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Plant micropropagation comprises biotechnological tools used for the conservation and mass propagation of orchid species. However, there are few reports of its use for orchids of the subtribe Pleurothallidinae. The present study evaluated the effects of 6-benzylaminopurine (BAP) on shoots formation and development of Dryadella zebrina (Porsch) Luer. For this, seeds were in vitro germinated, and the plantlets were submitted to different concentrations of BAP (0, 3, 6, 9, 12, and 15 µM). The plantlets derived from the treatments 0, 6, and 15 µM of BAP were collected after 60 days in culture and subjected to light microscopy analysis. Our results indicated that BAP increased the formation of the new shoots of D. zebrina, especially in treatment with 6 µM, and its use is indicated for the in vitro multiplication phase. The anatomical analyses of the roots showed a deleterious effect of 15 µM BAP on the meristematic region, with the presence of more vacuolated cells in this zone. Our results represent the first successful report of in vitro propagation for the genus Dryadella and may serve as a basis for further studies of in vitro propagation of phylogenetically related species.

Bamboos (Bambusoideae, Poaceae) are multiple-purpose perennial grasses, which display a growing production chain in Brazil. One of the main bottlenecks is high-quality supplying of plantlets, then requiring efficient mass propagation methods, such as micropropagation. Contamination by microorganisms is recurrent in bamboo in vitro cultures, although some of those manifestations are considered endophytes harboring plant growth promotion potential. The isolation of endophytic bacteria from in vitro cultures of Dendrocalamus asper and Bambusa oldhamii was performed to assess their potential growth-promoting effect in co-cultivation with in vitro plants of Guadua chacoensis, an economically promising bamboo species. Among the total bacterial collection (32 isolates), all of them showed growth-promotion potential as indole compounds-producers. Sequences of 16S rRNA genes from eight selected isolates were newly generated, and the BLASTn similarity test recovered four bacterial genera (Bacillus, Brevibacillus, Serratia, and Atlantibacter) and six species. The co-cultivation experiment was carried out with three isolates selected based on their low- (Ba16), medium- (Ba03), and high-yield (Ba24) production of indole compounds, and Bayesian inferences strongly supported them as Bacillus subtilis, Serratia marcescens, and Brevibacillus parabrevis, respectively. The co-cultivation with three bacterial isolates, and their dilution levels, did not influence shoot or root growth and, however, did not cause apparent impairment for G. chacoensis in vitro cultures. Taken together, the isolation of endophytic microorganisms from field-growth bamboo clump and its co-cultivation with in vitro cultures of bamboos is possible, encouraging a continuous discovery and improvement of micropropagation techniques.

The Serendipitaceae family was erected in 2016 to accommodate the Sebacinales 'group B' clade, which contains peculiar species of cultivable root-associated fungi involved in symbiotic associations with a wide range of plant species. Here we report the isolation of a new Serendipita species which was obtained from protocorms of the terrestrial orchid Epidendrum fulgens cultivated in a greenhouse. This species is described based on phylogenetic analysis and on its microscopic and ultrastructural features in pure culture and in association with the host's protocorms. Its genome size was estimated using flow cytometry, and its capacity to promote the germination of E. fulgens seeds and to associate with roots of Arabidopsis thaliana was also investigated. Serendipita restingae sp. nov. is closely related to Serendipita sp. MAFF305841, isolated from Microtis rara (Orchidaceae), from which it differs by 14.2% in the ITS region and by 6.5% in the LSU region. It produces microsclerotia formed of non-monilioid hyphae, a feature that was not reported for the Sebacinales hitherto. Serendipita restingae promoted the germination of E. fulgens seeds, forming typical mycorrhizal pelotons within protocorm cells. It was also able to colonize the roots of Arabidopsis thaliana under in vitro conditions. Arabidopsis plants grown in association with S. restingae increased their biomass more than fourfold. Serendipita restingae is the first Serendipitaceae species described for the Americas.

Little is known about the reproduction of Dendrocalamus asper because it flowers only every 100 to 120 years. In the present work we describe some reproductive features of this bamboo and character...

Bamboos are an important worldwide non-timber forest product with current rising interest due to their environmentally friendly applications. Besides the consolidated uses of the sweet shoots and culms for structural uses, Dendrocalamus asper is an imposing ornamental bamboo for horticulture. The present work aimed to establish in vitro calli culture and plant regeneration through somatic embryogenesis starting from young inflorescences of the giant bamboo, D. asper. Pre-anthesis inflorescences were collected, disinfested, and subjected to callus induction on MS basal medium supplemented by 0 µM, 9 µM, 18 µM, 27 µM, and 36 µM of 2,4-D in combination with 9 µM of 2-iP or 9 µM Kin. The different obtained calli types were characterized and subcultured in 0 µM, 4.5 µM, 9 µM, and 18 µM of 2,4-D in combination with 9 µM of both cytokinins for multiplication and differentiation. Additionally, the explant incision and its inoculation orientation onto culture media were tested for callus induction improvement. The 2,4-D was essential for callus induction, and its combination with both cytokinins resulted in embryogenic callus induction and further somatic embryos regeneration. The subsequent reduction of this auxin to 4.5 µM resulted in somatic embryo maturation. Somatic embryos transferred to a plant growth regulator-free medium resulted in plantlet conversion. The present work showed the feasibility of using inflorescences as explants and the efficiency of using the 2-iP in combination with 2,4-D to callus induction and in vitro bamboo plant regeneration through somatic embryogenesis.

Despite their great economic importance, relatively little is known about bamboo sexual reproduction because they usually spread through rhizomes and have long intervals between flowering periods. Bambusa tuldoides is no exception; the intervals between flowering periods are about 23 years and often do not result in successful caryopsis production. The aim of the present work was to characterize Bambusa tuldoides sexual reproduction at three stages of flower development and investigate possible male sterility. Pollen was cultured onto several types of culture medium in order to encourage germination, but not a single of the thousands of observed pollen germinated under any condition. Anthers and microspores were analyzed by scanning electron microscopy, transmission electron microscopy, and optical microscopy techniques. Anther dehiscence appeared to be normal when compared to other species. In contrast, microspores began to develop abnormally starting as early as the first flower development stage: retraction of the cytoplasm and rupture of the nuclear and mitochondria membrane. As the interior machinery of the microspores degenerated, starch accumulated within numerous amyloplasts during stages two to four of flower development. The sporoderms of these microspores were similarly incomplete: though they possessed an exine, they lacked an intine. The results here obtained suggest that the non-viability of these abnormal pollen grains prevents the development of Bambusa tuldoides caryopses.

