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Este estudo objetivou avaliar a influência das citocininas durante o estágio de multiplicação in vitro e a combinação entre carvão ativado com o ácido indol-3-butírico (AIB) durante o enraizamento in vitro de Handroanthus impetiginosus. Para a multiplicação in vitro, os segmentos nodais dos brotos obtidos a partir do estabelecimento de plântulas in vitro foram inoculados em meio de cultura Woody Plant Medium (WPM), com e sem as seguintes concentrações de 6-benzilaminopurina (BAP), cinetina (KIN) e tidiazuron (TDZ): 0,5; 1,0; 2,0; 4,0 e 8,0 µmol L-1. Para o enraizamento in vitro, brotos foram inoculados em meio WPM contendo combinações entre AIB (1,0; 3,0; 6,0 e 9,0 μmol L-1) e carvão ativado (1,0; 2,0 e 3,0 g L‑1), bem como suas ausências no meio de cultura. O maior número de brotos e gemas foi alcançado após usar 8,0 μmol L-1 de TDZ, enquanto que o melhor enraizamento ocorreu após a adição de 2,0 g L-1 de carvão ativado, independente da concentração de AIB. Nossos resultados mostram que usar TDZ em níveis adequados para o estágio de multiplicação in vitro seguido pelo uso de 2,0 g L-1 de carvão ativado para obter brotos bem enraizados é eficiente para a micropropagação de ipê-roxo de maneira rápida e com baixo custo, além de fornecer conhecimento sobre como manter esta espécie ameaçada de extinção em um ambiente in vitro podendo ajudar outros estudos de conservação.

Hancornia speciosa is a native fruit species from Brazil with important economic applications. However, the use of this species has been challenged by difficulties in its storage. Cryopreservation is a safe and efficient tool for long-term storage. Nevertheless, cryopreservation may promote reactive oxygen species (ROS) formation with subsequent lipid peroxidation, which causes membrane damage. The use of in vitro antioxidant compounds could help eliminate ROS and consequently improve explant survival. In the current study, the lateral buds of H. speciosa were precultivated in proline or glycine betaine (GB)-containing solutions, and the effect of these compounds on the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) and on lipid peroxidation was measured after sample warming. The present findings indicated that oxidative stress induced by cryopreservation was significantly reduced by the precultivation of lateral buds with proline and GB. SOD activity increased, and subsequently, hydrogen peroxide (H2O2) production increased followed by the immediate activation of CAT and APX. The enzyme activity increased for 7 following rewarming, indicating that precultivation with 0.1 M proline was fundamental to achieving high levels of shoot regeneration from H. speciosa lateral buds.

Background Coffee production relies on plantations with varieties from Coffea arabica and Coffea canephora species. The first, the most representative in terms of coffee consumption, is mostly propagated by seeds, which leads to management problems regarding the plantations maintenance, harvest and processing of grains. Therefore, an efficient clonal propagation process is required for this species cultivation, which is possible by reaching a scalable and cost-effective somatic embryogenesis protocol. A key process on somatic embryogenesis induction is the auxin homeostasis performed by Gretchen Hagen 3 (GH3) proteins through amino acid conjugation. In this study, the GH3 family members were identified on C. canephora genome, and by performing analysis related to gene and protein structure and transcriptomic profile on embryogenic tissues, we point a GH3 gene as a potential regulator of auxin homeostasis during early somatic embryogenesis in C. arabica plants. Results We have searched within the published C. canephora genome and found 17 GH3 family members. We checked the conserved domains for GH3 proteins and clustered the members in three main groups according to phylogenetic relationships. We identified amino acids sets in four GH3 proteins that are related to acidic amino acid conjugation to auxin, and using a transcription factor (TF) network approach followed by RT-qPCR we analyzed their possible transcriptional regulators and expression profiles in cells with contrasting embryogenic potential in C. arabica. The CaGH3.15 expression pattern is the most correlated with embryogenic potential and with CaBBM, a C. arabica ortholog of a major somatic embryogenesis regulator. Conclusion Therefore, one out of the GH3 members may be influencing on coffee somatic embryogenesis by auxin conjugation with acidic amino acids, which leads to the phytohormone degradation. It is an indicative that this gene can serve as a molecular marker for coffee cells with embryogenic potential and needs to be further studied on how much determinant it is for this process. This work, together with future studies, can support the improvement of coffee clonal propagation through in vitro derived somatic embryos.

