Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 135, Issue 3, pp 523–533 | Cite as

Efficient somatic embryogenesis and bulblet regeneration of the endangered bulbous flower Griffinia liboniana

  • Ziming Ren
  • Xuesi Lv
  • Dong Zhang
  • Yiping XiaEmail author
Original Article


Using flower organs as primary explants and via somatic embryogenesis, we developed an efficient protocol for bulblet regeneration from in vitro-derived seedlings (bulblets) of Griffinia liboniana. Callus induction was tested on five types of floral organ (perianth, filament, pedicel, ovary and anther) in the presence of three combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (6-BA). Filament constituted the most responsive primary explant for regenerative callus induction, and the highest frequencies of callus induction (63.0 ± 1.9%) and numbers of differentiated buds (3.7 ± 0.3 buds/callus) were found on Murashige and Skoog (1962) medium (MS) supplemented with 1.0 mg L−1 2,4-D and 1.0 mg L−1 6-BA. Starting with in vitro-derived bulblets (0.8–1.5 cm in diameter), somatic embryo (SE) formation occurred within 6 weeks, followed by 8 weeks for SE germination and development on PGR-free media. The highest percentage (78.9 ± 2.2%) of embryogenesis was obtained on MS media supplemented with 0.5 mg L−1 6-BA and 1.5 mg L−1 2,4-D, with an average of 28.0 ± 2.1 bulblets/explant. Well-rooted bulblets were successfully acclimated to ex vitro conditions. A stable ploidy level of the regenerated bulblets was confirmed by flow cytometry (FCM) analysis. This is the first report about micropropagation methods of G. liboniana and constitutes an efficient and reusable method for bulblet regeneration of this endangered species. Additionally, this protocol enables large-scale vegetative production, germplasm preservation and genetic engineering of endangered Griffinia species.


Griffinia Floral organ Callus Somatic embryogenesis Histological analysis Regenerated bulblets Flow cytometry 



2,4-Dichlorophenoxyacetic acid


1-Naphthaleneacetic acid




Plant growth regulators


Murashige and Skoog


Flow cytometry


Somatic embryogenesis


Starch granule


Cell division


Embryogenic cell


Non-embryogenic cell



This work was supported by the Technology Project of Zhejiang Province, P. R. China (Project No. 2016C02056-13). Thank Dr. Yiping Xia provides valuable guidance in every stage of the writing of this paper.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Department of Horticulture, College of Agriculture and BiotechnologyZhejiang UniversityHangzhouPeople’s Republic of China

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