An easy and inexpensive method of determining the photosynthetic pathway in grasses using a dye widely used in microscopy. To evaluate the efficiency of a new histochemical test for determination of the photosynthetic pathway in grasses (Poacea). Leaves of 58 grass species were sectioned transversally, and the sections treated with a 2% sodium hypochlorite solution to clarify the tissue. After discoloration, sections were washed with distilled water and double-stained with astra blue and safranin (1% each in 50% ethanol) for 1 min. Sections were then mounted between microscopy glass slides and coverslips using water. Grass species showing red staining of the bundle sheath cells were considered C4, and species with translucent bundle sheath were considered C3. The results of the histochemical test were then compared with results from carbon isotope composition analysis and the relevant scientific literature. Observations from the histochemical test were congruent with results from δ13C isotope composition analysis, and with data previously presented in the scientific literature. The proposed histochemical test proved efficient for characterization of the photosynthetic pathway in the tested grasses; however, the method should be further tested in a greater number of grass species, encompassing, preferably, all Poacea subfamilies. Future studies may elucidate if the proposed method can effectively be used in other botanical families. Furthermore, additional investigations may determine whether the phenolic compounds indicated by the histochemical test are exclusive to the bundle sheath of C4 grasses and if possible relations exist between these phenolic compounds and the C4 photosynthetic pathway in grasses.

Bamboos are one of the largest Poaceae groups and comprise about 1600 species distributed worldwide. Many of these species are sources of food, handicrafts, construction, charcoal, paper, and fiber. However, the obtainment of financially viable and high-quality seedlings of bamboo is not an easy task. Micropropagation has been successfully used to obtain large-scale bamboo seedlings and is one of the most promising techniques to overcome the bottlenecks in the propagation of these species. Two morphogenetic routes are being used in bamboo micropropagation: organogenesis and somatic embryogenesis (SE). In SE, one or more somatic cells differentiate into somatic embryos, resembling the structure of the zygotic embryo, with bipolar axes that are not vascular connected with the source tissue. Historically, SE has been neglected in comparison to organogenesis, possibly due to its greater complexity and difficulty in control and modulation. So far, ~30 bamboo species have established SE protocols, most of which from genera Bambusa and Dendrocalamus. This chapter discusses the principal advances in bamboo SE, with emphasis on complementary approaches as morpho-anatomy, biochemistry, and molecular biology. Also, we outline the main constraints and limitations of this promising technique and new paths to develop more efficient protocols.

Here we propose a protocol for embryogenic cultures induction, proliferation and maturation for the Brazilian conifer Podocarpus lambertii, and investigated the effect of abscisic acid (ABA) and glutathione (GSH) supplementation on the maturation phase. ABA, zeatin (Z) and salicylic acid (SA) endogenous levels were quantified. Number of somatic embryos obtained in ABA-supplemented treatment was signifi- cant higher than in ABA-free treatment, showing the relevance of ABA supplementation during somatic embryos maturation. Histological analysis showed the stereotyped sequence of developmental stages in conifer somatic embryos, reaching the late torpedo-staged embryo. GSH supplementation in maturation culture medium improved the somatic embryos number and morphological features. GSH 0 mM and GSH 0.1 mM treatments correlated with a decreased ABA endogenous level during maturation, while GSH 0.5 mM treatment showed constantlevels. Alltreatments resulted in decreased Z endogenous levels, supporting the concept that cytokinins are important during the initial cell division but not for the later stages of embryo development. The lowest SA levels found in GSH 0.5 mM treatment were coincident with early embryonic development, and this treatment resulted in the highest development of somatic embryos. Thus, a correlation between lower SA levels and improved somatic embryo formation can be hypothesized

Guadua chacoensis (Poaceae) is a woody bamboo native from the Atlantic forest biome. Morphogenetic and physiological studies are scarce in bamboos, and tissue culture-based biotechnologies tools can be used to investigate ultrastructure and physiological processes as well as to mass-propagate specific genotypes. This study evaluated the effect of light and dark conditions on chloroplast biogenesis as well as in the endogenous levels of zeatin (Z), abscisic acid (ABA), gibberellic acid (GA4), and jasmonic acid (JA) during in vitro culture of G. chacoensis. An increase was observed, followed by a decrease in starch content in response to light treatment, and in contrast, in darkness, an accumulation of starch which is associated to amyloplast formation at day 30 was observed. No etioplast formation was observed even in the dark and this was associated with the presence of fully developed chloroplast at the beginning of the experiment. Z levels quantified showed distinct behavior, as in light, no difference in the levels was observed, except at day 10, and in darkness, the levels increased along the evaluation time. ABA, GA4, and JA biosynthesis increased along the time of evaluation in light condition. In contrast, in darkness, the levels remained unaltered, excepted for an increase in JA levels at day 10, suggesting a strong relationship between light and these phytohormone levels. Possible reasons why light’s presence and absence cause changes in chloroplast ultrastructure and hormone endogenous levels in in vitro cultivated culms of G. chacoensis are discussed. Keywords Bamboos · Micropropagation · Transmission electron microscopy · Plant physiology · Phytohormone Made available in DSpace on 2019-03-02T00:37:13Z (GMT). No. of bitstreams: 1 Polesi2019ArticleChloroplastUltrastructureAndHo.pdf: 1356355 bytes, checksum: 25b47e78e8251e1ff16794227f651cc5 (MD5) Previous issue date: 2019-01-10

The plastome of Linum usitatissimum was completely sequenced allowing analyses of evolution of genome structure, RNA editing sites, molecular markers, and indicating the position of Linaceae within Malpighiales. Flax (Linum usitatissimum L.) is an economically important crop used as food, feed, and industrial feedstock. It belongs to the Linaceae family, which is noted by high morphological and ecological diversity. Here, we reported the complete sequence of flax plastome, the first species within Linaceae family to have the plastome sequenced, assembled and characterized in detail. The plastome of flax is a circular DNA molecule of 156,721 bp with a typical quadripartite structure including two IRs of 31,990 bp separating the LSC of 81,767 bp and the SSC of 10,974 bp. It shows two expansion events from IRB to LSC and from IRB to SSC, and a contraction event in the IRA-LSC junction, which changed significantly the size and the gene content of LSC, SSC and IRs. We identified 109 unique genes and 2 pseudogenes (rpl23 and ndhF). The plastome lost the conserved introns of clpP gene and the complete sequence of rps16 gene. The clpP, ycf1, and ycf2 genes show high nucleotide and aminoacid divergence, but they still possibly retain the functionality. Moreover, we also identified 176 SSRs, 20 tandem repeats, and 39 dispersed repeats. We predicted in 18 genes a total of 53 RNA editing sites of which 32 were not found before in other species. The phylogenetic inference based on 63 plastid protein-coding genes of 38 taxa supports three major clades within Malpighiales order. One of these clades has flax (Linaceae) sister to Chrysobalanaceae family, differing from earlier studies that included Linaceae into the euphorbioid clade.

