Nanoparticle-assisted Polymerase Chain Reaction (NanoPCR): Optimization of PCR detection of Leifsonia xyli subsp. xyli by the addition of nanoparticles

Leifsonia xyli subsp. xyli (Lxx) causes ratoon stunting disease (RSD) in sugarcane, and is one of major causes of production losses. The detection of Lxx bacteria in sugarcane is made mainly through molecular biology techniques, especially polymerase chain reaction (PCR). However, PCR presents some barriers to provide reliable results. The present work brings a Nanoparticle-assisted Polymerase Chain Reaction (NanoPCR) assay for the detection of Lxx in its latent infection on micropropagated sugarcane. This assay was based in the addition of Gold and Titanium dioxide nanoparticles to conventional PCR and evaluation of its effects. It was observed that the reactions performed with Titanium dioxide nanoparticles provided the formation of singular well-defined bands under electrophoresis, consistent with the expected molecular weight, without occurrence of non-specific bands or presenting false negatives occurrence, negative effects that were observed in the control assay. While the performed NanoPCR adding AuNP also provided the formation of well-defined bands, been able to inhibit the occurrence of false negatives, but wasn’t able to eliminate the occurrence of non-specific amplifications. The results indicate that NanoPCR by the addition of Gold and Titanium dioxide nanoparticles to conventional PCR increased the detection of Lxx

Control of contaminants during introduction and establishment of Bambusa vulgaris in vitro

The aim of this work was to test techniques to reduce microbial contamination in the phases of introduction and establishment of the in vitro cultivation of Bambusa vulgaris through two experiments. The first experiment was carried out in a completely randomized design using a factorial arrangement (pre-treatment of nodal segments using or not a solution of Derosal 500 SC® and Chloramphenicol × culture medium with half or full concentration of salts × culture medium with presence or absence of sucrose × culture medium with presence or absence of Plant Preservative MixtureTM). In a second experiment, carried out in a completely randomized design, the effect of different fungicides associated to Chloramphenicol in a liquid culture medium was tested. It was possible to verify that the isolated effects of the pre-treatment by immersion of the nodal segments in a solution of 4 mL L-1 of Derosal 500 SC® and 200 mg L-1 of Chloramphenicol for 30 minutes and explants placed in a sucrose-free medium reduced fungal contamination. In the second experiment, the treatment that reduced fungal contamination corresponded to explants placed for seven days in a liquid medium with half the concentration of salts, sucrose-free, with 2 mL L-1 of Plant Preservative MixtureTM and with 4 mL L-1 of  Derosal 500 SC® and 200 mg L-1 of Chloramphenicol

Aspectos do estabelecimento in vitro de handroanthus chrysotrichus (bignoniaceae) para a produção de mudas / Aspects of the in vitro establishment of handroanthus chrysotrichus (bignoniaceae) for the production of seedlings

Handroanthus chrysotrichus é uma espécie arbórea utilizada na arborização e que apresenta dificuldades na reprodução sexuada. O objetivo do trabalho foi avaliar aspectos do estabelecimento in vitro de H. Chrysotrichus em meio alternativo. O experimento foi realizado no Laboratório de Pesquisas Aplicadas à Biofábrica do Centro de Tecnologias Nordeste. Sementes Handroanthus chrysotrichus foram desinfestadas e inoculadas em frascos contendo os diferentes tipos de meio de cultivo: sais do meio MS (Murashige Skoog, 1962) suplementado com sacarose e inositol; fertilizante comercial Kristalon® laranja e meio sem sais (apenas com água destilada). Em seguida os meios foram gelificados com 5,5 g.L-1 de ágar. Os meios de cultura com ausência de sais ou com o fertilizante Kristalon® laranja foram mais efetivos no controle da contaminação in vitro de H. Chrysotrichus. Os meios supracitados influenciaram positivamente o percentual de germinação e de emergência, provavelmente pela redução no potencial osmótico do meio de cultura. As sementes inoculadas no meio contendo Kristalon® laranja apresentaram maior velocidade de germinação e de emergência, além originar plântulas com maior altura. As plântulas cultivadas nos meios com sais MS e Kristalon® apresentaram maior produção de folhas. De acordo com os resultados obtidos neste trabalho, conclui-se que o meio alternativo contendo o fertilizante comercial Kristalon® proporcionou bons resultados, podendo ser indicado para a simplificação do meio de cultivo na fase de estabelecimento in vitro de H. chrysotrichus

