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

Genome-wide and comparative phylogenetic analysis of senescence-associated NAC transcription factors in sunflower (Helianthus annuus)

Leaf senescence delay impacts positively in grain yield by maintaining the photosynthetic area during the reproductive stage and during grain filling. Therefore a comprehensive understanding of the gene families associated with leaf senescence is essential. NAC transcription factors (TF) form a large plant-specific gene family involved in regulating development, senescence, and responses to biotic and abiotic stresses. The main goal of this work was to identify sunflower NAC TF (HaNAC) and their association with senescence, studying their orthologous to understand possible functional relationships between genes of different species. To clarify the orthologous relationships, we used an in-depth comparative study of four divergent taxa, in dicots and monocots, with completely sequenced genomes (Arabidopsis thaliana, Vitis vinifera, Musa acuminata and Oryza sativa). These orthologous groups provide a curated resource for large scale protein sequence annotation of NAC TF. From the 151 HaNAC genes detected in the latest version of the sunflower genome, 50 genes were associated with senescence traits. These genes showed significant differential expression in two contrasting lines according to an RNAseq assay. An assessment of overexpressing the Arabidopsis line for HaNAC001 (a gene of the same orthologous group of Arabidopsis thaliana ORE1) revealed that this line displayed a significantly higher number of senescent leaves and a pronounced change in development rate. This finding suggests HaNAC001 as an interesting candidate to explore the molecular regulation of senescence in sunflower

Potato snakin-1 gene enhances tolerance to Rhizoctonia solani and Sclerotinia sclerotiorum in transgenic lettuce plants

Snakin-1 is a cysteine-rich antimicrobial peptide (AMP) isolated from potato tubers, with broad-spectrum activity. It belongs to the Snakin/GASA family, whose members have been studied because of their diverse roles in important plant processes, including defense. To analyze if this defensive function may lead to disease tolerance in lettuce, one of the most worldwide consumed leafy vegetable, we characterized three homozygous transgenic lines overexpressing Snakin-1. They were biologically assessed by the inoculation with the fungal pathogens Rhizoctonia solani and Sclerotinia sclerotiorum both in vitro and in planta at the greenhouse. When in vitro assays were performed with R. solani on Petri dishes containing crude plant extracts it was confirmed that the expressed Snakin-1 protein has antimicrobial activity. Furthermore, transgenic lines showed a better response than wild type in in vivo challenges against R. solani both in chamber and in greenhouse. In addition, two of these lines showed significant in vivo protection against the pathogen S. sclerotiorum in challenge assays on adult plants. Our results show that Snakin-1 is an interesting candidate gene for the selection/breeding of lettuce plants with increased fungal tolerance.Instituto 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: Trotz, Paulina M. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Lopez, Nilda Ester. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Vazquez Rovere, Cecilia. 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: Lopez Bilbao, Marisa Gisela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Translation of third and second harmonic generation microscopy into the clinic for the assessment of fresh lung tumor tissue

We show a portable third and second harmonic generation microscope in the clinic generating 3D real-time high resolution images of fresh lung tumor tissue providing immediate pathological feedback for clinicians potentially reducing endoscopy/operation time

Compact portable multiphoton microscopy reveals histopathological hallmarks of unprocessed lung tumor tissue in real time

During lung cancer operations a rapid and reliable assessment of tumor tissue can reduce operation time and potentially improve patient outcomes. We show that third harmonic generation (THG), second harmonic generation (SHG) and two-photon excited autofluorescence (2PEF) microscopy reveals relevant, histopathological information within seconds in fresh unprocessed human lung samples. We used a compact, portable microscope and recorded images within 1 to 3 seconds using a power of 5 mW. The generated THG/SHG/2PEF images of tumorous and nontumorous tissues are compared with the corresponding standard histology images, to identify alveolar structures and histopathological hallmarks. Cellular structures (tumor cells, macrophages and lymphocytes) (THG), collagen (SHG) and elastin (2PEF) are differentiated and allowed for rapid identification of carcinoid with solid growth pattern, minimally enlarged monomorphic cell nuclei with salt-and-pepper chromatin pattern, and adenocarcinoma with lipidic and micropapillary growth patterns. THG/SHG/2PEF imaging is thus a promising tool for clinical intraoperative assessment of lung tumor tissue

Bringing third and second harmonic generation microscopy into the clinic for the assessment of fresh lung tissue

Third and second harmonic generation (THG/SHG) microscopy is a novel imaging technique that has been suggested as a promising clinical tool, mainly for cancer diagnosis. Here, we test for the first time a compact, transportable ex-vivo THG/SHG microscope (Tritos Diagnostics B.V.) for clinical use. In lung surgery, immediate feedback on the nature of the excised tissue is important. We will present the first results of an ongoing study in which we image both lung tumor tissue immediately after excision and frozen section biopsies, and compare the images to the golden standard histology. Prior to this study, we performed a set of experiments collecting 3D images of mouse lung tissue to show that the combination of THG and SHG microscopy reveals key lung morphology components

Bringing third and second harmonic generation microscopy into the clinic for the assessment of fresh lung tissue

Third and second harmonic generation (THG/SHG) microscopy is a novel imaging technique that has been suggested as a promising clinical tool, mainly for cancer diagnosis. Here, we test for the first time a compact, transportable ex-vivo THG/SHG microscope (Tritos Diagnostics B.V.) for clinical use. In lung surgery, immediate feedback on the nature of the excised tissue is important. We will present the first results of an ongoing study in which we image both lung tumor tissue immediately after excision and frozen section biopsies, and compare the images to the golden standard histology. Prior to this study, we performed a set of experiments collecting 3D images of mouse lung tissue to show that the combination of THG and SHG microscopy reveals key lung morphology components

Bringing third and second harmonic generation microscopy into the clinic for the assessment of fresh lung tissue

Third and second harmonic generation (THG/SHG) microscopy is a novel imaging technique that has been suggested as a promising clinical tool, mainly for cancer diagnosis. Here, we test for the first time a compact, transportable ex-vivo THG/SHG microscope (Tritos Diagnostics B.V.) for clinical use. In lung surgery, immediate feedback on the nature of the excised tissue is important. We will present the first results of an ongoing study in which we image both lung tumor tissue immediately after excision and frozen section biopsies, and compare the images to the golden standard histology. Prior to this study, we performed a set of experiments collecting 3D images of mouse lung tissue to show that the combination of THG and SHG microscopy reveals key lung morphology components