Advances in the Micropropagation and Genetic Transformation of Agave Species.

The Agave genus is composed of approximately 210 species distributed from south United States to Colombia and Venezuela. Numerous Agave species have been used for the preparation of alcoholic beverages and have attracted interest in the pharmaceutical and food industry. Despite their economic importance, there are few initiatives for the improvement and selection of characteristics of interest. This is mainly due to its morphology, long lifecycles, and monocarpic nature. Micropropagation is a feasible alternative to the improvement of Agave species. It has been used for multiple purposes, including massive propagation, induction of somaclonal variation to enhance agronomic characteristics of interest, maintenance of specific genotypes, and genetic transformation using molecular techniques. In this report, we summarize the most outstanding findings regarding the micropropagation of Agave species mediated by multiple regeneration responses. We also describe the media and growth regulators for each of the previously described methods. In addition, we discuss how micropropagation has allowed the development of transformation protocols. Exploitation of this technology may be a feasible strategy to introduce genes and improve certain traits. Genetic transformation also offers an opportunity for studying molecular mechanisms. This represents advantages for optimizing production in the field and for implementing breeding programs.

Trends on Microalgae-Fungi Consortia Research: An Alternative for Biofuel Production?

The utilization of microalgae and fungi on an industrial scale is a challenge for researchers. Based on the question "how fungi have contributed to microalgae research?," we verified the scientific trends on microalgae-fungi consortia focused on biofuels production by searching for articles on the Web of Science and Scopus databases through the terms "microalgae*" or phytoplankton and "fung*." We found 1,452 articles published between 1950 and 2020; since 2006, the publication numbers have increased rapidly. The articles were published in 12 languages, but most were written in English (96.3%). Among 72 countries, China (360 articles), USA (344), and Germany (155) led the publication rank. Among the 10 most-prolific authors, 8 were Chinese, like 5 of the most-productive institutions, whereas the National Cheng Kung University was on the top of the list. The sources that published the most on the subject were: Bioresource Technology (96), PLoS ONE (28), and Science of the Total Environment (26). The keyword analysis emphasized the magnitude of applications in microalgae-fungi consortia research. Confirming this research question, biofuels appeared as a research trend, especially biodiesel, biogas, and related terms like lipid, lipid accumulation, anaerobic digestion, and biogas upgrading. For 70 years, articles have been published, where China and the United States seem to dominate the research scenario, and biodiesel is the main biofuel derived from this consortium. However, microalgae-based biofuel biorefinery is still a bottleneck on an industrial scale. Recent environmental challenges, such as greenhouse gas mitigation, can be a promising field for that microalgae-fungi application.

A new circular economy approach for integrated production of tomatoes and mushrooms.

Spent mushroom Substrate is the by-product generated at the end of the mushroom growing cycle. It can be used in agriculture for different purposes, including seedling production, soil conditioning or application as an organic fertilizer. Tomato is one of the world́s most important crops, requiring considerable care, in terms of both nutrition and disease control. The objective of this study was to investigate the viability of spent mushroom substrate as a nutrient source for tomato seedlings and develop an integrated tomato and mushroom co-production system. For seedling production, different compositions were evaluated with spent mushroom substrate from Pleurotus ostreatus or substrate colonized with Agaricus bisporus. The parameters evaluated comprised germination rate, seedling quality and physicochemical analysis. A tomato and mushroom integrated production system was developed using a 40-liter pot divided into upper (spent mushroom substrate and soil), middle (spent mushroom substrate from P. ostreatus) and lower (gravel) layers. For seedlings production, plants treated with the substrate colonized with A. bisporus presented a superior root length (10.1 cm) and aerial part length (6.6 cm). Co-production of tomato and mushrooms was also shown to be viable. In this co-cultivation system between tomato and mushroom, the treatment with the substrate colonized with A. bisporus differed from others, with this treatment presenting high yields of tomato (2.35 kg/plant pot) and mushrooms (1.33 kg/plant pot) within the same bucket. With this co-production system, the tomato production time was reduced by 60 days and prolonged continuous mushroom production by 120 days. These findings show a sustainable approach to manage different agroindustrial residues, encouraging the use of these residues for olericulture and fungiculture production.

Insights from Leishmania (Viannia) guyanensis in vitro behavior and intercellular communication.

