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

A highly efficient organogenesis protocol based on zeatin riboside for in vitro regeneration of eggplant

[EN] Background Efficient organogenesis induction in eggplant (Solanum melongena L.) is required for multiple in vitro culture applications. In this work, we aimed at developing a universal protocol for efficient in vitro regeneration of eggplant mainly based on the use of zeatin riboside (ZR). We evaluated the effect of seven combinations of ZR with indoleacetic acid (IAA) for organogenic regeneration in five genetically diverse S. melongena and one S. insanum L. accessions using two photoperiod conditions. In addition, the effect of six different concentrations of indolebutyric acid (IBA) in order to promote rooting was assessed to facilitate subsequent acclimatization of plants. The ploidy level of regenerated plants was studied. Results In a first experiment with accessions MEL1 and MEL3, significant (p < 0.05) differences were observed for the four factors evaluated for organogenesis from cotyledon, hypocotyl and leaf explants, with the best results obtained (9 and 11 shoots for MEL1 and MEL3, respectively) using cotyledon tissue, 16 h light / 8 h dark photoperiod conditions, and medium E6 (2 mg/L of ZR and 0 mg/L of IAA). The best combination of conditions was tested in the other four accessions and confirmed its high regeneration efficiency per explant when using both cotyledon and hypocotyl tissues. The best rooting media was R2 (1 mg/L IBA). The analysis of ploidy level revealed that between 25 and 50% of the regenerated plantlets were tetraploid. Conclusions An efficient protocol for organogenesis of both cultivated and wild accessions of eggplant, based on the use of ZR, is proposed. The universal protocol developed may be useful for fostering in vitro culture applications in eggplant requiring regeneration of plants and, in addition, allows developing tetraploid plants without the need of antimitotic chemicals.This research was funded by the Spanish Ministerio de Ciencia, Innovacion y Universidades, Agencia Estatal de Investigacion and Fondo Europeo de Desarrollo Regional (grant RTI-2018-094592-B-100 from MCIU/AEI/FEDER, UE) and by Universitat Politecnica de Valencia. The Spanish Ministerio de Educacion, Cultura y Deporte funded a predoctoral fellowship granted to Edgar Garcia-Fortea (FPU17/02389). The Generalitat Valenciana and Fondo Social Europeo funded a post-doctoral fellowship granted to Mariola Plazas (APOSTD/2018/014). The Japan Society for the Promotion of Science funded a post-doctoral fellowship granted to Pietro Gramazio (FY 2019 Postdoctoral Fellowship for Research in Japan [Standard]). The funding bodies were not involved in the design of the study, collection, analysis, interpretation of data, or drafting of the manuscript.García-Fortea, E.; Lluch-Ruiz, A.; Pineda Chaza, BJ.; García-Pérez, A.; Bracho-Gil, JP.; Plazas Ávila, MDLO.; Gramazio, P.... (2020). A highly efficient organogenesis protocol based on zeatin riboside for in vitro regeneration of eggplant. BMC Plant Biology. 20(1):1-16. https://doi.org/10.1186/s12870-019-2215-yS116201FAO. FAOSTAT Food and Agriculture. 2019. http://www.fao.org/faostat. Accessed 18 July 2019.Gürbüza N, Uluişikb S, Frarya A, Frary A, Doğanlar S. Health benefits and bioactive compounds of eggplant. Food Chem. 2018;268:602–10. https://doi.org/10.1016/j.foodchem.2018.06.093.Rivas-Sendra A, Corral-Martínez P, Camacho-Fernández C, Seguí-Simarro JM. Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis. Plant Cell Tissue Organ Cult. 2015;122:759–65. https://doi.org/10.1007/s11240-015-0791-6.Shelton AM, Hossain MJ, Paranjape V, Azad AK, Rahman ML, Khan ASMMR, Prodhan MZH, Rashid MA, Majumder R, Hossain MA, Hussain SS, Huesing JE, McCandless L. Bt eggplant project in Bangladesh: history, present status, and future direction. Front Bioeng Biotechnol. 2018;6:106. https://doi.org/10.3389/fbioe.2018.00106.Muren RC. Haploid plant induction from unpollinated ovaries in onion. Hortscience. 1989;24:833–4.Campion B, Bohanec B, Javornik B. Gynogenic lines of onion (Allium cepa L.): evidence of their homozygosity. Theor Appl Genet. 1995;91:598–602. https://doi.org/10.1007/BF00223285.Geoffriau E, Kahane R, Rancillac M. Variation of gynogenesis ability in onion (Allium cepa L.). Euphytica. 1997;94:37–44. https://doi.org/10.1023/A:1002949606450.Cardoso JC, Teixeira da Silva JA. Gerbera micropropagation. Biotechnol Adv. 2013;31:1344–57. https://doi.org/10.1016/J.BIOTECHADV.2013.05.008.Gleddie S, Keller W, Setterfield G. Somatic embryogenesis and plant regeneration from leaf explants and cell suspensions of Solanum melongena (eggplant). Can J Bot. 1983;61:656–66. https://doi.org/10.1139/b83-074.Sharma P, Rajam MV. Genotype, explant and position effects on organogenesis and somatic embryogenesis in eggplant ( Solanum melongena L.). J Exp Bot. 1995;46:135–41. https://doi.org/10.1093/jxb/46.1.135.Franklin G, Sheeba CJ, Lakshmi SG. Regeneration of eggplant (Solanum melongena L.) from root explants. Vitr Cell Dev Biol – Plant. 2004;40:188–91. https://doi.org/10.1079/IVP2003491.Taher D, Solberg S, Prohens J, Chou Y, Rakha M, Wu T. World vegetable center eggplant collection: origin, composition, seed dissemination and utilization in breeding. Front Plant Sci. 2017;8:1484. https://doi.org/10.3389/fpls.2017.01484.Altpeter F, Springer NM, Bartley LE, Blechl AE, Brutnell TP, Citovsky V, Conrad LJ, Gelvin SB, Jackson DP, Kausch AP, Lemaux PG, Medford JI, Orozco-Cárdenas ML, Tricoli DM, Van Eck J, Voytas DF, Walbot V, Wang K, Zhang ZJ, Stewart CN. Advancing crop transformation in the era of genome editing. Plant Cell. 2016;28:1510–20. https://doi.org/10.1105/tpc.16.00196.Haque E, Taniguchi H, Hassan MM, Bhowmik P, Karim MR, Śmiech M, Zhao K, Rahman M, Islam T. Application of CRISPR/Cas9 genome editing technology for the improvement of crops cultivated in tropical climates: recent progress, prospects, and challenges. Front Plant Sci. 2018;9:617. https://doi.org/10.3389/fpls.2018.00617.Limera C, Sabbadini S, Sweet JB, Mezzetti B. New biotechnological tools for the genetic improvement of major woody fruit species. Front Plant Sci. 2017;8:1418. https://doi.org/10.3389/fpls.2017.01418.Gilissen LJW, van Staveren MJ, Creemers-Molenaar J, Verhoeven HA. Development of polysomaty in seedlings and plants of Cucumis sativus L. Plant Sci. 1993;91:171–9. https://doi.org/10.1016/0168-9452(93)90140-U.Smulders MJM, Rus-Kortekaas W, Gilissen LJW. Development of polysomaty during differentiation in diploid and tetraploid tomato (Lycopersicon esculentum) plants. Plant Sci. 1994;97:53–60. https://doi.org/10.1016/0168-9452(94)90107-4.Mishiba KI, Mii M. Polysomaty analysis in diploid and tetraploid Portulaca grandiflora. Plant Sci. 2000;156:213–9. https://doi.org/10.1016/S0168-9452(00)00257-0.Meric C, Dane F. Determination of ploidy levels in Ipheion uniflorum (R. C. Graham) Rafin (Liliaceae). Acta Biol Hung. 2005;56:129–36. https://doi.org/10.1556/ABiol.56.2005.1-2.13.Letham DS. Purification and probable identity of a new cytokinin in sweet corn extracts. Life Sci. 1966;5:551–4. https://doi.org/10.1016/0024-3205(66)90175-5.Narasimhulu SB, Kirti PB, Prakash S, Chopra VL. Rapid and high frequency shoot regeneration from hypocotyl protoplasts of Brassica nigra. Plant Cell Tissue Organ Cult. 1993;32:35–9. https://doi.org/10.1007/BF00040113.Bhadra SK, Hammatt N, Power JB, Davey MR. A reproducible procedure for plant regeneration from seedling hypocotyl protoplasts of Vigna sublobata L. Plant Cell Rep. 1994;14:175–9. https://doi.org/10.1007/BF00233785.Hossain M, Imanishi S, Egashira H. An improvement of tomato protoplast culture for rapid plant regeneration. PCTOC. 