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

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

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

Peculiarities of the Transformation of Asteraceae Family Species: The Cases of Sunflower and Lettuce

The Asteraceae family is the largest and most diversified family of the Angiosperms, characterized by the presence of numerous clustered inflorescences, which have the appearance of a single compound flower. It is estimated that this family represents around 10% of all flowered species, with a great biodiversity, covering all environments on the planet, except Antarctica. Also, it includes economically important crops, such as lettuce, sunflower, and chrysanthemum; wild flowers; herbs, and several species that produce molecules with pharmacological properties. Nevertheless, the biotechnological improvement of this family is limited to a few species and their genetic transformation was achieved later than in other plant families. Lettuce (Lactuca sativa L.) is a model species in molecular biology and plant biotechnology that has easily adapted to tissue culture, with efficient shoot regeneration from different tissues, organs, cells, and protoplasts. Due to this plasticity, it was possible to obtain transgenic plants tolerant to biotic or abiotic stresses as well as for the production of commercially interesting molecules (molecular farming). These advances, together with the complete sequencing of lettuce genome allowed the rapid adoption of gene editing using the CRISPR system. On the other hand, sunflower (Helianthus annuus L.) is a species that for years was considered recalcitrant to in vitro culture. Although this difficulty was overcome and some publications were made on sunflower genetic transformation, until now there is no transgenic variety commercialized or authorized for cultivation. In this article, we review similarities (such as avoiding the utilization of the CaMV35S promoter in transformation vectors) and differences (such as transformation efficiency) in the state of the art of genetic transformation techniques performed in these two species.Instituto de BiotecnologíaFil: Darqui, Flavia Soledad. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Darqui, Flavia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Radonic, Laura Mabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Radonic, Laura Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Beracochea, Valeria Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Beracochea, Valeria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: 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; Argentina.Fil: Hopp, Horacio Esteban. 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.Fil: Lopez Bilbao, Marisa Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Genetic mapping of EST-SSR, SSR and InDel to improve saturation of genomic regions in a previously developed sunflower map

In order to saturate a sunflower genetic map and facilitate marker-assisted selection (MAS) breeding for stress response, it is necessary to enhance map saturation with molecular markers localized in linkage groups associated to genomic regions involved in these traits. This work describes the identification and characterization of 1,134 simple sequence repeat (SSR) containing expressed sequence tags (EST) from unigenes available databases. Twelve of these functional markers as well as 41 public SSR markers were successfully localized in linkage groups, thus contributing to the saturation of specific regions on a reference genetic-linkage-map derived from recombinant inbred lines (RIL) mapping population from the cross between PAC2 x RHA266 lines. The enriched map includes 547 markers (231 SSR, 9 EST-SSR, 3 insertions/deletions (InDel) and 304 amplified fragment length polymorphisms (AFLP) distributed in 17 linkage groups (LG), spanning genetic size to 1,942.3 cM and improving its mean density to 3.6 cM per locus. As consequence, no gaps longer than 13.2 cM remain uncovered throughout the entire map, which increases the feasibility of detecting genes or traits of agronomic importance in sunflower

Transcriptomic Survey of the Midgut of Anthonomus grandis (Coleoptera: Curculionidae)

Anthonomus grandis Boheman is a key pest in cotton crops in the New World. Its larval stage develops within the flower bud using it as food and as protection against its predators. This behavior limits the effectiveness of its control using conventional insecticide applications and biocontrol techniques. In spite of its importance, little is known about its genome sequence and, more important, its specific expression in key organs like the midgut. Total mRNA isolated from larval midguts was used for pyrosequencing. Sequence reads were assembled and annotated to generate a unigene data set. In total, 400,000 reads from A. grandis midgut with an average length of 237 bp were assembled and combined into 20,915 contigs. The assembled reads fell into 6,621 genes models. BlastX search using the NCBI-NR database showed that 3,006 unigenes had significant matches to known sequences. Gene Ontology (GO) mapping analysis evidenced that A. grandis is able to transcripts coding for proteins involved in catalytic processing of macromolecules that allows its adaptation to very different feeding source scenarios. Furthermore, transcripts encoding for proteins involved in detoxification mechanisms such as p450 genes, glutathione-S-transferase, and carboxylesterases are also expressed. This is the first report of a transcriptomic study in A. grandis and the largest set of sequence data reported for this species. These data are valuable resources to expand the knowledge of this insect group and could be used in the design of new control strategies based in molecular information.Instituto de Microbiología y Zoología AgrícolaFil: Salvador, Ricardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Perez Principi, Ricardo Dario. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Berretta, Marcelo Facundo. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Fernandez, Paula Del Carmen. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Paniego, Norma Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Sciocco, Alicia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies

Citrus are among the most prevailing fruit crops produced worldwide. The implementation of effective and reliable breeding programs is essential for coping with the increasing demands of satisfactory yield and quality of the fruit as well as to deal with the negative impact of fast-spreading diseases. Conventional methods are time-consuming and of difficult application because of inherent factors of citrus biology, such as their prolonged juvenile period and a complex reproductive stage, sometimes presenting infertility, self-incompatibility, parthenocarpy, or polyembryony. Moreover, certain desirable traits are absent from cultivated or wild citrus genotypes. All these features are challenging for the incorporation of the desirable traits. In this regard, genetic engineering technologies offer a series of alternative approaches that allow overcoming the difficulties of conventional breeding programs. This review gives a detailed overview of the currently used strategies for the development of genetically modified citrus. We describe different aspects regarding genotype varieties used, including elite cultivars or extensively used scions and rootstocks. Furthermore, we discuss technical aspects of citrus genetic transformation procedures via Agrobacterium, regular physical methods, and magnetofection. Finally, we describe the selection of explants considering young and mature tissues, protoplast isolation, etc. We also address current protocols and novel approaches for improving the in vitro regeneration process, which is an important bottleneck for citrus genetic transformation. This review also explores alternative emerging transformation strategies applied to citrus species such as transient and tissue localized transformation. New breeding technologies, including cisgenesis, intragenesis, and genome editing by clustered regularly interspaced short palindromic repeats (CRISPR), are also discussed. Other relevant aspects comprising new promoters and reporter genes, marker-free systems, and strategies for induction of early flowering, are also addressed. We provided a future perspective on the use of current and new technologies in citrus and its potential impact on regulatory processes.Instituto de Biotecnología y Biología Molecula