Evaluación del potencial biotecnológico de un promotor derivado del virus de la distorsión de la hoja de maracuyá (PLDV), un begomovirus que infecta maracuyá / Evaluation of the biotechnological potential of a promoter derived from passion fruit leaf distortion virus (PLDV), a begomovirus that infects passionfruit

RESUMEN Los avances biotecnológicos en plantas requieren la bioprospección de nuevos promotores para la expresión de genes de interés agronómico, en particular, es necesario caracterizar nuevos promotores con expresión tejido específica. El objetivo de esta investigación fue evaluar la actividad de expresión del promotor del gen AV1 que codifica para la proteína de la cápside (CP) del virus de la distorsión de la hoja de maracuyá (Passion fruit leaf distortion virus, PLDV) mediante ensayos transitorios de biobalística de baja presión. Se realizó un análisis de la región promotora del gen AV1 empleando herramientas bioinformáticas. Se construyó una fusión traduccional (CP-PLDV-GUS), que porta la región promotora del gen AV1 de PLDV fusionada al gen reportero uidA (GUS). CP-PLDV-GUS fue bombardeado sobre hojas de plántulas de tabaco cultivadas in vitro empleando una pistola de genes. Como control positivo se utilizó el plásmido pBI121 que porta el gen GUS bajo el control del promotor 35S de CaMV. Se llevaron a cabo 11 repeticiones, donde la unidad experimental fue la hoja y la variable de respuesta, la expresión transitoria del gen GUS representado por el número de puntos azules observados en las hojas bombardeadas. Como resultado, el análisis estadístico no paramétrico demostró que existe evidencia muestral suficiente para confirmar que, tanto el promotor AV1 del PLDV y 35S de CaMV presentan una actividad de expresión semejante. Finalmente, el promotor del gen AV1 de PLDV mostró una fuerte actividad de expresión del gen reportero en las células del mesófilo de las hojas, el cual podría ser usado para conferir expresión tejido específica en plantas transgénicas.

ABSTRACT Biotechnological advances in plants require the bioprospecting of new promoters for the gene´s expression of agronomic interest, in particular, it is necessary to characterize new promoters with tissue-specific expression The objective of this research was to evaluate the expression activity of the AV1 gene promoter that codes for the capsid protein (CP) of the Passion fruit leaf distortion virus (PLDV) by means of transient tests of low pressure biobalistics. An analysis of the promoter region was carried out using bioinformatics tools. A CP-PLDV-GUS translational fusion was constructed, which carries the promoter region of the AV1 gene of PLDV fused to the uidA reporter gene (GUS). CP-PLDV-GUS was bombarded on leaves of tobacco seedlings grown in vitro using a gene gun. As a positive control pBI121 carrying the GUS gene under the control of the 35S promoter of CaMV was used. It was carried out 11 repetitions where the experimental unit was the leaf and the response variable the transient expression of the GUS gene represented by number of blue dots observed in the bombarded leaves. As a result, the non-parametric statistical analysis showed that there is sufficient sample evidence to confirm that both the AV1 promoter of PLDV and 35S of CaMV exhibit similar expression activity. Finally, the promoter of the AV1 gene of PLDV showed a strong activity of expression of the reporter gene in the leaf mesophyll cells, which could be used to confer tissue-specific expression in transgenic plants.

Transformation of coffee (Coffea Arabica L. cv. Catimor) with the cry1ac gene by biolistic, without the use of markers / Transformação do café (Coffea arabica L. cv. Catimor) com o gene cry1ac por bombardeamento, sem usar marcadores

The transformation of coffee plantlets with the cry1ac gene of Bacillus thuringiensis was achieved by biolistic using either the whole pUBC plasmid or only the ubi-cry1ac-nos genetic cassette. The cry1ac gene was inserted into coffee plants in order to confer resistance to the leaf miner Leucoptera coffeella, an insect responsible for considerable losses in coffee crops. Bearing in mind that the genetic cassettes used for this study lack reporter genes and/or selection marker genes, the parameters for the transformation procedure by biolistic were previously standardised with a plasmid carrying the gus reporter gene. The presence of the cry1ac gene in young plantlet tissues was determined by PCR, Southern blot and reverse transcription-PCR. Our results show that the obtainment of viable coffee plantlets, transformed by bombardment with the cry1ac gene and without selection markers nor reporter genes, is feasible.