In this study, an efficient method for regenerating plants from nodal cultures of seedlings was developed for Aspidosperma polyneuron. Mature seeds were surface-sterilized and embryos were germinated in Woody Plant Medium (WPM). Epicotyl and hypocotyl nodal segments, excised from 3-week-old in vitro-grown seedlings, were cultured in WPM medium supplemented with 6-benzyladenine (BA) (2.5, 5.0, and 10 mM), alone or combined with indole-3-butyric acid (IBA) or a-naphthaleneacetic acid (NAA) (0.5 mM) for culture initiation and three subcultures. For root induction, IBA (2.5, 5.0, and 10 mM) pulse treatments (15 minutes) were initially applied, followed by transfer to growth regulator-free WPM for five weeks. Regenerated plants were first transplanted to trays containing soil and Plantmax® substrate (3:1) and later to polyethylene bags for acclimatization in a greenhouse. Hypocotyl explants exhibited superior shoot regeneration rate and rooting percentages compared with those of epicotyl explants. The highest numbers of shoots per explant (7-8) were obtained at the third subculture when using the culture medium supplemented with 10 mM BA. When combined with BA, the addition of 0.5 mM IBA or NAA did not affect the regeneration of shoots. An IBA pulse treatment of 5-10 mM for 15 minutes induced 60% of rooting. The regenerated plants were acclimatized and successfully established in a greenhouse, with a 90% survival rate observed after three months. Based on the results of this study, the micropropagation of Aspidosperma polyneuron from in vitro seedlings is feasible and could be a useful tool for the conservation and propagation of this important endangered species.

Grapevine is one of the major fruit crops worldwide and requires phytochemical use due to susceptibility to numerous pests, including downy mildew. The pyramiding of previous identified QTL resistance regions allows selection of genotypes with combined resistance loci in order to build up sustainable resistance. This study investigates resistance response of pyramided plants containing Rpv1 and Rpv3 loci to Plasmopara viticola infection process. Phenotypic characterization showed complete resistance and lack of necrotic hypersensitive response spots. Principal Component Analysis revealed infected 96 hpi (hours post-inoculation) samples with the most distant proteomes of the entire dataset, followed by the proteome of infected 48 hpi samples. Quantitative and qualitative protein differences observed using 2-DE gels coupled to nanoHPLC-ESI-MS/MS analysis showed a lack of transient breakdown in defense responses (biphasic modulation) accompanying the onset of disease. Forty-one proteins were identified, which were mainly included into functional categories of redox and energy metabolism. l -ascorbate degradation pathway was the major altered pathway and suggests up-regulation of anti-oxidant metabolism in response to apoplastic oxidative burst after infection. Overall, these data provide new insights into molecular basis of this incompatible interaction and suggests several targets that could potentially be exploited to develop new protection strategies against this pathogen. Biological significance This study provide new insights into the molecular basis of incompatible interaction between Plasmopara viticola and pyramided Rpv1 / Rpv3 grapevine and suggests several targets that could potentially be exploited to develop new protection strategies against this pathogen. This is the first proteomic characterization of resistant grapevine available in the literature and it presents contrasting proteomic profiles of that of susceptible plants. The resistance against downy mildew in grapevine has been a long sought and the availability of resistance loci is of major importance. This is the first molecular characterization of resistance provided by Rpv 1 and Rpv 3 genes.

Regeneration of nodular cultures involves the production of new individuals from organogenic nodules. Despite the existence of well-established protocols for bromeliads species, many gaps in understanding the mechanisms of this route still remain, particularly the regeneration of new shoots, a stage little explored. Therefore, this work aimed to monitor the morpho and histodifferentiation processes of shoot regeneration from nodular cultures of Billbergia zebrina. To accomplish this, nodular cultures previously obtained from nodal segments of B. zebrina were transferred to regeneration induction medium consisting of semi-solid MS medium supplemented with 1.0 µM of naphthalene acetic acid and 2.0 µM of benzylaminopurine, respectively. Cultures were maintained in a growth chamber over 8 weeks, and samples were collected weekly for analysis under light, transmission electron, and scanning electron microscopy. Cells in the shoot formation region presented characteristics of mitotically active cells, such as the presence of numerous plasmodesmata and conspicuous microtubules. New shoots maintained vascular connection to the node. Cells binding the nodule vascular bundle toward the shoot showed degeneration of organelles, which, in turn, originated vascular neoelements. The dome presented a depressed or prominent surface, depending on shoot development. Rounded stomata were conspicuous in the leaf primordia and generally higher in number as compared to the epidermal surface. This study elucidated the nodular culture in vitro system in aspects associated to the regeneration of shoots and it is anticipated that such data will lead to the optimization of present nodular cultures based protocols with the aim of conserving endangered bromeliad species.

The plastome of B. orellana reveals specific evolutionary features, unique RNA editing sites, molecular markers and the position of Bixaceae within Malvales. Annatto (Bixa orellana L.) is a native species of tropical Americas with center of origin in Brazilian Amazonia. Its seeds accumulate the apocarotenoids, bixin and norbixin, which are only found in high content in this species. The seeds of B. orellana are commercially valued by the food industry because its dyes replace synthetic ones from the market due to potential carcinogenic risks. The increasing consumption of B. orellana seeds for dye extraction makes necessary the increase of productivity, which is possible accessing the genetic basis and searching for elite genotypes. The identification and characterization of molecular markers are essential to analyse the genetic diversity of natural populations and to establish suitable strategies for conservation, domestication, germplasm characterization and genetic breeding. Therefore, we sequenced and characterized in detail the plastome of B. orellana. The plastome of B. orellana is a circular DNA molecule of 159,708 bp with a typical quadripartite structure and 112 unique genes. Additionally, a total of 312 SSR loci were identified in the plastome of B. orellana. Moreover, we predicted in 23 genes a total of 57 RNA-editing sites of which 11 are unique for B. orellana. Furthermore, our plastid phylogenomic analyses, using the plastome sequences available in the plastid database belonging to species of order Malvales, indicate a closed relationship between Bixaceae and Malvaceae, which formed a sister group to Thymelaeaceae. Finally, our study provided useful data to be employed in several genetic and biotechnological approaches in B. orellana and related species of the family Bixaceae.

