Editorial : Plant transformation

Plant transformation provides a key tool for much basic research, such as the study of gene functions and interactions, protein–protein interactions, developmental processes, as well as applications for crop improvement and the development of plant bioreactors to produce vaccines. Efficient and reproducible transformation technologies are not only essential for the development of transgenic plants but also critical for other applications like transient gene expression studies and gene editing.Instituto de BiotecnologíaFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Hopp, Horacio Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hopp, Horacio Esteban. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Spangenberg, German. La Trobe University. Agriculture Victoria. AgriBio; AustraliaFil: Herrera-Estrella, Luis. Texas Tech University. Institute of Genomics for Crop Abiotic Stress Tolerance. Plant and Soil Science Department; Estados UnidosFil: Herrera-Estrella, Luis. Centro de Investigación y de Estudios Avanzados. Unidad de Genómica Avanzada. Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO); Méxic

The ALS gene as genetic target in CRISPR/ cas approaches: what have we learned so far?

Specific mutations in the conserved domains of the acetolactate synthase (ALS) gene conduct to different key amino acid substitutions that can confer herbicide resistance in different plant species. This outcome has been widely exploited to produce herbicide-resistant agronomic crops as well as to direct many genome editing studies. Therefore, the ALS gene has become a model sequence target to improve our technological skills for more precise CRISPR/Cas nucleotide base substitution in plants, which is essential for modulation/modification of gene function as opposed to the more general gene knock out obtained by indels in conventional genome editing studies. This review summarizes the main knowledge and experiences attained from the use of the ALS gene as a target in CRISPR/Cas studies.Instituto de BiotecnologíaFil: Darqui, Flavia Soledad. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Lopez Bilbao, Marisa Gisela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Isolation and characterization of the tissue and development-specific potato snakin-1 promoter inducible by temperature and wounding

Snakin-1 (StSN1) is a broad-spectrum antimicrobial peptide isolated from Solanum tuberosum. Homologous proteins have been identified in a wide range of species but there is no apparent consensus in the roles they play. A 1394 bp fragment of the 5’upstream region of StSN1 gene, designated PStSN1, was isolated from the potato genome and sequenced. Bioinformatics analyses revealed a total of 55 potential regulatory motifs related to tissue-specificity, stress, defence and hormone responsiveness, among others. PStSN1 spatial and temporal activity was studied in transgenic Arabidopsis plants expressing a reporter gene under this promoter control (PStSN1::GUS). Histochemical staining revealed PStSN1::GUS expression in the root vasculature, cotyledons, young leaves and floral organs. Moreover, GUS staining was detected in young developmental stages gradually decreasing as the plant aged. Stress treatments on transgenic plants showed that PStSN1 activity was induced by high/low temperature and wounding. The characterization of PStSN1 in a model plant establishes a framework for the understanding of its possible biological functions and provides a potential tool for plant modification through genetic engineering.Instituto de BiotecnologíaFil: Almasia, Natalia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Nahirñak, Vanesa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Vazquez Rovere, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project

BACKGROUND: Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative strategies to high-throughput sequencing projects for the identification of novel genes differentially expressed in sunflower as a source of organ-specific genetic markers that can be functionally associated to important traits. RESULTS: Differential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4). The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60%) did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences putatively related to responses to important agronomic traits and key regulatory and physiological genes. CONCLUSIONS: The application of suppressed subtracted hybridization technology not only enabled the cost effective isolation of differentially expressed sequences but it also allowed the identification of novel sequences in sunflower from a relative small number of analyzed sequences when compared to major sequencing projects

Overexpression of snakin-1 gene enhances resistance to Rhizoctonia solani and Erwinia carotovora in transgenic potato plants

Snakin‐1 (SN1), a cysteine‐rich peptide with broad‐spectrum antimicrobial activity in vitro, was evaluated for its ability to confer resistance to pathogens in transgenic potatoes. Genetic variants of this gene were cloned from wild and cultivated Solanum species. Nucleotide sequences revealed highly evolutionary conservation with 91–98% identity values. Potato plants (S. tuberosum subsp. tuberosum cv. Kennebec) were transformed via Agrobacterium tumefaciens with a construct encoding the S. chacoense SN1 gene under the regulation of the ubiquitous CaMV 35S promoter. Transgenic lines were molecularly characterized and challenged with either Rhizoctonia solani or Erwinia carotovora to analyse whether constitutive in vivo overexpression of the SN1 gene may lead to disease resistance. Only transgenic lines that accumulated high levels of SN1 mRNA exhibited significant symptom reductions of R. solani infection such as stem cankers and damping‐off. Furthermore, these overexpressing lines showed significantly higher survival rates throughout the fungal resistance bioassays. In addition, the same lines showed significant protection against E. carotovora measured as: a reduction of lesion areas (from 46.5 to 88.1% with respect to the wild‐type), number of fallen leaves and thickened or necrotic stems. Enhanced resistance to these two important potato pathogens suggests in vivo antifungal and antibacterial activity of SN1 and thus its possible biotechnological application.Instituto de BiotecnologíaFil: Almasia, Natalia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina.Fil: Bazzini, Ariel Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina.Fil: Vazquez Rovere, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Almasia, Natalia Ines. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Área de Biotecnología; ArgentinaFil: Hopp, Horacio Esteban. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Área de Biotecnología; Argentin

