Enabling genome editing in tropical maize lines through an improved, morphogenic regulator-assisted transformation protocol.

The recalcitrance exhibited by many maize (Zea mays) genotypes to traditional genetic transformation protocols poses a significant challenge to the large-scale application of genome editing (GE) in this major crop species. Although a few maize genotypes are widely used for genetic transformation, they prove unsuitable for agronomic tests in field trials or commercial applications. This challenge is exacerbated by the predominance of transformable maize lines adapted to temperate geographies, despite a considerable proportion of maize production occurring in the tropics. Ectopic expression of morphogenic regulators (MRs) stands out as a promising approach to overcome low efficiency and genotype dependency, aiming to achieve 'universal' transformation and GE capabilities in maize. Here, we report the successful GE of agronomically relevant tropical maize lines using a MR-based, Agrobacterium-mediated transformation protocol previously optimized for the B104 temperate inbred line. To this end, we used a CRISPR/Cas9-based construct aiming at the knockout of the VIRESCENT YELLOW-LIKE (VYL) gene, which results in an easily recognizable phenotype. Mutations at VYL were verified in protoplasts prepared from B104 and three tropical lines, regardless of the presence of a single nucleotide polymorphism (SNP) at the seed region of the VYL target site in two of the tropical lines. Three out of five tropical lines were amenable to transformation, with efficiencies reaching up to 6.63%. Remarkably, 97% of the recovered events presented indels at the target site, which were inherited by the next generation. We observed off-target activity of the CRISPR/Cas9-based construct towards the VYL paralog VYL-MODIFIER, which could be partly due to the expression of the WUSCHEL (WUS) MR. Our results demonstrate efficient GE of relevant tropical maize lines, expanding the current availability of GE-amenable genotypes of this major crop.

Antibiofilm Activity on Candida albicans and Mechanism of Action on Biomembrane Models of the Antimicrobial Peptide Ctn[15-34].

Ctn[15-34], the C-terminal fragment of crotalicidin, an antimicrobial peptide from the South American rattlesnake Crotalus durissus terrificus venom, displays remarkable anti-infective and anti-proliferative activities. Herein, its activity on Candida albicans biofilms and its interaction with the cytoplasmic membrane of the fungal cell and with a biomembrane model in vitro was investigated. A standard C. albicans strain and a fluconazole-resistant clinical isolate were exposed to the peptide at its minimum inhibitory concentration (MIC) (10 µM) and up to 100 × MIC to inhibit biofilm formation and its eradication. A viability test using XTT and fluorescent dyes, confocal laser scanning microscopy, and atomic force microscopy (AFM) were used to observe the antibiofilm effect. To evaluate the importance of membrane composition on Ctn[15-34] activity, C. albicans protoplasts were also tested. Fluorescence assays using di-8-ANEPPS, dynamic light scattering, and zeta potential measurements using liposomes, protoplasts, and C. albicans cells indicated a direct mechanism of action that was dependent on membrane interaction and disruption. Overall, Ctn[15-34] showed to be an effective antifungal peptide, displaying antibiofilm activity and, importantly, interacting with and disrupting fungal plasma membrane.

Exogenous Nitric Oxide Delays Plant Regeneration from Protoplast and Protonema Development in Physcomitrella patens.

Nitric oxide (NO) has been recognized as a major player in the regulation of plant physiology and development. NO regulates cell cycle progression and cell elongation in flowering plants and green algae, although the information about NO function in non-vascular plants is scarce. Here, we analyze the effect of exogenous NO on Physcomitrella patens protonema growth. We find that increasing concentrations of the NO donor sodium nitroprusside (SNP) inhibit protonema relative growth rate and cell length. To further comprehend the effect of NO on moss development, we analyze the effect of SNP 5 and 10 µM on protoplast regeneration and, furthermore, protonema formation compared with untreated plants (control). Isolated protoplasts were left to regenerate for 24 h before starting the SNP treatments that lasted five days. The results show that SNP restrains the protoplast regeneration process and the formation of new protonema cells. When SNP treatments started five days after protoplast isolation, a decrease in cell number per protonema filament was observed, indicating an inhibition of cell cycle progression. Our results show that in non-vascular plants, NO negatively regulates plant regeneration, cell cycle and cell elongation.