This is the first study to describe in a timescale morphohistological and ultrastructural characteristics of fruit (cypsela) and seed development in Trichocline catharinensis, which was completed 21 days after anthesis (DAA). At anthesis, we identified an ovary with three differentiated regions, including the inner epidermis, inner part, and outer epidermis. The mature ovule showed an integument with the outer epidermis, integumentary parenchyma, and endothelium. Cells around the endothelium form the periendothelial zone with thick cell walls that showed Periodic acid-Schiff (PAS)–positive reaction. The periendothelial zone and endothelium showed degradation of the cells during embryogenesis. The main stages of embryo development from fecundation through mature seed were identified. The ripe cypsela showed the pericarp (exocarp), seed coat (exotesta), and remaining endosperm surrounding the embryo. Mature embryos were straight with shoot apical meristem (SAM), and root apical meristem (RAM) was separated by the hypocotyl. Light microscopy (LM) and transmission electron microscopy (TEM) analyses indicate cells with characteristics of meristem cells, as well as proteins and lipid bodies and mitochondria with few cristae in cotyledon cells. Our findings provide insight into taxonomic and physiological studies by detailing cypsela and seed ontogenesis from an endemic and vulnerable Asteraceae from southern Brazil. This study is also a starting point for establishing the biological criteria for seed harvesting and future studies of seed physiology and conservation of plant genetic resource.

Here we evaluated and characterized the growth dynamics of A. angustifolia embryogenic cultures (EC) submitted to different cryotreatment incubation times through morphological and time-lapse cell tracking analyzes. The EC submitted to cryopreservation protocol were evaluated by regrowth rates, and ultrastructural characterization by transmission electron microscopy (TEM). The results indicated that A. angustifolia EC support all the cryoprotection times evaluated, without cell proliferation inhibition, but with noticeable genotype-dependent response in all tested cell lines. The use of 1M DMSO showed non-inhibitory effects to EC regrowth independent of cell line or cryotreatment incubation time. However, after cryopreservation, Cr01 cell line regrowth was 100 % for all cryotreatments incubation times evaluated (30, 60, 120, 240 min), while Cr02 cell line only showed 100 % regrowth in 240 min of cryotreatment. The 100 % cell regrowth obtained in both cell lines indicates that the proposed protocol can be successful applied to A. angustifolia EC cryopreservation. Cell tracking analysis showed a survival and initial proliferation of embryogenic cells, with the first cell regrowth signs after 30 days in culture. TEM analysis revealed a conspicuous cell wall thickening in embryogenic cells after cryotreatment and after thawing, which may be related to osmotic stress response caused by the cryopreservation process. An increased heterochromatin presence was also observed in cryotreated or after thawing cells, may possibly be acting as a cell defense mechanism, decreasing the DNA vulnerability to cleavage and preserving the cell integrity.

Tissue culture comprises several techniques for the mass propagation of selected genotypes. In the case of palms, the most promising and efficient micropropagation technique is somatic embryogenesis. Ever since somatic embryogenesis was first induced in oil palm several decades ago, 19 palm species have been induced to develop somatic embryos from several types of explants. Optimization of somatic embryo multiplication protocols has been the central focus of research, but embryo maturation and conversion remains poorly-studied in many palm species. This review summarizes the advances made in both the study and optimization of palm somatic embryogenesis (SE) protocols. Explant choice and inoculation method, media components, culture method, histology of early somatic embryogenesis, gene expression, biochemical changes, embryo maturation and conversion, and acclimatization of regenerated plantlets are presented and critically discussed.

Micropropagation of banana by means of different morphogenetic routes is an important source of genetically uniform, pest- and disease-free planting materials of Musa spp. Morphogenesis of in vitro grown plants are affected by light quality, light quantity and photoperiod. Light-emitting diode (LED) lighting systems have several unique advantages, including their peak spectral output, which closely coincides with the red absorption peak of chlorophyll and the reported wavelengths for maximum photosynthetic efficiency. The present study was aimed to investigate the effect of two LED lighting treatments (white LED and deep red/white LED) compared to conventional fluorescent lamps on the stomata formation and chlorophyll (Chl) levels in micropropagated banana plantlets through organogenesis. Both LED lighting evaluated increased the levels of total Chl, Chl-a and Chl-b in banana in vitro plantlets, indicating no statistical difference between them, but being higher then plantlets subjected to fluorescent lamps. The number of stomata was also increased equally by both LED lighting treatments, indicating increased formation of stomata in both abaxial and adaxial leaf surfaces when compared to fluorescent lamps. Although all treatments presented 100 % survival in the acclimatization phase, LED lighting show promising for micropropagation of banana due to its known superior light quality, low energy consumed, little heat generated and long operating lifetime.

Varronia curassavica, a subshrubby medicinal species associated with restinga in the Atlantic Forest, has been exploited by local people and the pharmaceutical industry. Indeed, restingas have experienced a continuous process of degradation, and thus, with species and ecosystem both at risk, efforts to support conservation actions are required. The present study aimed to evaluate aspects of V. curassavica reproductive biology. To accomplish this, morphological characterization was performed by monitoring flowering events. The availability of nectar and pollen, as well as the frequency and behavior of floral visitors and dispersers, was also evaluated. This species exhibits both heterostyly and protogyny. Anthesis is diurnal, and flowers last less than a day. The high number of flower and fruit abortions suggests that mechanisms, such as self-incompatibility intra-morphs and easily detached flowers, contribute to reduced fruit production. The high diversity of floral visitors indicate a generalist pollination syndrome. Diptera, Hymenoptera and Lepidoptera were the main pollinators, and nectar was the main resource sought by these insects. Fruits were dispersed by birds and ants. It can be concluded that the interaction of V. curassavica with several species is a key factor in its own survival and for maintaining the biological diversity of restinga.

P. lambertii is a Brazilian conifer indigenous to one of the 25 biodiversity hotspots of the world, the subtropical moist forest ecoregion of the Atlantic Forest Biome. Its conservation status is classified as near threatened, being desirable the development and use of biotechnological tools, such as somatic embryogenesis (SE). SE is a complex, multifactorial model system of molecular, biochemical, physiological and developmental steps of plant embryogenesis, and comprises a high performance in vitro regenerative system for both the mass clonal propagation of an elite genotype and/or the mass propagation of endangered plant germplasm. Protocols for plant regeneration based on SE have been established for many conifers species, but species from the closely related families Araucariaceae and Podocarpaceae are in general considered recalcitrant to SE. Mostly of the SE protocols for conifers have been developed for members of family Pinaceae. In this context, here we describe the first SE protocol for a species from family Podocarpaceae, the P. lambertii. This protocol is based on induction with immature zygotic embryos, followed by establishment and multiplication of proembryogenic masses on plant growth regulator-free culture medium, and maturation phases I and II. Procedures including explant preparation, PEM proliferation, and somatic embryo development are described in detail.

