Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens.

Microalgae are photosynthetic microorganisms widely used for the production of highly valued compounds, and recently they have been shown to be promising as a system for the heterologous expression of proteins. Several transformation methods have been successfully developed, from which the Agrobacterium tumefaciens-mediated method remains the most promising. However, microalgae transformation efficiency by A. tumefaciens is shown to vary depending on several transformation conditions. The present study aimed to establish an efficient genetic transformation system in the green microalgae Dunaliella tertiolecta using the A. tumefaciens method. The parameters assessed were the infection medium, the concentration of the A. tumefaciens and co-culture time. As a preliminary screening, the expression of the gusA gene and the viability of transformed cells were evaluated and used to calculate a novel parameter called Transformation Efficiency Index (TEI). The statistical analysis of TEI values showed five treatments with the highest gusA gene expression. To ensure stable transformation, transformed colonies were cultured on selective medium using hygromycin B and the DNA of resistant colonies were extracted after five subcultures and molecularly analyzed by PCR. Results revealed that treatments which use solid infection medium, A. tumefaciens OD600 = 0.5 and co-culture times of 72 h exhibited the highest percentage of stable gusA expression. Overall, this study established an efficient, optimized A. tumefaciens-mediated genetic transformation of D. tertiolecta, which represents a relatively easy procedure with no expensive equipment required. This simple and efficient protocol opens the possibility for further genetic manipulation of this commercially-important microalgae for biotechnological applications.

Effects of outdoor cultures on the growth and lipid production of Phaeodactylum tricornutum using closed photobioreactors.

One of the principal challenges for large scale production of microalgae is the high costs of biomass production. Aiming for minimize this problem, microalgal biodiesel production should focus on outdoors cultures, using available solar light and allowing lower energy cost process. Testing species that proved to be common and easy to culture may be a good approach in this process. The present work reports indoor-outdoor cultures of Phaeodactylum tricornutum using different bioreactors types, using cell growth, biochemical composition, and the profiles of the fatty acids produced as the parameters to test the optimization processes. The results show that the use of outdoor cultures is a good choice to obtain P. tricornutum biomass with a good potential for biodiesel production. The microalgae produced reached better growth efficiency, major lipid content and showed an increment in the percentage of saturated fatty acids (required on the biodiesel production) respect indoor cultures. These results are important to show the relevance of using outdoor cultures as a way to improve the efficiency and the energetic balance of the biodiesel production with P. tricornutum algae.

Fertirrigación con diferentes formas de nitrógeno en el cultivo de tomate en un suelo arcilloso / Tomato crop fertigation with different nitrogen form in clayed soil / Fertirrigação com diferentes formas de nitrogênio no cultivo de tomate em um solo argiloso

Se estudió el efecto de nitrógeno nítrico, amoniacal y ureico, suministrados en distintas etapas de desarrollo del cultivo de tomate, sobre el rendimiento y la calidad de fruto. El trabajo se realizó en campo bajo riego por goteo, en un suelo vertisol pélico y clima cálido y semiárido. Tres tratamientos de fertilización (T1, T2 y testigo) fueron distribuidos en un diseño de bloques completos al azar con tres repeticiones. T1 y T2 se fertilizaron con 250-50-190kg·ha-1 de N, P y K, respectivamente; en estos el N se suministró en diferentes proporciones de N-ureico, N-amoniacal y N-nítrico, en distintas etapas de desarrollo del cultivo; el T3 se aplicó como los productores del Valle de Culiacán, Sinaloa, México (450-118-413kg·ha-1 de N-P-K), con 75% del N como nitrato. La concentración de N-NO3 en el extracto celular de peciolos en los T1 y T2 fluctuó de 500 a 1360ppm, y en T3 fue de 300-1175ppm. Estos valores estuvieron relacionados con la dosis de N total suministrado pero no con la proporción de NH4/NO3 aplicada, y tampoco influyeron en la producción de fruto de exportación. La producción de fruto fue estadísticamente igual en las dosis de fertilización alta y moderada, con un ahorro del 75% de N-nítrico. En calidad poscosecha de frutos, la fertilización reducida y la aplicación de N-ureico y N-amoniacal en altas proporciones no afectó la firmeza, ºBrix ni la pérdida de peso de los frutos.