Microalgae protoplasts isolation and fusion for biotechnology research / Aislamiento y fusión de protoplastos de microalgas para aplicaciones biotecnológicas

ABSTRACT Protoplasts are microbial or vegetable cells lacking a cell wall. These can be obtained from microalgae by an enzymatic hydrolysis process in the presence of an osmotic stabilizer. In general, protoplasts are experimentally useful in physiological, genetic and biochemical studies, so their acquisition and fusion will continue to be an active research area in modern biotechnology. The fusion of protoplasts in microalgae constitutes a tool for strain improvement because it allows both intra and interspecific genetic recombination, resulting in organisms with new or improved characteristics of industrial interest. In this review we briefly describe the methodology for obtaining protoplasts, as well as fusion methods and the main applications of microalgal platforms.

RESUMEN Los protoplastos son células microbianas o vegetales que carecen de pared celular. Estos pueden obtenerse a partir de microalgas por un proceso de hidrólisis enzimática en presencia de un estabilizador osmótico. En general, los protoplastos son experimentalmente útiles en estudios fisiológicos, genéticos y bioquímicos, por lo que su obtención y fusión continuarán siendo un área de investigación activa en la biotecnología moderna. La fusión de protoplastos en microalgas constituye una herramienta para el mejoramiento de cepas pues permite la recombinación genética intra e interespecífica, logrando así organismos con nuevas características de interés industrial. En esta revisión, describimos brevemente la metodología para obtener protoplastos, métodos de fusión y las principales aplicaciones de las plataformas basadas en microalgas.

A protoplast generation and transformation method for soybean sudden death syndrome causal agents Fusarium virguliforme and F. brasiliense.

BACKGROUND: Soybean production around the globe faces significant annual yield losses due to pests and diseases. One of the most significant causes of soybean yield loss annually in the U.S. is sudden death syndrome (SDS), caused by soil-borne fungi in the Fusarium solani species complex. Two of these species, F. virguliforme and F. brasiliense, have been discovered in the U.S. The genetic mechanisms that these pathogens employ to induce root rot and SDS are largely unknown. Previous methods describing F. virguliforme protoplast generation and transformation have been used to study gene function, but these methods lack important details and controls. In addition, no reports of protoplast generation and genetic transformation have been made for F. brasiliense. RESULTS: We developed a new protocol for developing fungal protoplasts in these Fusarium species and test the protoplasts for the ability to take up foreign DNA. We show that wild-type strains of F. virguliforme and F. brasiliense are sensitive to the antibiotics hygromycin and nourseothricin, but strains transformed with resistance genes displayed resistance to these antibiotics. In addition, integration of fluorescent protein reporter genes demonstrates that the foreign DNA is expressed and results in a functional protein, providing fluorescence to both pathogens. CONCLUSIONS: This protocol provides significant details for reproducibly producing protoplasts and transforming F. virguliforme and F. brasiliense. The protocol can be used to develop high quality protoplasts for further investigations into genetic mechanisms of growth and pathogenicity of F. virguliforme and F. brasiliense. Fluorescent strains developed in this study can be used to investigate temporal colonization and potential host preferences of these species.

Elucidating genomic patterns and recombination events in plant cybrid mitochondria.

KEY MESSAGE: Cybrid plant mitochondria undergo homologous recombination, mainly BIR, keep a single allele for each gene, and maintain exclusive sequences of each parent and a single copy of the homologous regions. The maintenance of a dynamic equilibrium between the mitochondrial and nuclear genomes requires continuous communication and a high level of compatibility between them, so that alterations in one genetic compartment need adjustments in the other. The co-evolution of nuclear and mitochondrial genomes has been poorly studied, even though the consequences and effects of this interaction are highly relevant for human health, as well as for crop improvement programs and for genetic engineering. The mitochondria of plants represent an excellent system to understand the mechanisms of genomic rearrangements, chimeric gene formation, incompatibility between nucleus and cytoplasm, and horizontal gene transfer. We carried out detailed analyses of the mtDNA of a repeated cybrid between the solanaceae Nicotiana tabacum and Hyoscyamus niger. The mtDNA of the cybrid was intermediate between the size of the parental mtDNAs and the sum of them. Noticeably, most of the homologous sequences inherited from both parents were lost. In contrast, the majority of the sequences exclusive of a single parent were maintained. The mitochondrial gene content included a majority of N. tabacum derived genes, but also chimeric, two-parent derived, and H. niger-derived genes in a tobacco nuclear background. Any of these alterations in the gene content could be the cause of CMS in the cybrid. The parental mtDNAs interacted through 28 homologous recombination events and a single case of illegitimate recombination. Three main homologous recombination mechanisms were recognized in the cybrid mitochondria. Break induced replication (BIR) pathway was the most frequent. We propose that BIR could be one of the mechanisms responsible for the loss of the majority of the repeated regions derived from H. niger.