1995;42:141–6. https://doi.org/10.1007/BF00034230.Yadav NR, Sticklen MB. Direct and efficient plant regeneration from leaf explants of Solanum tuberosum l. cv. Bintje. Plant Cell Rep. 1995;14:645–7. https://doi.org/10.1007/BF00232730.Chen L, Adachi T. Plant regeneration via somatic embryogenesis from cotyledon protoplast of tomato (Lycopersicon esculentum Mill.). Breed Sci. 1994;44:257–62. https://doi.org/10.1270/jsbbs1951.44.257.Richwine AM, Tipton JL, Thompson GA. Establishment of aloe, gasteria, and haworthia shoot cultures from inflorescence explants. HortScience. 1995;30:1443–4. https://doi.org/10.21273/HORTSCI.30.7.1443.Rolli E, Brunoni F, Bruni R. An optimized method for in vitro propagation of african baobab (Adansonia digitata L.) using two-node segments. Plant Biosyst. 2016;150:750–6. https://doi.org/10.1080/11263504.2014.991362.Farooq QUA, Fatima A, Murtaza N, Hussain FF. In vitro propagation of olive cultivars ‘Frontio’, ‘Earlik’, ‘Gemlik’. Acta Hortic. 2017:249–56. https://doi.org/10.17660/ActaHortic.2017.1152.34.Singh AK, Verma SS, Bansal KC. Plastid transformation in eggplant (Solanum melongena L.). Transgenic Res. 2010;19:113–9. https://doi.org/10.1007/s11248-009-9290-z.Muktadir MA, Habib MA, Khaleque Mian MA, Yousuf Akhond MA. Regeneration efficiency based on genotype, culture condition and growth regulators of eggplant (Solanum melongena L.). Agric Nat Resour. 2016;50:38–42. https://doi.org/10.1016/J.ANRES.2014.10.001.Rotino GL. Haploidy in eggplant. Dordrecht: Springer; 1996. p. 115–41. https://doi.org/10.1007/978-94-017-1858-5_8.Emrani Dehkehan M, Moieni A, Movahedi Z. Effects of zeatin riboside, mannitol and heat stress on eggplantn (Solanum melongena L.) anther culture. Imam Khomeini Int Univ Biotechnol Soc. 2017;6:16–26. https://doi.org/10.30479/IJGPB.2017.1370.Magioli C, de Oliveira DE, Rocha APM, Mansur E. Efficient shoot organogenesis of eggplant ( Solanum melongena L.) induced by thidiazuron. Plant Cell Rep. 1998;17:661–3. https://doi.org/10.1007/s002990050461.Scoccianti V, Sgarbi E, Fraternale D, Biondi S. Organogenesis from Solanum melongena l. (eggplant) cotyledon explants is associated with hormone-modulated enhancement of polyamine biosynthesis and conjugation. Protoplasma. 2000;211:51–63. https://doi.org/10.1007/BF01279899.Rahman M, Asaduzzaman M, Nahar N, Bari M. Efficient plant regeneration from cotyledon and midrib derived callus in eggplant (Solanum melongena L.). J Bio-Science. 2006;14:31–8. https://doi.org/10.3329/jbs.v14i0.439.Bhat SV, Jadhav A, Pawar BD, Kale AA, Chimote V, Pawar SV. In vitro shoot organogenesis and plantlet regeneration in brinjal (Solanum melongena L.). N Save Nat to Surviv. 2013;8:821–4.Swathy PS, Rupal G, Prabhu V, Mahato KK, Muthusamy A. In vitro culture responses, callus growth and organogenetic potential of brinjal (Solanum melongena L.) to he-ne laser irradiation. J Photochem Photobiol B Biol. 2017;174:333–41. https://doi.org/10.1016/j.jphotobiol.2017.08.017.Acquadro A, Barchi L, Gramazio P, Portis E, Vilanova S, Comino C, et al. Coding SNPs analysis highlights genetic relationships and evolution pattern in eggplant complexes. PLoS One. 2017;12:e0180774. https://doi.org/10.1371/journal.pone.0180774.Ranil RHG, Prohens J, Aubriot X, Niran HML, Plazas M, Fonseka RM, Vilanova S, Fonseka HH, Gramazio P, Knapp S. Solanum insanum L. (subgenus Leptostemonum bitter, Solanaceae), the neglected wild progenitor of eggplant (S. melongena L.): a review of taxonomy, characteristics and uses aimed at its enhancement for improved eggplant breeding. Genet Resour Crop Evol. 2017;64:1707–22. https://doi.org/10.1007/s10722-016-0467-z.Souza FVD. Garcia-Sogo B, Souza AS, San-Juán AP, Moreno V. Morphogenetic response of cotyledon and leaf explants of melon (Cucumis melo L.) cv. Amarillo Oro. Braz Arch Biol Technol. 