises moleculares. Verificou-se a presença de endófitos no interior das microplantas, mesmo após 30 dias de cultivo em meio de cultura MS, suplementado com diferentes antibióticos, e analisou-se o crescimento da parte aérea e radicular das microplantas. Decorrido o período de 30 dias de cultivo, constatou-se ainda a presença de bactérias endofíticas, no entanto, as microplantas apresentaram acentuada redução no crescimento da parte aérea e radicular, evidenciando assim a influência da microbiota endofítica no crescimento das microplantas in vitro e a importância da manutenção do equilíbrio endofítos/microplanta.

RESUMO

ABSTRACT: This research aimed to evaluate the antibiotic therapy effects in the growing of micropropagated pineapple plants as well as in the community of the endophytic bacterial of the species. It was performed molecular analyzes and verified the presence of endophytes within microplants even after 30 days of culture on MS medium supplemented with different antibiotics and analyzed the development growth of shoots and roots of microplants. After the period of 30 days of cultivation, even being checked the presence of endophytic bacteria, it was verified that the microplants showed reduction of the growing of shoots and roots, thereby demonstrating the influence of in vitro endophytic microorganisms in the development of microplants, and the importance of maintaining it in equilibrium with endophytes/ microplants.

RESUMO: Com o intuito de avaliar os efeitos da antibioticoterapia no crescimento de plantas micropropagadas de abacaxizeiros, bem como na comunidade bacteriana endofítica da espécie, realizaram-se análises moleculares. Verificou-se a presença de endófitos no interior das microplantas, mesmo após 30 dias de cultivo em meio de cultura MS, suplementado com diferentes antibióticos, e analisou-se o crescimento da parte aérea e radicular das microplantas. Decorrido o período de 30 dias de cultivo, constatou-se ainda a presença de bactérias endofíticas, no entanto, as microplantas apresentaram acentuada redução no crescimento da parte aérea e radicular, evidenciando assim a influência da microbiota endofítica no crescimento das microplantas in vitro e a importância da manutenção do equilíbrio endofítos/microplanta.

RESUMO

Micropropagation has been applied in the recovery and rejuvenation of adult trees, which is achieved by various subcultures in the multiplication phase. This strategy has brought questions about the endophytic microbiota associated with these plants along its manipulation. Therefore, the aim of this study was to evaluate the composition of the endophytic bacterial communities associated with two explants sources [the canopy branches (CB) and the trunk base of the tree (TB)] under prolonged in vitro cultivation. In addition we analyzed the bacterial community dynamic along the subcultures in different micropropagation phases. Bacterial DNA was extracted from samples of mini-stumps (in vivo) from CB and TB and in micro-stumps produced by in vitro cultivations of these explants sources--both originated from one single matrix plant of Eucalyptus benthamii. In vitro establishment occurred in two dates and the evaluation of endophytic bacterial communities was made in vivo and in vitro samples (on 10th, 13th and 16th subcultures), when elongated shoots and roots were analyzed. Analysis was performed by PCR-DGGE based on the V6 region of ribosomal gene 16S rDNA. Bands profiles showed differences in communities between in vivo and in vitro samples, and also distinctions of communities assessed in the subcultures, elongated and rooted samples. Distinctions in the composition of endophytic bacterial communities were greater in CB micro-stumps. These results indicate a differential colonization of explants by endophytic bacteria, with predominance of common (ever-present) endophytes in TB samples and casual, here named opportunistic, in CB samples.

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RESUMO

UNLABELLED: The direct induction of adventitious buds and somatic embryos from explants is a morphogenetic process that is under the influence of exogenous plant growth regulators and its interactions with endogenous phytohormones. We performed an in vitro histological analysis in peach palm (Bactris gasipaes Kunth) shoot apexes and determined that the positioning of competent cells and their interaction with neighboring cells, under the influence of combinations of exogenously applied growth regulators (NAA/BAP and NAA/TDZ), allows the pre-procambial cells (PPCs) to act in different morphogenic pathways to establish niche competent cells. It is likely that there has been a habituation phenomenon during the regeneration and development of the microplants. This includes promoting the tillering of primary or secondary buds due to culturing in the absence of NAA/BAP or NAA/TDZ after a period in the presence of these growth regulators. Histological analyses determined that the adventitious roots were derived from the dedifferentiation of the parenchymal cells located in the basal region of the adventitious buds, with the establishment of rooting pole, due to an auxin gradient. Furthermore, histological and histochemical analyses allowed us to characterize how the PPCs provide niches for multipotent, pluripotent and totipotent stem-like cells for vascular differentiation, organogenesis and somatic embryogenesis in the peach palm. The histological and histochemical analyses also allowed us to detect the unicellular or multicellular origin of somatic embryogenesis. Therefore, our results indicate that the use of growth regulators in microplants can lead to habituation and to different morphogenic pathways leading to potential niche establishment, depending on the positioning of the competent cells and their interaction with neighboring cells. KEY MESSAGE: Our results indicate that the use of growth regulators in microplants can lead to habituation and to different morphogenic pathways leading to potential niche establishment, depending on the positioning of the competent cells and their interaction with neighboring cells.