BACKGROUND: Pentavalent antimonial-based chemotherapy is the first-line approach for leishmaniasis treatment and disease control. Nevertheless antimony-resistant parasites have been reported in some endemic regions. Treatment refractoriness is complex and is associated with patient- and parasite-related variables. Although amastigotes are the parasite stage in the vertebrate host and, thus, exposed to the drug, the stress caused by trivalent antimony in promastigotes has been shown to promote significant modification in expression of several genes involved in various biological processes, which will ultimately affect parasite behavior. Leishmania (Viannia) guyanensis is one of the main etiological agents in the Amazon Basin region, with a high relapse rate (approximately 25%). METHODS: Herein, we conducted several in vitro analyses with L. (V.) guyanensis strains derived from cured and refractory patients after treatment with standardized antimonial therapeutic schemes, in addition to a drug-resistant in vitro-selected strain. Drug sensitivity assessed through Sb(III) half-maximal inhibitory concentration (IC50) assays, growth patterns (with and without drug pressure) and metacyclic-like percentages were determined for all strains and compared to treatment outcomes. Finally, co-cultivation without intercellular contact was followed by parasitic density and Sb(III) IC50 measurements. RESULTS: Poor treatment response was correlated with increased Sb(III) IC50 values. The decrease in drug sensitivity was associated with a reduced cell replication rate, increased in vitro growth ability, and higher metacyclic-like proportion. Additionally, in vitro co-cultivation assays demonstrated that intercellular communication enabled lower drug sensitivity and enhanced in vitro growth ability, regardless of direct cell contact. CONCLUSIONS: Data concerning drug sensitivity in the Viannia subgenus are emerging, and L. (V.) guyanensis plays a pivotal epidemiological role in Latin America. Therefore, investigating the parasitic features potentially related to relapses is urgent. Altogether, the data presented here indicate that all tested strains of L. (V.) guyanensis displayed an association between treatment outcome and in vitro parameters, especially the drug sensitivity. Remarkably, sharing enhanced growth ability and decreased drug sensitivity, without intercellular communication, were demonstrated.

Interaction of Aspergillus flavus and A. parasiticus with Salmonella spp. isolated from peanuts.

Although Aspergillus flavus and Aspergillus parasiticus are the main microorganisms of concern in peanuts, due to aflatoxin contamination, several Salmonella outbreaks from this product have been reported over the last ten decades. Thus, it is important to understand the relationship between microorganisms to predict, manage and estimate the diversity in the peanut supply chain. The purpose of this study was to evaluate aflatoxin production during the co-cultivation of Aspergillus section Flavi and Salmonella both isolated from peanuts. Three strains of A. section Flavi: A. flavus producing aflatoxin B, A. flavus non-producing aflatoxin and A. parasiticus producing aflatoxin B and G were co-cultivated with seven serotypes of Salmonella of which six were isolated from the peanut supply chain (S. Muenster, S. Miami, S. Glostrup, S. Javiana, S. Oranienburg and S. Yoruba) and one was S. Typhimurium ATCC 14028. First of all, each Salmonella strain was inoculated by pour plate (ca. 5 log cfu/mL) in PDA (potato dextrose agar). Then, each pre-cultured fungus was inoculated in the center of the petri dish. The plates were incubated at 30 °C and the fungal colony diameter was measured once a day for 7 days. As a control each Aspergillus strain was cultivated in the absence of Salmonella culture. All three strains of Aspergillus with absence of Salmonella (control) reached the maximum colony diameter and their growth rate was influenced when co-cultivated (p < 0.05) with all Salmonella serotypes tested. The maximum inhibition in the colony diameter was 20% for A. flavus aflatoxin B producer and A. parasiticus, and 18% for A. flavus non- aflatoxin producer when cultivated with Salmonella. However, no significant difference (p < 0.05) in reduction of colony diameter was observed among the Salmonella serotypes. Aflatoxin production was determined previously, by using the agar plug technique on thin layer chromatography (TLC). The production of aflatoxin G by A. parasiticus in co-cultivation with Salmonella was not observed. On the other hand, A. flavus preserved their characteristics of aflatoxin B production. The quantification of aflatoxin reduction by Salmonella interaction was evaluated using HPLC method. There was a maximum reduction of aflatoxin production of 88.7% and 72.9% in A. flavus and A. parasiticus, respectively, when cultivated with Salmonella. These results indicate that some serotypes of Salmonella may interfere with aflatoxin production and fungal growth of A. flavus and A. parasiticus in the peanut supply chain.