Pineapple presents a significant portion of the global fruit market. Recently, a micropropagation protocol was established based on nodule cluster cultures (NC), which showed high regenerative efficiency. Aiming to expand the understanding of gene regulation in this protocol, global DNA methylation levels (GDML) and fresh mass increase ratio (FMIR) during NC multiplication and differentiation to microshoots were evaluated after 0, 9, 18, 27, and 36 days in culture. In addition, genetic fidelity of randomly chosen regenerated plants was analyzed by means of amplified fragment length polymorphism (AFLP), flow cytometric analysis of ploidy level, as well as observation of phenotypic features. An association between GDML and the density of new shoot apical meristems formed in cultures was observed, which was, in turn, related to FMIR and the age of each subculture. The analysis by means of AFLP and DNA ploidy level, as well as phenotypic observations indicated the homogeneity of regenerated plantlets.

Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. is an important fungal pathogen known to cause glomerella leaf spot (GLS). The objective of this study was to analyze the infection of C. gloeosporioides on leaves of apple cv. Fuji (resistant) and cv. Gala (susceptible) and apply proteomics techniques to study the apple defense responses 48 h after inoculation (h.a.i.). On both of cultivars, C. gloeosporioides started to germinate at 3 h.a.i. on adaxial surface and produced appressoria adhering to epidermal cell juxtapositions. Histological analysis showed more stratified parenchyma in leaves of cv. Fuji than cv. Gala associated with differences in the chemical composition of cell walls. Total and unique proteins expressed by cvs. Fuji and Gala at 3, 12, 24 and 48 h.a.i. were detected by comparative proteomes analysis. A total of 42 unique proteins expressed at 24 and 48 h.a.i. were identified by MALDI/TOF mass spectrometry, and most of these proteins were identified as directly involved in defense responses.

This study aimed to establish a cryopreservation protocol for embryogenic cultures of A. angustifolia, enabling the ex situ conservation of the species. Embryogenic cultures were established from immature seeds and treated with variations of the cryoprotectant solutions SuDG, SoD and PVS2 prior to immersion in liquid nitrogen. Cell viability was evaluated after 30, 60 and 90 days of re-growth. The highest re-growth without morphological alterations and with normal biochemical composition was obtained with the PVS2 solution with 40 min immersion in ethanol (-20 °C). This procedure opens new horizons for the ex situ conservation of the species genetic. Este estudo objetivou estabelecer um protocolo de criopreservação para culturas embriogênicas de A. angustifolia, possibilitando a conservação ex situ da espécie. Culturas embriogênicas foram estabelecidas a partir de sementes imaturas e tratadas com variações das soluções crioprotetoras SuDG, SoD e PVS2 antes da imersão em nitrogênio líquido. A viabilidade celular foi avaliada após 30, 60 e 90 dias de recrescimento. O maior recrescimento sem alterações morfológicas e composição bioquímica normal foi obtido com a solução PVS2 com 40 min de imersão em etanol (-20 °C). Este procedimento abre novos horizontes para a conservação ex situ dos recursos genéticos da espécie.

The content and quality of phenolic compounds in apple pulp, peel, and leaves constitute important nutraceutical properties of apples in the human diet and are equally important to plant ecology. Moreover, increased understanding of the dynamics of polyphenol synthesis in different apple tissues of different genotypes may assist in apple breeding programs. Thus, the present study aimed to analyze the levels of total phenolics in apple pulp, peel, and leaves from cultivars Aori27, Elstar, Fuji, and Mellow at harvest time. Significant differences in the total phenolic content were found between cultivars for all evaluated tissues, but the differences between cultivars were not consistent among tissues, indicating the potential existence of tissue-specific genetic regulation mechanisms, as well as cultivar-specific dynamics. Additionally, tissues of two cultivars, Fuji and Mellow, were analyzed in the last three months of fruit development, showing a similar overall evolution in the phenolic concentration for all tissues, but some notable cultivar-specific characteristics for apple pulp and peel. These results are relevant for apple breeding when we consider as selection criteria the nutraceutical properties of apple polyphenols, the speed and degree of pulp enzymatic browning, and the physiological and agro-ecological roles of polyphenols in different organs and tissues of apple trees.

In Brazil winemaking is expanding not only in the renewal of vineyards in traditional production areas, but also in the introduction of new crops in the newer winemaking regions. Promising cha- racteristics were observed in the quality of the grapes and wines produced in altitude regions of Santa Catarina. The aim of this study is to characterize the geographic and climatic conditions which affect the phenology and vegetative growth of grapevine in Sao Joaquim, in the mountains region of Santa Catarina. The vineyards are located in areas with undulated and strongly undu- lated relief, predominantly in soils originating from basaltic rocks, in climate conditions classified as Cfb according to the Koppen, as Region I according to Winkler classification. The phenological stage of the grapevines in the Sao Joaquim region begins later and lasts longer in comparison to other winegrowing regions in Brazil. Decrease in photoperiod and low temperatures (

SummaryThe present work studied the induction, multiplication and development of nodular cluster cultures (NCC) of Vriesea reitzii, an endangered bromeliad species native to the Brazilian Atlantic Forest Biome, in different culture media under different culture conditions. Leaf basal segments of in vitro-grown plantlets were used as explants and inoculated into Murashige and Skoog (MS) liquid medium supplemented with different types and concentrations of plant growth regulators (PGRs) in the presence or absence of light, followed by sub-culturing in or on PGR-free liquid or solidified culture media to promote multiplication. The induction of NCC, which occurred from the basal region of the explants, was observed in all culture media tested. Morphological features and regenerative efficiency were ascribed to the different culture media, based mainly on medium type, and the concentration and combination of PGRs, as well as on exposure to light or dark conditions. Green-coloured NCC with a granular texture s...

Regrowth capacity and genetic stability of plants recovered following cryopreservation are associated with changes in DNA epigenetics, particularly in DNA methylation levels. In this study, global DNA methylation profiles associated with frequency of regrowth of peach palm (Bactris gasipaes) somatic embryos following cryopreservation using droplet-vitrification were investigated. Somatic embryo clusters (SEC) subjected to plant vitrification solution 3 (PVS3) for different durations (0, 60, 120, 180, and 240 min) were evaluated for regrowth capacity. The highest frequency of regrowth (52.4 %) was obtained when SEC were incubated in PVS3 for 120 min prior to droplet-vitrification cryopreservation. Global DNA methylation profiles were influenced by both cryoprotectants and droplet-vitrification cryopreservation. Incubation of SEC in PVS3 for limited durations not only reduced frequency of regrowth, but also increased DNA methylations levels when compared with proliferating SEC grown in a temporary immersion system. Although SEC subjected to cryopreservation exhibited the highest DNA methylation variation, 120 min SEC incubation in a PVS3 solution resulted in the recovery of initial global methylation profiles after 24 weeks of regrowth.

