Skip to main content

Plant regeneration of Picea asperata Mast. by somatic embryogenesis


Key message

This study provides a novel desiccation indicator for somatic embryos and an efficient system for somatic seedling production in Picea asperata Mast.


Picea asperata Mast. is a widely distributed native spruce in China with outstanding wood properties and adaptability. In this study, we established an effective system to propagate plantlets through somatic embryogenesis in P. asperata. The effects of cone collection date, seed family, and plant growth regulators on the initiation of embryonal masses (EM) were analyzed. Besides the significant effects of seed family and cone collection date on EM initiation, the highest induction frequency was obtained with modified Litvay medium (MLV) containing 10 µM 2,4-dichlorophenoxyacetic acid and 5 µM 6-benzylaminopurine. The best tissue proliferation in suspension cultures was obtained with an inoculum density of 1:7 (v/v, sedimented cells:liquid medium) after 9 days in culture. The best medium for somatic embryo (SE) maturation was MLV supplemented with 105 µM (±)-abscisic acid, 5% polyethylene glycol 4000, 3% sucrose, 0.1% activated charcoal (AC), 0.05% l-glutamine, 0.1% casein acid hydrolysate, and 0.4% gellan gum. In this medium, 415 ± 103 SEs with well-developed cotyledons were obtained per gram (fresh weight) of EM. After partial desiccation treatment under low light intensity, somatic embryos, depending on their color change, were divided into green cotyledonary embryos (GCEs) and non-green cotyledonary embryos (NGCEs). These embryos, along with non-desiccated SEs as controls, were germinated on 1/2 MLV supplemented with 2% sucrose, 0.2% AC, 0.05% l-glutamine, and 0.6% gellan gum. Only GCEs, with red radicles, produced plantlets at the highest rates (>74.5%) among all of the treatments. This efficient system of somatic seedling production in P. asperata includes screened culture factors and a novel desiccation indicator to ensure SE quality before germination.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8



2,4-Dichlorophenoxyacetic acid




Abscisic acid


Activated charcoal

PEG 4000:

Polyethylene glycol 4000


Somatic embryo


Embryonal masses


Embryogenic cell line


Green cotyledonary embryo


Non-green cotyledonary embryo


Fresh weight


Modified Litvay medium


Analysis of variance


  • Attree SM, Moore D, Sawhney VK, Fowke LC (1991) Enhanced maturation and desiccation tolerance of white spruce [Picea glauca (Moench) Voss] somatic embryos: effects of a non-plasmolysing water stress and abscisic acid. Ann Bot 68:519–525

    Google Scholar 

  • Bozhkov PV, von Arnold S (1998) Polyethylene glycol promotes maturation but inhibits further development of Picea abies somatic embryos. Physiol Plant 104:211–224

    CAS  Article  Google Scholar 

  • Chalupa V (1985) Somatic embryogenesis and plantlet regeneration from cultured immature and mature embryos of Picea abies (L.) Karst. Commun Inst For Cech 14:57–63

    Google Scholar 

  • Filonova LH, Bozhkov PV, von Arnold S (2000) Developmental pathway of somatic embryogenesis in Picea abies as revealed by time-lapse tracking. J Exp Bot 51:249–264

    CAS  Article  PubMed  Google Scholar 

  • García-Mendiguren O, Montalbán I, Goicoa T, Ugarte M, Moncaleán P (2016) Environmental conditions at the initial stages of Pinus radiata somatic embryogenesis affect the production of somatic embryos. Trees Struct Funct 30:949–958

    Article  Google Scholar 

  • Grossnickle SC (2011) Tissue culture of conifer seedlings-20 years on: viewed through the lens of seedling quality. In: Riley LE, Haase DL, Pinto JR, technical coordinators (eds) National Proceedings: Forest and Conservation Nursery Associations-2010. Proc. RMRS-P-65. USDA Forest Service, Rocky Mountain Research Station, Fort Collins, pp 139–146

  • Gupta PK, Holmstrom D (2005) Double staining technology for distinguishing embryogenic cultures. In: Jain SM, Gupta PK (eds) Protocol for somatic embryogenesis in woody plants. Springer, The Netherlands, pp 573–575

