Marcílio de Almeida

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

AbstractThe 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.n 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.UNLABELLEDnThe 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.nnnKEY MESSAGEnOur 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.

Anatomical and biochemical characterization of the calcium effect on eucalyptus urophylla callus morphogenesis in vitro

Calcium chloride concentrations from 0.0 to 12.12 mM were added to the culture medium and calcium content in calluses were determined directly by X-ray fluorescence spectrometry, a non-destructive method, allowing the processing of the same tissue for histological analysis. A multivariate statistical analysis (PCA – Principal Components Analysis) grouped the treatments into 5 blocks and indicated the most responsive group. Lack of calcium supply caused a complete absence of a morphogenic process and tissue collapse. An increase in calcium concentration gave higher total protein and sugar contents, an increase in peroxidase specific activity and changes in the histological characteristics. It was possible to verify that calcium stimulated globular somatic embryo formation at concentration of 6.62 mM.

Micropropagation of Eucalyptus benthamii to form a clonal micro-garden

Eucalyptus benthamii is an important component of forestry plantations in cold regions, but it is difficult to obtain clonal plants of this species, especially by low rooting. In this study, we developed a method for cloning selected genotypes of E. benthamii using a micropropagation technique, enabling the formation of a clonal micro-garden. Nodal segments from sprouts of mini-stumps in the clonal mini-garden were used as explants. After in vitro establishment of the explants, we tested two selected clones (BP101 and BP118), three culture media (Wood Plant Medium (WPM), Correia and colleagues JADS medium, and Murashige and Skoog medium), and two plant growth regulators (6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA)) for the multiplication of adventitious buds. Additionally, combinations of two other plant growth regulators (BAP and gibberellic acid (GA3)) were tested for the elongation of shoots. The in vitro and ex vitro rooting of micro-plantlets prior to acclimatization were compared. The in vitro bud multiplication of E. benthamii depended on the clone, culture medium, and concentration of plant growth regulators. The best results were obtained with WPM supplemented with 0.5xa0mgxa0L−1 BAP and 0.05xa0mgxa0L−1 NAA. The elongation of shoots depended on the clone and plant growth regulator, and the best results were obtained with nutrient medium free of GA3 and BAP. Histological analysis showed that both in vitro and ex vitro rooting were successful, resulting in normal development of adventitious roots showing a vascular connection with the vascular cambium. The new protocol is efficient for micro-plantlet production of E. benthamii and can be used for the formation of a clonal micro-garden for other Eucalyptus or tree species.

In vitro morphogenic response of leaf sheath of Phyllostachys bambusoides

Nodal segments from secondary branches of saplings of Phyllostachys bambusoides were inoculated in MS medium to assess the in vitro morphogenic response of leaf sheath through the induction to callogenesis by Picloram (4-amino-3,5,6-trichloropicolinic acid) at different concentrations of carbohydrate under the same conditions with presence or absence of luminosity. In our experiment, secondary explants were kept in MS medium containing 8.0 mg·L−1 of Picloram for the callus formation. Calluses were transferred in MS medium supplemented with sucrose, fructose and glucose (control, 2%, 4% and 6%). Results show that Picloram induced the callogenesis in leaf sheath. The secondary embryogenesis was formed in yellow-globular callus. The sucrose as carbohydrate source in the absence of light was more efficient to induce rhizogenesis. Glucose was more efficiency in the presence of light. Callogenic induction and further embryogenesis evidenced the competence and determination of leaf sheath cells.

TDZ pulsing evaluation on the in vitro morphogenesis of peach palm

Peach palm (Bactris gasipaes Kunth.) cropping is an excellent alternative to native species exploitation; nevertheless, the problems with seed germination and conventional propagation justify the use of in vitro culturing. Aiming to asses TDZ pulsing effect on B. gasipaes morphogenesis, explants obtained from unarmed microplants were maintained in two treatments, half of them in MS free medium (without growth regulator) and the other half in MS with TDZ (0.36xa0μM). Both groups were transferred to growth regulator-free MS medium following 14xa0days of culture. After 84xa0days of culture, TDZ pulsing increased the growth and development of the shoots, restricted the growth and development of the roots, with no influence on adventitious bud induction or somatic embryogenesis. Furthermore, development of prickles, thickening of roots and chlorotic leaves were noted under TDZ pulsing. Leaf sheath histological analysis showed an epidermal origin and no vascularization of these prickles.

Low temperature, IBA concentrations and optimal time for adventitious rooting of Eucalyptus benthamii mini-cuttings

Eucalyptus benthamii is a forest species of economic interest that has difficulty with seed production and also is considered to have difficulty with adventitious rooting using propagation techniques, such as cutting or mini-cutting. We aimed to assess the adventitious rooting percentage under different storage times in low temperatures and at various IBA (indole-3-butyric acid) concentrations to determine the optimal time of permanence for rooting Eucalyptus benthamii minicuttings in a greenhouse. Shoots collected from mini-stumps cultivated in a semi-hydroponic system were used to obtain the mini-cuttings. For the first experiment, the mini-cuttings were stored at 4°C for 0 (immediate planting), 24, 48, 72, 96 and 120 h. The second experiment evaluated the rooting dynamic to determine the optimal time of permanence for minicuttings in a greenhouse. The basal region of the mini-cutting was treated with various IBA solutions: 0 (free of IBA), 1,000, 2,000, 3,000 and 4,000 mg·L−1. Every seven days (0 (immediate planting), 7, 14, 21 and 28 days), destructive sampling of the mini-cuttings was performed to evaluate the histology of the adventitious rooting. Eucalyptus benthamii minicuttings should be rooted immediately after the collection of the shoots. The 2,000 mg·L−1 IBA concentration induced a greater speed and percentage of adventitious rooting, and an interval of 35 to 42 days was indicated for permanence of the mini-cuttings in the greenhouse. Exposure to low temperature induced adventitious root formation with diffuse vascular connections.

