Introduction of the Nicotiana protein kinase (NPK1) gene by combiningAgrobacterium-mediated transformation and recurrent somatic embryogenesis to enhance salt tolerance in cauliflower

Abstract

Key message The results underlined that constitutively expressingNPK1can significantly contribute to enhanced salt stress tolerance in cauliflower, suggesting that this could be a promising basis for the creation of new stress tolerance cruciferous vegetable lines. 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 ofNPK1transgene to NaCl salt stress tolerance in cauliflower KFRM4 lines was studied. TheAgrobacterium tumefaciens-mediated transformation protocol, using EHA101(pSHX004) vector harbouring theNPK1and phosphinothricinN-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 bothNPK1andbartransgenes 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 ofNPK1gene expression estimated by RT-qPCR, and the L2 line with the highestNPK1expression 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 thebargene 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.