    Chapter  Google Scholar 

  • Hakman I, von Arnold S (1985) Plantlet regeneration through somatic embryogenesis in Picea abies (Norway Spruce). J Plant Physiol 121:149–158

    CAS  Article  Google Scholar 

  • Harry IS, Thorpe TA (1991) Somatic embryogenesis and plant regeneration from mature zygotic embryos of red spruce. Bot Gaz 152:446–452

    CAS  Article  Google Scholar 

  • Hay EI, Charest PJ (1999) Somatic embryo germination and desiccation tolerance in conifers. In: Jain SM, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants. Springer, The Netherlands, pp 61–96

    Chapter  Google Scholar 

  • Hazubska-Przybył T, Wawrzyniak M, Obarska A, Bojarczuk K (2015) Effect of partial drying and desiccation on somatic seedling quality in Norway and Serbian spruce. Acta Physiol Plant 37:1–9

    Article  Google Scholar 

  • Ishii K (1995) Somatic embryogenesis in Picea glehnii and P. jezoensis. In: Mohan Jain S, Gupta PK, Newton RJ (eds) Somatic Embryogenesis in Woody Plants. Kluwer Academic Publishers, Dordrecht, pp 55–65

    Chapter  Google Scholar 

  • Jing D, Zhang J, Xia Y, Kong L, Ouyang F, Zhang S, Zhang H, Wang J (2016) Proteomic analysis of stress-related proteins and metabolic pathways in Picea asperata somatic embryos during partial desiccation. Plant Biotechnol J. doi:10.1111/pbi.12588

    PubMed  Google Scholar 

  • Johansen DA (1940) Plant microtechnique. McGraw-Hill Book Company, New York

    Google Scholar 

  • Joshi R, Kumar P (2013) Regulation of somatic embryogenesis in crops: a review. Agric Rev 34:1–20

    Google Scholar 

  • Klimaszewska K, Trontin JF, Becwar MR, Devillard C, Park YS, Lelu-Walter MA (2007) Recent progress in somatic embryogenesis of four Pinus spp. Tree For Sci Biotechnol 1:11–25

    Google Scholar 

  • Klimaszewska K, Overton C, Stewart D, Rutledge RG (2011) Initiation of somatic embryos and regeneration of plants from primordial shoots of 10-year-old somatic white spruce and expression profiles of 11 genes followed during the tissue culture process. Planta 233:635–647

    CAS  Article  PubMed  Google Scholar 

  • Klimaszewska K, Hargreaves C, Lelu-Walter MA, Trontin JF (2016) Advances in conifer somatic embryogenesis since year, 2000. In: Maria AG, Lambardi M (eds) In Vitro Embryogenesis in Higher Plants. Springer, New York, pp 131–166

    Chapter  Google Scholar 

  • Kong L, Yeung EC (1992) Development of white spruce somatic embryos: II. Continual shoot meristem development during germination. In Vitro Cell Dev Biol Plant 28:125–131

    Article  Google Scholar 

  • Kong L, Yeung EC (1995) Effects of silver nitrate and polyethylene glycol on white spruce (Picea glauca) somatic embryo development: enhancing cotyledonary embryo formation and endogenous ABA content. Physiol Plant 93:298–304

    CAS  Article  Google Scholar 

  • Krüssmann G (1983) Handbuch der Nadelgehölze, 2nd edn. Paul Pareyc, Hamburg

    Google Scholar 

  • Lelu-Walter MA, Thompson D, Harvengt L, Sanchez L, Toribio M, Pâques LE (2013) Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction. Tree Genet Genomes 9:883–899

    Article  Google Scholar 

  • Li Q, Zhang K, Luo JX (2007) A preliminary research on the variation of growth traits and clonal selection in clonal seed orchard of Picea asperata. J Sichuan For Sci Technol 28:21–27

    Google Scholar 

  • Liao YK, Juan IP (2015) Improving the germination of somatic embryos of Picea morrisonicola Hayata: effects of cold storage and partial drying. J For Res 20:114–124

    CAS  Article  Google Scholar 

  • Lin J, Hu Y (2000) Atlas of structure in the gymnosperms from China. Science Press, Beijing

