Skip to main content
SpringerLink
Log in

Enhancement of somatic embryogenesis in camphor tree (Cinnamomum camphora L.): osmotic stress and other factors affecting somatic embryo formation on hormone-free medium

  • Original Paper
  • Published:
Trees Aims and scope Submit manuscript

Abstract

The aim of this study was to improve the direct somatic embryogenesis and initiate embryogenic callus formation in camphor tree (Cinnamomum camphora L.) on hormone-free medium. The influence of osmotic stress pretreatment of immature zygotic embryos (0.5 and 1.0 M solution of sucrose for 12, 24, 48, 72, 96, 120, and 144 h at 4 or 25°C) before cultured on hormone-free medium, on embryogenesis efficiency was assessed. The embryogenesis frequency was improved from 16.29 to 93.27%, while the average number of somatic embryos per explant increased from 3 to 12.57. Activated charcoal (AC), medium renewal, basal medium, light conditions and sucrose concentration in culture medium were also evaluated for their effect on somatic embryogenesis. AC addition and 10-day medium renewal did not increase embryogenesis efficiency significantly, and Murashige and Skoog (MS) medium proved to be more beneficial for somatic embryo formation than others. No differences were found between embryogenesis frequencies when cultured in darkness or under light, but culturing under light yielded more embryos. After the sucrose solution pretreatment, high level concentration of sucrose in induction medium was not needed for somatic embryogenesis, for it had a negative effect on somatic embryo formation when the concentration of sucrose was higher than 50 g l−1. The derived embryogenic lines were maintained via repetitive embryogenesis on hormone-free medium. Low ratio formation of embryogenic callus was observed on the surface of somatic embryos both on induction and proliferation medium. Plantlets derived from somatic embryos grew vigorously with normal appearance similar to germinated zygotic embryos.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

ABA:

Abscisic acid

AC:

Activated charcoal

B5:

Gamborg et al. (1968)

BA:

Benzylaminopurine

CH:

Casein hydrolysate

IBA:

Indole-3-butyric acid

BIM:

MS basal medium with 500 mg l−1 CH and 500 mg l−1 glutamine

MS:

Murashige & Skoog (1962)

PGR:

Plant growth regulator

TDZ:

Thidiazuron

WPM:

Woody plant medium (1980)

References

  • Ara H, Jaiswal U, Jaiswal VS (2005) Mango (Mangifera indica L.). In: Jain S, Gupta P (eds) Protocol for somatic embryogenesis in woody plants. Springer, Netherlands, pp 229–246

    Chapter  Google Scholar 

  • Babu KN, Sajina A, Minoo D, John CZ, Mini PM, Tushar KV, Rema J, Ravindran PN (2003) Micropropagation of camphor tree (Cinnamomum camphora). Plant Cell Tissue Organ Cult 74:179–183. doi:10.1023/A:1023988110064

    Article  Google Scholar 

  • Baillie AM, Epp DJ, Hutcheson D, Keller WA (1992) In vitro culture of isolated microspores and regeneration of plants in Brassica campestris. Plant Cell Rep 11:234–237. doi:10.1007/BF00235072

    Article  Google Scholar 

  • Blanc G, Michaux-Ferriere N, Teisson C, Lardet L, Carron MP (1999) Effects of carbohydrate addition on the induction of somatic embryogenesis in Hevea brasiliensis. Plant Cell Tissue Organ Cult 59:103–112. doi:10.1023/A:1006437731011

    Article  CAS  Google Scholar 

  • Burnett L, Yarrow S, Huang B (1992) Embryogenesis and plant regeneration from isolated microspores of Brassica rapa L. spp. oleifera. Plant Cell Rep 11:215–218. doi:10.1007/BF00232537

    Article  Google Scholar 

  • Cangahuala-Inocente GC, Dal Vesco LL, Steinmacher D, Torres AC, Guerra MP (2007) Improvements in somatic embryogenesis protocol in Feijoa (Acca sellowiana (Berg) Burret): induction, conversion and synthetic seeds. Sci Hortic (Amsterdam) 111:228–234. doi:10.1016/j.scienta.2006.10.030

    Article  CAS  Google Scholar 

  • Chandler SF, Lu CY (2005) Biotechnology in ornamental horticulture. In Vitro Cell Dev Biol Plant 41:591–601. doi:10.1079/IVP2005681