Cryopreservation is a process whereby biological structures are preserved in liquid nitrogen (−196 °C) without losing their viability. Many cryopreservation techniques use the Plant Vitrification Solution 2 (PVS2) for cryoprotection. This study will therefore evaluate the influence of different exposure times to the cryoprotectant PVS2 and discuss the importance of the mobilization of reserves and the antioxidant metabolism during the germination of cryopreserved Passiflora ligularis embryos. The composition of P. ligularis seeds was analytically determined. We tested the germination capacity and the Germination Speed Index (GSI) of embryos (that is, seeds without external tegument) which were exposed to different PVS2 exposure times (0, 30, 60 and 120 min) at 30 days after thawing. Proline content, hydrogen peroxide, activity of isocitrate lyase (ICL), malate synthase (MSy), lipid peroxidation and antioxidant enzyme activities (SOD, CAT, APX) were measured at 7, 14 and 21 days after cryopreservation. The germination from cryopreserved embryos was maximal (85%) after 60 min PVS2 exposure with a GSI of 0.6. At 60 min, the highest activity of the enzymes involved in the glyoxylate cycle, ICL and MSy were recorded. We hypothesize that a 60 min exposure to PVS2 accelerates the reserve mobilization which correlates positively with germination. Until 60 min, there was a positive correlation between the PVS2 exposure time and the proline content, as well as the activity of antioxidant enzymes (SOD, CAT, APX), and a negative correlation with the lipid peroxidation. This study enables us to optimize the long-term conservation of this species. In conclusion, fundamental research is necessary to optimize the cryopreservation procedure, and this study offers an effective and efficient workflow which can be extrapolated to other (oil-rich) species.

This study aimed to assess the influence of culture medium and cytokinin type on in vitro multiplication of an Eucalyptus grandis x E. urophylla hybrid clone via temporary immersion bioreactor (TIB®). JADS, modified MS at a (NO3ˉ):(NH +) ratio, and WPM were used as liquid media; and 6-benzilaminopurine (BAP), kinetin (KIN), and thidiazuron (TDZ) were the cytokinins used in the study. According to the results, explants under the influence of modified MS presented better results for all traits analyzed in this study. BAP was the most suitable plant growth regulator for axillary bud shoot proliferation. By the leaf histological analysis, most of the shoots grown in the presence of BAP either exhibited normal morphology or only a few hyperhydricity symptoms. All these results were obtained at 19 days after in vitro cultivation, a shorter period when compared with other works in the literature, showing the protocol efficiency in the multiplication of this hybrid clone using TIB®.