Improved protease activity of Pixian broad bean paste with cocultivation of Aspergillus oryzae QM-6 and Aspergillus niger QH-3

BACKGROUND: The preparation of broad bean koji is a key process in the production of Pixian broad bean paste (PBP). Protease is essential for the degradation of proteins during PBP fermentation. To obtain broad bean koji with high protease activity using the cocultivated strains of Aspergillus oryzae QM-6 (A. oryzae QM-6) and Aspergillus niger QH-3 (A. niger QH-3), the optimization of acid and neutral protease activities was carried out using Box­Behnken design with response surface methodology (RSM). RESULTS: The optimum conditions were found to be as follows: inoculation proportion (X1), 3:1 (A. oryzae QM-6: A. niger QH-3, w/w); culture temperature (X2), 33°C; inoculum size (X3), 0.5% (w/w); incubation time (X4), 5 d. The acid and neutral protease activities were 605.2 ± 12.4 U/g and 1582.9 ± 23.7 U/g, respectively, which were in good agreement with the predicted values. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles revealed that the broad bean koji extracellular proteins in the case of cocultivation were richer compared to those in the case of A. oryzae QM-6 or A. niger QH-3 strain only. In addition, the free amino acids (FAAs) in the fermentation product were 55% higher in the cocultivation process than in that involving only A. oryzae QM-6, further confirming the diversity of proteases in the fermentation products. CONCLUSIONS: The optimal conditions of koji-making in PBP were obtained using RSM. The cocultivation of A. oryzae and A. niger increases the overall enzyme activities in the culture medium and the FAAs content, which would thus have potential application in the PBP industry.

Phytoremediation of barium-affected flooded soils using single and intercropping cultivation of aquatic macrophytes.

Aquatic macrophytes are potentially useful for phytoremediation on flooded areas. A field study in Brazil was conducted to evaluate Eleocharis acutangula (E), Cyperus papyrus (C) and Typha domingensis (T) in monocropping and intercropping, aiming to phytoremediate barium-polluted flooded soils. The treatments were: monocroppings (E, C and T); double intercroppings (EC, ET and CT); and triple intercropping (ECT). The 180-d field trial was performed in a flooded area with high barium content, with a randomized complete block design and three replicates. Plant stand size, biomass yield, and Ba concentration aboveground/Ba concentration in roots (translocation factor - TF) as well as Ba mass aboveground/Ba mass in roots (mass translocation factor - mTF) were determined. Most of the treatments did not differ on dry biomass, except for EC, which showed the lowest yield. Consistently with its biology, E. acutangula in monocropping showed the largest plant stand. Otherwise, intercroppings with T. domingensis achieved the highest amounts of barium absorbed from the soil and transferred most of the barium content from belowground to aboveground (mTF > 1.0), especially ET, which showed the highest mTF among the intercroppings (2.03). Remarkably, TF values did not reflect such phytoextraction ability for CT and ECT. Thus, mTF was more appropriate than TF to assess phytoextraction capacity. Furthermore, it was demonstrated that intercropping can increase barium uptake from flooded soils. Particularly, the intercropping ET constituted the most cost-effective treatment, with the cyperaceous species providing high plant coverage while T. domingensis facilitated barium removal by translocating it to the aboveground biomass.

In vitro establishment of Comanthera curralensis, sempre viva native of Chapada Diamantina - Bahia / Estabelecimento in vitro de Comanthera curralensis, sempre viva, nativa da Chapada Diamantina - Bahia

The goal of the present study was to evaluate the germination, initial growth, and in vitro co-cultivation of Comanthera curralensis Moldenke, a "sempre viva" native of the Chapada Diamantina state of Bahia. Full strength (MS) and half-strength MS (MS1/2) growth media supplemented with two different sucrose concentrations (15 and 30g L-1) were tested for germination and initial plant growth. Three different plant densities were tested by in vitro culture (8, 10 and 12 plants per container). MS1/2 medium with 15g L-1 sucrose resulted in a higher percentage of germination and plant growth for the in vitro establishment of C. curralensis. The use of 12 plants per container is indicated for cost reduction in C. curralensis in vitro production.(AU)

Este trabalho teve como objetivo avaliar a germinação, o crescimento inicial e o co-cultivo in vitro de Comanthera curralensis Moldenke, uma "sempre viva" nativa da Chapada Diamantina-BA. Para germinação e crescimento inicial, foram testados os meios de cultura MS completo e MS1/2 suplementados com duas concentrações de sacarose (15 e 30gL-1); no cultivo in vitro, foram testadas três quantidades de plantas por recipiente (8,10 e 12). A utilização do meio MS1/2 com 15gL-1 de sacarose proporcionou maiores porcentagem de germinação crescimento das plantas no estabelecimento in vitro de C. curralensis , e o uso de 12 plantas por recipiente é indicado para a redução de custos na produção in vitro da espécie.(AU)

In vitro establishment of Comanthera curralensis , "sempre viva" native of Chapada Diamantina - Bahia / Estabelecimento in vitro de Comanthera curralensis, "sempre viva", nativa da Chapada Diamantina - Bahia

ABSTRACT: The goal of the present study was to evaluate the germination, initial growth, and in vitro co-cultivation of Comanthera curralensis Moldenke, a "sempre viva" native of the Chapada Diamantina state of Bahia. Full strength (MS) and half-strength MS (MS1/2) growth media supplemented with two different sucrose concentrations (15 and 30g L-1) were tested for germination and initial plant growth. Three different plant densities were tested by in vitro culture (8, 10 and 12 plants per container). MS1/2 medium with 15g L-1 sucrose resulted in a higher percentage of germination and plant growth for the in vitro establishment of C. curralensis. The use of 12 plants per container is indicated for cost reduction in C. curralensis in vitro production.