Bactris gasipaes (Arecaceae), also known as peach palm, was domesticated by Amazonian Indians and is cultivated for its fruit and heart-of-palm, a vegetable grown in the tree's inner core. Currently, the conservation of this species relies on in situ conditions and field gene banks. Complementary conservation strategies, such as those based on in vitro techniques, are indicated in such cases. To establish an appropriate cryopreservation protocol, this study aimed to evaluate the ultrastructural features of B. gasipaes embryogenic cultures submitted to vitrification and subsequent cryogenic temperatures. Accordingly, somatic embryo clusters were submitted to Plant Vitrification Solution 3 (PVS3). In general, cells submitted to PVS3 had viable cell characteristics associated with apparently many mitochondria, prominent nucleus, and preserved cell walls. Cells not incubated in PVS3 did not survive after the cryogenic process in liquid nitrogen. The best incubation time for the vitrification technique was 240 min, resulting in a survival rate of 37 %. In these cases, several features were indicative of quite active cell metabolism, including intact nuclei and preserved cell walls, an apparently many of mitochondria and lipid bodies, and the presence of many starch granules and condensed chromatin. Moreover, ultrastructure analysis revealed that overall cellular structures had been preserved after cryogenic treatment, thus validating the use of vitrification in conjunction with cryopreservation of peach palm elite genotypes, as well as wild genotypes, which carry a rich pool of genes that must be conserved.

Biotechnology tools based on tissue culture techniques allow mass clonal propagation of plants with desired genetic and sanitary features. In the present work a pineapple micropropagation protocol was established based on nodule cluster cultures (NC) associated with different culture systems. Leaf segments from in vitro -grown plantlets cultivated in culture medium supplemented with 2 μM NAA and 8 μM 2-iP or BAP resulted in the highest rates of NC induction. NC fresh weight increased ratio was more efficient in a culture medium supplemented with 2 μM NAA and 2 μM BAP in twin-flasks temporary immersion system (TIS-TF). This culture medium was also efficient for the microshoots development in RITA ® temporary immersion system (TIS-RITA ® ). Microshoots elongation in permanent immersion system (PIS) was more efficient in a culture medium supplemented with 10 μM GA 3 . Shoots longer than 2 cm showed 92% survival rate when acclimatized. Free polyamine endogenous levels was measured and showed higher contents for the TIS-TF, followed by TIS-RITA ® , and PIS, respectively. Putrescine levels also followed this pattern, being absent in PIS, which, on the other hand, showed higher levels of spermine and spermidine. The results indicate the establishment of efficient protocol for pineapple micropropagation, with a high multiplication and regenerative rates and great potential for agriculture application. The biochemical analysis suggests that besides culture medium composition, flask design and culture system also drastically affects the plant's metabolism toward multiplication and plant regeneration.

Elaeis guineensis (African oil palm) and E. oleifera (American oil palm) are bred to attain high oil yields, disease resistances, and decelerated shoot elongation. We cultivated immature zygotic embryos from backcrossed and F1 interspecific progenies on media containing 110, 150, or 200 mg·l-1 2,4-diclorophenoxyacetic acid (2,4-D) to obtain embryogenic cultures. These were set to multiply on medium containing 8 mg·l-1 2,4-D or lower concentrations of auxins and finally we induced plantlets regeneration, from each zygotic embryo, independently, in order to have the clones organized according to their respective origins. Reductions in auxins induced cultures to revert from highly embryogenic into competent for embryogenesis and finally to organogenic degenerated callus lines. Histology and the expression of SOMATIC EMBRYOGENESIS RECEPTOR KINASE, DEHYDRIN, DEFENSIN, TRANSPOSASE, and LIPID TRANSFER PROTEIN were analyzed on four callus lines representative of morphological aspects consistently observed. The highest number of embryogenic cultures was obtained on 150 mg·l-1 2,4-D. Maturation and multiplication of somatic embryos through secondary embryogenesis occurred simultaneously on 8 mg·l-1 2,4-D. LIPID TRANSFER PROTEIN expression was detected in one of the embryogenic cultures and correlated with protoderm onset. Three six-week cycles on induction medium yielded 1.5 shoots above 6 cm per poly-embryogenic complex, which performed better than embryoids individualized mechanically. Rooting was observed for 77% and 82% of shoots from these two types of explants, respectively. Rooted plantlets ready for acclimatization were obtained nine months after shoot induc

Given their distribution, importance, and richness, Myrtaceae species comprise a model system for studying the evolution of tropical plant diversity. In addition, chloroplast (cp) genome sequencing is an efficient tool for phylogenetic relationship studies. Feijoa [Acca sellowiana (O. Berg) Burret; CN: pineapple-guava] is a Myrtaceae species that occurs naturally in southern Brazil and northern Uruguay. Feijoa is known for its exquisite perfume and flavorful fruits, pharmacological properties, ornamental value and increasing economic relevance. In the present work, we reported the complete cp genome of feijoa. The feijoa cp genome is a circular molecule of 159,370 bp with a quadripartite structure containing two single copy regions, a Large Single Copy region (LSC 88,028 bp) and a Small Single Copy region (SSC 18,598 bp) separated by Inverted Repeat regions (IRs 26,372 bp). The genome structure, gene order, GC content and codon usage are similar to those of typical angiosperm cp genomes. When compared to other cp genome sequences of Myrtaceae, feijoa showed closest relationship with pitanga (Eugenia uniflora L.). Furthermore, a comparison of pitanga synonymous (Ks) and nonsynonymous (Ka) substitution rates revealed extremely low values. Maximum Likelihood and Bayesian Inference analyses produced phylogenomic trees identical in topology. These trees supported monophyly of three Myrtoideae clades.

In this work, it was observed a straight relationship between the manipulation of the reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio, nitric oxide emission and quality and number of early somatic embryos in Araucaria angustifolia, a Brazilian endangered native conifer. In low concentrations GSH (0.01 and 0.1mM) is a potential NO scavenger in the culture medium. Furthermore, it can increase the number of early SE formed in cell suspension culture media in a few days. However, the maintenance in this low redox state lead to a loss of early somatic embryos polarization. In gelled culture medium, high levels of GSH (5mM) allows the development of globular embryos presenting a high NO emission on embryo apex, stressing its importance in the differentiation and cell division. Taken together these results indicate that the modification of the embryogenic cultures redox state might be an effective strategy to develop more efficient embryogenic systems in A. angustifolia.

