PLANT TISSUE CULTURE Improved Agrobacterium tumefaciens-mediated transformation of soybean [Glycine max (L.) Merr.] following optimization of culture conditions and mechanical techniques Alkesh Hada 1,2 & Veda Krishnan 1 & M. S. Mohamed Jaabir 2 & Archana Kumari 3 & Monica Jolly 1 & Shelly Praveen 1 & Archana Sachdev 1 Received: 22 August 2017 /Accepted: 26 September 2018 / Editor: John Finer # The Society for In Vitro Biology 2018 Abstract In the present study, Agrobacterium tumefaciens-mediated transformation of Glycine max (L.) Merr. (soybean) cv. DS-9712 using half-seed explants was optimized for eight different parameters, including seed imbibition, medium pH, infection mode (sonication and vacuum infiltration), co-cultivation conditions, concentrations of supplementary compounds, and selection. Using this improved protocol, maximum transformation of 14% and regeneration efficiencies of 45% were achieved by using explants prepared from mature seeds imbibed for 36 h, infected with A. tumefaciens strain EHA105 at an optical density (OD 600 ) of 0.8, suspended in pH 5.4 medium containing 0.2 mM acetosyringone and 450 mg L -1 L-cysteine, followed by sonication for 10 s, vacuum infiltration for 2 min, and co-cultivated for 3 d on 35 mg L -1 kanamycin-containing medium. Independent transgenic lines were confirmed to be transgenic after ß-glucuronidase histochemical assays, polymerase chain reaction, and southern hybridization analysis. The protocol developed in the present study showed high regeneration efficiency within a relatively short time of 76 d. This rapid and efficient protocol might overcome some hurdles associated with the genetic manipulation of soybean. Keywords Agrobacterium tumefaciens . Half-seed explants . Soybean transformation . Regeneration Introduction Glycine max (L.) Merr. (soybean) is widely used as an impor- tant source of protein, oil, and meal for both human and ani- mal consumption, as well as for other industrial uses; there- fore, genetic improvement of soybean is a worldwide goal. Significant efforts to add desired traits into elite cultivars have been expended to improve the quality of cultivated soybean varieties by conventional breeding, as well as through biotech- nological approaches. Using comparative genomics, informa- tion flow about this legume has accelerated exponentially with the discovery of genes responsible for biotic/abiotic resistance and enhanced nutritional value; thus, making soybean serve as a Bmodel plant for functional genomics in legumes,^ in addi- tion to being of great economic relevance. However, the acute obstacle, even after two decades of concentrated effort, lies in the lack of robust transformation and regeneration protocols for large-scale production of elite transgenic cultivars. To realize the potential of biotechnology in developing stable soybean transgenics having novel desirable traits and to study the transgene regulation, efficient methods of gene transfer are required; these include particle bombardment, microinjection, electroporation, direct DNA uptake, and Agrobacterium-medi- ated transformation. Among these, particle bombardment and Agrobacterium-mediated transformation procedures have been Alkesh Hada and Veda Krishnan contributed equally to this research. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11627-018-9944-8) contains supplementary material, which is available to authorized users. * Archana Sachdev arcs_bio@yahoo.com 1 Division of Biochemistry, Indian Agricultural Research Institute, New Delhi 110012, India 2 Department of Biotechnology, National College (Autonomous), Tiruchirappalli, Tamil Nadu 620001, India 3 Division of Pathology, Indian Agricultural Research Institute, New Delhi 110012, India In Vitro Cellular & Developmental Biology - Plant https://doi.org/10.1007/s11627-018-9944-8