Production of engineered long-life and male sterile Pelargonium plants

[EN] Background: Pelargonium is one of the most popular garden plants in the world. Moreover, it has a considerable economic importance in the ornamental plant market. Conventional cross-breeding strategies have generated a range of cultivars with excellent traits. However, gene transfer via Agrobacterium tumefaciens could be a helpful tool to further improve Pelargonium by enabling the introduction of new genes/traits. We report a simple and reliable protocol for the genetic transformation of Pelargonium spp. and the production of engineered long-life and male sterile Pelargonium zonale plants, using the pSAG12::ipt and PsEND1::barnase chimaeric genes respectively. Results: The pSAG12::ipt transgenic plants showed delayed leaf senescence, increased branching and reduced internodal length, as compared to control plants. Leaves and flowers of the pSAG12::ipt plants were reduced in size and displayed a more intense coloration. In the transgenic lines carrying the PsEND1::barnase construct no pollen grains were observed in the modified anther structures, which developed instead of normal anthers. The locules of sterile anthers collapsed 3¿4 days prior to floral anthesis and, in most cases, the undeveloped anther tissues underwent necrosis. Conclusion: The chimaeric construct pSAG12::ipt can be useful in Pelargonium spp. to delay the senescence process and to modify plant architecture. In addition, the use of engineered male sterile plants would be especially useful to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals and related plant species. These characteristics could be of interest, from a commercial point of view, both for pelargonium producers and consumers.This work was funded by grants AGL2009-13388-C03-01 and BIO2009-08134 from the Spanish Ministry of Science and Innovation (MICINN). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). In the past five years we have received funding from the Spanish Ministry of Science and Innovation (MICINN) and the article-processing charge will be pay with funds from two granted projects. The authors received salaries from two different institutions: The Polytechnic University of Valencia (UPV) or the High Spanish Council of Scientific Research (CSIC). We are not currently applying for a patent related with the content of this manuscript. All the mentioned organisms/institutions do not gain or lose financially from the publication of this manuscript either now or in the future.García Sogo, B.; Pineda Chaza, BJ.; Roque Mesa, EM.; Antón Martínez, MT.; Atarés Huerta, A.; Borja, M.; Beltran Porter, JP.... (2012). Production of engineered long-life and male sterile Pelargonium plants. BMC Plant Biology. 12:156-171. https://doi.org/10.1186/1471-2229-12-1561561711

Método para la obtención de cultivares de tomate con frutos partenocárpicos (sin semillas) y mayor calidad organoléptica

Método para la obtención de cultivares de tomate con frutos partenocárpicos (sin semillas) y mayor calidad organoléptica. El método se basa en la transferencia y expresión del gen LFY de Arabidopsis thaliana en plantas transgénicas de tomate. Los frutos de las plantas transgénicas con el gen LFY mantienen el mismo tamaño y peso que los del cultivar original, pero carecen de semillas, tienen más carne, menos pulpa y una forma ligeramente apuntillada. El análisis de calidad refleja un incremento del 60% en el contenido en sólidos solubles (la media alcanza 6,12 ºBrix) y del 60% en ácidos valorables (la media llega al 0,72%), lo que indica una mejora de la calidad organoléptica de los frutos en comparación con los del cultivar original no transgénico. Además, los frutos de las plantas transgénicas tienen otros atributos que indican una mayor calidad, tales como un mayor contenido en azúcares (sobre todo glucosa y fructosa) y licopeno, una sustancia que tiene propiedades antioxidantes.Peer reviewedUniversidad Politécnica de Valencia, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Consejo Superior de Investigaciones Científicas (España), Universidad de AlmeríaB2 Patente con examen previ

Nuevo cultivar con frutos y sépalos convertidos en frutos de alto interés para su consumo fresco y procesado industrial

Número de publicación: 2 341 527 21 Número de solicitud: 200900003 51 Int. Cl.: C12N 15/82 (2006.01) A01H 5/00 (2006.01Nuevo cultivar con frutos y sépalos convertidos en frutos de alto interés para su consumo fresco y procesado industrial. En la presente invención se describen secuencias de nucleótidos capaces de incrementar la expresión de un gen de desarrollo reproductivo lo que tiene como resultado la generación de cultivares con un fruto de alto interés para su consumo fresco y procesado industrial caracterizado por poseer características mejoradas respecto de los cultivares conocidos de variedades comerciales. Estos nuevos cultivares tienen el cáliz de la flor carnoso y convertido en fruto. El fruto verdadero y el cáliz tienen mayores niveles de azúcares y licopeno y un mayor contenido en grados Brix. Además, exhiben una mayor tasa de cuajado de fruto y tienen inhibida la zona de abscisión del fruto, lo que facilita la recolección mecánica.Universidad de Almerí

