Introduction of the Nicotiana protein kinase (NPK1) gene by combining Agrobacterium -mediated transformation and recurre
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ORIGINAL ARTICLE
Introduction of the Nicotiana protein kinase (NPK1) gene by combining Agrobacterium‑mediated transformation and recurrent somatic embryogenesis to enhance salt tolerance in cauliflower Suzana Pavlović1 · Jelena Savić2 · Jelena Milojević2 · Branka Vinterhalter2 · Zdenka Girek1 · Slađan Adžić1 · Bogoljub Zečević1 · Nevena Banjac2 Received: 27 July 2020 / Accepted: 10 October 2020 © Springer Nature B.V. 2020
Abstract Cauliflower is exposed to various biotic and abiotic stresses, including increased salinity due to the intensive irrigation of crops. Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules that play important roles in regulating innate immune responses in plants. Based on involvement of tobacco MAP kinase kinase kinase (NPK1) in stress response, the effect of the expression of NPK1 transgene to NaCl salt stress tolerance in cauliflower KFRM4 lines was studied. The Agrobacterium tumefaciens-mediated transformation protocol, using EHA101(pSHX004) vector harbouring the NPK1 and phosphinothricin N-acetyltransferase (bar) genes, the cyclic somatic embryogenesis regeneration pathway, the application of acetosyringone (AS) during co-cultivation and a delayed phosphinothricine (PPT) selection procedure provided sufficient transformation efficiency of 7.33% without escapes. PCR analysis indicated the integration of both NPK1 and bar transgenes in regenerated cauliflower lines. Transgenic cauliflower lines, exposed to NaCl stress in vitro, showed higher growth rates, greater ability to retain chlorophyll and carotenoids, and increased osmotic regulation capacity compared with non-transformed control plants. The tolerance level of transformed lines correlated with the level of NPK1 gene expression estimated by RT-qPCR, and the L2 line with the highest NPK1 expression displayed the greatest tolerance to NaCl stress. None of the obtained cauliflower transformed lines grown in greenhouses showed any morphological or yield differences compared with non-transformed plants. Furthermore, the expression of the bar gene facilitated the tolerance of transformed lines to the total herbicide PPT, applied at concentrations 2–3 times higher than those routinely used for weed control in the crop field. Key message The results underlined that constitutively expressing NPK1 can significantly contribute to enhanced salt stresstolerance in cauliflower, suggesting that this could be a promising basis for the creation of new stress tolerancecruciferous vegetable lines. Keywords Brassica oleracea var. botrytis l. · Cyclic somatic embryogenesis · Agrobacterium tumefaciens-mediated genetic transformation · NPK1 gene · Herbicide resistance · Salt stress
Introduction Communicated by Sergio J. Ochatt. * Suzana Pavlović [email protected] 1
Institute for Vegetable Crops, Karađorđeva 71, Smederevska Palanka 11420, Serbia
Institute for Biological Research “Siniša Stanković”, University of Belgrade, Despot Stefan Boulevard 142, Belgrade 11000, Serbia
2
Cauliflower (Brassica
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