Yeast extract elicitation increases vinblastine and vincristine yield in protoplast derived tissues and plantlets in Catharanthus roseus

Abstract Catharanthus roseus (L.) G. Don, Apocynaceae, is an immensely important medicinal plant, produces a variety of anticancerous compounds. The yield of two most investigated alkaloids vinblastine and vincristine is unfortunately very low. A vast array of technologies including elicitation have recently been used to enrich Catharanthus alkaloid in culture. Yeast extract is a biotic elicitor, the polysaccharide and the peptide moiety have been recognized as a signalling element in enriching secondary metabolites. In this study, the yeast extract elicitation on vinblastine and vincristine was studied in various protoplast derived tissues and plantlets. Four different yeast extract treatments (T1 = 0.5 g/l, T2 = 1.0 g/l, T3 = 1.5 g/l and T4 = 2.0 g/l) were prepared and used. The alkaloid was quantified and a comparative account of yield were presented by the use of High performance thin layer chromatography. The yeast extract amendment in medium improved vinblastine and vincristine yield in cultivating tissues, maximum being in germinating embryos and in in vitro raised leaf. The highest yield was in T3 (1.5 mg/l) in which 22.74% vinblastine and 48.49% vincristine enrichment was noted in germinating embryos; the enhancement was however, treatment-specific. Antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase activities were investigated as addition of yeast extract caused cellular stress and had enriched level of alkaloids.

Fusión intergénica de protoplastos de Saccharomyces cerevisiae y Hanseniaspora guillermondii / Intergenic fusion of Saccharomyces cerevisiae and Hanseniaspora guillermondii protoplasts

La fusión de protoplastos ha facilitado la obtención de nuevas cepas de levaduras con propiedades biotecnológicas muy interesantes. El principal objetivo de esta investigación fue obtener una levadura híbrida intergénica con potencialidades enológicas características de dos géneros diferentes. Para ello se fusionaron protoplastos de Saccharomyces cerevisiae autóctona de la región zuliana con Hanseniaspora guillermondii CECT 11102 de origen comercial. Saccharomyces es una levadura que produce altas concentraciones de etanol pero el perfil aromático es sencillo y común. Hanseniaspora no resiste las concentraciones de etanol, pero puede generar aromas agradables e intensos. La identificación de las levaduras antes y después de la fusión de protoplastos se realizó con la técnica PCR-RFLP del gen 5.8S rADN y las regiones intergénicas adyacentes ITS1 e ITS2 del ADN extraído, sometiendo los productos amplificados a un análisis de restricción con las enzimas HinfI, HaeIII, CfoI y DdeI. El polietilenglicol fue usado para inducir la fusión de protoplastos. La cepa híbrida presentó características de ambas levaduras parentales debido a que resistió altas concentraciones de etanol como S. cerevisiae y fue capaz de metabolizar el salicín como H. guillermondii. El análisis molecular PCR-RFLP de la levadura híbrida mostró un patrón de bandas diferente al de las levaduras parentales.

Protoplast fusion has facilitated the development of new yeast strains with very interesting biotechnological properties. The main objective of this research was to obtain hybrid yeast with potentialities of two different genera, that could be used in the wine manufacture. Saccharomyces cerevisiae protoplasts from the Zulian region were fused with Hanseniaspora guillermondii CECT 11102 of commercial origin. Saccharomyces is a yeast that produces high levels of ethanol but the aromatic profile is simple and common. Hanseniaspora does not withstand ethanol concentrations, but it can generate pleasant and intense aromas. Identification of yeast before and after the fusion of protoplasts was performed using the PCR-RFLP technique of the 5.8S rDNA gene and the adjacent ITS1 and ITS2 intergenic regions of the extracted DNA, subjecting the amplified products to a restriction with the enzymes HinfI, HaeIII, CfoI and DdeI. Polyethylene glycol was used to induce fusion of protoplasts. The hybrid strain showed characteristics of both parental yeasts because it resisted high concentrations of ethanol as S. cerevisiae and was able to metabolize the salicin as H. guillermondii. PCR-RFLP molecular analysis of hybrid yeast showed a different band pattern than those pertaining to parental yeasts.