Método para la obtención de cultivares de tomate con frutos partenocárpicos (sin semillas) y mayor calidad organoléptica

Número de publicación: ES2323028 A1 (03.07.2009) También publicado como: ES2323028 B2 (08.07.2010) Número de Solicitud: Consulta de Expedientes OEPM (C.E.O.) P200501603 (23.06.2005)El método se basa en la transferencia y expresión del gen LFY de Arabidopsis thaliana en plantas transgénicas de tomate. Los frutos de las plantas transgénicas con el gen LFY mantienen el mismo tamaño y peso que los del cultivar original, pero carecen de semillas, tienen más carne, menos pulpa y una forma ligeramente apuntillada. El análisis de calidad refleja un incremento del 60 % en el contenido en sólidos solubles (la media alcanza 6,12 ºBrix) y del 60 % en ácidos valorables (la media llega al 0,72 %), lo que indica una mejora de la calidad organoléptica de los frutos en comparación con los del cultivar original no transgénico. Además, los frutos de las plantas transgénicas tienen otros atributos que indican una mayor calidad, tales como un mayor contenido en azúcares (sobre todo glucosa y fructosa) y licopeno, una sustancia que tiene propiedades antioxidantes.Universidad de Almería. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria. Consejo Superior de Investigaciones Científicas (CSIC

Alq mutation increases fruit set rate and allows the maintenance of fruit yield under moderate saline conditions

[EN] Arlequin (Alq) is a gain-of-function mutant whose most relevant feature is that sepals are able to become fruit-like organs due to the ectopic expression of the ALQ-TAGL1 gene. The role of this gene in tomato fruit ripening was previously demonstrated. To discover new functional roles for ALQ-TAGL1, and most particularly its involvement in the fruit set process, a detailed characterization of Alq yield-related traits was performed. Under standard conditions, the Alq mutant showed a much higher fruit set rate than the wild type. A significant percentage of Alq fruits were seedless. The results showed that pollination-independent fruit set in Alq is due to early transition from flower to fruit. Analysis of endogenous hormones in Alq suggests that increased content of cytokinins and decreased level of abscisic acid may account for precocious fruit set. Comparative expression analysis showed relevant changes of several genes involved in cell division, gibberellin metabolism, and the auxin signalling pathway. Since pollination-independent fruit set may be a very useful strategy for maintaining fruit production under adverse conditions, fruit set and yield in Alq plants under moderate salinity were assessed. Interestingly, Alq mutant plants showed a high yield under saline conditions, similar to that of Alq and the wild type under unstressed conditions.This work was supported by the research grants AGL2015-64991-C3-3-R and AGL2015-64991-C3-1-R from the Spanish Ministry of Economy and Competitiveness (MINECO/FEDER). The PhD grant to CRA (BES-2013-063778) was funded by the Spanish Ministry of Economy and Competitiveness.The authors thank Dr Isabel Lopez-Diaz and Dr Esther Carrera for their help in hormone quantification carried out at the Plant Hormone Quantification Service, IBMCP,Valencia, Spain. The authors thank David Harry Rhead for reviewing the manuscript in the English language.Ribelles Alfonso, C.; García Sogo, B.; Yuste-Lisbona, FJ.; Atarés Huerta, A.; Castañeda, L.; Capel, C.; Lozano, R.... (2019). Alq mutation increases fruit set rate and allows the maintenance of fruit yield under moderate saline conditions. Journal of Experimental Botany. 70(20):5731-5744. https://doi.org/10.1093/jxb/erz342S57315744702

Tomato CRABS CLAW paralogues interact with chromatin remodelling factors to mediate carpel development and floral determinacy

CRABS CLAW (CRC) orthologues play a crucial role in floral meristem (FM) determinacy and gynoecium formation across angiosperms, the key developmental processes for ensuring successful plant reproduction and crop production. However, the mechanisms behind CRC mediated FM termination are far from fully understood. Here, we addressed the functional characterization of tomato (Solanum lycopersicum) paralogous CRC genes. Using mapping-by-sequencing, RNA interference and CRISPR/Cas9 techniques, expression analyses, protein-protein interaction assays and Arabidopsis complementation experiments, we examined their potential roles in FM determinacy and carpel formation. We revealed that the incomplete penetrance and variable expressivity of the indeterminate carpel-inside-carpel phenotype observed in fruit iterative growth (fig) mutant plants are due to the lack of function of the S. lycopersicum CRC homologue SlCRCa. Furthermore, a detailed functional analysis of tomato CRC paralogues, SlCRCa and SlCRCb, allowed us to propose that they operate as positive regulators of FM determinacy by acting in a compensatory and partially redundant manner to safeguard the proper formation of flowers and fruits. Our results uncover for the first time the physical interaction of putative CRC orthologues with members of the chromatin remodelling complex that epigenetically represses WUSCHEL expression through histone deacetylation to ensure the proper termination of floral stem cell activity.Peer reviewe

