Plantlet regeneration from primary callus cultures of Lilium brownii F.E.Br. ex Miellez var. giganteum G. Y. Li & Z. H. Chen, a rare bulbous germplasm

  • Yun Wu
  • Yi-Di Ma
  • Yue Li
  • Lin Zhang
  • Yi-Ping XiaEmail author


Lilium brownii F.E.Br. ex Miellez var. giganteum G. Y. Li & Z. H. Chen, an endangered valuable genetic resource, was used to establish and optimize a callus propagation system and to investigate the effects of internal and external phytohormones for the purpose of germplasm conservation. Of the combinations and concentrations of auxins and cytokinins examined, Murashige and Skoog (MS) medium supplemented with 8 g L−1 agar, 30 g L−1 sucrose, 0.45 μM 2,4-dichlorophenoxyacetic acid, 2.69 to 5.37 μM α-naphthaleneacetic acid, and 0.44 μM 6-benzyladenine, 0.45 μM thidiazuron, and 0.28 μM zeatin riboside generated the best results, effectively promoting callus proliferation. Four callus types could be discriminated, of which type A (yellowish, granular) and type B (yellow, medium-granular) were dry, friable, and grew well. Periodic acid-Schiff staining revealed small and regular cells, with numerous starch granules surrounding each nucleus. In culture, callus clumps produced an average of 14.33 shoots under “MS + 7-d-dark–light” treatment with 100% regeneration frequency. Bulblets formed within 60 d after shoot transfer to bulblet formation medium. Type A and B callus was likely to be embryogenic, according to morphology, cytology, and high shoot regenerating capacity. Examination of endogenous phytohormone levels showed that the abscisic acid to indole-3-acetic acid (ABA/IAA) ratio gradually increased with increasing diameter of callus clumps treated with all exogenous phytohormones, except zeatin riboside, leading to the hypothesis that callus induction competence was closely associated with endogenous ABA/IAA ratio. This first report should assist further genetic studies of this rare Lilium and other bulbous plants.


Lilium Callus status Endogenous phytohormones Morphogenesis Bulblet formation 



The authors would thank American Journal Experts for editing and improving the English language of this manuscript.

Authors’ contribution statement

YPX, YW, YDM, YL, and LZ conceived and designed the experiments. YDM, YL, and YW performed the experiments and analyzed the data. YW, YL, and YDM wrote the manuscript. LZ and YPX revised the manuscript. All authors approved the final manuscript.

Funding information

This work was funded by National Natural Science Foundation of China (No. 31772337).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11627_2018_9955_Fig5_ESM.png (4.1 mb)
Figure S1.

Morphological characters and habitats of Lilium brownii var. giganteum in North Island of Shengsi Archipelago, Zhejiang Province. (a) Habitats in the island. (b) The beginning bud. (c) The late bud. (d) The beginning of flower blooming. (e) The late of flower blooming. (f) The whole plant. (g) The bulb underground. (h) The seed capsule. (PNG 4154 kb)

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High Resolution Image (TIF 37106 kb)
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Figure S2.

The effects of different medium on embryogenic callus regeneration after 90 days. (a) ‘MS + 7-d-Dark–Light + 8 g L−1 agar + 60 g L−1 sucrose’ treatment. (b) ‘MS + 7-d-Dark–Light + NAA 0.11 μM + 6-BA 2.22 μM + 8 g L−1 agar + 60 g L−1 sucrose’ treatment. (c) ‘MS + 7-d-Dark–Light + NAA 0.11 μM + 6-BA 4.44 μM’ treatment. (d) ‘MS + 7-d-Dark–Light + NAA 0.11 μM + 6-BA 8.88 μM’ treatment. (e) ‘MS + 7-d-Dark–Light + NAA 0.54 μM + 6-BA 2.22 μM’ treatment. (f) ‘MS + 7-d-Dark–Light + NAA 0.54 μM + 6-BA 4.44 μM’ treatment. (g) ‘MS + 7-d-Dark–Light + NAA 0.54 μM + 6-BA 8.88 μM’ treatment. (h) ‘MS + 7-d-Dark–Light + NAA 2.69 μM + 6-BA 2.22 μM’ treatment. (i) ‘MS + 7-d-Dark–Light + NAA 2.69 μM + 6-BA 4.44 μM’ treatment. (j) ‘MS + 7-d-Dark–Light + NAA 2.69 μM + 6-BA 8.88 μM’ treatment. The red arrows indicate the typical semitransparent and swollen leaves. (PNG 5783 kb)

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Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  • Yun Wu
    • 1
  • Yi-Di Ma
    • 1
    • 2
  • Yue Li
    • 1
  • Lin Zhang
    • 1
  • Yi-Ping Xia
    • 1
    Email author
  1. 1.Department of Horticulture, College of Agriculture and BiotechnologyZhejiang UniversityHangzhouPeople’s Republic of China
  2. 2.China Jiliang UniversityHangzhouPeople’s Republic of China

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