Anatomical analysis of peach palm (Bactris gasipaes) leaves cultivated in vitro, ex vitro and in vivo

The present work characterized and compared the anatomical structures of the leaves of Bactris gasipaes (Arecaceae) plants grown under different cultivation conditions (in vitro, ex vitro and in vivo) with the goal of identifying the origins of the difficulties encountered in acclimatizing micro-plants. The Quant program was used to determine leaf tissue thicknesses and areas, and histochemical tests were performed on leaf sections and analyzed using light microscopy. Stomatal and trichome densities were determined using the epidermal impression method and by scanning electronic microscopy. Our results indicated that there were no discernible alterations of the anatomical characteristics of the leaves of micro-plants cultivated under differing conditions and that the thickening of the mesophyll and the vascular fibers indicated adaptive responses to ex vitro conditions. As such, the observed difficulties in acclimatizing peach palm micro-plants to ex vitro conditions cannot be attributed to plant anatomical characteristics acquired during in vitro cultivation.

Vegetative rescue and cloning of Eucalyptus benthamii selected adult trees

Eucalyptus benthanii presents great aptitude for cultivation in American continent, and due to its origin from western Sydney, NSW, Australia, is especially recommended for regions with cold climates and frequent frosts. Taking their optimal silvicultural performance into account, selected genotypes will certainly represent an excellent alternative for future plantations. The cloning of superior genotypes is accomplished by vegetative propagation of mature trees, and requires physiologically juvenile or rejuvenated material. Special techniques are necessary to reverse the juvenility and adult tree recovery and promote favorable growth and rooting conditions. In the specific case of Eucalyptus sp., the most common vegetative rescue method used by forestry companies to obtain shoots (i.e., propagules) is the coppicing of adult trees, a technique which provides excellent juvenile shoots, favoring the adventitious rooting in cuttings. Cloning techniques are required to vegetative rescue and germoplasm banks formation of selected trees. The present study aimed to evaluate the vegetative rescue after the selection of phenotypes with superior silvicultural characteristics to clone adult trees of E. benthamii, evaluating which technique presented the best cloning result. The cuttings from canopy branches, epicormic shoots from the first (lowest) canopy branches, shoots obtained from girdling and shoots from the pruning of canopy branches were submitted to micropropagation, cutting, and microcutting techniques for each shoot type. Among the key findings for vegetative rescue and cloning, the percentage of in vitro establishment of epicormic shoot (79.2xa0%), ex vitro rooting (75.0xa0%) and acclimatization (27.8xa0%) were suitable. The cuttings of sprouts from girdling stood out as having rooting (18.7xa0% from greenhouse and 14.3xa0% from mini-incubator) and acclimation (11.2xa0% from greenhouse and 9.0xa0% from mini-incubator). The others vegetative propagations techniques tested did not result in shoot survival, rooting or acclimation.

Alterações morfológicas foliares em abacaxizeiros cv. IAC "Gomo-de-mel" micropropagados e aclimatizados em diferentes condições de luminosidade

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.

Miniestaquia de Eucalyptus benthamii: efeito do genótipo, AIB, zinco, boro e coletas de brotações

-1 de Zn, S3 - 0,5 mg L -1 de B, S4 - 0,5 mg L -1 de Zn e B, S5 - 1,0 mg L -1 de Zn e B, S6 - 2,0 mg L -1 ABSTRACT: The aim of this study was to evaluate the induction of adventitious rooting in Eucalyptus benthamii mini-cuttings regarding to genotype, Zn and B concentrations, shoot collections and IBA application. Shoots for the mini-cuttings confection were collected from mini-stumps fertigated with nutrient solutions containing different concentrations of Zn and B (S1 - free of Zn and B, S2 - 0.5 mg L -1 Zn, S3 - 0.5 mg L -1 B, S4 - 0.5 mg L -1 Zn and B, S5 - 1.0 mg L -1 Zn and B, S6 - 2.0 mg L -1 Zn and B). The basal portion of the mini-cuttings was immersed for 10 s in a solution containing 2,000 mg L -1 IBA. A free IBA solution was used as control. The mini-cuttings survival in greenhouse, shade-house and in full sun area were evaluated. In full sun area, only the rooted mini-cuttings were evaluated, and of these, the total length of the root system was measured. The adventitious rooting of Eucalyptus benthamii mini-cuttings depended of the genotype, Zn and B concentrations, shoots collections and IBA application. In general, the adventitious rooting percentage was low, and the genotypes were considered difficult to propagation by mini-cuttings technique. The mini-cuttings collected of mini-stumps fertigated with nutrient solutions S5 (1.0 mg L -1 Zn and B) and S6 (2.0 mg L -1 Zn and B) associated with the presence of IBA in the concentration of 2,000 mg L -1 presented the greater adventitious rooting percentage.