Biotechnological improvement of ornamental plants

The discovery of commercial transgenic varieties of orange petunias sold in Europe and the United States although they had never reached the approved status, and the consequent recommendation to destroy them, was the trigger to discuss about biotechnological improvement of ornamental plants. Inside the restricted world of 26 vegetal transgenic species, according to the ISAAA’s reports (http://www.isaaa.org), there are three ornamental species: carnation, rose and the Beijing University developed petunia; all of them with the same trait, a change in their colour. On the other hand, in 2014, the whole-genome sequence of carnation appeared which was the first and until now the only one among ornamental species. In this context, we review the publications from the last five years in petunia, rose, chrysanthemum and carnation. In these papers there are detailed descriptions of modification of the cascade of genes and transcription factors involved in stress situations, in different developmental stages and their regulation through different plant hormones. This knowledge will allow breeding for better and new varieties with changes in their abiotic or biotic stress tolerance, altered growth or yield and modified product quality as colour or fragrance.A descoberta de variedades transgênicas de petúnias laranja vendidas na Europa e nos Estados Unidos que nunca alcançaram o status aprovado e a consequente recomendação de destruí-las foi o fator desencadeador para a discussão sobre a melhoria biotecnológica de plantas ornamentais. Dentro do mundo estrito de 26 espécies transgênicas vegetais, de acordo com os relatórios do ISAAA (http://www.isaaa.org), existem três espécies ornamentais: cravo, rosa e as petúnias desenvolvidas pela Universidade de Pequim que tem como característica mudança na cor. Por outro lado, em 2014 foi realizado pela primeira vez o sequenciamento completo do genoma do cravo que é o único sequenciado entre as espécies ornamentais. Neste contexto, revisamos as publicações dos últimos cinco anos em petúnia, rosa, crisântemo e cravo. Nestes trabalhos, há descrições detalhadas da modificação da cascata de genes e fatores de transcrição envolvidos em situações de estresse, diferentes estágios do crescimento e sua regulação através de diferentes hormônios vegetais. Este conhecimento contribuirá diretamente no melhoramento vegetal, o qual permitirá o desenvolvimento de novas variedades que sejam resistentes a diferentes situações de estresse abiótico ou biótico, alterações nos fatores que contribuem para o crescimento ou produtividade e modificações nos parâmetros de qualidade (como cor ou fragrância).Instituto de BiotecnologíaFil: Darqui, Flavia Soledad. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Radonic, Laura Mabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: López Bilbao, Marisa Gisela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Identification of Single Nucleotide Polymorphisms and analysis of Linkage Disequilibrium in sunflower elite inbred lines using the candidate gene approach

Background. Association analysis is a powerful tool to identify gene loci that may contribute to phenotypic variation. This includes the estimation of nucleotide diversity, the assessment of linkage disequilibrium structure (LD) and the evaluation of selection processes. Trait mapping by allele association requires a high-density map, which could be obtained by the addition of Single Nucleotide Polymorphisms (SNPs) and short insertion and/or deletions (indels) to SSR and AFLP genetic maps. Nucleotide diversity analysis of randomly selected candidate regions is a promising approach for the success of association analysis and fine mapping in the sunflower genome. Moreover, knowledge of the distance over which LD persists, in agronomically meaningful sunflower accessions, is important to establish the density of markers and the experimental design for association analysis. Results. A set of 28 candidate genes related to biotic and abiotic stresses were studied in 19 sunflower inbred lines. A total of 14,348 bp of sequence alignment was analyzed per individual. In average, 1 SNP was found per 69 nucleotides and 38 indels were identified in the complete data set. The mean nucleotide polymorphism was moderate ( = 0.0056), as expected for inbred materials. The number of haplotypes per region ranged from 1 to 9 (mean = 3.54 1.88). Model-based population structure analysis allowed detection of admixed individuals within the set of accessions examined. Two putative gene pools were identified (G1 and G2), with a large proportion of the inbred lines being assigned to one of them (G1). Consistent with the absence of population sub-structuring, LD for G1 decayed more rapidly (r 2= 0.48 at 643 bp; trend line, pooled data) than the LD trend line for the entire set of 19 individuals (r2= 0.64 for the same distance). Conclusion. Knowledge about the patterns of diversity and the genetic relationships between breeding materials could be an invaluable aid in crop improvement strategies. The relatively high frequency of SNPs within the elite inbred lines studied here, along with the predicted extent of LD over distances of 100 kbp (r2∼0.1) suggest that high resolution association mapping in sunflower could be achieved with marker densities lower than those usually reported in the literature.Fil:Fusari, C.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Lia, V.V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Hopp, H.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Heinz, R.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Transcriptomic identification of candidate genes involved in sunflower responses to chilling and salt stresses based on cDNA microarray analysis

