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Induction and early identification of tetraploid black locust by hypocotyl in vitro

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Abstract

Tetraploid black locust (Robinia pseudoacacia L.) has advantages over diploid varieties in growth and stress resistance. Previous studies had shown that tetraploid black locust can be induced only by treating shoot tips of the germinating seeds with colchicine. In order to establish a simpler and more efficient tetraploid induction method in black locust, we induced tetraploid plants by treating the hypocotyls from in vitro–grown seedlings with different concentrations of colchicine, and then provided a method for tetraploid identification based on stomatal size and density in this study. The highest induction efficiency was 53.33% by treating hypocotyl explants with 70 mg·L−1 colchicine for 2 d. Tetraploid black locust was significantly different from diploid in morphological characteristics. Leaf area, leaf thickness, chlorophyll content, stomatal length, and stomatal width were greater in tetraploid plants than in diploid plants, whereas the opposite was true for leaf aspect ratio, stomatal index, and stomatal density. The differences in stomatal size and density between diploid and tetraploid were nearly two times; therefore, size and density of stomata can be used as a stable and distinct marker to rapidly screen tetraploids. The results of this study suggested that the hypocotyl was an ideal material to induce tetraploid black locust in vitro and confirmed that stomatal size and density could be an effective and reliable marker to identify ploidy level in black locust.

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Acknowledgments

We thank the glasshouse staff for the management of plants, and other students in our lab for their friendly help.

Funding

This work was funded by the National Nature Science Foundation of China (31570677), the National Key R&D Program of China (2017YFD0600503), and the State Forestry Administration of science and technology development center project (XPC-2015-3).

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Authors and Affiliations

  1. Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, Engineering Technology Research Center of Black Locust of National Forestry and Grassland Administration, National Engineering Laboratory For Tree Breeding, College of Biological Science and Technology, Beijing Forestry University, Beijing, 100083, People’s Republic of China

    Xiuyu Li, Zijie Zhang, Yunhui Ren, Yue Feng, Qi Guo, Li Dong, Yuhan Sun & Yun Li

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  1. Xiuyu Li
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  2. Zijie Zhang
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  3. Yunhui Ren
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  4. Yue Feng
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  5. Qi Guo
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  6. Li Dong
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  7. Yuhan Sun
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  8. Yun Li
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Contributions

YL, XL, and YR conceived and designed research. YF, QG, and LD conducted experiments. XL and ZZ analyzed data and wrote the manuscript. YS contributed stock plants. All authors have read and approved the final manuscript.

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Correspondence to Yun Li.

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The authors declare that they have no competing interests.

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Editor: Bin Tian

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Li, X., Zhang, Z., Ren, Y. et al. Induction and early identification of tetraploid black locust by hypocotyl in vitro. In Vitro Cell.Dev.Biol.-Plant 57, 372–379 (2021). https://doi.org/10.1007/s11627-020-10133-5

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  • DOI: https://doi.org/10.1007/s11627-020-10133-5

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