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.
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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
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
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
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
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
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
Chandler SF, Lu CY (2005) Biotechnology in ornamental horticulture. In Vitro Cell Dev Biol Plant 41:591–601. doi:10.1079/IVP2005681
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Lakshmanan P, Taji A (2000) Somatic embryogenesis in leguminous plants. Plant Biol 2:136–148. doi:10.1055/s-2000-9159
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
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
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
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
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
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
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
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
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
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
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
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
Rodriguez-Sotres R, Black M (1994) Osmotic potential and abscisic acid regulate triacylglycerol synthesis in developing wheat embryos. Planta 192:9–15
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
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
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
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
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
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
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
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.
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Communicated by D. Treutter.
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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
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DOI: https://doi.org/10.1007/s00468-009-0345-9