title: A high efficient protocol for soybean root transformation by Agrobacterium rhizogenes and most stable reference genes for RT-qPCR analysis.
abstract: A 55% transformation efficiency was obtained by our optimized protocol; and we showed that GmELF1 - β and GmELF1 - α are the most stable reference genes for expression analyses under this specific condition. Gene functional analyses are essential to the validation of results obtained from in silico and/or gene-prospecting studies. Genetic transformation methods that yield tissues of transient expression quickly have been of considerable interest to researchers. Agrobacterium rhizogenes-mediated transformation methods, which are employed to generate plants with transformed roots, have proven useful for the study of stress caused by root phytopathogens via gene overexpression and/or silencing. While some protocols have been adapted to soybean plants, transformation efficiencies remain limited; thus, few viable plants are available for performing bioassays. Furthermore, mRNA analyses that employ reverse transcription quantitative polymerase chain reactions (RT-qPCR) require the use of reference genes with stable expression levels across different organs, development steps and treatments. In the present study, an A. rhizogenes-mediated soybean root transformation approach was optimized. The method delivers significantly higher transformation efficiency levels and rates of transformed plant recovery, thus enhancing studies of soybean abiotic conditions or interactions between phytopathogens, such as nematodes. A 55% transformation efficiency was obtained following the addition of an acclimation step that involves hydroponics and different selection processes. The present study also validated the reference genes GmELF1-β and GmELF1-α as the most stable to be used in RT-qPCR analysis in composite plants, mainly under nematode infection.
authors.affiliation: Department of Biochemistry and Biotechnology, Universidade Estadual de Londrina, Londrina, Brazil.|Brazilian Agricultural Research Corporation (Empresa Brasileira de Pesquisa Agropecuária-EMBRAPA Soybean), Londrina, Brazil.
authors.forename: K M
authors.initials: KM
authors.lastname: Kuma
authors.affiliation: Genetics and Molecular Biology Department, Universidade Estadual de Londrina, Londrina, Brazil.|Brazilian Agricultural Research Corporation (Empresa Brasileira de Pesquisa Agropecuária-EMBRAPA Soybean), Londrina, Brazil.
authors.forename: V S
authors.initials: VS
authors.lastname: Lopes-Caitar
authors.affiliation: Brazilian Agricultural Research Corporation (Empresa Brasileira de Pesquisa Agropecuária-EMBRAPA Soybean), Londrina, Brazil.
authors.forename: C C T
authors.initials: CC
authors.lastname: Romero
authors.affiliation: Department of Biochemistry and Biotechnology, Universidade Estadual de Londrina, Londrina, Brazil.|Brazilian Agricultural Research Corporation (Empresa Brasileira de Pesquisa Agropecuária-EMBRAPA Soybean), Londrina, Brazil.
authors.forename: S M H
authors.initials: SM
authors.lastname: Silva
authors.affiliation: Brazilian Agricultural Research Corporation (Empresa Brasileira de Pesquisa Agropecuária-EMBRAPA Soybean), Londrina, Brazil.
authors.forename: M K
authors.initials: MK
authors.lastname: Kuwahara
authors.affiliation: Universidade Estadual do Norte do Paraná, Bandeirantes, Brazil.
authors.forename: M C C G
authors.initials: MC
authors.lastname: Carvalho
authors.affiliation: Brazilian Agricultural Research Corporation (Empresa Brasileira de Pesquisa Agropecuária-EMBRAPA Soybean), Londrina, Brazil.
authors.forename: R V
authors.initials: RV
authors.lastname: Abdelnoor
authors.affiliation: Brazilian Agricultural Research Corporation (Empresa Brasileira de Pesquisa Agropecuária-EMBRAPA Soybean), Londrina, Brazil.
authors.forename: W P
authors.initials: WP
authors.lastname: Dias
authors.affiliation: Brazilian Agricultural Research Corporation (Empresa Brasileira de Pesquisa Agropecuária-EMBRAPA Soybean), Londrina, Brazil. francismar.marcelino@embrapa.br.
authors.forename: F C
authors.initials: FC
authors.lastname: Marcelino-Guimarães
mesh_terms: D060054:Agrobacterium / Q000235:genetics*; D005821:Genetic Techniques; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D013025:Glycine max / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
keywords: Reference genes; Soybean composite plants; Transgenic roots; Transient transformation