RESUMO: Este trabalho teve como objetivo avaliar a germinação, o crescimento inicial e o co-cultivo in vitro de Comanthera curralensis Moldenke, uma "sempre viva" nativa da Chapada Diamantina-BA. Para germinação e crescimento inicial, foram testados os meios de cultura MS completo e MS1/2 suplementados com duas concentrações de sacarose (15 e 30gL-1); no cultivo in vitro, foram testadas três quantidades de plantas por recipiente (8,10 e 12). A utilização do meio MS1/2 com 15gL-1 de sacarose proporcionou maiores porcentagem de germinação crescimento das plantas no estabelecimento in vitro de C. curralensis , e o uso de 12 plantas por recipiente é indicado para a redução de custos na produção in vitro da espécie.

Co-cultivation of Aspergillus nidulans Recombinant Strains Produces an Enzymatic Cocktail as Alternative to Alkaline Sugarcane Bagasse Pretreatment.

Plant materials represent a strategic energy source because they can give rise to sustainable biofuels through the fermentation of their carbohydrates. A clear example of a plant-derived biofuel resource is the sugar cane bagasse exhibiting 60-80% of fermentable sugars in its composition. However, the current methods of plant bioconversion employ severe and harmful chemical/physical pretreatments raising biofuel cost production and environmental degradation. Replacing these methods with co-cultivated enzymatic cocktails is an alternative. Here we propose a pretreatment for sugarcane bagasse using a multi-enzymatic cocktail from the co-cultivation of four Aspergillus nidulans recombinant strains. The co-cultivation resulted in the simultaneous production of GH51 arabinofuranosidase (AbfA), GH11 endo-1,4-xylanase (XlnA), GH43 endo-1,5-arabinanase (AbnA) and GH12 xyloglucan specific endo-ß-1,4-glucanase (XegA). This core set of recombinant enzymes was more efficient than the alternative alkaline method in maintaining the cellulose integrity and exposing this cellulose to the following saccharification process. Thermogravimetric and differential thermal analysis revealed residual byproducts on the alkali pretreated biomass, which were not found in the enzymatic pretreatment. Therefore, the enzymatic pretreatment was residue-free and seemed to be more efficient than the applied alkaline method, which makes it suitable for bioethanol production.

Quantitative transient GUS expression in J-104 rice calli through manipulation of in vitro culture conditions

This paper purposes suitable conditions for callus induction and co-cultivation with Agrobacterium tumefaciens of J-104 rice cultivar. It was evaluated the effect of different concentrations of 2.4-D and agar, and the inclusion of L-proline and L-glutamine in callus culture medium. The use of 2.5 mg/L 2.4-D and 0.8% agar allowed the highest percentage of embryogenic calli. Callus formation was improved considerably with 500 mg/L of L-proline and L-glutamine in the culture medium. Different factors were studied throughout co-cultivation of calli with A. tumefaciens: inoculation time, co-cultivation temperature, concentration of acetosyringone and co-cultivation period. Transient GUS expression was quantified by fluorometry in all co-cultivated calli. The best results were obtained with the following conditions: 10 min as inoculation time, 100µM acetosyringone in co-cultivation medium, temperature of 20ºC, and 3 days as co-cultivation period.

Se describen las condiciones óptimas para la callogénesis y cocultivo de callos con Agrobacterium tume-faciens de la variedad de arroz J-104. Se determinó el efecto de diferentes concentraciones de 2.4-D, agar y de L-prolina y L-glutamina en el medio de cultivo de callos. El uso de 2,5 mg/L de 2.4-D y 0,8% de agar permitió lograr el porcentaje más alto de callos embriogénicos. La formación de callos fue mejorada considerablemente con la adición de 500 mg/L de L-prolina e igual concentración de L-glutamina en el medio de cultivo. Se estudiaron diferentes factores en el cocultivo de los callos con A. tumefaciens: tiempo de inoculación, concentración de acetosiringona, temperatura y tiempo de cocultivo. Para comparar el efecto de cada factor sobre la expresión GUS se cuantificó la actividad transitoria mediante fluorimetría. Los valores más altos de actividad fluorimétrica fueron obtenidos con las siguientes condiciones: 10 min de inoculación, 100µM de acetosiringona en el medio de cocultivo y 3 días de cocultivo a 20 ºC.