An Improved In Vitro Protocol for Agrobacterium rhizogenes-Mediated Transformation of Recalcitrant Plants for Root Biology Studies: A Case Study of Tea Plants (Camellia sinensis var. sinensis)
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Tea is a widely consumed nonalcoholic beverage, made from tender tea leaves, which contain extraordinarily high amount of specialized metabolites. These specialized metabolites are beneficial to human health and crucial in tea brisk, umami taste, and pleasant scent. Agrobacterium-assisted genetic transformation studies on tea plant are largely limited due to recalcitrant nature of tea plant. Release of oxidation phenolics that are dominant from explant tissues and the antibacterial nature of polyphenols during the transformation are among the possible reasons for tea plant recalcitrance. Our main objective in this study was to create an in vitro system for selection of transgenic roots from composite plants for possible production of specialized metabolites. Three-week-old in vitro-germinated seedlings were used in generating composite plants. The hypocotyl and the stems were wounded using a sonicator and a sterilized needle and infected with Agrobacterium rhizogenes harboring pBI121 plasmid and maintained in aseptic conditions. Molecular assay of roots from antibiotic selected plants showed amplification of rol B C and gus confirming the integration of Agrobacterium rhizogenes T-DNA region into the root system. From our data, it was clear that hairy root generation in tea can be achieved using A. rhizogenes. With this method, composite tea plants containing wild-type shoots with transgenic roots can be developed for gene functional characterization as well as specialized metabolite production and root-shoot interaction research in plants.
KeywordsClimate change Recalcitrant Agrobacterium rhizogenes Specialized metabolites Transformation Cotyledon Composite plants
We thank all our lab members Jing-Yi Pang, Dong-Wei Zhao, Zhen Yan, and Meng-Xian Zhang for their technical support.
This work supported by the National Natural Science Foundation of China (Grant Number 31370687 and 31770734) and funding was provided by Research Fund for the Doctoral Program of Higher Education (Grant No. 20123418110002) to Shu Wei.
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