pp 1–12 | Cite as

Dynamics of physiological and miRNA changes after long-term proliferation in somatic embryogenesis of Picea balfouriana

  • Qingfen Li
  • Cheng Deng
  • Tianqing Zhu
  • Juanjuan Ling
  • Hanguo Zhang
  • Lisheng Kong
  • Shougong Zhang
  • Junhui Wang
  • Xiaoyang ChenEmail author
Original Article


Key message

We found that embryogenic tissues of Picea balfouriana adopt different physiological pathways for long-term proliferation with 6-benzylaminopurine treatment and identified the miRNAs closely associated with proliferation.


The long-term maintenance of somatic embryo production capacity in conifer embryogenic tissue (ET) is essential for the production of vigorous somatic seedlings. However, this ability is often lost after several months of proliferation in many conifer species including Picea balfouriana. Cytokinins are known to influence several important physiological processes during plant growth and development, including somatic embryogenesis (SE). In this study, we found that the 6-benzylaminopurine (BA) concentration influenced the yields of P. balfouriana somatic embryos and their germination response. Only ET of P. balfouriana proliferated on medium supplemented with 3.6 µM BA produced somatic embryos that germinated into normal plants. Most hormone levels increased in ET after prolonged proliferation. Moreover, antioxidant enzyme activities and polyamine contents were also significantly changed after 8 months of culture, which might be modulated by accumulated zeatin riboside (ZR). Finally, some selected microRNAs and their target genes were confirmed to be involved in the proliferation of ET of P. balfouriana and they also might be regulated by accumulated ZR. These findings may facilitate efforts to clarify basic physiological processes after the long-term proliferation stage of SE in conifers and delay the decreased production capacity of somatic embryos.


Embryogenic tissue BA Hormones Antioxidant enzymes Polyamines miRNA 





Abscisic acid


Embryogenic tissue


Fresh weight


Indole-3-acetic acid


Plant growth regulator






Quantitative reverse transcription-polymerase chain reaction


Somatic embryogenesis


Superoxide dismutase






Zeatin riboside



The authors thank the Forestry Station of Xianggelila Country in Yunnan Province for cone sampling in the P. balfouriana seed orchard. This study was supported by grants from the General Financial Grant from China Postdoctoral Science Foundation (2017M622717) and the Fundamental Research Funds for the Central Nonprofit Research Institution of the Chinese Academy of Forestry (TGB2013011).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

468_2018_1793_MOESM1_ESM.doc (39 kb)
Supplementary material 1 (DOC 39 KB)
468_2018_1793_MOESM2_ESM.doc (40 kb)
Supplementary material 2 (DOC 40 KB)
468_2018_1793_MOESM3_ESM.tif (1 mb)
Supplementary material 3 Endogenous hormone levels during different stages of proliferation at three BA concentrations (S1 was the ETs initiated from initiation medium and then proliferated on medium with 10 μM 2,4-D and 5 μM BA for 2 weeks; S2 was the ETs proliferated on medium as above for 8 months; S3 was the ETs proliferated for 8 months and then cultured for 1 week, on medium without PGRs). ad Changes in IAA (a), GA3 (b), ABA (c), and ZR (d) levels. The black uppercase or lowercase letters show the significance of differences among the three stages under each BA treatment (lowercase and uppercase indicate significant (P < 0.05) and very significant (P < 0.01), respectively). The pink and blue lowercase letters show the significance of differences among three BA treatments in the S2 and S3 stages, respectively. (TIF 1035 KB)
468_2018_1793_MOESM4_ESM.tif (2 mb)
Supplementary material 4 Ratios of different hormones among the three concentrations of BA. ac Changes in the ratios of ABA/ZR (a), ABA/IAA (b), and IAA/ZR (c). (TIF 2090 KB)
468_2018_1793_MOESM5_ESM.tif (678 kb)
Supplementary material 5 Activities of antioxidant enzymes during different stages of proliferation under BA treatments. a, b Changes in the activity of SOD (a) and POD (b). (TIF 677 KB)
468_2018_1793_MOESM6_ESM.tif (499 kb)
Supplementary material 6 Polyamine contents at different stages of proliferation under BA treatments. ac Changes in Put (a), Spd (b), and Spm (c). (TIF 499 KB)
468_2018_1793_MOESM7_ESM.tif (155 kb)
Supplementary material 7 Changes of physiological index and miRNAs levels in ETs of P. balfouriana in response to BA treatments. a Cell line 4A. b, d, f Proliferation of ETs on proliferation media containing different concentrations of BA. ETs proliferated on medium with 2.5 μM BA (b), 3.6 μM BA (d), and 5.0 μM BA (f). c, e, g Prematuration of ETs. Somatic embryos stained red (acetocarmine) and suspensors stained blue (Evan’s blue). ETs were cultured on 1/2 LM without PGRs after proliferation for 8 months on medium with 2.5 μM BA (c), 3.6 μM BA (e), and 5.0 μM BA (g). S1 was collected when the ETs proliferated on initiation medium with 10 μM 2,4-D and 5 μM BA for 2 weeks; S2 was collected when the ETs proliferated on medium containing different concentrations of BA for 8 months; S3 was collected when the ETs matured on 1/2 LM without PGRs (prematuration) for 1 week. “vs” means compared with. (TIF 155 KB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Forestry and Landscape ArchitectureSouth China Agricultural UniversityGuangzhouChina
  2. 2.State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of State Forestry Administration, Research Institute of ForestryChinese Academy of ForestryBeijingChina
  3. 3.State Key Laboratory of Tree Genetics and BreedingNortheast Forestry UniversityHarbinChina
  4. 4.Department of Biology, Centre for Forest BiologyUniversity of VictoriaVictoriaCanada

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