Abstract
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This is the first report of a highly efficient Agrobacterium tumefaciens-mediated transformation protocol for Acanthaceae and its utilization in revealing important roles of cytokinin in regulating heterophylly in Hygrophila difformis.
Abstract
Plants show amazing morphological differences in leaf form in response to changes in the surrounding environment, which is a phenomenon called heterophylly. Previous studies have shown that the aquatic plant Hygrophila difformis (Acanthaceae) is an ideal model for heterophylly study. However, low efficiency and poor reproducibility of genetic transformation restricted H. difformis as a model plant. In this study, we reported successful induction of callus, shoots and the establishment of an efficient stable transformation protocol as mediated by Agrobacterium tumefaciens LBA4404. We found that the highest callus induction efficiency was achieved with 1 mg/L 1-Naphthaleneacetic acid (NAA) and 2 mg/L 6-benzyladenine (6-BA), that efficient shoot induction required 0.1 mg/L NAA and 0.1 mg/L 6-BA and that high transformation efficiency required 100 µM acetosyringone. Due to the importance of phytohormones in the regulation of heterophylly and the inadequate knowledge about the function of cytokinin (CK) in this process, we analyzed the function of CK in the regulation of heterophylly by exogenous CK application and endogenous CK detection. By using our newly developed transformation system to detect CK signals, contents and distribution in H. difformis, we revealed an important role of CK in environmental mediated heterophylly.
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Acknowledgments
This work was supported by grants to Prof. Hongwei Hou from Project of State Key Laboratory of Freshwater Ecology and Biotechnology (2019FB11), Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences (AB2018020), National Key R & D Program (2017YFE0128800, 2018YFD0900801) and General Project of Natural Science Foundation (31870384). This work was also supported by JSPS KAKENHI (JP16H01472, JP16K07408, 18H04787 and 18H04844 to S.K.) and by the MEXT Supported Program for the Strategic Research Foundation at Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant Number S1511023 to S.K. This work was also supported by NSERC Discovery Grant (RGPIN-2017-04381 to Elizabeth A. Schultz).
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HH planned and designed the research. GL, SH and JY performed experiments and data analysis. GL, SH, XZ, SK, EAS, HH contributed to the writing and editing of this article.
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Fig. S2. Plant and leaf morphology upon control and lovastatin treatment.(A) Top view of plants grown in 26 °C, 60% RH conditions. Note that plants grown in this condition and treated with 0.1% (w/v) ethanol was set as control.(B) Top view of plants treated with 5 µM lovastatin.(C) Leaf shape of control.(D) Leaf shape of plants treated with 5 µM lovastatin.Bars =1 cm in (A)-(D)
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Li, G., Hu, S., Yang, J. et al. Establishment of an Agrobacterium mediated transformation protocol for the detection of cytokinin in the heterophyllous plant Hygrophila difformis (Acanthaceae). Plant Cell Rep 39, 737–750 (2020). https://doi.org/10.1007/s00299-020-02527-x
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DOI: https://doi.org/10.1007/s00299-020-02527-x