Isolation and regeneration of transiently transformed protoplasts from gametophytic blades of the marine red alga Porphyra yezoensis

Despite the recent progress of transient gene expression systems in a red alga Porphyra yezoensis by particle bombardment, a stable transformation system has yet to establish in any marine red macrophytes. One of the reasons of the difficulty in genetic transformation in red algae is the lack of systems to select and isolate transformed cells from gametophytic blades. Thus, toward the establishment of the stable transformation system in P. yezoensis, we have developed a procedure by which transiently transformed gametophytic cells were prepared from particle bombarded-gametophytic blade as regeneratable protoplasts. Using mixture of marine bacterial enzymes, yield of protoplasts was high as reported elsewhere; however, these protoplasts did not develop. In contrast, protoplasts prepared from gametophytes treated with allantoin were normally developed, in which the overexpression of a â-glucuronidase reporter gene had no effect on the regeneration of protoplasts. Therefore, the use of allantoin in protoplast preparation sheds a new light on the realization of an efficient isolation and selection of study transformed cells from gametophytic blades.

Isolation and fusion of protoplasts from the phytopathogenic fungus Sclerotium rolfsii (Sacc. )

Sclerotium rolfsii (Sacc.) is a serious plant pathogenic fungus and lacks perfect (basidial) stage in production. Protoplast fusion technology was employed to reconstruct fusants from this fungus. Two strains designated as A and R were used. Maximum protoplast yields of 3.8x10(5) /g mycelia and 2.8x10(5) /g mycelia were formed in strains A and R respectively. Osmotic stabilizer sucrose 1M gave maximum yield. Lysing enzyme at the rate of 15mg/ml was found best for yield. Fusion of protoplasts from strains A and R was carried out in fusion media containing PEG 4000 30 percent (w/v) with 0.2mM CaCl2. Four fusants F1, F2, F3 and F4 were recovered. Morphological, physiological and pathogenic characters of fusants were compared with parent strains on carrots, beans and tomato.

Factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum / Fatores que afetam a produção e regeneração de protoplastos de Colletotrichum lindemuthianum

The present work reports factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum. The usefulness of protoplast isolation is relevant for many different applications and has been principally used in procedures involving genetic manipulation. Osmotic stabilizers, lytic enzymes, incubation time and mycelial age were evaluated in terms of their effects on protoplast yield. The optimal condition for protoplast production included the incubation of young mycelia (48 h) in 0.6 mol l-1 NaCl as the osmotic stabilizer, with 30 mg ml-1 Lysing Enzymes from Trichoderma harzianum for 3 h of incubation. In these conditions protoplasts production was higher than 10(6) protoplatos ml-1 in the digestion mixture, number suitable enough for experiments of transformation in fungi. Sucrose concentrations of 1.2 mol l-1 and 1 mol l-1 were the most suitable osmotic stabilizers for the regeneration after 48 h, with rates of 16.35 percent and 14.54 percent, respectively. This study produced an efficient method for protoplast production and reverted them into a typical mycelial morphology using a Colletotrichum lindemuthianum LV115 isolate.

O presente trabalho apresenta os fatores que afetam a produção e regeneração de protoplastos de Colletotrichum lindemuthianum. O isolamento de protoplastos é muito relevante para diferentes aplicações, principalmente, em procedimentos que envolvem a manipulação genética. Estabilizadores osmóticos, enzimas líticas, tempo de incubação e idade micelial foram testados com relação ao efeito na liberação de protoplastos. As condições otimizadas para produção de protoplastos foram incubação de micélio jovem (48 h) em estabilizador osmótico NaCl 0.6 mol l-1, acrescido de 30 mg ml-1 da enzima Lysing Enzymes de Trichoderma harzianum incubado, durante 3 h. Nessas condições, a obtenção de protoplastos foi maior que 10(6) protoplatos ml-1 na mistura de digestão, número suficientemente adequado para experimentos de transformação em fungos. Sacarose nas concentrações de 1.2 mol l-1 e 1 mol l-1 foram os estabilizadores mais apropriados para a regeneração, após 48 h, sendo as taxas de regeneração de 16.35 por cento e 14.54 por cento, respectivamente. Este estudo produziu um método eficiente para produção e reversão de protoplastos à morfologia micelial típica de Colletotrichum lindemuthianum utilizando o isolado LV115.