Bioemulsifier produced by Yarrowia lipolytica using residual glycerol as a carbon source

Bioemulsifier is bioactive molecules produced by different microorganisms with reducing power and surface and interfacial tension. Among the microorganisms producing this molecule is yeast, which can produce different bioemulsifiers in different substrates. Undoubtedly, this biomolecule has excellent potential for industrial applications, but high production costs are the biggest problem in production. Aiming at cost reduction the present study using crude residual glycerol for biosurfactant production by Yarrowia lipolytica. Then isolates were grown in residual glycerol compound medium, rotating 200 rpm at 28ºC for 48 hours. Bioemulsifier production was observed by analysis of dry biomass, pH, surface tension and emulsification index. The results indicated that the emulsion produced from biosurfactant using glycerol as a carbon source by Y. lipolytica has the potential for bioemulsifier production. All isolates obtained similar results for all analyzes, indicating that this species has a linear production among the isolates. Biomass reached 10.08 ± 0.62 g.L-1, there was a sharp drop in pH reaching 4.6, surface tension averaged 41.7 mN.m-1 and emulsification index reached 56%. The isolates tested show potential for bioemulsifier production using glycerol as an unconventional carbon source

Effects of the bendazol fungicide on in vitro development of the nim (Azadirachta indica A. JUSS)

Two experiments were conducted to evaluate Bendazol fungicidal effects in neem micropropagation. In these experiments, the nodal segment explants from in vitro plants were used. In the first experiment, the explants remained in DKW culture medium for a period of 30 days containing different concentrations of Bendazol (M1 -50, M2 - 100, M3 - 200, and M4 - 400 mg.L-1). The control treatment (M0) was prepared with DKW medium + BAP (0.225 mg.L-1). In the second experiment, the explants were maintained for only one week in media supplemented with Bendazol or BAP, and then they were transferred and kept in free Bendazol/BAP media for three weeks. In each experiment, the design was completely randomized with five treatments, 10 replicates per treatment, and one explant/cultivation flask.  The variables analyzed included the formation of calluses and roots, lateral bud development, shoot height, contamination and plant death. There was no significant difference in tree variables (shoot, callus formation and shoot height) between treatments in both experiments. There was no death, plant contamination and rooting during the experiment. The results indicate that Bendazol can be used at low doses for in vitro neem cloning thereby replacing BAP and ultimately reducing production costs

Identificação molecular de Leifsonia xyli subsp. xyli nas variedades RB 863129 e RB 92579 de cana-de-açúcar submetidas à termoterapia

A cultura da cana-de-açúcar é uma das mais importantes atividades agrícolas do Brasil, ocupando atualmente a 3ª colocação em área plantada no país. Apesar da importância econômica da cana-de-açúcar para o país, o crescimento da cultura é limitado pelo raquitismo-da-soqueira, principal doença da cultura, causada pela bactéria Leifsonia xyli subsp. xyli (Lxx). A técnica mais difundida para o controle do raquitismo-da-soqueira é a termoterapia de rebolos. O estudo objetivou avaliar a efetividade da termoterapia de rebolos para o controle de Lxx nas variedades RB 863129 e RB 92579 de cana-de-açúcar. Os rebolos foram submetidos aos tratamentos de combinação de temperatura e tempo, que consistiu em 52°C por 30 minutos, 50°C por 2 horas e um tratamento controle, onde os rebolos não foram submetidos a tratamento térmico. Em seguida, os rebolos foram cultivados em caixas contendo substrato Basaplant® e transferidos para a estufa, onde permaneceram por 50 dias. Após este período, foi avaliada a porcentagem total de emissão de brotações e a fitossanidade em relação à presença de DNA genômico de Lxx. Nas condições deste estudo foi verificado que, para as variedades RB 863129 e RB 92579 de cana-de-açúcar, todos os tratamentos testados foram diagnosticados como positivos para Lxx, bem como, comprometeram a emissão de brotações

