Marcos Fernando Basso

Fisiologia foliar e qualidade enológica da uva em videiras infectadas por vírus

Leaf physiology and enologic grape quality of virus-infected plants Viruses may induce metabolic and structural disarray in plant cells to varying degrees depending on viral species and plant susceptibility. With this focus, grapevine plants (Vitis vinifera) cvs. Cabernet Franc and Cabernet Sauvignon, symptomless and showing symptoms of virus-infection, in two commercial vineyards were comparatively analyzed. The parameters were: 1. photosynthetic potential (light-saturated rate of photosynthesis, saturating light intensities, light compensation point, dark respiration rate, apparent quantum yield, chlorophyll a and b); 2. foliar carbon metabolism (total soluble sugars and starch), and 3. enologic quality of the produced grapes (total soluble solids - °Brix, density, pH and titerable total acidity in the must; total color intensity and total polyphenols in berry skins) were recorded. In symptomatic plants Grapevine leafroll-associated virus 2 (GLRaV-2) and Rupestris stem pitting-associated virus (RSPaV) have been detected by ELISA carried out for six viruses. The virus infections induced significant reductions in chlorophyll content and in photosynthetic potential of both cultivars. Leaves of infected plants also showed significant accumulation of carbohydrates, suggesting a blockage of carbon transport out of these tissues. Concerning the enologic quality and based on technological parameters of the must (°Brix, density, titerable total acidity and polyphenols), grapes of infected plants showed at harvest a significantly lower level of maturation. These viruses directly affect the productive capacity as well as the quality of the grapes produced by these cultivars.

Detecção e identificação molecular de vírus associados a videiras sintomáticas e assintomáticas

The vegetative propagation of grapevine facilitates multiple viral infections, with different symptoms which vary according to combinations of cultivar or host species with viral species. The aims of this research were to detect and identify the viral species infecting two grapevine species/cultivars: one symptomatic and one symptomless. DsRNA from both samples was assayed by RT-PCR using 17 pairs of specific primers for detection of the Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine fleck virus (GFkV), Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine chrome mosaic virus (GCMV), Rupestris stem pitting-associated virus (RSPaV) and Grapevine leafroll-associated virus 1-4 (GLRaV-1 to -4), besides three degenerate primer pairs. For each primer pair at least one amplicon was cloned and sequenced. Symtomatic and symptomless plants were multiple infected by RSPaV, GLRaV-2 and/or GLRaV-3. The nucleotide sequences of seven isolates of RSPaV, three of GLRaV-2 and two of GLRaV-3 showed identities higher than 90% with the homologous viral species and allowed to identify possible viral strains in infected samples. These results highlight the necessity of viral diagnosis based on specific assays to determine grapevine sanitary status.

Produção de antissoro policlonal utilizando a proteína capsidial recombinante do Rupestris stem pitting-associated virus

RSPaV is the causal agent of pitting in the grapevine woody cylinder. The aim of this research was to produce polyclonal antiserum against recombinant RSPaV coat protein (CP) and evaluate its specificity and sensibility. The CP gene (780bp) of RSPaV was previously characterized. This gene was subcloned into the EcoRI site of the pRSET-B expression vector and the recombinant plasmid was used to induce the expression of the CP in E. coli cells. The CP, fused to a 6-His-tag, was purified from E. coli total protein extract by affinity chromatography using Ni-NTA resin. Identity of the purified protein was confirmed by SDS-PAGE and Western blot, using antibodies against the histidine tail. The in vitro-expressed recombinant CP presented a MW of ca. 31kDa. The purified protein was quantified and 2.55mg used for the immunization of a rabbit. The obtained polyclonal antiserum reacted with different RSPaV isolates extracted from grapevines in indirect ELISA.