The sodium transporter encoded by the HKT1;2 gene modulates sodium/potassium homeostasis in tomato shoots under salinity

[EN] Excessive soil salinity diminishes crop yield and quality. In a previous study in tomato, we identified two closely linked genes encoding HKT1-like transporters, HKT1;1 and HKT1;2, as candidate genes for a major quantitative trait locus (kc7.1) related to shoot Na+/K+ homeostasis - a major salt tolerance trait - using two populations of recombinant inbred lines (RILs). Here, we determine the effectiveness of these genes in conferring improved salt tolerance by using two near-isogenic lines (NILs) that were homozygous for either the Solanum lycopersicum allele (NIL17) or for the Solanum cheesmaniae allele (NIL14) at both HKT1 loci; transgenic lines derived from these NILs in which each HKT1;1 and HKT1;2 had been silenced by stable transformation were also used. Silencing of ScHKT1;2 and SlHKT1;2 altered the leaf Na+/K+ ratio and caused hypersensitivity to salinity in plants cultivated under transpiring conditions, whereas silencing SlHKT1;1/ScHKT1;1 had a lesser effect. These results indicate that HKT1;2 has the more significant role in Na+ homeostasis and salinity tolerance in tomato.We thank Dr Espen Granum for critically reading the manuscript, Maria Isabel Gaspar Vidal and Elena Sanchez Romero for technical assistance, the Instrumental Technical Service at EEZ-CSIC for DNA sequencing and ICP-OES mineral analysis and Michael O'Shea for proofreading the text. In addition, we thank Dr Ana P. Ortega who assisted in preliminary experiments. This work was supported by ERDF-cofinanced grants, AGL2010-17090 and AGL2013-41733-R (A.B.), AGL2015-64991-C3-3-R (V.M.) and AGL2014-56675-R (M.J.A.) from the Spanish "Ministerio de Economia, Industria y Competitividad'; CVI-7558, Proyecto de Excelencia, from Junta de Andalucia (A.B); and the Australian Research Council (ARC) for Centre of Excellence (CE14010008) and Future Fellowship (FT130100709) funding (M.G.). N.J-P. was supported by an FPI program BES-2011-046096 and her stay in M.G.'s lab by a short-stay EEBB-I-14-08682, both from the Spanish from "Ministerio de Economia Industria y Competitividad'. The authors have no conflict of interest to declare.Jaime-Perez, N.; Pineda Chaza, BJ.; García Sogo, B.; Atarés Huerta, A.; Athman, A.; Byrt, CS.; Olias, R.... (2017). The sodium transporter encoded by the HKT1;2 gene modulates sodium/potassium homeostasis in tomato shoots under salinity. Plant Cell & Environment. 40(5):658-671. https://doi.org/10.1111/pce.12883S65867140

Approaching the genetic dissection of indirect adventitious organogenesis process in tomato explants

[EN] The screening of 862 T-DNA lines was carried out to approach the genetic dissection of indirect adventitious organogenesis in tomato. Several mutants defective in different phases of adventitious organogenesis, namely callus growth (tdc-1), bud differentiation (tdb-1,-2,-3) and shoot-bud development (tds-1) were identified and characterized. The alteration of the TDC-1 gene blocked callus proliferation depending on the composition of growth regulators in the culture medium. Calli from tds-1 explants differentiated buds but did not develop normal shoots. Histological analysis showed that their abnormal development is due to failure in the organization of normal adventitious shoot meristems. Interestingly, tdc-1 and tds-1 mutant plants were indistinguishable from WT ones, indicating that the respective altered genes play specific roles in cell proliferation from explant cut zones (TDC-1 gene) or in the organization of adventitious shoot meristems (TDS-1 gene). Unlike the previous, plants of the three mutants defective in the differentiation of adventitious shoot-buds (tdb-1,-2,-3) showed multiple changes in vegetative and reproductive traits. Cosegregation analyses revealed the existence of an association between the phenotype of the tdb-3 mutant and a T-DNA insert, which led to the discovery that the SlMAPKKK17 gene is involved in the shoot-bud differentiation process.Vicente Moreno and Rafael Lozano thank the Ministry of Science and Innovation (State Innovation Agency) for granting the projects PID2019-110833RB-C32 and PID2019-110833RB-C31. Benito Pineda's work in the context of this article has been funded by 'Aid for First Research Projects (PAID-06-18)' by the Vicerrectorado de Investigacion, Innovacion y Transferencia de la Universitat Politecnica de Valencia (UPV), Valencia, Spain'. The PhD fellowship for Jorge Sanchez-Lopez and Marybel Jaquez-Gutierrez were funded by the Universidad de Sinaloa and the CONACYT of Mexico.Sanchez-Lopez, J.; Atarés Huerta, A.; Jaquez-Gutierrez, M.; Ortiz-Atienza, A.; Capel, C.; Pineda Chaza, BJ.; García Sogo, B.... (2021). Approaching the genetic dissection of indirect adventitious organogenesis process in tomato explants. Plant Science. 302:1-14. https://doi.org/10.1016/j.plantsci.2020.110721S11430

