Influence of Silver Nitrate on Somatic Embryogenesis Induction in Arabica Coffee (Coffea arabica L.).

Plant somatic embryogenesis (SE) has been defined as the formation of embryos from a single or group of haploid or somatic cells [1, 2]. Low frequency (LFSE) and high frequency somatic embryogenesis (HFSE) have been described. In the first type, somatic embryos are induced directly from pro-embryogenic cells of explants, while in the second, they originate from embryogenic callus [1]. It has been suggested that in LFSE the origin of somatic embryos is unicellular, whereas in HFSE has been described as unicellular or multicellular [3]. SE is a powerful biotechnological tool used to propagate elite plants or to conserve important genotypes [4]. Moreover, SE offers an efficient in vitro regeneration approach as a fundamental step in plant genetic improvement for studying basic aspects of ontogenesis of somatic embryos [5]. In Coffea spp., the first studies of SE have been reported at the beginning of 1970 [6]. Since then, a large quantity of LFSE and HFSE protocols have been optimized demonstrating that coffee is not a recalcitrant species for SE [4]. In the LFSE the somatic embryos are obtained faster (approximately 70 days) using only one medium meanwhile in HFSE several media are used and somatic embryo formation takes 9-10 months [4]. Although, in LFSE small number of somatic embryos are obtained (around 10 per explant) compared to hundreds of somatic embryos obtained per gram of embryogenic calli [4], the unicellular origin of somatic embryos in LFSE represents an advantage for the chemical and physical mutagenesis, genetic transformation and genetic editing, since prevents or reduces the appearance of chimeras [7]. In C. arabica and C. canephora many factors (such as genotype, explant type, the physiological state, age and growth conditions of the donor plants, the season of collection, nutrient composition of the medium, the volume of dissolved CO2 or O2 in the culture flask, and plant growth regulators) that affect LFSE induction have been studied [3, 8, 9, 10, 11, 12, 13]. However, few studies reported the effect of silver nitrate on LFSE using leaf explants of C. arabica L. and to the best of our knowledge it has not been analyzed using Caturra and Catuaí, which are two economic important producer cultivars in Costa Rica. Since SE is genotype dependent, the culture medium need to be modified for the different genotypes [7].Therefore, the objective of this study was to determine the influence of the benzyladenine (BAP), indole-3-acetic acid (IAA), and silver nitrate (AgNO3) on low frequency somatic embryogenesis using leaf explants of Coffea arabica L. cultivars Caturra and Catuaí

SNP markers found in non-coding regions can distinguish among low-variant genotypes of arabica and other coffee species.

Development of efficient and scalable methods for molecular identification of Coffea spp. are necessary to accelerate studies related to the characterization of germplasm for both conservation or breeding purposes, and the validation of coffee germplasm. The low genetic diversity of coffee hinders the establishment of protocols that facilitate the molecular characterization of a given genotype. In this study, nucleotide variability was analyzed at 22 loci in the genome of 19 coffee accessions using de novo primer sets and high-resolution melting (HRM). Single nucleotide polymorphisms (SNPs) variants were studied in coding regions of genes implicated in sucrose accumulation in the seed, Sucrose synthase 2 (SUS2), Ent-kaurene oxidase 1 (CaKO1), and Caffeoyl-coenzyme A 3-O-methyltransferase (CcOAOMT). The non-coding Internal transcribed spacer 2 (ITS2) region was also studied. Variability was shown at 103 positions both at the interspecies level (15 loci) and among varieties of Coffea arabica L. (4 loci). The HMR technique for identification of variants in genes CaKO1, SUS2, CcoAOMT, as well as in the ITS2 region proved to be a robust technique for germplasm characterization. More important this technique can be used for fingerprinting and traceability of coffee grain exports which is an increasing market-consumer demand

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

A comparative analysis of mixed-integer linear formulations for the multi-family capacitated lot-sizing problem

