The SAUR gene family in coffee: genome-wide identification and gene expression analysis during somatic embryogenesis.

BACKGROUND: Small auxin-up RNA (SAUR) genes form a wide family supposedly involved in different physiological and developmental processes in plants such as leaf senescence, auxin signaling and transport, hypocotyl development and tolerance to abiotic stresses. The transcription of SAUR genes is quickly induced by auxins, a group of phytohormones of major importance on embryo development. To better understand the distribution and expression profile of such still not explored family in Coffea sp., especially during the development of somatic embryogenesis (SE), SAUR members were characterized in silico using the available Coffea canephora genome data and analyzed for gene expression by RT-qPCR in C. arabica embryogenic samples. METHODS AND RESULTS: Over C. canephora genome 31 CcSAURs were distributed by 11 chromosomes. Out of these 31 gene members, 5 SAURs were selected for gene expression analysis in C. arabica embryogenic materials. CaSAUR12 and CaSAUR18 were the members highly expressed through almost all plant materials. The other genes had more expression in at least one of the developing embryo stages or plantlets. The CaSAUR12 was the only member to exhibit an increased expression in both non-embryogenic calli and the developing embryo stages. CONCLUSION: The identification of SAUR family on C. canephora genome followed by the analysis of gene expression profile across coffee somatic embryogenesis process on C. arabica represents a further additional step towards a better comprehension of molecular components acting on SE. Along with new research about this gene family such knowledge may support studies about clonal propagation methods via somatic embryogenesis to help the scientific community towards improvements into coffee crop.

Molecular characterization of Bacillus thuringiensis strains to control Spodoptera eridania (Cramer) (Lepidoptera: Noctuidae) population

Abstract The main objective of this study was to characterize the toxicity and genetic divergence of 18 Bacillus thuringiensis strains in the biological control of Spodoptera eridania. Bacterial suspensions were added to the S. eridania diet. Half of the selected B. thuringiensis strains caused high mortality seven days after infection. The genetic divergence of B. thuringiensis strains was assessed based on Enterobacterial Repetitive Intergenic Consensus (ERIC) and Repetitive Extragenic Palindromic (REP) sequences, and five phylogenetic groups were formed. Despite their genetic diversity B. thuringiensis strains did not show any correlation between the collection sites and toxicity to larvae. Some B. thuringiensis strains are highly toxic to S. eridania, thus highlighting the potential of their endotoxins as biopesticides.

Genome-wide analysis, transcription factor network approach and gene expression profile of GH3 genes over early somatic embryogenesis in Coffea spp.

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.

The cryoprotectant PVS2 plays a crucial role in germinating Passiflora ligularis embryos after cryopreservation by influencing the mobilization of lipids and the antioxidant metabolism.

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.

Gene expression in two contrasting hybrid clones of Eucalyptus camaldulensis x Eucalyptus urophylla grown under water deficit conditions.

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.

Behavior of lateral buds of Hancornia speciosa after cryopreservation by encapsulation-vitrification / Comportamento de gemas laterais de Hancornia speciosa após criopreservação por encapsulamento-vitrificação

Hancornia speciosa is a fruitful species from Cerrado biome with high economic potential. However, the intense and disordered extractivism have caused a reduction of its population in its endemic area. In addition, seed recalcitrance negatively affects the conventional conservation of the species. Aiming to find alternatives that enable the long-term conservation of this species, the study's objective was to assess the behavior of lateral bud's regrowth after cryopreservation procedures by encapsulation-vitrification technique. Sodium alginate capsules containing lateral buds were pre-cultured in liquid WPM supplemented with 1.0 M glycerol, and subsequently exposed to different concentrations of sucrose (0.3; 0.75 and 1.0 M) for 24 or 48 hours. The capsules were subjected to dehydration in silica gel or airflow hood for 0, 1, 2 and 3 hours before different incubation times in PVS2 (0, 15, 30, 60 and 120 minutes) at 0°C. A high regeneration percentage of lateral buds was observed after cryopreservation of capsules treated with 0.75 M sucrose plus 1.0 M glycerol (24 hours), associated with dehydration in an airflow hood (1 hour) and immersion in PVS2 (15 minutes). Encapsulation-vitrification allowed the long-term conservation, and provided high plant material survival rates after cryopreservation of Hancornia speciosa sensitive explants.

