Endophytic bacteria in long-term in vitro cultivated axenic pineapple microplants revealed by PCR-DGGE

In vitro propagated plants are believed to be free of microbes. However, after 5 years of in vitro culture of pineapple plants, without evidence of microbial contamination, the use of culture-independent molecular approach [classifying heterogeneous nucleic acids amplified via universal and specific 16S rRNA gene by polymerase chain reaction (PCR)], and further analysis by denaturing gradient gel electrophoresis (DGGE) revealed endophytic bacteria in roots, young and mature leaves of such plants. The amplification of 16S rRNA gene (Bacteria domain) with the exclusion of the plant chloroplast DNA interference, confirmed the presence of bacterial DNA, from endophytic microorganisms within microplant tissues. PCR-DGGE analysis revealed clear differences on bacterial communities depending on plant organ. Group-specific DGGE analyses also indicated differences in the structures of Actinobacteria, Alphaproteobacteria and Betaproteobacteria communities in each part of plants. The results suggest the occurrence of a succession of bacterial communities colonizing actively the microplants organs. This study is the first report that brings together evidences that pineapple microplants, previously considered axenic, harbor an endophytic bacterial community encompassing members of Actinobacteria, Alphaproteobacteria and Betaproteobacteria group which is responsive to differences in organs due to plant development.

Metabolic response induced by endophytic fungi and bacteria in H. marrubioides Epling in vitro microplants

Hyptis marrubioides Epling is a native plant from Brazilian Cerrado. In this paper, the response of in vitro microplants of this species to inoculation with bacterial and fungal endophytic isolates is evaluated. HPLC-DAD analysis showed the presence of 3,4-O-(Z)-dicaffeoylquinic acid and quercetin-7-O-glucoside as the main components. GC/MS analysis demonstrated that the sesquiterpenes τ-cadinol and caryophyllene oxide were only produced in microplants inoculated with endophytic bacteria, while methyl hexadecanoate, methyl heptadecanoate and methyl (Z,Z,Z) 9,12,15-octadecatrienoate and the triterpene methyl 3β-hydroxy-urs-12-en-28-oate were overexpressed only when the microplant was treated with endophytic fungi.

Morphological alterations in leave of micropropagated pineapple plants cv. IAC Gomo-de-mel acclimatizated in different conditions of luminosity

(Morphological alterations in leave of micropropagated pineapple plants cv. IAC Gomo-de-mel acclimatizated in different conditions of luminosity). Microprapagated plants usually show difficulties to adapt to ex vitro conditions, and many times are submitted to the rustication process to aim the reduction of all the impacts resulting from the environmental changes. Once the leaf and its annexes are important indicators of adaptability strategies of the plants to adverse environmental conditions, the objective of this work was to compare the leaf anatomy of pineapple cv. IAC Gomo-de-mel in vitro cultivated plants with microplants acclimatized in different conditions of luminosity, under mesh, with 50% of shading and directly exposed to sunlight, to verify the needed of rustication process on this cultivar. Evaluations of the leaf epidermis using light and electronic scanning microscopy showed an increase on scale density in both leaves surfaces of the ex vitro microplants, mainly related to the ones directly exposed to sunlight. Subsequent observations showed an increase on cuticle thickness, on wavy contours of epidermal cells, and on the distribution and quantity of mesophyll fibers, evidencing the light conditions interference in morphological characteristics of these microplants. These alterations had not harmed microplant development, showing that are not need of rustication stages on the acclimatization process of this cultivar.

Bacteriosomes in axenic plants: endophytes as stable endosymbionts

Observations of cells of axenic peach palm (Bactris gasipaes) microplants by light microscopy revealed movements of small particles within the cells. The phenomenon was characterized initially as Brownian movement, but electron microscopy revealed the presence of an intracellular bacterial community in these plants. Microscopy observations revealed the particular shapes of bacterial cells colonizing inner tissues of analyzed plants. Applying a molecular characterization by polymerase chain reaction and denaturing gradient gel electrophoresis, it was revealed the existence of bacterial rRNA within the plants. Sequencing of the rRNA identified three different phylogenetic groups; two bands had a high degree of similarity to sequences from Moraxella sp. and Brevibacillus sp., and a third sequence was similar to a non-cultivated cyanobacterium. The presence of those endosymbionts, called bacteriosomes, in axenic peach palm microplants raises the question of whether these stable endosymbionts were acquired in the process of evolution and how could they benefit the process of plants micropropagation.

Effects of arbuscular mycorrhizal fungi and phosphorus fertilization on post vitro growth of micropropagated Zingiber officinale roscoe

The rhizomes of Zingiber officinale Roscoe (ginger) are widely used for their medicinal and flavoring properties, whereas the influence of root symbionts on their growth is poorly understood. In this study, the effects of phosphate fertilization and inoculation with a mixture of arbuscular mycorrhizal fungi (AMF) (isolates Glomus clarum RGS101A, Entrophospora colombiana SCT115A and Acaulospora koskei SPL102A) on survival, growth and development of micropropagated ginger were investigated. After transplanting to post vitro conditions, the ginger microplants were subjected to the following treatments: a) AMF mixture, b) P addition (25 mg kg-1), c) AMF + P, and d) non-mycorrhizal control without P addition. After eight months of growth, survival ranged from 86 to 100 % in the AMF and AMF+P treatments versus 71 % survival in control and P treatments. In the AMF, P and AMF+P treatments, the shoot, root and rhizome biomass production were significantly larger than in the control plants. In the non-mycorrhizal control plants the leaf number, leaf area, number of shoots/plants, and shoot length were significantly lower than in the AMF, P and AMF+P treatments. Root colonization ranged from 81 to 93 % and was not affected by P application. The data confirmed the response of several growth variables of micropropagated ginger to mycorrhizal colonization and P addition.

Efeito da sacarose e sorbitol na conservação in vitro de Passiflora giberti N. E. Brown

This work objectified the study of sucrose and sorbitol effect in the in vitro conservation for Passiflora giberti N. E. Brown, access. Therefore, an experiment was conducted in a completely randomized design to compare control treatment (standard MS) to MS medium supplemented with three sucrose concentrations (0, 15 and 30 g L -1) combined with three sorbitol concentrations (10, 20 and 40 g L -1), in a total of 10 treatments with 20 replicas. The experiment evaluation was carried out at 30, 60, 90 and 120 days of incubation, whereas the height of shoots (cm), number of roots, number and color of leaves were observed. The results showed the possibility to maintain passion-fruit microplants for a four months period under slow growth in MS medium supplemented with 10 or 20 g L -1 of sorbitol, without sucrose, and kept under 16 hours photoperiod (22 μ E m -2 s -1) and temperature of 27 ± 1°C. Sucrose sustained the longest development of the microplants. Root formation was affected by the sorbitol in the concentration of 40 g L -1 and by the absence of sucrose in the culture medium.

Pre-procambial cells are niches for pluripotent and totipotent stem-like cells for organogenesis and somatic embryogenesis in the peach palm: a histological study

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.