A transformação das plântulas de café com o gene cry1ac de Bacillus thuringiensis foi realizada por biobalística, utilizando todo o pUBC plasmídeo ou só o cassete genético UBI-cry1ac-nos. O gene cry1ac foi inserido no cafeeiro a fim de conferir resistência à folha mineiro Leucoptera coffeella, um inseto responsável por perdas consideráveis nas culturas de café. Tendo em conta que ao plasmídeo e ao cassete genético utilizados para este estudo faltam genes repórteres e/ou de seleção, os parâmetros para o processo de transformação por biobalística foram previamente padronizados com um plasmídeo transportando o gene repórter gus. A presença do gene cry1ac em tecidos de jovens plântulas foi determinada por PCR, Southern blot e transcrição reversa-PCR. Nossos resultados mostram que a obtenção de plântulas de café, transformado por bombardeamento com o gene cry1ac sem genes de seleção genética nem repórteres é viável.

Transformation of coffee (Coffea Arabica L. cv. Catimor) with the cry1ac gene by biolistic, without the use of markers

The transformation of coffee plantlets with the cry1ac gene of Bacillus thuringiensis was achieved by biolistic using either the whole pUBC plasmid or only the ubi-cry1ac-nos genetic cassette. The cry1ac gene was inserted into coffee plants in order to confer resistance to the leaf miner Leucoptera coffeella, an insect responsible for considerable losses in coffee crops. Bearing in mind that the genetic cassettes used for this study lack reporter genes and/or selection marker genes, the parameters for the transformation procedure by biolistic were previously standardised with a plasmid carrying the gus reporter gene. The presence of the cry1ac gene in young plantlet tissues was determined by PCR, Southern blot and reverse transcription-PCR. Our results show that the obtainment of viable coffee plantlets, transformed by bombardment with the cry1ac gene and without selection markers nor reporter genes, is feasible.

A transformação das plântulas de café com o gene cry1ac de Bacillus thuringiensis foi realizada por biobalística, utilizando todo o pUBC plasmídeo ou só o cassete genético UBI-cry1ac-nos. O gene cry1ac foi inserido no cafeeiro a fim de conferir resistência à folha mineiro Leucoptera coffeella, um inseto responsável por perdas consideráveis nas culturas de café. Tendo em conta que ao plasmídeo e ao cassete genético utilizados para este estudo faltam genes repórteres e/ou de seleção, os parâmetros para o processo de transformação por biobalística foram previamente padronizados com um plasmídeo transportando o gene repórter gus. A presença do gene cry1ac em tecidos de jovens plântulas foi determinada por PCR, Southern blot e transcrição reversa-PCR. Nossos resultados mostram que a obtenção de plântulas de café, transformado por bombardeamento com o gene cry1ac sem genes de seleção genética nem repórteres é viável.

Detección de geminivirus asociados a la alstroemeria (alstroemeria l.) En villa guerrero, estado de México / Detection of geminivirus associated to alstroemeria (alstroemeria l.) In villa guerrero, state of Mexico / Detecção de geminivírus associados a alstroemeria (alstroemeria l.) Em villa guerrero, estado do México