Araucaria angustifolia, the Brazilian pine, is an endangered native conifer with economic and ecological importance. The female cone develops seeds containing the zygotic embryo, which, at cotyledonary stage, shows well-developed meristems. Little is known about the structure of gymnosperm meristems. In the present work, the composition and morphological organization of Araucaria angustifolia shoot and root apical meristems were studied during embryo development, using histochemical and microscope analyses. Histochemical evaluation revealed the presence of cellulose within the cell wall, cells with the presence of total proteins that react with Coomassie Brilliant Blue, starch grains, and large nuclei with evident nucleoli in the cytoplasm. Scanning electron microscopy showed apical meristem surface morphology, and both scanning and transmission microscopy revealed a thin and irregular cell wall with plasmodesmata and within the cells, mitochondria, many vacuoles, lipid bodies, Golgi bodies, and many amyloplasts with endoplasmic reticulum surrounding them and large nuclei. Similar to angiosperm cells, A. angustifolia meristem cells exhibit pluripotent characteristics, such as apparatus for intercellular communication and differentiation.

Angiosperm and gymnosperm plants evolved from a common ancestor about 300 million years ago. Apart from morphological and structural differences in embryogenesis and seed origin, a set of embryogenesis-regulating genes and the molecular mechanisms involved in embryo development seem to have been conserved alike in both taxa. Few studies have covered molecular aspects of embryogenesis in the Brazilian pine, the only economically important native conifer in Brazil. Thus eight embryogenesis-regulating genes, viz., ARGONAUTE 1, CUP-SHAPED COTYLEDON 1, WUSCHEL-related WOX, S-LOCUS LECTIN PROTEIN KINASE, SCARECROW-like, VICILIN 7S, LEAFY COTYLEDON 1, and REVERSIBLE GLYCOSYLATED POLYPEPTIDE 1, were analyzed through semi-quantitative RT-PCR during embryo development and germination. All the eight were found to be differentially expressed in the various developmental stages of zygotic embryos, seeds and seedling tissues. To our knowledge, this is the first report on embryogenesis-regulating gene expression in members of the Araucariaceae family, as well as in plants with recalcitrant seeds.

SummaryAmino acids play fundamental roles in plant morphogenesis. Among sources of organic nitrogen (N), glutamine has frequently been used during the establishment and maintenance of cell and tissue cultures. The aim of this study was analyse endogenous levels of glutamine during somatic and zygotic embryogenesis of Acca sellowiana (Feijoa or pineapple guava). The in vitro absorption of 3H-labelled glutamine was investigated. Zygotic embryos and embryogenic cultures (EC) were evaluated at 30 d and 70 d after explant inoculation onto the medium. Endogenous levels of glutamine were similar during zygotic and somatic embryogenesis, and showed a gradual decline until day-24 in culture. The highest rates of 3H-labelled glutamine uptake were observed during the first 2 h of incubation, resulting in values of 6.29 µmol g–1 fresh weight (FW) for zygotic embryos, 14.43 µmol g–1 FW for EC after 30 d, and 13.85 µmol g–1 FW for EC after 70 d. These results showed that the decreased levels of glutamine observed durin...

Pineapple cultivars are normally diploids, however some triploids have been found. The ‘Gigante de Tarauaca’ is a native pineapple that produces large fruits (as much as 15 kg) in the region of Tarauaca, Acre State, Northern Brazil. Hypothesizing that this feature is related with polyploidy, we investigated its genome by means of chromosomal counting and flow cytometry, using the traditional diploid cultivar Perola as a control. The chromosomal counting was performed by means of the Feulgen reaction, and the ploidy levels were compared in histograms by flow cytometry after the nuclei were isolated and treated with Propidium Iodide and RNAse. The somatic chromosome numbers and ploidy levels identified in the histograms by flow cytometry revealed the triploid nature of the ‘Gigante de Tarauaca’ (2n = 3x = 75) and the diploid status of ‘Perola’ (2n = 2x = 50). To the best of our knowledge this is the first report of the triploid nature of the ‘Gigante de Tarauaca’.

Biotechnological techniques comprise useful tools for the conservation of endangered plant genetic resources. In the present work, polymorphism and usefulness of amplified fragment length polymorphism (AFLP) markers in assessing the genetic diversity in populations of Billbergia zebrina were investigated in nodular cultures and adult plants of the species. AFLP markers revealed moderate-to-high genetic diversity based on the estimations of Nei's gene diversity (mean He = 0.28), Shannon index of diversity (mean HS = 0.48) and the number of polymorphic fragments (mean of 56.17 polymorphic fragments over six primer pairs). In comparison to published studies of population genetics performed in other bromeliad species, the present study suggests that natural populations of B. zebrina likely maintain high levels of genetic diversity, an important feature towards conservation of plant genetic resources. The results obtained reveal that AFLP markers comprise a powerful tool in order to assess the levels of genetic diversity in natural populations of this endangered species. Integrating AFLP markers with in vitro propagation techniques is understood as an adequate strategy for conservation programmes of this species.

Somatic embryogenesis is an in vitro morphogenetic route in which isolated cells or a small group of somatic cells give rise to bipolar structures resembling zygotic embryos. Lipids, carbohydrates, and proteins are major compounds in plant and animal metabolism. Comparative analysis along different developmental stages of Acca sellowiana (Myrtaceae) zygotic and somatic embryos, revealed a progressive increase in levels of total lipids. A high degree of similarity could be found in the total lipids composition between A. sellowiana somatic and zygotic embryos. High lipid levels were found in zygotic embryos in the torpedo and cotyledonary stages, and these levels increased according to the progression in the developmental stages. Somatic embryos obtained through direct embryogenesis route showed higher levels of lipids than in indirect somatic embryogenesis. The compounds most frequently were linoleic acid (C18:2), palmitic (C16:0) and oleic (C18:1). These results indicate a high similarity degree of accumulation of total lipids, regardless of zygotic or somatic embryogenesis.

