Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 135, Issue 1, pp 1–12 | Cite as

Assessment of genetic stability and analysis of alkaloids potential in micropropagated plants of Croomia japonica Miquel, an endangered, medicinal plant in China and Japan

  • Weimei JiangEmail author
  • Shijian Hua
  • Xinpeng Zhou
  • Penghao Han
  • Qixiang Lu
  • Yingxiong Qiu
Original Article


Croomia japonica (Stemonaceae) is an endangered species both in China and Japan. We have developed an efficient regeneration system through adventitious buds organogenesis in C. japonica using rhizome buds as explants. Multiple buds regenerated directly on the explants without calli within 2 months when explants were cultured on Murashige and Skoog’s (MS) medium with 8.88 µmol 6-benzylaminopurine (BAP) and 1.07 µmol α-naphthaleneacetic acid (NAA). The adventitious buds of newly forming were proliferated by subsequent subcultures on MS medium with 2.66 µmol BAP and 2.69 µmol NAA. We evaluated the kinds and concentrations of plant growth regulators on adventitious shoot regeneration and root induction and also inspected the adsorbent (polyvinyl pyrrolidone and activated charcoal) and antioxidant (ascorbic acid, AS) on the inhibition of tissue browning. The results showed that soaking the explants with 1.14 mmol AS was the best approach for controlling browning. The maximum number of stout shoots per explant was achieved on MS medium containing 8.88 µmol BAP. In vitro regenerated shoots were rooted on MS medium supplemented with three different concentrations of auxin. The highest rooting rate (84.0 ± 3.6%) was reached on MS medium with 5.71 µmol Indole-3-acetic acid (IAA). One-step culture was developed when the adventitious buds cultured on the medium were supplemented with 2.66 µmol BAP and 0.54–2.69 µmol NAA. Rooted plantlets were acclimatized to the green house and development with a 87% survival rate. Genetic stability assessment of in vitro plants compared to the wild plants was revealed by simple sequence repeat markers. Similarly, flow cytometric analysis confirmed that the ploidy level of in vitro plantlets was stable. Total alkaloid content in wild and in vitro plants was tested by acid dye colorimetric analysis with tuberostemonine as the reference. Our results showed that alkaloids content in 11-week cultures reached 40.7–88.4% of the content of wild plants. The protocol described here could be employed for effective mass propagation of C. japonica for commercial and conservational purposes.


Alkaloids Croomia japonica Endangered species Genetic stability In vitro propagation Molecular marker Stemonaceae 



Activated charcoal


Ascorbic acid




Bromocresol green


Indole-3-acetic acid


Indole-3-butyric acid


Murashige and Skoog medium


α-Naphthaleneacetic acid


Plant growth regulator


Polyvinyl pyrrolidone


Simple sequence repeat



This work was supported by the Zhejiang Provincial Natural Science Foundation (Grant No. LY14C020002) and the International Cooperation and Exchange of the National Natural Science Foundation of China (Grant Nos. 31511140095, 31561143015). All authors read and approved the final manuscript.

Author Contributions

WM Jiang conceived the study and wrote the manuscript; SJ Hua conducted culture experiments; XP Zhou performed experiment to measure the total alkaloids; PH Han performed experiment to analyze the ploidy level of regenerated plants; QX Lu performed DNA isolation and SSR analysis; YX Qiu revised the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11240_2018_1422_MOESM1_ESM.docx (12.6 mb)
Supplementary material 1 (DOCX 12887 KB)


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© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Key Laboratory of Conservation Biology for Endangered Wildlife of the Ministry of Education, College of Life SciencesZhejiang UniversityHangzhouChina
  2. 2.School of International StudiesZhejiang UniversityHangzhouChina

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