Production and regeneration of protoplasts from orchid Mycorrhizal Fungi Epulorhiza repens and Ceratorhiza sp

The aim of this work was to study the standardization of conditions to obtain and regenerate Epulorhiza repens and Ceratorhiza sp. protoplasts. For E. repens, the largest number of protoplasts (8.0 × 10(6) protoplasts/mL) was obtained in 0.6 M KCl, using 15 mg/mL of Lysing Enzymes, and 2-day-old fungal mycelium. When 0.5 M sucrose was used as osmotic stabilizer, the highest frequency of regeneration was achieved (8.5 percent); 80.0 percent of protoplasts were nucleated, and 20.0 percent anucleated. For Ceratorhiza sp., the largest number of protoplasts (4.0 × 10(7) protoplasts/mL) was achieved in 0.6 M NaCl, when 15 mg/mL of Lysing Enzymes and 15mg/mL of Glucanex, with 2-day-old fungal mycelium were used. The highest frequency of regeneration was 6.7 percent using 0.5 M sucrose as osmotic stabilizer; 88.8 percent of protoplasts were nucleated, and 11.2 percent anucleated.

O objetivo deste trabalho foi padronizar as condições de obtenção e regeneração de protoplastos de Epulorhiza repens e Ceratorhiza sp. Para o fungo E. repens, a maior produção de protoplastos, 8.0 x 10(6) protoplastos/mL, foi obtida em KCl 0.6 M, na presença de 15 mg/mL de "Lysing Enzymes" e micélio fúngico com 2 dias de idade. A maior freqüência de regeneração obtida foi de 8,5 por cento quando sacarose 0.5 M foi utilizada como estabilizador osmótico. Do total de protoplastos obtidos, 80 por cento eram nucleados e 20 por cento anucleados. Para Ceratorhiza sp., a maior produção de protoplastos, 4,0 x 10(7) protoplastos/mL, foi obtida em NaCl 0.6 M, na presença de 15 mg/mL de "Lysing Enzymes" e 15mg/mL de Glucanex, e micélio fúngico com 2 dias de idade. A maior freqüência de regeneração obtida foi de 6.7 por cento utilizando sacarose 0.5 M como estabilizador osmótico. Do total de protoplastos obtidos, 88.8 por cento eram nucleados e 1.2 por cento anucleados. O estabelecimento de protocolo otimizado para obtenção e regeneração de protoplastos dos fungos E. repens e Ceratorhiza sp. é importante, permitindo o estabelecimento de técnicas de transformação genética, o isolamento de mutantes, a determinação de cariótipo eletroforético e o cruzamento de linhagens.

Isolation and fusion of protoplasts from the phytopathogenic fungus sclerotium rolfsii(sacc.).

Sclerotium rolfsii (Sacc.) is a serious plant pathogenic fungus and lacks perfect (basidial) stage in production. Protoplast fusion technology was employed to reconstruct fusants from this fungus. Two strains designated as A and R were used. Maximum protoplast yields of 3.8x10(5)/g mycelia and 2.8x10(5)/g mycelia were formed in strains A and R respectively. Osmotic stabilizer sucrose 1M gave maximum yield. Lysing enzyme at the rate of 15mg/ml was found best for yield. Fusion of protoplasts from strains A and R was carried out in fusion media containing PEG 4000 30% (w/v) with 0.2mM CaCl2. Four fusants F1, F2, F3 and F4 were recovered. Morphological, physiological and pathogenic characters of fusants were compared with parent strains on carrots, beans and tomato.

Isolation and fusion of protoplasts from the phytopathogenic fungus Sclerotium rolfsii (Sacc.)

Sclerotium rolfsii (Sacc.) is a serious plant pathogenic fungus and lacks perfect (basidial) stage in production. Protoplast fusion technology was employed to reconstruct fusants from this fungus. Two strains designated as A and R were used. Maximum protoplast yields of 3.8x10(5) /g mycelia and 2.8x10(5) /g mycelia were formed in strains A and R respectively. Osmotic stabilizer sucrose 1M gave maximum yield. Lysing enzyme at the rate of 15mg/ml was found best for yield. Fusion of protoplasts from strains A and R was carried out in fusion media containing PEG 4000 30% (w/v) with 0.2mM CaCl2. Four fusants F1, F2, F3 and F4 were recovered. Morphological, physiological and pathogenic characters of fusants were compared with parent strains on carrots, beans and tomato.

Isolation of recombinant strains with enhanced pectinase production by protoplast fusion between Penicillium expansum and Penicillium griseoroseum

Protoplast fusion between complementary auxotrophic and morphological mutant strains of Penicillium griseoroseum and P. expansum was induced by polyethylene glycol and calcium ions (Ca2+). Fusant strains were obtained in minimal medium and a prototrophic strain, possibly diploid, was chosen for haplodization with the fungicide benomyl. Different recombinant strains were isolated and characterized for occurrence of auxotrophic mutations and pectinolytic enzyme production. The fusant prototrophic did not present higher pectinase production than the parental strains, but among 29 recombinants analyzed, four presented enhanced enzyme activities. The recombinant RGE27, which possesses the same auxotrophic and morphologic mutations as the P. griseoroseum parental strain, presented a considerable increase in polygalacturonase (3-fold) and pectin lyase production (1.2-fold).

