作者：Yosvanis Acanda1 ,Michel Canton1 ,Hao Wu1 , Janice Zale1
作者单位：Plant Pathology Department, University of Florida, IFAS, CREC, Lake Alfred, USA
摘要：In mature citrus transformation, the nptII gene is most commonly used for selection and it is confounded by the high number of non-transformed, escaped shoots that develop on semi-solid kanamycin selection medium, even at high concentrations. Selection in liquid medium with kanamycin in temporary immersion bioreactors might provide a better means of distinguishing between trans-formed and non-transformed shoots. A dose-response curve was constructed for wild-type Carrizo rootstock in liquid medium to evaluate the effects of kanamycin concentration on the number and the length of microshoots. Kanamycin at 200 mg/l was chosen as the optimal concentration for selec-
tion of transgenic mature citrus shoots in bioreactors. At this dose, most non-transgenic microshoots turned yellow and their lengths and numbers were significantly reduced in comparison to the no kanamycin controls. Selection of transgenic shoots in bioreactors was tested after Agrobac-terium transformations of mature Carrizo and Valencia using three different binary vectors containing nptII as the selectable marker. Shoots developed on semi-solid medium and were transferred to temporary immersion bioreactorscontaining liquid MS medium with 200 mg/l kanamycin.After two weeks of culture in bioreactors, 21 dark green shoots were visually selected on the basis of color from a total of 6882 microshoots, and 17 of them (81%) were con-firmed as transgenic with either the GUS histochemical assay, GFP fluorescence or PCR. Yellow shoots (5675) tobe discarded from pTLAB21 and pCAMBIA2301 transfor-mations were also tested for GUS or GFP expression and only one (0.01%) was positive. Kanamycin selection of mature transgenic shoots in temporary immersion bioreac-
tors permitted transgenics to be visually distinguished onthe basis of color, and reduced labor and consumable costs for PCR screening, particularly when reporter genes were not used.
关键词：Agrobacterium transformation , Temporary immersion bioreactor ,Kanamycin selecti
文献注录：Yosvanis Acanda1 ,Michel Canton1 ,Hao Wu1 , Janice Zale1. Kanamycin selection in temporary immersion bioreactors allows visual selection of transgenic citrus shoots [J]. Plant Cell Tiss Organ Cult (. 2017, 129: 351-357.
报/刊名：《Plant Cell Tiss Organ Cult (》，发表于2017 / 第 129 期。
文献类型： [J] （文献级别：SCI刊物）
页码：第 351-357 页 / 共7 页