En plantaciones de alstroemeria (Alstroemeria L.) de Villa Guerrero, Estado de México, se han detectado plantas con síntomas similares a los inducidos por geminivirus en otros cultivos hortícolas. En dichas plantaciones también se ha observado la presencia de la mosquita blanca, considerada como el vector más eficiente de estos virus. El objetivo del presente trabajo fue detectar la presencia de geminivirus en plantas de alstroemeria. Mediante PCR, usando los iniciadores MotCP2118/MotCP2123, se obtuvo un segmento de ~600pb, similar al del control positivo correspondiente a chile infectado con el begomovirus pepper huasteco yellow vein virus (PHYVV, antes pepper huasteco virus) en plantas sintomáticas, mientras que en las asintomáticas la detección fue negativa. Plantas de Nicotiana glutinosa, N. benthamiana, N. rustica, N. tabacum var. xanthi y Datura stramonium inoculadas por biobalística con ADN total obtenido de alstroemerias con síntomas y positivas a PHYVV mediante PCR, mostraron mosaicos leves y deformación de hojas, mientras que en plantas de Capsicum annuum se observaron mosaicos, necrosis en nervaduras y abultamientos en hojas. Con el ADN de estas plantas también se obtuvieron bandas correspondientes al PHYVV, pero en las monocotiledóneas bombardeadas, incluyendo alstroemeria, no fue detectado el fragmento. La secuencia de oligonucleótidos de los productos de PCR mostró 98% de homología con el begomovirus PHYVV. Aunque no fue posible reproducir en alstroemeria los síntomas observados en campo, sí se evidenció mediante PCR la presencia de un geminivirus similar al PHYVV en tejido de plantas sintomáticas.

In alstroemeria (Alstroemeria L.) plantations located in Villa Guerrero, Mexico State, plants with symptoms similar to those induced by geminivirus in other horticultural crops have been detected. In addition, the presence of whiteflies, which are considered the most efficient vectors of these viruses, has been observed in these plantations. The goal of this work was to detect the presence of this geminivirus species in alstroemeria plants. By means of PCR analysis using primers MotCP2118/MotCP2123, a fragment of ~600pb similar to the amplicon obtained from PHYVV-infected positive control was amplified only from symptomatic plants. Nicotiana glutinosa, N. benthamiana, N. rustica, N. tabacum var. xanthi and Datura stramonium plants were inoculated by bombardment with total DNA obtained from symptomatic alstroemerias and positive to PHYVV by means of PCR. Inoculated plants showed mild mosaics and deformation of leaves, whereas in the leaves of Capsicum annum plants, mosaics, vein necrosis and blisters were observed. Using DNA from these plants as template in PCR, amplicons corresponded to PHYVV were also obtained; however, in bombarded monocotyledons, including alstroemeria, this fragment was not detected. The sequence of oligonucleotides from the PCR products showed 98% homology to PHYVV geminivirus. Even though symptoms presented by alstroemeria plants in the field were not reproduced, the presence of a geminivirus similar to PHYVV in tissue of symptomatic plants was evidenced through PCR.

Em plantações de alstroemeria (Alstroemeria L.) de Villa Guerrero, Estado do México, se tem detectado plantas com sintomas similares aos induzidos por geminivírus em outros cultivos hortícolas. Em ditas plantações também tem sido observada a presença da mosquinha branca, considerada como o vetor mais eficiente destes vírus. O objetivo do presente trabalho foi detectar a presença de geminivírus em plantas de alstroemeria. Mediante PCR, usando os iniciadores MotCP2118/MotCP2123, obteve-se um segmento de ~600pb, similar ao do controle positivo correspondente a chile infectado com o begomovírus pepper huasteco yellow vein virus (PHYVV, antes pepper huasteco virus) em plantas sintomáticas, enquanto que nas assintomáticas a detecção foi negativa. Plantas de Nicotiana glutinosa, N. benthamiana, N. rustica, N. tabacum var. xanthi e Datura stramonium inoculadas por biobalística com DNA total obtido de alstroemerias com sintomas e positivas a PHYVV mediante PCR, mostraram mosaicos leves e deformação de folhas, enquanto que em plantas de Capsicum annuum se observaram mosaicos, necrose em nervaduras e protuberâncias em folhas. Com o DNA destas plantas também se obtiveram bandas correspondentes ao PHYVV, mas nas monocotiledóneas bombardeadas, incluindo alstroemeria, não foi detectado o fragmento. A sequência de oligonucleotídeos dos produtos de PCR mostrou 98% de homologia com o begomovírus PHYVV. Embora não foi possível reproduzir em alstroemeria os sintomas observados em campo, sím foi evidenciada, mediante PCR, a presença de um geminivírus similar ao PHYVV em tecido de plantas sintomáticas.