2006;49:21–7. https://doi.org/10.1590/S1516-89132006000100003.Abdalmajid M, Mohd RI, Mihdzar AK, Halimi MS. In vitro performances of hypocotyl and cotyledon explants of tomato cultivars under sodium chloride stress. Afr J Biotechnol. 2011;10:8757–64. https://doi.org/10.5897/AJB10.2222.Matand K, Wu N, Wu H, Tucker E, Love K. More improved peanut (Arachis hypogaea L.) protocol for direct shoot organogenesis in mature dry-cotyledonary and root tissues. J Biotech Res. 2013;5:24–34.Pierik RLM. In vitro culture of higher plants. Dordrecht: Kluwer Academic Publishers; 1997.Waman AA, Bohra P, Sathyanarayana BN, Umesha K, Mukunda GK, Ashok TH, Gowda B. Optimization of factors affecting in vitro establishment, ex vitro rooting and hardening for commercial scale multiplication of silk banana (Musa aab). Erwerbs-Obstbau. 2015;57:153–64. https://doi.org/10.1007/s10341-015-0244-8.Sarker R, Yesmin S, Hoque M. Multiple shoot formation in eggplant (Solanum melongena L.). Plant Tissue Cult Biotechnol. 2006;16:53–61. https://doi.org/10.3329/ptcb.v16i1.1106.Van Den Bulk RW, Lgffler HJM, Lindhout WH, Koornneef M. Somaclonal variation in tomato: effect of explant source and a comparison with chemical mutagenesis. Theor Appl Genet. 1990;80:817–25. https://doi.org/10.1007/BF00224199.Chen W, Tang CY, Kao YL. Ploidy doubling by in vitro culture of excised protocorms or protocorm-like bodies in Phalaenopsis species. Plant Cell Tissue Organ Cult. 2009;98:229–38. https://doi.org/10.1007/s11240-009-9557-3.Syfert MM, Castaneda-Alvarez NP, Khoury CK, Sarkinen T, Sosa CC, Achicanoy HA, Bernau V, Prohens J, Daunay MC, Knapp S. Crop wild relatives of the brinjal eggplant (Solanum melongena): Poorly represented in genebanks and many species at risk of extinction. Am J Bot. 2016;103:635–51. https://doi.org/10.3732/ajb.1500539.Muñoz-Falcón JE, Prohens J, Vilanova S, Nuez F. Diversity in commercial varieties and landraces of black eggplants and implications for broadening the breeders’ gene pool. Ann Appl Biol. 2009;154:453–65. https://doi.org/10.1111/j.1744-7348.2009.00314.x.Kaushik P, Prohens J, Vilanova S, Gramazio P, Plazas M. Phenotyping of eggplant wild relatives and interspecific hybrids with conventional and phenomics descriptors provides insight for their potential utilization in breeding. Front Plant Sci. 2016;7:677. https://doi.org/10.3389/fpls.2016.00677.Plazas M, Vilanova S, Gramazio P, Rodriguez-Burruezo A, Rajakapasha R, Ramya F, Niran L, Fonseka H, Kouassi B, Kouassi A, Kouassi A, Prohens J. Interspecific hybridization between eggplant and wild relatives from different genepools. J Am Soc Hortic Sci. 2016;141:34–44. https://doi.org/10.21273/JASHS.141.1.34.Kouassi B, Prohens J, Gramazio P, Kouassi AB, Vilanova S, Galán-Ávila A, Herraiz FJ, Kouassi A, Seguí-Simarro JM, Plazas M. Development of backcross generations and new interspecific hybrid combinations for introgression breeding in eggplant (Solanum melongena). Sci Hortic (Amsterdam). 2016;213:199–207. https://doi.org/10.1016/J.SCIENTA.2016.10.039.García-Fortea E, Gramazio P, Vilanova S, Fita A, Mangino G, Villanueva G, Arrones A, Knapp S, Prohens J, Plazas M. First successful backcrossing towards eggplant (Solanum melongena ) of a New World species, the silverleaf nightshade (S. elaeagnifolium ), and characterization of interspecific hybrids and backcrosses. Sci Hortic. 