    Article  Google Scholar 

  • Cheong EJ, Pooler MR (2004) Factors affecting somatic embryogenesis in Prunus incisa cv. February pink. Plant Cell Rep 22:810–815. doi:10.1007/s00299-004-0771-5

    Article  PubMed  CAS  Google Scholar 

  • Chung HH, Chen JT, Chang WC (2005) Cytokinins induce direct somatic embryogenesis of Dendrobium Chiengmai Pink and subsequent plant regeneration. In Vitro Cell Dev Biol Plant 41:765–769. doi:10.1079/IVP2005702

    Article  CAS  Google Scholar 

  • Chung HH, Chen JT, Chang WC (2007) Plant regeneration through direct somatic embryogenesis from leaf explants of Dendrobium. Biol Plant 51:346–350. doi:10.1007/s10535-007-0069-x

    Article  CAS  Google Scholar 

  • Czarnecka E, Edelman L, Schoffl F (1984) Comparative analysis of physical stress responses in soybean seedlings using cloned heat shock cDNAs. Plant Mol Biol 3:45–58. doi:10.1007/BF00023415

    Article  CAS  Google Scholar 

  • De Jong AJ, Schmidt ED, De Vries SC (1993) Early events in higher-plant embryogenesis. Plant Mol Biol 22:367–377. doi:10.1007/BF00014943

    Article  PubMed  Google Scholar 

  • Dias JS, Correia MC (2002) Effect of medium renovation and incubation temperature regimes on tronchuda cabbage microspore culture embryogenesis. Sci Hortic (Amsterdam) 93:205–214. doi:10.1016/S0304-4238(01)00334-X

    Article  Google Scholar 

  • Du L, Zhou S, Bao MZ (2007) Effect of plant growth regulators on direct somatic embryogenesis in camphor tree (Cinnamomum camphora L.) from immature zygotic embryos and embryogenic calli induction. For Stud China 9(4):267–271

    Article  CAS  Google Scholar 

  • Fehér A, Pasternak TP, Dudits D (2003) Transition of somatic plant cells to an embryogenic state. Plant Cell Tissue Organ Cult 74:201–228. doi:10.1023/A:1024033216561

    Article  Google Scholar 

  • Finer JJ (1987) Direct somatic embryogenesis and plant regeneration from immature embryos of hybrid sunflowers (Helianthus annuus L.) on a high sucrose-containing medium. Plant Cell Rep 6:372–374. doi:10.1007/BF00269564

    Article  CAS  Google Scholar 

  • FLEPPC (Florida Exotic Pest Plant Council) (2001) List of invasive species. Florida Exotic Pest Plant Council, Florida EPPC Newsletter 11(1):3–4. Available at http://www.fleppc.org (last Accessed: 3 January 2003)

  • Fuentes SRL, Calheiros MBP, Manetti J, Vieira LGE (2000) The effects of silver nitrate and different carbohydrate sources on somatic embryogenesis in Coffea canephora. Plant Cell Tissue Organ Cult 60:5–13. doi:10.1023/A:1006474324652

    Article  CAS  Google Scholar 

  • Gaj MD (2002) Stimulation of somatic embryo formation by mutagens and darkness in culture of immature zygotic embryos of Arabidopsis thaliana (L.) Heynh. Plant Growth Regul 37:93–98. doi:10.1023/A:1020392014493

    Article  CAS  Google Scholar 

  • Gaj MD (2004) Factors influencing somatic embryogenesis induction and plant regeneration with particular reference to Arabidopsis thaliana (L.) Heynh. Plant Growth Regul 43:27–47. doi:10.1023/B:GROW.0000038275.29262.fb

    Article  CAS  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158. doi:10.1016/0014-4827(68)90403-5

    Article  PubMed  CAS  Google Scholar 

  • Giri CC, Shyamkumar B, Anjaneyulu C (2004) Progress in tissue culture, genetic transformation and applications of biotechnology to trees: an overview. Trees Struct Funct 18:115–135. doi:10.1007/s00468-003-0287-6

    Article  Google Scholar 

  • Gomes FLAF, Heredia FF, Silva PBE, Facó O, Campos FDD (2006) Somatic embryogenesis and plant regeneration in Opuntia ficus indica (L.) Mill. (Cactaceae). Sci Hortic (Amsterdam) 108:15–21. doi:10.1016/j.scienta.2005.12.007