<p>Despite having a very broad spectrum of applicability in practice, the multi-family capacitated lot-sizing problem (MFCLSP) has been scarcely studied. The MFCLSP is an extension of the capacitated lot-sizing problem with setup times (CLST) in which items are organized into families based on similar setup structures. In this paper, we propose three formulations for the MFCLSP (MF-TRAD, MF-ARBNET, and MF-EXREQ), and develop a comprehensive comparative analysis to evaluate their performance using a generic solver (CPLEX). Solving large-scale problems to optimality has been shown to consume a great amount of computational time, which is very impractical for real-life applications. Because of that, this study focuses on analyzing the performance of these formulations in a limited, and reasonable, amount of time. The results show the MF-EXREQ model outperforms the other two models in both the time to the first feasible solution and the quality of the solutions generated throughout the solving process.</p

NTH2 1271_1272delTA Gene Disruption Results in Salt Tolerance in Saccharomyces cerevisiae

Trehalose is a common energy reservoir, and its accumulation results in osmotic protection. This sugar can accumulate through its synthesis or slow degradation of the reservoir by trehalase enzymes. Saccharomyces cerevisiae contains two neutral trehalases, NTH1 and NTH2, responsible for 75% and 25% of the enzymatic metabolism. We were interested in the loss-of-function of both enzymes with CRISPR/Cas9. The later NTH2 was of great importance since it is responsible for minor metabolic degradation of this sugar. It was believed that losing its functionality results in limited osmotic protection. We constructed an osmotolerant superior yeast capable of growing in 0.85 M NaCl after independent nth21271_1272delTA mutation by CRISPR/Cas9 technology, compared with nth1&nbsp;893_894insT and wild type. We suggest that this yeast model could give clues to breeding commercial yeast resulting in non-GMO salinity-tolerant strains

<i>NTH2</i> 1271_1272delTA Gene Disruption Results in Salt Tolerance in <i>Saccharomyces cerevisiae</i>

Trehalose is a common energy reservoir, and its accumulation results in osmotic protection. This sugar can accumulate through its synthesis or slow degradation of the reservoir by trehalase enzymes. Saccharomyces cerevisiae contains two neutral trehalases, NTH1 and NTH2, responsible for 75% and 25% of the enzymatic metabolism. We were interested in the loss-of-function of both enzymes with CRISPR/Cas9. The later NTH2 was of great importance since it is responsible for minor metabolic degradation of this sugar. It was believed that losing its functionality results in limited osmotic protection. We constructed an osmotolerant superior yeast capable of growing in 0.85 M NaCl after independent nth21271_1272delTA mutation by CRISPR/Cas9 technology, compared with nth1 893_894insT and wild type. We suggest that this yeast model could give clues to breeding commercial yeast resulting in non-GMO salinity-tolerant strains

Responses of Arabica coffee (Coffea arabica L. var. Catuaí) cell suspensions to chemically induced mutagenesis and salinity stress under in vitro culture conditions

Crop improvement of Coffea arabica L. (coffee) via mutagenesis could accelerate breeding programs; thus, the present study aimed to develop an in vitro protocol using the chemical mutagens sodium azide (NaN3) and ethyl methanesulfonate (EMS) on embryogenic cell suspensions of Arabica coffee variety Catuaí and, subsequently, to evaluate the responses of the resulting mutagenized tissues to salinity stress. Embryogenic suspension cultures were incubated with 0.0, 2.5, 5.0, or 10.0 mM NaN3 or 0.0, 185.2, 370.5, or 741.0 mM EMS. As the concentration of NaN3 or EMS increased, the survival of embryogenic suspension cultures decreased compared to controls. The median lethal dose (LD50) for NaN3 was 5 mM for 15 min and for EMS it was 185.2 mM for 120 min. Embryogenic suspension cultures treated with NaN3 or EMS were cultured on selective medium supplemented with 0, 50, 100, 150, 250, or 300 mM NaCl showed that 50 mM NaCl could be used as selection pressure. Plantlet growth and total amino acid content were affected by NaCl stress; some mutants had longer shoots and higher amino acid content than controls. Random amplified polymorphic DNA (RAPD) analysis was performed to determine whether the NaN3 or EMS treatments could induce genetic variability and resulted in identifiable polymorphic markers. A total of 18 10-mer primers were used to amplify genomic DNA of putative mutant and non-mutant arabica coffee embryogenic cultures and produced 50 scorable bands, of which 22% were polymorphic.Universidad de Costa Rica/[111-B5-140]/UCR/Costa RicaUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biologí