A new transformation-regeneration procedure in the model legume Lotus japonicus: root explants as a source of large numbers of cells susceptible to Agrobacterium-mediated transformation.
We describe herein a simple and efficient transformation procedure for the production of transgenic Lotus japonicus plants. In this new procedure, dedifferentiated root explants, used as starting material, are the source of a large number of cells that are competent for the regeneration procedure, with a high susceptibility to Agrobacterium infection. The application of this protocol resulted in a tenfold increase in the number of transformants produced by a single plant in comparison to the widely used hypocotyl transformation procedure. Furthermore, our procedure allowed the use of intact plants stored for a long time at 4 degrees C, thus providing a potential continuous supply of explants for transformation experiments. The overall time of incubation under tissue culture conditions required to obtain a plant transferable into soil is 4 months. The transgenic nature of the transformants was demonstrated by the detection of beta-glucuronidase (GUS) activity in the primary transformants and by molecular analysis. Stable transformation was indicated by Mendelian segregation of the hygromycin selectable marker and of the gusA activity after selfing of the transgenic plants.
Lombari
P
P
International Institute of Genetics and Biophysics, Via Marconi 12, 80125, Naples, Italy.
Ercolano
E
E
El Alaoui
H
H
Chiurazzi
M
M
D016960:Agrobacterium tumefaciens / Q000235:genetics; D002934:Cinnamates; D046508:Culture Techniques / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006921:Hygromycin B / Q000031:analogs & derivatives* / Q000494:pharmacology; D000070116:Lotus / Q000235:genetics / Q000502:physiology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D010937:Plant Growth Regulators / Q000494:pharmacology; D018517:Plant Roots / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D012038:Regeneration / Q000187:drug effects; D014170:Transformation, Genetic
D002934:Cinnamates; D004269:DNA, Bacterial; D010937:Plant Growth Regulators; C036483:T-DNA; D006921:Hygromycin B; C026273:hygromycin A; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D005966:Glucuronidase