Transient gene expression in western white pine using agroinfiltration
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ORIGINAL PAPER
Transient gene expression in western white pine using agroinfiltration Zhenguo Ma1 • Jun-Jun Liu1 • Arezoo Zamany1 • Holly Williams1
Received: 19 February 2018 / Accepted: 2 March 2018 Ó Her Majesty the Queen in right of Canada, Natural Resources Canada, Canadian Forest Service 2019
Abstract A method for transient gene expression was developed for western white pine (WWP, Pinus monticola Dougl. ex D.Don) using reporter gene uidA encoding bglucuronidase (GUS). GUS was transiently expressed in cross sections of primary and secondary needles, cotyledons, and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens. Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration (dpa). GUS activity expressed inside WWP cells was confirmed using light microscopy. In fluorometric assays, GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles, while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly. Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons, all tested WWP tissues using the protocol had high levels of transient GUS expression. Thus, heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach. The current protocol for efficient transient gene expression will
Project funding: The work was partly supported by the CFS-GRDI fund. The online version is available at http://www.springerlink.com Corresponding editor: Tao Xu. & Jun-Jun Liu [email protected] 1
Canadian Forest Service, Natural Resources Canada, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada
aid functional genomics study of WWP and its pathogens and related conifer species. Keywords Agrobacterium Agroinfiltration bglucuronidase Transient gene expression Western white pine Abbreviations GUS b-glucuronidase MU 4-methylumbelliferone MUG 4-methylumbelliferyl-b-d-glucuronide WWP Western white pine
Introduction Escherichia coli uidA encodes b-glucuronidase (GUS) and it is extensively used as a reporter gene for transient and stable transformation of plants (Fior and Gerola 2009). GUS is stable and highly sensitive, making it suitable for detection by fluorometric, spectrophotometric, or histochemical techniques (Jefferson et al. 1987). As a heterologous reporter gene, uidA has been transiently expressed in model plant species and other angiosperms such as tobacco (Shah et al. 2013), Arabidopsis (Kim et al. 2009), and bentgrass (Basu et al. 2003). In gymnosperms, GUS expression has been realized in the transformation of a few species such as loblolly pine (Pinus taeda) (Tang 2001) and WWP (Liu and Ekramoddoullah 2003). In stable gene expression, T-DNA is integrated into the plant genome, and expression of the transgene increases 10–14 days post Agrobacter
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