TDZ-induced axillary shoot proliferation of Rhododendron mucronulatum Turcz and assessment of clonal fidelity using DNA-based markers and flow cytometry
In order to induce in vitro axillary shoot proliferation from single-node explants of Rhododendron mucronulatum Turcz., two techniques of thidiazuron (TDZ) application were tested: (i) two-step procedure including cultivation on Anderson medium (AM) supplemented with varying TDZ concentrations (0.1 μM; 0.25 μM; 0.5 μM; 1.0 μM; 2.5 μM) for 8 wk followed by cultivation on hormone-free medium (AM0) for 6 wk and (ii) 4-h liquid-pulse treatment with different TDZ concentrations (7.5 μM, 15.0 μM, or 30.0 μM) followed by cultivation on AM0 for 8 wk. The highest number of axillary shoots per explant was achieved with 0.1-μM TDZ after the two-step procedure. The best response in terms of percent regeneration (87%), shoot length (13 mm), absence of structure anomalies, and the shortest shoot production cycle (8 wk) was obtained with 30.0-μM TDZ liquid-pulse treatment for 4 h. The clonal fidelity of regenerated shoots was evaluated by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers and flow cytometry. Genetic similarity of all regenerants between themselves and with the mother seedlings was 99%. Flow cytometric analysis revealed that all samples studied were diploid. The nuclear DNA content of microshoots obtained under the TDZ treatments varied from 1.26 to 1.32 pg per 2C. There were no significant differences in DNA content among mother seedlings and in vitro developed shoots triggered by 0.1- and 2.5-μM TDZ nor by those triggered by the 30.0-μM TDZ pulse treatment.
KeywordsAxillary shoot proliferation Thidiazuron Somaclonal variability Rhododendron mucronulatum
“Collection of living plants indoors and outdoors” was used in the study. The authors wish to thank Geoffrey Harper for assistance in preparing the manuscript.
TIN, research supervisor, contributed with scientific advice, wrote, and revised the final version of the manuscript. SVA performed RAPD and ISSR analyses. EVA assisted in the performance of experiments and read and approved the final manuscript. YGZ designed all the experiments, performed FCM, analyzed the data, and wrote the manuscript.
The reported study was funded by the Russian Foundation for Basic Research (RFBR) in according to research project № 17-04-00782. The work was carried out within the framework of the state task of the Central Siberian Botanical Garden SB RAS № AAAA-A17-117012610051-5. Plant material from collection № USU_440534
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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