Geminivirus data warehouse: a database enriched with machine learning approaches

BackgroundThe Geminiviridae family encompasses a group of single-stranded DNA viruses with twinned and quasi-isometric virions, which infect a wide range of dicotyledonous and monocotyledonous plants and are responsible for significant economic losses worldwide. Geminiviruses are divided into nine genera, according to their insect vector, host range, genome organization, and phylogeny reconstruction. Using rolling-circle amplification approaches along with high-throughput sequencing technologies, thousands of full-length geminivirus and satellite genome sequences were amplified and have become available in public databases. As a consequence, many important challenges have emerged, namely, how to classify, store, and analyze massive datasets as well as how to extract information or new knowledge. Data mining approaches, mainly supported by machine learning (ML) techniques, are a natural means for high-throughput data analysis in the context of genomics, transcriptomics, proteomics, and metabolomics.ResultsHere, we describe the development of a data warehouse enriched with ML approaches, designated geminivirus.org. We implemented search modules, bioinformatics tools, and ML methods to retrieve high precision information, demarcate species, and create classifiers for genera and open reading frames (ORFs) of geminivirus genomes.ConclusionsThe use of data mining techniques such as ETL (Extract, Transform, Load) to feed our database, as well as algorithms based on machine learning for knowledge extraction, allowed us to obtain a database with quality data and suitable tools for bioinformatics analysis. The Geminivirus Data Warehouse (geminivirus.org) offers a simple and user-friendly environment for information retrieval and knowledge discovery related to geminiviruses.

Indução de multibrotação in vitro em videira cv. Bordô

The Micropropagation allows mass propagation and may contribute to meet the demands of mother plants and seedlings of proven genetic quality and health of the vine. Different culture media plus or absence of plant growth regulators has been used frequently to assist in the induction of shoots. The study aimed to evaluate the means of culture and GZ and MS full or half the concentration of salts plus BAP for induction of multiple on the grapevine cv. Bordo. The experiment was conducted in a randomized design in a factorial 4 x 4, with four culture media (MS, MS/2, GZ, GZ/2) and four concentrations of BAP (0, 2.5, 5 and 10 μM) with four replications. It was evaluated at 30 days the number of micro-shoots per cutting, number of new micro-cuttings per shoot, number of leaves per shoot, mean length of shoots, number of roots by micro-cutting and length of the main root. The culture medium for more efficient induction of multiple in grapevine cv. Bordo and the development of the shoot is the MS medium plus 2.5 μM of BAP.

Chapter 8 – Transgenic Plants

Humankind has genetically manipulated plant crops for many years through conventional breeding. Until recently, breeding was the only way to introduce phenotypic characteristics of interest, which are determined by genes, to an individual plant or species. The desired phenotypic traits are transferred to the progeny through breeding and selection. However, such conventional methods of genetic manipulation have some limitations, including the sexual barrier between species, phylogenetic isolation barriers between and within genetic groups, a reduced gene pool, and linkage drag. All of these drawbacks are in addition to the lengthy time usually required for desirable traits to be transferred.

Induction of multi-sprouting in vitro in grapevine cv. Bordô.

The Micropropagation allows mass propagation and may contribute to meet the demands of mother plants and seedlings of proven genetic quality and health of the vine. Different culture media plus or absence of plant growth regulators has been used frequently to assist in the induction of shoots. The study aimed to evaluate the means of culture and GZ and MS full or half the concentration of salts plus BAP for induction of multiple on the grapevine cv. Bordo. The experiment was conducted in a randomized design in a factorial 4 x 4, with four culture media (MS, MS/2, GZ, GZ/2) and four concentrations of BAP (0, 2.5, 5 and 10 μM) with four replications. It was evaluated at 30 days the number of micro-shoots per cutting, number of new micro-cuttings per shoot, number of leaves per shoot, mean length of shoots, number of roots by micro-cutting and length of the main root. The culture medium for more efficient induction of multiple in grapevine cv. Bordo and the development of the shoot is the MS medium plus 2.5 μM of BAP.

Induction of multibrotacion in vitro in videira cv. Bordô

The Micropropagation allows mass propagation and may contribute to meet the demands of mother plants and seedlings of proven genetic quality and health of the vine. Different culture media plus or absence of plant growth regulators has been used frequently to assist in the induction of shoots. The study aimed to evaluate the means of culture and GZ and MS full or half the concentration of salts plus BAP for induction of multiple on the grapevine cv. Bordo. The experiment was conducted in a randomized design in a factorial 4 x 4, with four culture media (MS, MS/2, GZ, GZ/2) and four concentrations of BAP (0, 2.5, 5 and 10 μM) with four replications. It was evaluated at 30 days the number of micro-shoots per cutting, number of new micro-cuttings per shoot, number of leaves per shoot, mean length of shoots, number of roots by micro-cutting and length of the main root. The culture medium for more efficient induction of multiple in grapevine cv. Bordo and the development of the shoot is the MS medium plus 2.5 μM of BAP.