Micropropagation systems based on nodular cultures (NCs), are considered as an intermediary in vitro morphogenetic route, diverging from regenerative systems based on organogenesis and somatic embryogenesis. The aim of this study was to establish a regenerative protocol based on the induction and development of NCs in Vriesea reitzii , an endangered bromeliad from the Atlantic forest which also has ornamental value. Additionally structural analyses were performed in order to better understand this in vitro morphogenetic route. NCs were regenerated in MSB culture medium free of PGR or supplemented with different levels of NAA alone or in combination with in combination with 2-iP. The subculture of these NCs on MSB medium supplemented with 10 μM of GA 3 promoted the synchronized shoot elongation. A regenerative efficiency of 12.4 g g −1 of NCs was obtained, and this results in 5300 microshoots after 10 weeks in culture. The structural analyses of the NCs revealed that the regenerative process occurs from the proliferation of meristematic cell groups resulting in the development of multiple shoot meristems and buds. The development of NCs leads to the formation of monopolar structures called microshoots, which evolve to elongated shoots. Intermediary features shown in NCs are consistent with their classification as an intermediary system among organogenesis and somatic embryogenesis.

Over recent years nitric oxide (NO) not only has appeared as an important endogenous signaling molecule in plants and as a mediator in many developmental and physiological processes, but has also received recognition as a plant hormone. The impressive recent achievements in elucidating the role of NO in plants have come about by the application of NO donors. The aim herein was to study the effects of the different NO donors, sodium nitroprusside (SNP) and the nitrosyl ethylenediaminetetraacetate ruthenium(II) ([Ru(NO)(Hedta)]) complex on cellular growth in embryogenic suspension cultures of Araucaria angustifolia . Appraisal of our data revealed that [Ru(NO)(Hedta)] stimulated about 60% of cellular growth in embryogenic suspension cultures of A. angustifolia , with results similar to those observed with the SNP donor. Nevertheless, application of the NO scavenger PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) inhibited this cellular growth in both. Cellular growth was correlated with an increase in endogenous NO levels after 21 days of culture, especially in treatments with NO donors. Our results demonstrated that the [Ru(NO)Hedta] complex could possibly be used as a NO donor in plants.

Acca sellowiana (Berg.) Burr. is a native Myrtaceae from southern Brazil and Uruguay, now the subject of a domestication and breeding program. Biotechnological tools have been used to assist in this program. The establishment of a reliable protocol of somatic embryogenesis has been pursued, with a view to capturing and fixing genetic gains. The rationale behind this work relies on the fact that deepening comprehension of the general metabolism of zygotic embryogenesis may certainly improve the protocol for somatic embryogenesis. Thus, in the present work we studied the accumulation of protein, total sugars, starch, amino acids, polyamines (PAs), IAA and ABA, in different stages of A. sellowiana zygotic embryogenesis. Starch is the predominant storage compound during zygotic embryo development. Increased synthesis of amino acids in the cotyledonary stage, mainly of asparagine, was observed throughout development. Total free PAs showed increased synthesis, whereas total conjugated PAs were mainly observed in the early developmental stages. IAA decreased and ABA increased with the progression from early to late embryogenesis. Besides providing basic information on the morphophysiological and biochemical changes of zygotic embryogenesis, the results here obtained may provide adequate strategies towards the modulation of somatic embryogenesis in this species as well as in other woody angiosperms.

The γ-aminobutyric acid (Gaba) is a non-protein amino acid found in prokaryotes and eukaryotes. Its role in plant development has not been fully established. This study reports a quantification of the levels of endogenous Gaba, as well as investigation of its role in different stages of somatic embryogenesis in Acca sellowiana Berg. (Myrtaceae). Zygotic embryos were used as explants and they were inoculated into the culture medium contained different concentrations of Gaba (0,2, 4, 6, 8 and 10 µM). The highest concentrations of endogenous Gaba were detected between the third and nine days after inoculation, reaching the value of 12.77 µmol.g-1FW. High frequency of somatic embryogenesis was observed in response to 10 µM Gaba. This treatment also resulted in a large number of normal embryos, and the lowest percentage of formation of fused somatic embryos, phenotypic characteristic of most deformed embryos in all treatments. Also, all treatments promoted the formation of the somatic embryos with positive characteristics of development resumption, which however did not originate the seedlings. O ácido γ-aminobutírico (Gaba) é um aminoácido não protéico encontrado em procariontes e eucariontes. Seu papel em plantas ainda não está bem estabelecido. No presente estudo procurou-se quantificar os teores endógenos de Gaba, bem como investigar seu papel nos diferentes estágios da embriogênese somática em Acca sellowiana Berg. (Myrtaceae). Foram empregados embriões zigóticos como explantes e os mesmos foram inoculados em meio de cultura contendo diferentes concentrações de Gaba: 0 (controle), 2, 4, 6, 8 e 10 µM. As maiores concentrações de gaba endógeno foram detectadas no período compreendido entre o 3º e o 9º dia após a inoculação, tendo alcançado, neste último dia, o valor de 12,77 µmol.g-1FW. Alta freqüência de embriogênese somática foi observada em resposta a 10 µM de Gaba. Este tratamento também resultou em grande número de embriões normais, bem como o menor percentual de formação de embriões cupuliformes, característica fenotípica da maioria dos embriões deformados em todos os tratamentos. Em todos os tratamentos ocorreram embriões somáticos que apresentaram características positivas quanto à retomada de desenvolvimento, mas que não resultaram na formação de plântulas.

SummaryThis report describes a reliable method for callus induction, isolation, and the culture of anther-derived protoplasts from the apple rootstock, ‘M9’, and provides information on the development of a protoplast-to-plant regeneration system. Callus induction occurred in anthers when cultivated on Nitsch and Nitsch (NN) or on AT3 media. Suspension cells were established from friable, anther-derived callus formed on NN basal medium. Protoplasts were isolated from callus and anther-derived suspension cells. The highest viability value for freshly isolated protoplasts (62.67 ± 1.53%) was achieved using a 4 h digestion period with suspension cells on Kao and Michayluk (KM) basal medium diluted in an enzyme solution primarily composed of [1% (w/v)] cellulase ‘Onozuka’ RS in combination with [1% (w/v)] macerase and [2% (w/v)] pectolyse Y-23. Protoplasts were cultured using three methods: agarose bead, hanging drop, or liquid culture. The first protoplast division was observed after 8 d of cultivation. The ...

This paper describes a proteome analysis and changes in endogenous abscisic acid (ABA) contents during seed development of Araucaria angustifolia (Bert.) O. Ktze. Megagametophytes and embryonic axis tissues exhibited a similar ABA variation pattern during seed development, reaching maximum values at the pre-cotyledonary stage. The embryonic axis protein content increased until the cotyledonary stage with following stabilization at mature seed. The two-dimensional electrophoresis at the torpedo developmental stage showed approximately 230 polypeptides against 340 in the mature stage. Peptide mass fingerprinting analyses identified three polypeptides, corresponding to an AtSAC4, a late embryogenesis abundant (LEA) and a storage protein, respectively.