Assuntos

RESUMO

Pejibaye is a good alternative for the extractive exploration of species such as juçara and açaí. However, when it is produced by seeds its planting is heterogeneous, which makes micropropagation a good alternative for cultivation in large scale. This study aimed to evaluate the influence of the interaction between ammonium nitrate and potassium nitrate on peach palm somatic embryos rooting in vitro cultivated, for optimization of the micropropagation protocol. The embryos were inoculated in MS medium with different concentrations of NH4NO3 and KNO3. Morphophisyologic parameters of root development were measured at 120 and 240 days of cultivation. At 120 days, at lower nitrogen concentrations, roots were stimulated to grow and major root branching occurred at low concentrations of NH4NO3 and high concentrations of KNO3. At 240 days, there was a reduction of the root growing, with more percentage of thin roots. It was concluded that up to 120 days, the microplants should be cultivated in culture medium with lower concentrations of NH4NO3 and higher concentrations of KNO3 than those recommended by MS medium, and after this period they should be cultivated at the recommended doses.

A pupunheira tem se mostrado boa alternativa à exploração extrativista de espécies como juçara e açaí. Porém, quando produzida via sementes apresenta plantio heterogêneo, o que torna a micropropagação ótima alternativa para seu cultivo em larga escala. O experimento objetivou avaliar a influência da interação entre nitrato de amônio e nitrato de potássio no enraizamento de microplantas obtidas a partir do desenvolvimento in vitro de embriões somáticos de pupunheiras, visando otimizar seu protocolo de micropropagação. Os embriões foram inoculados em meio MS com diferentes concentrações de NH4NO3 e KNO3. Aos 120 e 240 dias de cultivo, foram avaliados parâmetros morfofisiológicos do desenvolvimento radicular. Aos 120 dias, nas concentrações mais baixas de nitrogênio, houve estímulo ao crescimento das raízes e a maior ramificação radicular ocorreu com baixas concentrações de NH4NO3 e altas de KNO3. Aos 240 dias, notou-se redução do crescimento radicular e raízes finas prevalecentes. Conclui-se que até 120 dias as microplantas devem ser mantidas em meio com concentrações menores de NH4NO3 e maiores de KNO3 que as empregadas no meio MS, voltando para as concentrações usuais após esse período.

RESUMO

Pejibaye is a good alternative for the extractive exploration of species such as juçara and açaí. However, when it is produced by seeds its planting is heterogeneous, which makes micropropagation a good alternative for cultivation in large scale. This study aimed to evaluate the influence of the interaction between ammonium nitrate and potassium nitrate on peach palm somatic embryos rooting in vitro cultivated, for optimization of the micropropagation protocol. The embryos were inoculated in MS medium with different concentrations of NH4NO3 and KNO3. Morphophisyologic parameters of root development were measured at 120 and 240 days of cultivation. At 120 days, at lower nitrogen concentrations, roots were stimulated to grow and major root branching occurred at low concentrations of NH4NO3 and high concentrations of KNO3. At 240 days, there was a reduction of the root growing, with more percentage of thin roots. It was concluded that up to 120 days, the microplants should be cultivated in culture medium with lower concentrations of NH4NO3 and higher concentrations of KNO3 than those recommended by MS medium, and after this period they should be cultivated at the recommended doses.

