Abstract
In this study, recalcitrance of tea plant ( Camellia sinensis) to Agrobacterium-mediated genetic transformation was investigated with an emphasis on specialized compounds in tea. Chemical constituents in tea leaves and calli were extracted into liquid Luria–Bertani (LB) medium to determine their biological activities on Agrobacterium growth, virulence, and plant transformation efficiency. Compared to the control Agrobacterium grown in LB medium, tea leaf extract containing 6.5 mg mL−1 catechins resulted in an 84.6 % reduction of Agrobacterium growth, a 73–36 % suppression of expression for the six virulence (vir) genes, browning of infected tobacco explant wounds, and an absence of transient or stable transformation events. Tea callus extract, containing 0.22 mg mL−1 catechins, did not significantly affect Agrobacterium growth or tobacco transgenic hairy root generation, whereas it enhanced the expression of some vir genes. Treatment with authentic catechin mixtures (other than caffeine) dissolved in LB resulted in suppression of Agrobacterium growth, vir gene expression, and tobacco transformation efficiency. Our data suggest that catechins are the key active constituents in tea leaves. Transient transformation efficiencies of tea leaves were much lower than those of tobacco leaves as indicated by the GUS (β-glucuronidase) assay, probably a result of inhibition by the catechins present in tea leaves. Lower transformation efficiencies of tea calli suggested that additional plant factor(s) might also exert inhibitory effects on tea plant transformation. Agrobacterium rhizogenes ATCC 15834 induced transgenic roots from the tea explants with 15–20 % efficiency. Our data suggested catechins inhibition of tea gene transformation could be overcome by using optimized strains of Agrobacterium.
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Abbreviations
- HPLC:
-
High performance liquid chromatography
- AS:
-
Acetosyringone
- GUS:
-
β-glucuronidase
- EDTA:
-
Ethylene diamine tetraacetic acid
- PCR:
-
Polymerase chain reaction
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Acknowledgments
We thank Prof Li-Ping Gao at the Anhui Agricultural University and Prof. Jianliang Lu at the Zhejiang University for providing tea calli and the Agrobacterium strains. This work was financially supported by the National Science Foundation in China (#31070614 and #31370687), the Doctoral Programs of Higher Education of the Ministry of Education (#20123418110002), the Program for Changjiang Scholars and Innovative Research Team in Universities (IRT1101), and the “Twelfth Five-Year” National Key Basic Research and Development Project (973) in China (2012CB722903).
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Song, DP., Feng, L., Rana, M.M. et al. Effects of catechins on Agrobacterium-mediated genetic transformation of Camellia sinensis . Plant Cell Tiss Organ Cult 119, 27–37 (2014). https://doi.org/10.1007/s11240-014-0511-7
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DOI: https://doi.org/10.1007/s11240-014-0511-7