Fusões de protoplastos entre linhagens mutantes auxotróficas e morfológicas complementares de Penicillium griseoroseum e P. expansum foram induzidas por polietilenoglicol e íons cálcio (Ca2+). Fusionantes foram obtidos em meio mínimo e uma linhagem prototrófica, possivelmente diplóide, foi selecionada para a haploidização com o fungicida benomil. Diferentes linhagens recombinantes foram isoladas e caracterizadas quanto à presença de mutações auxotróficas e a produção de enzimas pectinolíticas. O fusionante prototrófico não apresentou maior atividade de pectinases em relação às linhagens parentais, entretanto, entre 29 recombinantes analisados, quatro apresentaram maiores atividades enzimáticas. O recombinante RGE27, o qual possui as mesmas mutações auxotróficas e morfológicas que a linhagem parental de P. griseoroseum, apresentou um aumento considerável na produção de poligalacturonase (3 vezes) e de pectina liase (1,2 vezes).

Protoplast formation and regeneration from Streptomyces clavuligerus NRRL 3585 and clavulanic acid production

Protoplasts of the wild type Streptomyces clavuligerus NRRL 3585 (ATCC 27064) were formed from spores cultures obtained in the lag, exponential and stationary growth phases by using 0.5% glycine in the culture medium. The protoplasts were obtained by treatment of the cells with lysozyme (EC-3.2.1.17) 40,000 U (1mg/mL), in an osmotic solution for 90 min at 28ºC. The frequency of regenerated protoplasts in the lag phase was 1.7x10³ CFU/mL (28.97%), in the beginning of the exponential phase 0.4x10² CFU/mL (31.67%), in the exponential growth phase 2.5x10³ CFU/mL (46.30%) and 1.0x10(5) CFU/mL in stationary phase (48.45%). Antibiotic production and activity of regenerated protoplasts were observed in all phases, except in the lag phase. The protoplast formation and regeneration techniques resulted in a new isolate strain of Streptomyces clavuligerus that produced approximately 2.5 fold more clavulanic acid.

Protoplastos foram formados a partir de esporos da amostra selvagem de Streptomyces clavuligerus durante a fase lag, exponencial e estacionária de crescimento, utilizando glicina a 0.5% como meio de cultura. Os protoplastos foram obtidos pelo tratamento das células com lisozima (EC-3.2.1.17) 40.000 U (1mg/mL) em solução osmótica de sorbitol e TES, por 90 min a 28ºC. A freqüência de protoplastos regenerados na fase lag foi de 1,7x10³ UFC/mL (28,97%), no início da fase exponencial correspondeu a 0,4x10² UFC/mL (31,67%), no final da fase exponencial observou-se 2,5x10³ UFC/mL (46,30%) e para a fase estacionária de crescimento apresentou 1,0x10(5) UFC/mL (48,45%). A produção do antibiótico e a atividade antibiótica dos protoplastos regenerados foram observadas em todas as fases de crescimento, exceto na fase lag. As técnicas de formação de protoplastos e regeneração resultaram em uma nova linhagem de Streptomyces clavuligerus produzindo 2,5 vezes mais ácido clavulânico.

Embryogenic calli induction from nucellar tissue of Citrus cultivars

Nucellar tissues of seven Citrus varieties were introduced onto three growth media to produce embryogenic callus. The media tested were: EME [MT, modified, with the addition of malt extract (500 mg.L-1)]; 1/2-EME [half concentration of MT macronutrients + half concentration of BH3 macronutrients + 500 mg.L-1 malt extract + 1.55 g.L-1 of glutamine]; and EBA [EME + 0.44 muM 6-benzyladenine + 0.04 muM 2,4 D]. Soft friable calli were obtained from 'Cravo' and 'Ponkan' mandarins (Citrus reticulata, Blanco), 'Murcott' tangor (Citrus reticulata Blanco x Citrus sinensis L. Osbeck), 'Serra d'água' and 'Valencia' sweet oranges (Citrus sinensis, L. Osbeck) 120 days after callus induction. 'Natal' and 'Pera' sweet oranges (Citrus sinensis, L. Osbeck) produced hard non-friable calli in this period. EME and 1/2-EME media had the best results for 'Cravo' mandarin, 'Ponkan' mandarin and 'Serra d'água' sweet orange, whereas EBA was the best media composition to induce soft friable calli on 'Murcott' tangor and 'Valencia' sweet orange. Friable callus cultures of 'Cravo' and 'Ponkan' mandarins, and 'Murcott' tangor yielded high quality protoplasts after isolation. Abbreviations: a.c. - activated charcoal; BA - 6-benzyladenine; IAA - indole-acetic acid; 2,4-D - 2,4-diclorophenoxyacetic acid; MT - Murashige & Tucker basal medium.

