RESUMEN
An efficient system for the establishment and multiplication of highly prolific embryogenic cell cultures of grapevine (Vitis sp.) was developed. Using anther-derived pro-embryogenic masses as starting material, cell suspensions of different grapevine cultivars (Tempranillo, Cabernet-Sauvignon) and rootstocks (Kober 125 AA, Kober 5 BB, 110 Richter) were initiated in liquid medium containing NOA (1.0 mg l(-1)) and BAP (0.25 mg l(-1)) as growth regulators. Conditioned medium was recovered and utilised for establishing new, highly totipotent cell cultures. The suspensions obtained, showed embryogenic competence resulting in somatic embryo induction and subsequent plant regeneration. In this study, a simplified establishment procedure for grapevine embryogenic cell suspension allowing the fast multiplication of embryogenic material is described. Evidence for the promoting effect of the protein fraction derived from conditioned medium, on cell proliferation was found. In bioassays, addition of ss-D: -GlcY affect cell proliferation suggesting that arabinogalactan proteins are required for growth processes in grapevine cell cultures.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Desarrollo Embrionario , Regeneración , Vitis/embriología , Vitis/fisiología , Proliferación Celular , Medios de Cultivo Condicionados , Proteínas de Plantas/metabolismo , Semillas/embriología , Vitis/citologíaRESUMEN
The nucleotide sequences of RNAs 1 and 2 of a German isolate of Raspberry ringspot virus (RpRSV) infecting grapevine (RpRSV-Grapevine), as well as partial sequences of another grapevine isolate from Switzerland (RAC815) were determined. The sequences of the protease-polymerase region encoded by RNA1, and the movement protein and coat protein genes encoded by RNA 2, of these isolates were compared with those of other isolates available in databases. The coat proteins of the grapevine isolates formed a sister group to all those from other RpRSV isolates, but whether this resulted from divergence or recombination was uncertain.
Asunto(s)
Nepovirus/genética , Vitis/virología , Secuencia de Bases , Datos de Secuencia Molecular , Nepovirus/aislamiento & purificación , Nepovirus/patogenicidad , Filogenia , ARN Viral/genética , Recombinación Genética , Homología de Secuencia de Aminoácido , Proteínas Virales/genéticaRESUMEN
The complete nucleotide sequence of the RNAs 1 and 2 of the nepovirus Raspberry ringspot virus cherry isolate (RpRSV-ch) from grapevine was determined. The RNA 1 is 7935 nucleotides (nt) long excluding the poly(A) tail, and contains one long open reading frame (ORF) encoding a polypeptide of 2367 amino acids. This ORF is preceeded by a 136nt 5' non-coding region, and followed by a 695nt 3' non-coding region. Conserved amino acid motifs, characteristic of the viral protease cofactor, the NTP-binding protein, proteinase and polymerase, were found in the sequence of the RNA 1-encoded polyprotein. The RNA 2 is 3915nt long excluding the poly(A) tail, and contains one long ORF encoding a polypeptide of 1106 amino acids. This ORF is preceeded by a 203nt 5' non-coding region, and followed by a 390nt 3' non-coding region. When compared to the corresponding sequences of other nepoviruses, a maximum level of 34% identity was found between the RNA 1-encoded polypetides of RpRSV-ch and other nepoviruses. For the RNA 2-encoded polypeptide, 88% identity was found between RpRSV-ch and RpRSV-S, a Scottish isolate of RpRSV from raspberry, and a maximum 29% identity between RpRSV-ch and other nepoviruses.
Asunto(s)
Nepovirus/genética , Nepovirus/aislamiento & purificación , Vitis/virología , Regiones no Traducidas 3'/genética , Regiones no Traducidas 5'/genética , Secuencias de Aminoácidos/genética , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Poliproteínas/genética , ARN Viral/genética , ARN Viral/aislamiento & purificación , Análisis de Secuencia de ADN , Transcripción Genética , Proteínas Virales/genéticaRESUMEN
The movement protein genes from several isolates of ArMV and GFLV of different geographical origins were amplified by RT/PCR using degenerate primers, cloned and sequenced. A single pair of degenerate primers was designed from these sequences to allow the simultaneous amplification of parts of the movement protein genes of ArMV and GFLV. Their use in an immunocapture-RT/PCR for the detection of ArMV or GFLV in infected grapevines proved to be ten times more sensitive than the corresponding ArMV or GFLV ELISA tests. A Sph1 restriction site found in the sequences corresponding to the amplified products from the GFLV isolates, but not in the amplified products from the ArMV isolates, allowed the differentiation between ArMV and GFLV in the infected grapevines by a Sph1 restriction digestion of the amplified products.
Asunto(s)
Cartilla de ADN , ADN Viral/análisis , Nepovirus/aislamiento & purificación , Enfermedades de las Plantas/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rosales/virología , Animales , Secuencia de Bases , Clonación Molecular , Europa (Continente) , Nepovirus/genética , Conejos , Mapeo Restrictivo , Sensibilidad y Especificidad , Alineación de Secuencia , Homología de Secuencia , Especificidad de la Especie , Túnez , Proteínas Virales/genéticaRESUMEN
The RNAs 2 of an Arabis mosaic virus (ArMV) and a grapevine fanleaf virus (GFLV) isolate, originating from South West of Germany near Neustadt an der Weinstrasse (NW), were sequenced. They are 3820 and 3775 nucleotides long respectively, and both contain one open reading frame encoding a polypeptide of 1110 amino acids. Their 5' non-coding regions contain conserved and repeated sequences, which are able to form stem-loop structures. Nucleotide sequence comparisons between the full-length RNAs 2 revealed homology levels of 84 and 82% between the ArMV-NW and the ArMV-L and -U, respectively, 90% between GFLV-NW and GFLV-F13, and 72% between ArMV-NW and GFLV-NW. Amino acid sequence comparisons showed that the greatest difference was found between the 2A proteins of the different ArMV isolates, the 2A protein of the ArMV-NW showing more similarity to the 2A protein of GFLV-NW than to those of ArMV-L2 or -U2.