    Article  CAS  Google Scholar 

  • Hansen M, Svinnset K (1993) Microspore culture of swede (Brassica napus ssp. rapifera) and the effects of fresh and conditioned media. Plant Cell Rep 12:496–500. doi:10.1007/BF00236094

    Article  Google Scholar 

  • Jimenez VM (2005) Involvement of plant hormones and plant growth regulators on in vitro somatic embryogenesis. Plant Growth Regul 47:91–110. doi:10.1007/s10725-005-3478-x

    Article  CAS  Google Scholar 

  • Kamada H, Kobayashi K, Kiyosue T, Harada H (1989) Stress-induced somatic embryogenesis in carrot and its application to synthetic seed production. In Vitro Cell Dev Biol 25:1163–1166. doi:10.1007/BF02621268

    Article  Google Scholar 

  • Kamada H, Ishikawa K, Saga H, Harada H (1993) Induction of somatic embryogenesis in carrot by osmotic stress. Plant Tissue Cult Lett 10:38–44

    CAS  Google Scholar 

  • Karami O, Deljou A, Esna-Ashari M, Ostad-Ahmadi P (2006) Effect of sucrose concentrations on somatic embryogenesis in carnation (Dianthus caryophyllus L.). Sci Hortic (Amsterdam) 110:340–344. doi:10.1016/j.scienta.2006.07.029

    Article  CAS  Google Scholar 

  • Kiyosue T, Takano K, Kamada H, Harada H (1990) Induction of somatic embryogenesis in carrot by heavy metal ions. Can J Bot 68:2301. doi:10.1139/b90-293

    Article  CAS  Google Scholar 

  • Lakshmanan P, Taji A (2000) Somatic embryogenesis in leguminous plants. Plant Biol 2:136–148. doi:10.1055/s-2000-9159

    Article  CAS  Google Scholar 

  • Land Protection (2001) NRM facts pest series. Department of Natural Resources and Mines, State of Queensland, Australia. Available http://www.nrm.qld.gov.au (Accessed 9 January 2003)

  • Lou H, Kako S (1995) Role of high sugar concentrations in inducing somatic embryogenesis from cucumber cotyledons. Sci Hortic (Amsterdam) 64:11–20. doi:10.1016/0304-4238(95)00833-8

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–479. doi:10.1111/j.1399-3054.1962.tb08052.x

    Article  CAS  Google Scholar 

  • Neto VBD, Otoni WC (2003) Carbon sources and their osmotic potential in plant tissue culture: does it matter? Sci Hortic (Amsterdam) 97:193–202. doi:10.1016/S0304-4238(02)00231-5

    Article  Google Scholar 

  • Odjakova MK, Conger BV (1999) The influence of osmotic pretreatment and inoculum age on the initiation and regenerability of switchgrass suspension cultures. In Vitro Cell Dev Biol Plant 35:442–444. doi:10.1007/s11627-999-0065-2

    Article  Google Scholar 

  • Osuna P, Barrow JR (2004) Regeneration of black grama (Bouteloua eriopoda Torr. Torr) plants via somatic embryogenesis. In Vitro Cell Dev Biol Plant 40:299–302. doi:10.1079/IVP2003533

    Article  CAS  Google Scholar 

  • Pan MJ, van Staden J (1998) The use of charcoal in in vitro culture: a review. Plant Growth Regul 26:155–163. doi:10.1023/A:1006119015972

    Article  CAS  Google Scholar 

  • Pasternak T, Prinsen E, Ayaydin F, Miskolczi P, Potters G, Asard H, Van Onckelen H, Dudits D, Fehér A (2002) The role of auxin. pH and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa (Medicago sativa L.). Plant Physiol 129:1807–1819. doi:10.1104/pp.000810

    Article  PubMed  CAS  Google Scholar 

  • Perera PIP, Hocher V, Verdeil JL, Doulbeau S, Yakandawala DMD, Weerakoon LK (2007) Unfertilized ovary: a novel explant for coconut (Cocos nucifera L.) somatic embryogenesis. Plant Cell Rep 26:21–28. doi:10.1007/s00299-006-0216-4

    Article  PubMed  CAS  Google Scholar 

  • Pinto G, Silva S, Park YS, Neves L, Araújo, Santos C (2008) Factors influencing somatic embryogenesis induction in Eucalyptus globules Labill.: basal medium and anti-browning agents. Plant Cell Tissue Organ Cult 95:79–88. doi:10.1007/s11240-008-9418-5