The effect of nitrogen form (urea, nitrate and ammonium) provided at various stages of development of the tomato crop on the yield and quality of fruit was studied. The work was conducted under field conditions in a pellustert soil, drip irrigation and a warm and semi-arid climate. Three fertilization treatments (T1, T2 and control) were arranged in a randomized complete-block design with three replicates. T1 and T2 were fertilized with 250-50-190kg·ha-1 N, P and K, respectively; which N was supplied in different ratios of ureic-N, ammonium-N and nitric-N along growing tomato plants; T3 was provided in a similar way as is generally employed by the tomato producers in the Culiacan Valley, Sinaloa, Mexico (450-118-413kg·ha-1 N-P-K), with 75% of N in nitrate form. The concentration of N-NO-3 in the petiole cell extract in T1 and T2 ranged from 500 to 1360ppm, and in the control it was 300-1175ppm. These values were related with the total N doses supplied but not with the ratio of NH4/NO3 provided, neither influenced on the total amount of exportation fruit. The fruit production was statistically similar in the high and moderated fertilizer doses with a fertilizer savings of 75% of nitrate N. With respect to the post-harvest quality in the tomato fruit, moderated fertilization associated with high application of ureic-N and ammonium-N did not affect the fruit firmness, ºBrix or the weight loss in the fruit.

Estudou-se o efeito do nitrogênio nítrico, amoniacal e uréico, subministrados em distintas etapas de desenvolvimento do cultivo de tomate, sobre o rendimento e a qualidade do fruto. O trabalho se realizou no campo sob irrigação por gotejamento, em um solo vertisol pélico e clima cálido e semi-árido. Três tratamentos de fertilização (T1, T2 e testemunho) foram distribuídos em um desenho de blocos completos aleatório com três repetições. T1 e T2 se fertilizaram com 250-50-190kg•ha-1 de N, P e K, respectivamente; em estes o N se subministrou em diferentes proporções de N-uréico, N-amoniacal e N-nítrico, em distintas etapas de desenvolvimento do cultivo; o T3 se aplicou como os produtores do Vale de Culiacán, Sinaloa, México (450-118-413kg•ha-1 de N-P-K), com 75% do N como nitrato. A concentração de N-NO3 no extrato celular de pecíolos nos T1 e T2 flutuou de 500 a 1360ppm, e em T3 foi de 300-1175ppm. Estes valores estiveram relacionados com a dose de N total subministrado, mas não com a proporção de NH4/NO3 aplicada, e tampouco influíram na produção do fruto de exportação. A produção do fruto foi estatisticamente igual nas doses de fertilização alta e moderada, poupando 75% de N-nítrico. Em qualidade pos-colheita de frutos, a fertilização reduzida e a aplicação de N-uréico e N-amoniacal em altas proporções não afetou a firmeza, oBrix nem a perda de peso dos frutos.

Efecto del calcio y potencial osmótico de la solución nutritiva en la pudrición apical, composición mineral y rendimiento de tomate / Effect of calcium and osmotic potential of the nutritive solution on the tomato blossom-end rot, mineral composition and yield / Efeito do calcio e potencial osmótico da solução nutritiva na podridão apical, composição mineral e rendimento de tomate