    Google Scholar 

  • Litvay JD, Verma DC, Johnson MA (1985) Influence of a loblolly pine (Pinus taeda L.). Culture medium and its components on growth and somatic embryogenesis of the wild carrot (Daucus carota L.). Plant Cell Rep 4:325–328

    CAS  Article  PubMed  Google Scholar 

  • Luo JX, Sun P, Li XQ (2001) Advances in genetic improvement of spruce in abroad and breeding strategies of Picea asperata Mast. J Sichuan For Sci Technol 22:31–40

    Google Scholar 

  • Maruyama TE, Hosoi Y (2012) Post-maturation treatment improves and synchronizes somatic embryo germination of three species of Japanese pines. Plant Cell Tissue Org Cult 110:45–52

    Article  Google Scholar 

  • Montalbán I, Setién-Olarra A, Hargreaves C, Moncaleán P (2013) Somatic embryogenesis in Pinus halepensis Mill.: an important ecological species from the Mediterranean forest. Trees Struct Funct 27:1339–1351

    Article  Google Scholar 

  • Park YS (2002) Implementation of conifer somatic embryogenesis in clonal forestry: technical requirements and deployment considerations. Ann For Sci 59:651–656

    Article  Google Scholar 

  • Park YS, Barrett JD, Bonga JM (1998) Application of somatic embryogenesis in high-value clonal forestry: deployment, genetic control, and stability of cryopreserved clones. In Vitro Cell Dev Biol Plant 34:231–239

    Article  Google Scholar 

  • Pullman GS, Gupta PK, Timmis R, Carpenter C, Kreitinger M, Welty E (2005) Improved Norway spruce somatic embryo development through the use of abscisic acid combined with activated carbon. Plant Cell Rep 24:271–279

    CAS  Article  PubMed  Google Scholar 

  • Ramarosandratana A, Harvengt L, Bouvet A, Calvayrac R, Paques M (2001) Effects of carbohydrate source, polyethylene glycol and gellan gum concentration on embryonal-suspensor mass (ESM) proliferation and maturation of maritime pine somatic embryos. In Vitro Cell Dev Biol Plant 37:29–34

    CAS  Article  Google Scholar 

  • Roberts DR, Sutton BCS, Flinn BS (1990) Synchronous and high frequency germination of interior spruce somatic embryos following partial drying at high relative humidity. Can J Bot 68:1086–1090

    Article  Google Scholar 

  • Salajova T, Salaj J (2001) Somatic embryogenesis and plantlet regeneration from cotyledon explants isolated from emblings and seedlings of hybrid firs. J Plant Physiol 158:747–755

    CAS  Article  Google Scholar 

  • Stasolla C, Yeung EC (2003) Recent advances in conifer somatic embryogenesis: improving somatic embryo quality. Plant Cell Tissue Organ Cult 74:15–35

    CAS  Article  Google Scholar 

  • Stasolla C, Kong L, Yeung EC, Thorpe TA (2002) Maturation of somatic embryos in conifers: morphogenesis, physiology, biochemistry, and molecular biology. In Vitro Cell Dev Biol Plant 38:93–105

    CAS  Article  Google Scholar 

  • von Arnold S, Clapham D (2008) Spruce embryogenesis. In: Suárez MF, Bozhkov PV (eds) Plant embryogenesis. Humana Press, Totowa, pp 31–47

    Chapter  Google Scholar 

  • Vooková B, Kormuťák A (2006) Comparison of induction frequency, maturation capacity and germination of Abies numidica during secondary somatic embryogenesis. Biol Plant 50:785–788

    Article  Google Scholar 

Download references


This study was supported by a grant from the China Twelfth Five-Year Plan for Science Technology Support (2012BAD01B01). The authors acknowledge Xiao Long Shan Forestry Research Institute for cone collection and thank Prof. Iain Charles Bruce (Peking University, China) for critical reading of the manuscript.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Junhui Wang.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Communicated by K. Klimaszewska.

Y. Xia and J. Zhang contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 66 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Xia, Y., Zhang, J., Jing, D. et al. Plant regeneration of Picea asperata Mast. by somatic embryogenesis. Trees 31, 299–312 (2017).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Spruce
  • Embryonal masses
  • Somatic embryo maturation
  • Somatic embryo germination
  • Partial desiccation
  • Indicator