Nucelos de sete variedades de Citrus foram introduzidos em três meios de cultura para produção de calos embriogênicos. Os meios de cultura testados foram: EME [MT, modificado pela adição de extrato de malte (500 mg.L-1)]; 1/2-EME [1/2 concentração de macronutrientes no meio MT + 1/2 concentração de macronutrientes no meio BH3 + 500 mg.L-1 extrato de malte + 1,55 g.L-1 de glutamina]; e EBA [EME + 0,44 miM 6-benziladenina + 0,04 miM 2,4 D]. Calos friáveis foram obtidos nas variedades tangerinas 'Cravo' e 'Ponkan' (Citrus reticulata, Blanco), tangor 'Murcote' (Citrus reticulata Blanco x Citrus sinensis L. Osbeck), laranja 'Valencia' (Citrus sinensis L. Osbeck) e lima 'Serra d'água' (Citrus sinensis L. Osbeck) 120 dias após a instalação do ensaio. As laranjas 'Natal' e 'Pera' (Citrus sinensis L. Osbeck) produziram calos duros de crescimento lento neste período. Os meios EME e 1/2-EME mostraram os melhores resultados para as tangerinas 'Cravo' e 'Ponkan' e lima 'Serra d'água', enquanto que EBA foi o melhor meio de cultura para indução de calos friáveis de tangor 'Murcote' e laranja 'Valencia'. Calos friáveis das variedades, tangerinas 'Cravo' e 'Pokan' e tangor 'Murcote' foram subcultivados e apresentaram bom rendimento e qualidade de protoplastos após isolamento.

Embryogenic calli induction from nucellar tissue of Citrus cultivars

Nucellar tissues of seven Citrus varieties were introduced onto three growth media to produce embryogenic callus. The media tested were: EME [MT, modified, with the addition of malt extract (500 mg.L-1)]; 1/2-EME [half concentration of MT macronutrients + half concentration of BH3 macronutrients + 500 mg.L-1 malt extract + 1.55 g.L-1 of glutamine]; and EBA [EME + 0.44 muM 6-benzyladenine + 0.04 muM 2,4 D]. Soft friable calli were obtained from 'Cravo' and 'Ponkan' mandarins (Citrus reticulata, Blanco), 'Murcott' tangor (Citrus reticulata Blanco x Citrus sinensis L. Osbeck), 'Serra d'água' and 'Valencia' sweet oranges (Citrus sinensis, L. Osbeck) 120 days after callus induction. 'Natal' and 'Pera' sweet oranges (Citrus sinensis, L. Osbeck) produced hard non-friable calli in this period. EME and 1/2-EME media had the best results for 'Cravo' mandarin, 'Ponkan' mandarin and 'Serra d'água' sweet orange, whereas EBA was the best media composition to induce soft friable calli on 'Murcott' tangor and 'Valencia' sweet orange. Friable callus cultures of 'Cravo' and 'Ponkan' mandarins, and 'Murcott' tangor yielded high quality protoplasts after isolation. Abbreviations: a.c. - activated charcoal; BA - 6-benzyladenine; IAA - indole-acetic acid; 2,4-D - 2,4-diclorophenoxyacetic acid; MT - Murashige & Tucker basal medium.

Nucelos de sete variedades de Citrus foram introduzidos em três meios de cultura para produção de calos embriogênicos. Os meios de cultura testados foram: EME [MT, modificado pela adição de extrato de malte (500 mg.L-1)]; 1/2-EME [1/2 concentração de macronutrientes no meio MT + 1/2 concentração de macronutrientes no meio BH3 + 500 mg.L-1 extrato de malte + 1,55 g.L-1 de glutamina]; e EBA [EME + 0,44 miM 6-benziladenina + 0,04 miM 2,4 D]. Calos friáveis foram obtidos nas variedades tangerinas 'Cravo' e 'Ponkan' (Citrus reticulata, Blanco), tangor 'Murcote' (Citrus reticulata Blanco x Citrus sinensis L. Osbeck), laranja 'Valencia' (Citrus sinensis L. Osbeck) e lima 'Serra d'água' (Citrus sinensis L. Osbeck) 120 dias após a instalação do ensaio. As laranjas 'Natal' e 'Pera' (Citrus sinensis L. Osbeck) produziram calos duros de crescimento lento neste período. Os meios EME e 1/2-EME mostraram os melhores resultados para as tangerinas 'Cravo' e 'Ponkan' e lima 'Serra d'água', enquanto que EBA foi o melhor meio de cultura para indução de calos friáveis de tangor 'Murcote' e laranja 'Valencia'. Calos friáveis das variedades, tangerinas 'Cravo' e 'Pokan' e tangor 'Murcote' foram subcultivados e apresentaram bom rendimento e qualidade de protoplastos após isolamento.