La pudrición apical (BER, por sus siglas en inglés, de blossom end-rot), un desorden físico común en la producción de tomate, es causada por diversos factores y las medidas para su control no están bien definidas. Se realizaron dos estudios en instalación tipo casa sombra en el Valle de Culiacán, Sinaloa, México, para conocer los efectos de la concentración de Ca, del potencial osmótico (Ys) de la solución nutritiva (SN) y su interacción sobre número de frutos con pudrición apical (NFBER), el contenido de nutrientes y el rendimiento de frutos de dos híbridos de tomate, B-52 tipo bola y Aníbal tipo ‘saladette’. En el experimento 1 se evaluaron nueve tratamientos resultantes de la combinación de variar la concentración de Ca (4,5; 6,75 y 9,0meq·l-1) y el Ys (-0,047; -0,072 y -0,097MPa) de la SN. En el experimento 2 se evaluaron otras concentraciones de Ca (7, 9 y 11meq·l-1) y diferentes Ys (-0,036; -0,048 y -0,072MPa). En el experimento 1 el incremento de Ca en la solución disminuyó significativamente el NFBER, mientras que niveles decrecientes de Ys lo aumentaron. En el experimento 2 los frutos con BER tuvieron contenidos significativamente menores de Ca y P y mayores de Mg, comparados con los frutos sin BER. No hubo diferencias significativas en el rendimiento de frutos comerciales debido a los factores Ca y Ys en los dos estudios, por lo que es posible reducir hasta en 50% la inversión en fertilizantes.

Blossom-end rot (BER), a physical common disorder in tomato production, is caused by many factors and the control measures are not well defined. In order to study the effects of Ca concentration and osmotic potential (Ys) of the nutrient solution (NS), and their interaction, on the number of fruits with blossom-end rot (NFBER), nutrient contents and yield in two tomato hybrids, B-52 ball type and Anibal ‘saladette’ type, two experiments were carried out under net house conditions at the Culiacan Valley, Sinaloa, México. In experiment 1, nine treatments resulting from the combination of varying Ca concentrations (4.5, 6.75 and 9.0meq·l-1) and Ys in the NS (-0.047, -0.072 and -0.097MPa) were evaluated. In experiment 2 other concentrations of Ca (7, 9 and 11meq·l-1) and different Ys (-0.036, -0.048 and -0.072MPa) were tested. In experiment 1, increasing levels of Ca in NS significantly reduced NFBER, while decreasing levels of Ys increased it. In experiment 2 the fruits with BER had significantly lower contents of Ca and P, and higher Mg as compared to the fruits without BER. No significant effects in tomato fruit yield were obtained with the Ca and Ys factors in the two experiments; thus, it is possible to reduce by 50% the investment in fertilizer.

A podridão apical (BER, por suas siglas em inglês, de blossom end-rot), uma desordem física comum na produção de tomate, é causada por diversos fatores e as medidas para seu controle não estão bem definidas. Realizaram-se dois estudos em instalação tipo casa sombra no Valle de Culiacán, Sinaloa, México, para conhecer os efeitos da concentração de Ca, do potencial osmótico (Ys) da solução nutritiva (SN) e sua interação sobre número de frutos com podridão apical (NFBER), o conteúdo de nutrientes e o rendimento de frutos de dois híbridos de tomate, B-52 tipo bola e Aníbal tipo ‘saladette’. No experimento 1 se avaliaram nove tratamentos resultantes da combinação de variar a concentração de Ca (4,5; 6,75 e 9,0meq·l-1) e o Ys (-0,047; -0,072 e -0,097MPa) da SN. No experimento 2 se avaliaram outras concentrações de Ca (7, 9 e 11meq·l¹) e diferentes Ys (-0,036; -0,048 e -0,072MPa). No experimento 1 o incremento de Ca na solução diminuiu significativamente o NFBER, enquanto que níveis decrescentes de Ys o aumentaram. No experimento 2 os frutos com BER tiveram conteúdos significativamente menores de Ca e P e maiores de Mg, comparados com os frutos sem BER. Não houve diferenças significativas no rendimento de frutos comerciais devido aos fatores Ca e Ys nos dois estudos, pelo que é possível reduzir até em 50% a inversão em fertilizantes.