The physiological and molecular responses to water stress are mediated by a range of mechanisms, many of which involve abscisic acid (ABA)-dependent signaling pathways. In addition, plants contain drought response genes that can be induced by ABA-independent routes, mediated by secondary messengers such as Ca2+, or regulated by epigenetic modifications. The complex processes involved in the response to water stress can be investigated using molecular techniques to evaluate the expression patterns of genes of interest and to infer the behavior of different genotypes and species. In the present study, we first analyzed the stability of a set of reference genes for normalization of the gene expression with real-time quantitative polymerase chain reaction (RT-qPCR), since there were no results related to the genotype used in this study. We verified that although there were some variations between algorithms used, the three most stable reference genes were SAND, PP2A-3 and EF-1α. The expressions of genes encoding for proteins associated with drought-tolerance responses, namely 9-cis-epoxycarotenoid dioxygenase 3 (EgrNCED3), pyrabactin resistance 1 (EgrPYR1), dehydration-responsive element-binding 2.5 (EgrDREB2.5) transcription factors, calcium-dependent protein kinase 26 (EgrCDPK26), methyl transferase 1 (EgrMET1) and deficient in DNA methylation 1 (EgrDDM1) protein, were determined by RT-qPCR in leaf samples from drought sensitive (VM05) and drought tolerant (VM01) clones of the hybrid Eucalyptus camaldulensis x Eucalyptus urophylla grown under water stress and irrigation conditions. When the two clones were maintained under conditions of water deficiency, VM01 exhibited higher expression levels of EgrNCED3 and EgrPYR1 genes than VM05 at all sampling times, implying that ABA biosynthesis and subsequent induction of the ABA-dependent cascade mediated by the PYR1-ABA receptor complex were enhanced in the tolerant clone. Under water-stress conditions, this clone also presented increased expression of the EgrDREB2.5 gene, representative of an ABA-independent cascade, and of the EgrCPK26 gene, related to stomatal opening and closure. On the other hand, the expression levels of EgrMET1 and EgrDDM1 genes in the sensitive clone were higher than in the tolerant clone under all conditions, showing a putative impact of epigenetic modifications on tolerance to water deficiency. The results obtained indicate that the superior ability of the VM01-tolerant clone to perceive water deficiency and activate drought-resistance genes is associated with the high expression levels of EgrNCED3, EgrPYR1 and EgrDREB2.5 under water-stress conditions. These findings will facilitate future research on the functional characterization of stress-related response genes, the identification of molecular markers, the evaluation of drought tolerance and genetic transformation in tree species.

Hancornia speciosa Gomes is a fruit species belonging to the Apocynaceae family and holds social, cultural and economic potential mostly due to its use in composition of many food industry products and the consumption its fruits in natura . Several aspects regarding their propagation need further studies, since the species is undergoing a continuous process of domestication. The objective was to obtain an in vitro protocol for indirect organogenesis and rooting with subsequent acclimatization of H. speciosa plants. To obtain indirect organogenesis, internodal segments were inoculated in WPM culture medium gelled with 7 g L -1 agar, added with 30 g L -1 sucrose, 0.4 g L -1 PVP, and supplied with different concentrations of 2,4-D (0.0; 2.46; 7.38; 12.30; and 17.22 µM), BAP (0.0; 4.92; 9.84; 14.76; and 19.68 µM), and TDZ (0.0; 4.92; 9.84; 14.76; and 19.68 µM). For the in vitro rooting, shootings with approximately 6.0 cm diameter length kept for 15 days in WPM medium with no plant growth regulator and, afterwards, subjected to treatments with different auxins (Control; 9.84 µM IBA; 9.84 µM NAA; and 9.84 µM IAA) as well as the combination among them, in order to verify their effects on percentage of rooting (%), number of roots, and average length of the largest root (cm). The formation of calluses was observed in all explants subjected to the concentrations of the regulators tested. The highest shooting regeneration occurred with 7.38 µM 2,4-D (73%). The highest percentage of shoot rooting (80%) and roots with the largest length (1.3 cm) were found in the culture medium with the combination of 4.92 µM NAA and 4.92 µM IBA. The in vitro regeneration of H. speciosa is feasible. The acclimatization of rooted shoots in Trospstrato® was accomplished with successful and 100% survival of plant material was observed during this stage.