Optimization of coffee (Coffea arabica) transformation parameters using uidA and hpt genes: effect of osmotic pre-treatment, helium pressure and target distance / Optimización de los parámetros de transformación del café (Coffea arabica) utilizando los genes uidA y hpt: efecto del pretratamiento osmótico, presión de helio y distancia objetivo

The aim of this work was to optimize the biolistic delivery parameters that affect the DNA delivery and stable expression of marker genes into coffee tissues (Coffea arabica. L. cvs. Caturra and Catuaí). The effect of osmotic preculture length, osmotic concentration of medium, Helium pressure and target distance on transient expression of the uidA gene in coffee leaves and somatic embryos were tested. The highest transient uidA expression was obtained when Caturra (18.3±2.8) and Catuaí (6.8±2.0) leaves and Catuaí embryos (80.0±7.4) were cultured for 5h on Yasuda medium complemented with 0.5M Mannitol +0.5M Sorbitol. The combination of 1100psi and a target distance of 9cm resulted in the highest number of blue spots per Caturra leaf segment (23.6±3.9), whereas for the Catuaí variety the combination of 1100psi and a target distance of six (10.2±1.9) and nine (8.2±1.9) cm gave the highest number of blue spots per leaf segment. The optimized protocol was tested with pCAMBIA 1 301 (uidA gene and the hpt gene), pCAMBIA 1 305.2 (uidA version GUSPlus ™ and the hpt gene) and pCAMBIA 1 301-BAR (uidA gene and the bar gene). The highest number of blue spots was obtained when Caturra (54.6±5.7) and Catuaí (28.9±4.3) leaves were bombarded with pCAMBIA 1 305.2. Selection of bombarded coffee tissues with 100mg/l hygromicyn caused the oxidation of tissues. Rev. Biol. Trop. 57 (Suppl. 1): 151-160. Epub 2009 November 30.

La presente investigación tuvo como objetivo optimizar los parámetros que afectan la incorporación y expresión de genes marcadores mediante biobalística en segmentos de hoja y embriones somáticos de café (Coffea arabica. L. cvs. Caturra y Catuaí). La mayor expresión transitoria del gen uidA en segmentos de hoja de Caturra (18.3±2.8) y Catuaí (6.8±2.0) y embriones somáticos de Catuaí (80.0±7.4) se obtuvo al cultivar los explantes por cinco horas previo al bombardeo en el medio Yasuda complementado con 0.5M mannitol+0.5M sorbitol. Asimismo, se obtuvo una mayor expresión transitoria del gen uidA al bombardear los segmentos de hoja de Caturra y Catuaí y embriones somáticos de Catuaí con una presión de helio de 1 100psi y una distancia de bombardeo de 6 o 9 cm.

Un método de transformación genética de maíz para conferirle resistencia ulterior a enfermedades virales / A method for genetic transformation of maize for resistance to viral diseases

A system for the genetic transformation of maize was developed for two Costa Rican varieties: CR-7 and Diamantes 8843, that can allow the subsequent transfer of viral-derived genes in order to confer resistance to the disease caused by maize rayado fino virus (MRFV). The method is based on particle bombardment of organogenic calli derived from shoot tips. On the other hand, the molecular construction pRFcp-bar, containing the coat protein gene of MRFV and the marker gene bar, was elaborated. For the visual selection of the transformed material was used also the plasmid pDM803 that contains the reporter gene uidA (GUS). The results indicate that devices evaluated: the PIG ("Particle Inflow Gun") and the Bio-Rad are both enough efficient to transfer foreign genes to the genome of the maize.