FUSÃO DE PROTOPLASTOS DE Saccharomyces cerevisiae AVALIADA POR FLOCULAÇÃO E PRODUÇÃO DE H2S

In order to obtain yeast strains with important characteristics for the wine industry, protoplast fusions were performed. Attention was driven to two important traits: flocculation and of H2S production. Because both are traits occuring at low frequency in wine yeast (1% each), their natural occurence is unlike. Crossing was made through protoplast fusion because strains were sexualy incompatible. Initially, auxotrophic contrasting mutants were obtained, their marks were tested for reversion and finally, their growth rates were determined. Protoplasts were obtained after treatment with Novozym 234 PEG and CaCl2. The rate of fusion was 2,7x10-4, and 54,6% of the resulting colonies were able to grow on minimal media. After several transfers to YEPD media, the flocculant and H2S- productive strains were selected.

Com o objetivo de obter linhagens de leveduras com características de importância para a indústria vinícola, empregou-se a técnica da fusão de protoplastos. A atenção foi dirigida a duas características: floculação e não produção de H2S. Uma vez que cada característica é de baixa frequência (1% cada) em leveduras vinícolas, torna-se pouco provável a ocorrência natural de uma linhagem com ambas as características. O cruzamento foi realizado via fusão de protoplastos pois as linhagens apresentaram incompatibilidade sexual. Inicialmente foram obtidos os mutantes auxotróficos contrastantes e posteriormente suas marcas foram avaliadas quanto à reversão e, em seguida foram determinadas suas curvas de crescimento. Obteve-se a protoplastização por tratamento com Novozym 234 e a fusão foi mediada por PEG 40% e CaCl2 1,2 M. A taxa de fusão de protoplastos foi de 2,7x10-4, sendo que das colônias resultantes, 54,6% continuaram o crescimento após transferência para MM, "minimal media". Após várias transferências em meio YEPD foram selecionadas as linhagens floculantes e H2S-.

FUSÃO DE PROTOPLASTOS DE Saccharomyces cerevisiae AVALIADA POR FLOCULAÇÃO E PRODUÇÃO DE H2S

In order to obtain yeast strains with important characteristics for the wine industry, protoplast fusions were performed. Attention was driven to two important traits: flocculation and of H2S production. Because both are traits occuring at low frequency in wine yeast (1% each), their natural occurence is unlike. Crossing was made through protoplast fusion because strains were sexualy incompatible. Initially, auxotrophic contrasting mutants were obtained, their marks were tested for reversion and finally, their growth rates were determined. Protoplasts were obtained after treatment with Novozym 234 PEG and CaCl2. The rate of fusion was 2,7x10-4, and 54,6% of the resulting colonies were able to grow on minimal media. After several transfers to YEPD media, the flocculant and H2S- productive strains were selected.

Com o objetivo de obter linhagens de leveduras com características de importância para a indústria vinícola, empregou-se a técnica da fusão de protoplastos. A atenção foi dirigida a duas características: floculação e não produção de H2S. Uma vez que cada característica é de baixa frequência (1% cada) em leveduras vinícolas, torna-se pouco provável a ocorrência natural de uma linhagem com ambas as características. O cruzamento foi realizado via fusão de protoplastos pois as linhagens apresentaram incompatibilidade sexual. Inicialmente foram obtidos os mutantes auxotróficos contrastantes e posteriormente suas marcas foram avaliadas quanto à reversão e, em seguida foram determinadas suas curvas de crescimento. Obteve-se a protoplastização por tratamento com Novozym 234 e a fusão foi mediada por PEG 40% e CaCl2 1,2 M. A taxa de fusão de protoplastos foi de 2,7x10-4, sendo que das colônias resultantes, 54,6% continuaram o crescimento após transferência para MM, "minimal media". Após várias transferências em meio YEPD foram selecionadas as linhagens floculantes e H2S-.