2019;246:563–73. https://doi.org/10.1016/j.scienta.2018.11.018.Murashige T, Skoog F. A revised medium for rapid growth and bio agsays with tobacco tissue cultures. Physiol Plant. 1962;15:473–9.Dpooležel J, Binarová P, Lcretti S. Analysis of nuclear DNA content in plant cells by flow cytometry. Biol Plant. 1989;31:113–20. https://doi.org/10.1007/BF02907241.Ihaka R, Gentleman R. R: a language for data analysis and graphics. J Comput Graph Stat. 1996;5:299–314. https://doi.org/10.1080/10618600.1996.10474713

Abamectin and emamectin in grapes of Vitis vinifera L. from a district of the Valley of Ica-Peru

Context: In 14 districts of the valley of Ica-Peru, Vitis vinifera L. plants are cultivated that produce grapes for consumption as table grapes and raisins (dried grapes); at the same time, for the production of wines and Piscos. Aims: To determine the levels of abamectin and emamectin in grapes of Vitis vinifera L from a district of the Valley of Ica-Peru. Methods: 30 lots (30 kg) of Moscatel grape variety V. vinifera L. were collected from six countryside (artisanal and organic cultivation) of the San Juan Bautista district. The extraction of abamectin (ABM) and emamectin benzoate (EMB) from the grapes was carried out with acetonitrile; it was quantified by means of Liquid Chromatography coupled to Mass Spectrometry (HPLC-MS). The maximum permissible limit values (MRL) were established at 0.010 ppm for both insecticides. Results: The determined levels of abamectin and emamectin in grapes were 0.0012-0.015 ppm and 0.0013-0.013 ppm, respectively. Values higher than the maximum permissible limits of abamectin were found in batches A2 (0.0102 ppm), C1 (0.015 ppm), C5 (0.0113 ppm), and F2 (0.012 ppm); emamectin benzoate in lots B1 (0.0113 ppm), B4 (0.013 ppm) and C4 (0.012 ppm). Using the Shapiro-Wilk, Anderson Darling and Student’s t tests, it was found that the global means of the values of the two insecticides in grapes are lower than the MRL. According to the global analysis of variance, the means of the concentrations of both insecticides were not different between the six sampling zones (countryside). Conclusions: The insecticides abamectin and emamectin are below the maximum permissible limit values (0.010 ppm) in Moscatel grapes of Vitis vinifera L., so the residual effect would not have implications for human health

Constraints on the χ_(c1) versus χ_(c2) polarizations in proton-proton collisions at √s = 8 TeV

The polarizations of promptly produced χ_(c1) and χ_(c2) mesons are studied using data collected by the CMS experiment at the LHC, in proton-proton collisions at √s=8  TeV. The χ_c states are reconstructed via their radiative decays χ_c → J/ψγ, with the photons being measured through conversions to e⁺e⁻, which allows the two states to be well resolved. The polarizations are measured in the helicity frame, through the analysis of the χ_(c2) to χ_(c1) yield ratio as a function of the polar or azimuthal angle of the positive muon emitted in the J/ψ → μ⁺μ⁻ decay, in three bins of J/ψ transverse momentum. While no differences are seen between the two states in terms of azimuthal decay angle distributions, they are observed to have significantly different polar anisotropies. The measurement favors a scenario where at least one of the two states is strongly polarized along the helicity quantization axis, in agreement with nonrelativistic quantum chromodynamics predictions. This is the first measurement of significantly polarized quarkonia produced at high transverse momentum