    Article  CAS  Google Scholar 

  • Poupin MJ, Arce-Johnson P (2005) Transgenic trees for a new era. In Vitro Cell Dev Biol Plant 41:91–101. doi:10.1079/IVP2004587

    Article  CAS  Google Scholar 

  • Pullman GS, Namjoshi K, Zhang Y (2003) Somatic embryogenesis in loblolly pine (Pinus taeda L.): improving culture initiation with abscisic acid and silver nitrate. Plant Cell Rep 22:85–95. doi:10.1007/s00299-003-0673-y

    Article  PubMed  CAS  Google Scholar 

  • Pullman GS, Mein J, Johnson S, Zhang Y (2005) Gibberellin inhibitors improve embryogenic tissue initiation in conifers. Plant Cell Rep 23:596–605. doi:10.1007/s00299-004-0880-1

    Article  PubMed  CAS  Google Scholar 

  • Rodriguez-Sotres R, Black M (1994) Osmotic potential and abscisic acid regulate triacylglycerol synthesis in developing wheat embryos. Planta 192:9–15

    Google Scholar 

  • Saunders JW, Tsai CJ (1999) Production of somatic embryos and shoots from sugarbeet callus: effects of abscisic acid, other growth regulators, nitrogen source, sucrose concentration and genotype. In Vitro Cell Dev Biol Plant 35:18–24. doi:10.1007/s11627-999-0004-2

    Article  CAS  Google Scholar 

  • Shi XP, Dai XG, Liu GF, Zhang JW, Ning GG, Bao MZ (2009) Cyclic secondary somatic embryogenesis and efficient plant regeneration in camphor tree (Cinnamomum camphora L.). In Vitro Cell Dev Biol Plant (revised)

  • Vila SK, Rey HY, Mroginski LA (2007) Factors affecting somatic embryogenesis induction and conversion in “Paradise tree” (Melia azedarach L.). J Plant Growth Regul 26:268–277. doi:10.1007/s00344-007-9007-6

    Article  CAS  Google Scholar 

  • Yang YX, Liu GF, Bao MZ (2006) Somatic embryogenesis and plant regeneration from petioles of Parthenocissus tricuspidata Planch. In Vitro Cell Dev Biol Plant 42:520–524. doi:10.1079/IVP2006821

    Article  CAS  Google Scholar 

  • You XL, Yi JS, Choi YE (2006) Cellular change and callose accumulation in zygotic embryos of Eleutherococcus senticosus caused by plasmolyzing pretreatment result in high frequency of single-cell-derived somatic embryogenesis. Protoplasma 227:105–112. doi:10.1007/s00709-006-0149-3

    Article  Google Scholar 

  • Zhang W, Fu Q, Dai XG, Bao MZ (2008) The culture of isolated microspores of ornamental kale (Brassica oleracea var. acephala) and the importance of genotype to embryo regeneration. Sci Hortic (Amsterdam) 117:69–72. doi:10.1016/j.scienta.2008.03.023

    Article  CAS  Google Scholar 

  • Zhou SJ, Brown DCW (2006) High efficiency plant production of North American ginseng via somatic embryogenesis from cotyledon explants. Plant Cell Rep 25:166–173. doi:10.1007/s00299-005-0043-z

    Article  PubMed  CAS  Google Scholar 

  • Zhu JK, Hasegawa EM, Bressan RA (1997) Molecular aspects of osmotic stress in plants. Crit Rev Plant Sci 16:253–277. doi:10.1080/713608147

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This research is supported by research project from Ministry of Education Foundation of China (NCET-04-0733). We thank all the colleagues in our lab for constructive discussion and technical support.

Author information

Authors and Affiliations

  1. Key Laboratory of Horticultural Plant Biology of Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, 430070, Wuhan, People’s Republic of China

    Xueping Shi, Xigang Dai, Guofeng Liu & Manzhu Bao

Authors
  1. Xueping Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xigang Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Guofeng Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Manzhu Bao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Manzhu Bao.

Additional information

Communicated by D. Treutter.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shi, X., Dai, X., Liu, G. et al. Enhancement of somatic embryogenesis in camphor tree (Cinnamomum camphora L.): osmotic stress and other factors affecting somatic embryo formation on hormone-free medium. Trees 23, 1033–1042 (2009). https://doi.org/10.1007/s00468-009-0345-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00468-009-0345-9

Keywords

Search

Navigation