Albino T-DNA tomato mutant reveals a key function of 1-deoxy-D-xylulose-5-phosphate synthase (DXS1) in plant development and survival

[EN] Photosynthetic activity is indispensable for plant growth and survival and it depends on the synthesis of plastidial isoprenoids as chlorophylls and carotenoids. In the non-mevalonate pathway (MEP), the 1-deoxy-D-xylulose-5-phosphate synthase 1 (DXS1) enzyme has been postulated to catalyze the ratelimiting step in the formation of plastidial isoprenoids. In tomato, the function of DXS1 has only been studied in fruits, and hence its functional relevance during plant development remains unknown. Here we report the characterization of the wls-2297 tomato mutant, whose severe deficiency in chlorophylls and carotenoids promotes an albino phenotype. Additionally, growth of mutant seedlings was arrested without developing vegetative organs, which resulted in premature lethality. Gene cloning and silencing experiments revealed that the phenotype of wls-2297 mutant was caused by 38.6 kb-deletion promoted by a single T-DNA insertion affecting the DXS1 gene. This was corroborated by in vivo and molecular complementation assays, which allowed the rescue of mutant phenotype. Further characterization of tomato plants overexpressing DXS1 and comparative expression analysis indicate that DXS1 may play other important roles besides to that proposed during fruit carotenoid biosynthesis. Taken together, these results demonstrate that DXS1 is essentially required for the development and survival of tomato plants.This work was supported by research grants from the Spanish Ministry of Economy and Competitiveness and the UE-European Regional Development Fund (AGL2015-64991-C3-1-R, and AGL2015-64991-C3-3-R), and Junta de Andalucia (P12-AGR-1482). PhD fellowship to M.G.-A. was funded by the FPU Programme of the Spanish Ministry of Science and Innovation. The authors thank research facilities provided by the Campus de Excelencia Internacional Agroalimentario (CeiA3).Garcia-Alcazar, M.; Giménez Caminero, ME.; Pineda Chaza, BJ.; Capel, C.; García Sogo, B.; Sánchez Martín-Sauceda, S.; Yuste-Lisbona, FJ.... (2017). Albino T-DNA tomato mutant reveals a key function of 1-deoxy-D-xylulose-5-phosphate synthase (DXS1) in plant development and survival. Scientific Reports. 7:1-12. https://doi.org/10.1038/srep45333112

ENO regulates tomato fruit size through the floral meristem development network

A dramatic evolution of fruit size has accompanied the domestication and improvement of fruit-bearing crop species. In tomato (Solanum lycopersicum), naturally occurring cis-regulatory mutations in the genes of the CLAVATA-WUSCHEL signaling pathway have led to a significant increase in fruit size generating enlarged meristems that lead to flowers with extra organs and bigger fruits. In this work, by combining mapping-by-sequencing and CRISPR/Cas9 genome editing methods, we isolated EXCESSIVE NUMBER OF FLORAL ORGANS (ENO), an AP2/ERF transcription factor which regulates floral meristem activity. Thus, the ENO gene mutation gives rise to plants that yield larger multilocular fruits due to an increased size of the floral meristem. Genetic analyses indicate that eno exhibits synergistic effects with mutations at the LOCULE NUMBER (encoding SlWUS) and FASCIATED (encoding SlCLV3) loci, two central players in the evolution of fruit size in the domestication of cultivated tomatoes. Our findings reveal that an eno mutation causes a substantial expansion of SlWUS expression domains in a flower-specific manner. In vitro binding results show that ENO is able to interact with the GGC-box cis-regulatory element within the SlWUS promoter region, suggesting that ENO directly regulates SlWUS expression domains to maintain floral stem-cell homeostasis. Furthermore, the study of natural allelic variation of the ENO locus proved that a cis-regulatory mutation in the promoter of ENO had been targeted by positive selection during the domestication process, setting up the background for significant increases in fruit locule number and fruit size in modern tomatoes