Hancornia speciosa é uma frutífera do Cerrado com elevado potencial econômico. Entretanto, o extrativismo desordenado causou a redução populacional em sua área endêmica. Além disso, a recalcitrância da semente afeta negativamente sua conservação convencional. Buscando alternativas de conservação para essa espécie, objetivou-se avaliar a regeneração das gemas laterais após a técnica de encapsulamento-vitrificação. Cápsulas de alginato de sódio contendo gemas laterais foram pré-cultivadas em meio WPM acrescido de 1,0 M de glicerol e, posteriormente, imersas em diferentes concentrações de sacarose (0,3; 0,75 e 1,0 M) por 24 ou 48 horas. As cápsulas foram submetidas à desidratação em sílica gel ou em fluxo laminar por 0, 1, 2 e 3 horas antes de sua incubação em diferentes tempos de PVS2 (0, 15, 30, 60 e 120 minutos). Elevada porcentagem de regeneração de gemas laterais foi observada após a criopreservação de cápsulas tratadas com 0,75 M de sacarose + 1,0 M de glicerol por 24 horas, associado com a desidratação em fluxo laminar (1 hora) e imersão em PVS2 (15 minutos). O encapsulamento-vitrificação é eficiente para a conservação de longo prazo e permite a obtençao de altas taxas de sobrevivência após a criopreservação de explantes sensíveis (gemas laterais) de Hancornia speciosa.

Indirect in vitro organogenesis of Hancornia speciosa Gomes / Organogênese indireta in vitro de Hancornia speciosa Gomes

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.

Hancornia speciosa Gomes é uma frutífera pertencente à família Apocynaceae e possui grande potencial social, cultural e econômico, principalmente devido à utilização de seus frutos na composição de diversos produtos do setor alimentício e para consumo in natura. Vários aspectos de sua propagação necessitam de maiores estudos, uma vez que a espécie passa por um contínuo processo de domesticação. Objetivou-se obter um protocolo de organogênese indireta e de enraizamento in vitro, com posterior aclimatização das plantas de H. speciosa. Para a obtenção de organogênese indireta, segmentos internodais foram inoculados em meio de cultivo WPM gelificado com ágar 7 g L-1, acrescido de 30 g L-1 de sacarose, 0,4 g L-1 de PVP e suplementado com diferentes concentrações de 2,4-D (0,0; 2,46; 7,38; 12,30 e 17,22 µM), BAP (0,0; 4,92; 9,84; 14,76 e 19,68 µM) e TDZ (0,0; 4,92; 9,84; 14,76 e 19,68 µM). Para o enraizamento in vitro, foram utilizadas brotações com aproximadamente 6,0 cm de comprimento, mantidas por 15 dias em meio WPM isento de fitorreguladores e, em seguida, submetidas a tratamentos com diferentes auxinas (Controle; 9,84 µM de AIB; 9,84 µM de ANA e 9,84 µM de AIA), bem como as combinações entre estes reguladores (4,92 µM AIA + 4,92 µM AIB; 4,92 µM ANA + 4,92 µM AIB; 4,92 µM ANA + 4,92 µM AIA). Foi observada formação de calos em todos os explantes submetidos às concentrações dos reguladores testados. A maior regeneração de brotação ocorreu com 7,38 µM de 2,4-D (73%). Maior porcentagem de enraizamento das brotações (80%) e raízes com maior comprimento (1,3 cm) foi verificada em meio de cultivo contendo a combinação de 4,92 µM de ANA e 4,92 µM de AIB. É possível a regeneração in vitro de H. speciosa. A aclimatização foi realizada com sucesso, com 100% de sobrevivência das plantas.