A pupunheira tem se mostrado boa alternativa à exploração extrativista de espécies como juçara e açaí. Porém, quando produzida via sementes apresenta plantio heterogêneo, o que torna a micropropagação ótima alternativa para seu cultivo em larga escala. O experimento objetivou avaliar a influência da interação entre nitrato de amônio e nitrato de potássio no enraizamento de microplantas obtidas a partir do desenvolvimento in vitro de embriões somáticos de pupunheiras, visando otimizar seu protocolo de micropropagação. Os embriões foram inoculados em meio MS com diferentes concentrações de NH4NO3 e KNO3. Aos 120 e 240 dias de cultivo, foram avaliados parâmetros morfofisiológicos do desenvolvimento radicular. Aos 120 dias, nas concentrações mais baixas de nitrogênio, houve estímulo ao crescimento das raízes e a maior ramificação radicular ocorreu com baixas concentrações de NH4NO3 e altas de KNO3. Aos 240 dias, notou-se redução do crescimento radicular e raízes finas prevalecentes. Conclui-se que até 120 dias as microplantas devem ser mantidas em meio com concentrações menores de NH4NO3 e maiores de KNO3 que as empregadas no meio MS, voltando para as concentrações usuais após esse período.

RESUMO

Given the susceptibility of tomato plants to pests, the aim of the present study was to understand how hormones are involved in the formation of tomato natural defences against insect herbivory. Tomato hormone mutants, previously introgressed into the same genetic background of reference, were screened for alterations in trichome densities and allelochemical content. Ethylene, gibberellin, and auxin mutants indirectly showed alteration in trichome density, through effects on epidermal cell area. However, brassinosteroids (BRs) and jasmonates (JAs) directly affected trichome density and allelochemical content, and in an opposite fashion. The BR-deficient mutant dpy showed enhanced pubescence, zingiberene biosynthesis, and proteinase inhibitor expression; the opposite was observed for the JA-insensitive jai1-1 mutant. The dpy x jai1-1 double mutant showed that jai1-1 is epistatic to dpy, indicating that BR acts upstream of the JA signalling pathway. Herbivory tests with the poliphagous insect Spodoptera frugiperda and the tomato pest Tuta absoluta clearly confirmed the importance of the JA-BR interaction in defence against herbivory. The study underscores the importance of hormonal interactions on relevant agricultural traits and raises a novel biological mechanism in tomato that may differ from the BR and JA interaction already suggested for Arabidopsis.

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RESUMO

Shoots of E. camaldulensis seedlings from three distinct provenances were submitted to three different levels of in vitro water deficit induced by the addition of sorbitol in the growth media. Transversal sections from the leaf blades and stems were anatomically analysed using the historesin inclusion technique. Shoots from the different provenances presented distinct responses to the treatments. The Gilbert River provenance was the most sensible. The basic alterations found on the anatomical structure in response to the water deficit were: cell collapse, late tissue differentiation, vascular bundle and epidermis disorganization, and alterations on the mesophyll and epidermis thickness.

Três procedências distintas de E. camaldulensis foram submetidas à diferentes níveis de deficiência hídrica in vitro induzida através da adição de sorbitol em meio de cultura. Cortes transversais de limbos foliares e caules foram analisados anatomicamente através da técnica de emblocamento em historesina. As procedências apresentaram respostas diferenciadas, sendo Gilbert River a mais sensível. As alterações básicas da estrutura anatômica em função da deficiência hídrica são as seguintes: colapso das células, atraso na diferenciação dos tecidos, desorganização do feixe vascular e da epiderme e alterações na espessura do mesofilo e epiderme.

RESUMO

Shoots of E. camaldulensis seedlings from three distinct provenances were submitted to three different levels of in vitro water deficit induced by the addition of sorbitol in the growth media. Transversal sections from the leaf blades and stems were anatomically analysed using the historesin inclusion technique. Shoots from the different provenances presented distinct responses to the treatments. The Gilbert River provenance was the most sensible. The basic alterations found on the anatomical structure in response to the water deficit were: cell collapse, late tissue differentiation, vascular bundle and epidermis disorganization, and alterations on the mesophyll and epidermis thickness.

Três procedências distintas de E. camaldulensis foram submetidas à diferentes níveis de deficiência hídrica in vitro induzida através da adição de sorbitol em meio de cultura. Cortes transversais de limbos foliares e caules foram analisados anatomicamente através da técnica de emblocamento em historesina. As procedências apresentaram respostas diferenciadas, sendo Gilbert River a mais sensível. As alterações básicas da estrutura anatômica em função da deficiência hídrica são as seguintes: colapso das células, atraso na diferenciação dos tecidos, desorganização do feixe vascular e da epiderme e alterações na espessura do mesofilo e epiderme.