Basic procedure for the in vitro propagation of Brazilian trees for reforestation purposes

Important strategies can be used to avoid biodiversity loss by deforestation in tropical rainforests. Some use biotechnological techniques to support conservation initiatives. Plant tissue culture techniques are highly accepted biotechnological approaches for conservation of biodiversity. The work aimed to propose a basic operational model for the induction of in vitro germination of trees through plant tissue cultivation techniques. Fruits of 15 tree species, ten woody trees (Couroupita guianensis Aubl., Tabebuia heptaphylla (Vell.) Toledo, Tabebuia impetiginosa (Mart. ex DC.) Standl., Tabebuia roseoalba (Ridl.) Sandwith, Vochysia haenkeana (Spreng.) Mart., Vitex montevidensis Cham., Copaifera coriacea Mart., Spondias tuberosa Arruda, Schinus terebinthifolia Raddi, and Talisia esculenta (A. St.-Hil.) Radlk.) and five palm trees (Syagrus coronate (Mart.) Becc., Attalea oleifera Barb. Rodr., Elaeis guineensis Jacq., Colubrina glandulosa Perk., and Astrocaryum vulgare Mart.) were collected at different locations in the State of Pernambuco, Brazil. The in vitro germination used two different protocols, one designed for palm trees and one designed for woody trees. It was evaluated the parameters microbial contamination, survival, in vitro establishment, germination percentage and percentage of seeds converted to plants. The results showed that the set of methodologies proposed as a basic protocol for the in vitro introduction was able to achieve satisfactory results for 13 of the 15 tested species. The protocol proposed a high potential for use in the rescue of seeds through in vitro plant tissue culture. The described technique is an efficient tool for the propagation of trees used in reforestation programs

Influência da sacarose no crescimento e no perfil de pigmentos fotossintéticos em duas espécies arbóreas cultivadas in vitro / Influence of sucrose on growth and profile of photosynthetics pigments in two arboreal species cultivated in vitro

O uso de técnicas de cultivo in vitro com fins de produção de mudas é uma realidade biotecnológica que permite a manutenção da biodiversidade de muitas florestas. Todavia, altas concentrações de sacarose no meio de cultura podem influenciar negativamente o crescimento das plantas. Objetivou-se avaliar a influência de diferentes concentrações de sacarose no crescimento e perfil de pigmentos fotossintéticos em duas espécies arbóreas da Mata Atlântica cultivadas in vitro. Segmentos nodais de Handroanthus impetiginosus e Jacaranda brasiliana foram obtidos de plantas estabelecidas in vitro e inoculados em meio WPM (Wood Plant Medium), acrescido de 0,1 g.L-1 de inositol e 5,5 g.L-1 de ágar e diferentes concentrações de sacarose: 0,0; 15,0 e 30,0 g.L-1. Aos 50 dias de cultivo foi avaliado o comprimento da parte aérea, número de folhas, teor de clorofilas, razão clorofila a/b e carotenoides. Os dados foram submetidos ao Teste de Tukey a 5% de probabilidade. A adição de sacarose no meio de cultivo influenciou positivamente a emissão de folhas, entretanto não promoveram diferenças significativas no comprimento das brotações de J. Brasiliana. O teor de clorofilas totais foi proporcional à concentração de sacarose no meio de cultura. Não houve influência das concentrações de sacarose no conteúdo de carotenoides e na razão clorofilas a/b. Com base nos resultados, a redução em 50% na concentração da sacarose no meio de cultivo não implica em perdas na qualidade das brotações e permite a redução dos custos para a produção de mudas dessas espécies