This work evaluated the influence of seal type and substrate type on pre-acclimatization, and symptomatological aspects relating to acclimatization of ipê-roxo. For the pre-acclimatization, ipê-roxo plants obtained through an in vitro zygotic embryo culture were aseptically inoculated in test tubes with different types of seal (cotton plug, plastic cap + parafilm and plastic lid) and different substrates (agar, vermiculite and Plantmax®) supplemented with a WPM culture medium with 1 gL-1 of activated charcoal, 10 mgL-1 of citric acid and 30 gL-1 of sucrose added. For the acclimatization, in vitro ipê-roxo plants were transplanted into 56-cm3 plastic tubes containing vermiculite and Plantmax® at a 2:1 ratio which had been previously autoclaved at 121oC and 1 atm for 20 minutes. Once transplanted, the plants were irrigated with different concentrations of the MS medium (25%, 50%, 75%, 100% and 150%), using distilled water as control. After 30 days, the effects of seal type and substrate type on pre-acclimatization were examined. Cotton can be recommended where Plantmax® or vermiculite is used as substrate. In the acclimatization it was noted that where plants were irrigated with distilled water and concentrations < 50% they showed symptoms of nutritional deficiency. Similar symptoms were observed when using MS medium at a concentration equal to or greater than 100%. The 75% MS concentration was found to be the most effective at maintaining the nutritional vigor of ipê-roxo seedlings in the process of acclimatization.

Este trabalho teve por objetivo avaliar a influência do tipo de vedação e substrato na pré-aclimatização e os aspectos sintomatológicos na aclimatização de Ipê-roxo. Para pré-aclimatização, plantas de Ipê-roxo obtidas in vitro por meio de cultura de embriões zigóticos foram inoculadas assepticamente em tubos de ensaio contendo diferentes tipos de vedação (algodão, tampa plástica + parafilme e tampa plástica) e diferentes substratos (ágar, vermiculita e Plantmax®) suplementados com meio de cultivo WPM acrescidos de 1 gL-1 de carvão ativado, 10 mgL-1 de ácido cítrico e 30 gL-1 de sacarose. Para a aclimatização, plantas de Ipê-roxo obtidas in vitro foram transplantadas para tubetes de 56 cm³; contendo vermiculita e Plantmax®na proporção 2:1, previamente autoclavado a 121ºC e 1 atm, por 20 minutos. Após o transplantio, as plantas foram irrigadas com diferentes concentrações do meio MS (25%, 50%, 75%, 100% e 150%), sendo utilizada como controle água destilada. Após 30 dias, foram verificados efeitos do tipo de vedação e substrato na pré-aclimatização. O algodão pode ser recomendado quando se utiliza Plantmax®ou vermiculita como substrato. Na aclimatização, verificou-se que as plantas quando irrigadas com água destilada e concentrações < 50% apresentaram sintomas de deficiência nutricional. Foram observados sintomas semelhantes com a utilização de MS em concentração igual ou superior a 100%. A concentração 75% do meio MS foi a mais eficiente na manutenção do vigor nutricional de mudas de Ipê-roxo em processo de aclimatização. This work evaluated the influence of seal type and substrate type on pre-acclimatization, and symptomatological aspects relating to acclimatization of ipê-roxo. For the pre-acclimatization, ipê-roxo plants obtained through an in vitro zygotic embryo culture were aseptically inoculated in test tubes with different types of seal (cotton plug, plastic cap + parafilm and plastic lid) and different substrates (agar, vermiculite and Plantmax®) supplemented with a WPM culture medium with 1 gL-1 of activated charcoal, 10 mgL-1 of citric acid and 30 gL-1 of sucrose added. For the acclimatization, in vitro ipê-roxo plants were transplanted into 56-cm³plastic tubes containing vermiculite and Plantmax®at a 2:1 ratio which had been previously autoclaved at 121ºC and 1 atm for 20 minutes. Once transplanted, the plants were irrigated with different concentrations of the MS medium (25%, 50%, 75%, 100% and 150%), using distilled water as control. After 30 days, the effects of seal type and substrate type on pre-acclimatization were examined. Cotton can be recommended where Plantmax®or vermiculite is used as substrate. In the acclimatization it was noted that where plants were irrigated with distilled water and concentrations < 50% they showed symptoms of nutritional deficiency. Similar symptoms were observed when using MS medium at a concentration equal to or greater than 100%. The 75% MS concentration was found to be the most effective at maintaining the nutritional vigor of ipê-roxo seedlings in the process of acclimatization.