<p>Abstract</p> <p>Background</p> <p>Considering that sunflower production is expanding to arid regions, tolerance to abiotic stresses as drought, low temperatures and salinity arises as one of the main constrains nowadays. Differential organ-specific sunflower ESTs (expressed sequence tags) were previously generated by a subtractive hybridization method that included a considerable number of putative abiotic stress associated sequences. The objective of this work is to analyze concerted gene expression profiles of organ-specific ESTs by fluorescence microarray assay, in response to high sodium chloride concentration and chilling treatments with the aim to identify and follow up candidate genes for early responses to abiotic stress in sunflower.</p> <p>Results</p> <p>Abiotic-related expressed genes were the target of this characterization through a gene expression analysis using an organ-specific cDNA fluorescence microarray approach in response to high salinity and low temperatures. The experiment included three independent replicates from leaf samples. We analyzed 317 unigenes previously isolated from differential organ-specific cDNA libraries from leaf, stem and flower at R1 and R4 developmental stage. A statistical analysis based on mean comparison by ANOVA and ordination by Principal Component Analysis allowed the detection of 80 candidate genes for either salinity and/or chilling stresses. Out of them, 50 genes were up or down regulated under both stresses, supporting common regulatory mechanisms and general responses to chilling and salinity. Interestingly 15 and 12 sequences were up regulated or down regulated specifically in one stress but not in the other, respectively. These genes are potentially involved in different regulatory mechanisms including transcription/translation/protein degradation/protein folding/ROS production or ROS-scavenging. Differential gene expression patterns were confirmed by qRT-PCR for 12.5% of the microarray candidate sequences.</p> <p>Conclusion</p> <p>Eighty genes isolated from organ-specific cDNA libraries were identified as candidate genes for sunflower early response to low temperatures and salinity. Microarray profiling of chilling and NaCl-treated sunflower leaves revealed dynamic changes in transcript abundance, including transcription factors, defense/stress related proteins, and effectors of homeostasis, all of which highlight the complexity of both stress responses. This study not only allowed the identification of common transcriptional changes to both stress conditions but also lead to the detection of stress-specific genes not previously reported in sunflower. This is the first organ-specific cDNA fluorescence microarray study addressing a simultaneous evaluation of concerted transcriptional changes in response to chilling and salinity stress in cultivated sunflower.</p

Simplified methodology for large scale isolation of homozygous transgenic lines of lettuce

Background: Lettuce is a globally important leafy vegetable and a model plant for biotechnology due to its adaptability to tissue culture and stable genetic transformation. Lettuce is also crucial for functional genomics research in the Asteraceae which includes species of great agronomical importance. The development of transgenic events implies the production of a large number of shoots that must be differentiated between transgenic and non-transgenic through the activity of the selective agent, being kanamycin the most popular. Results: In this work we adjusted the selection conditions of transgenic seedlings to avoid any escapes, finding that threshold concentration of kanamycin was 75 mg/L. To monitor the selection system, we studied the morphological response of transgenic and non-transgenic seedlings in presence of kanamycin to look for a visual morphological marker. Several traits like shoot length, primary root length, number of leaves, fresh weight, and appearance of the aerial part and development of lateral roots were affected in non-transgenic seedlings after 30 d of culture in selective media. However, only lateral root development showed an early, qualitative and reliable association with nptII presence, as corroborated by PCR detection. Applied in successive transgenic progenies, this method of selection combined with morphological follow-up allowed selecting the homozygous presence of nptII gene in 100% of the analyzed plants from T2 to T5. Conclusions: This protocol allows a simplified scaling-up of the production of multiple homozygous transgenic progeny lines in the early generations avoiding expensive and time-consuming molecular assays.Inst. de BiotecnologíaFil: Darqui, Flavia Soledad. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Radonic, Laura Mabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Lopez, Nilda Ester. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: López Bilbao, Marisa Gisela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin