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Efficient induction of microspore embryogenesis using abscisic acid, jasmonic acid and salicylic acid in Brassica napus L

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The stress hormones abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) play an important role in the regulation of physiological processes and are often used in tissue culture to promote somatic embryogenesis and to enhance the quality of somatic embryos. Despite many studies on Brassica napus microspore culture, the effects of stress hormones (ABA, JA and SA) on microspore embryogenesis are not well explored. In this study, the effects of three incubation periods (6, 12 and 24 h) at different levels of ABA, JA and SA (0, 0.2, 0.5, 1.0, 2.0 and 5.0 mg l−1) on microspore embryogenesis of rapeseed (B. napus L.) cv. ‘Regent’ were investigated. ABA (0.5 mg l−1 for 12 h) enhanced microspore embryogenesis by about threefold compared with untreated cultures and increased normal plantlet regeneration by 68 %. ABA treatment also effectively reduced secondary embryo formation at all concentrations tested but enhanced callusing at high levels, for example 67 % at 1.0 mg l−1 for 24 h. Highest embryo yield (286.0 embryos Petri dish−1) was achieved using 1.0 mg l−1 JA for 24 h and highest normal plantlet regeneration (54 %) was observed in cultures exposed to 0.5 mg l−1 JA for 12 h. JA (5.0 mg l−1 for 24 h) also reduced the germination of microspore-derived embryos on regeneration medium by 21 %. SA at 0.2 and 0.5 mg l−1 for 6 h increased microspore embryogenesis (184.0 and 193.4 embryos Petri dish−1) relative to the control (136.2 embryos Petri dish−1). However, SA did not improve normal regeneration, secondary embryo formation or callusing. Microspore embryogenesis and plant regeneration could be improved by ABA, JA as well as SA when the appropriate level and duration of incubation were selected.

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Fig. 1

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Abscisic acid


Allene oxide cyclase


Allene oxide synthase


Acetylsalicylic acid

GA3 :

Gibberellic acid


Jasmonic acid


Late embryogenesis abundant

Lox :



Microspore-derived embryo


Methyl jasmonate


Nitric oxide


Programmed cell death


Protocorm like body


Reactive oxygen species


Salicylic acid


  • Abdollahi MR, Corral-Martínez P, Mousavi A, Salmanian AH, Moeini A, Seguí-Simarro JM (2009) An efficient method for transformation of pre-androgenic, isolated Brassica napus microspores involving microprojectile bombardment and Agrobacterium-mediated transformation. Acta Physiol Plant 31:1313–1317

    Article  Google Scholar 

  • Ahmadi B, Ghadimzadeh M, Moghaddam AF, Alizadeh K, Teixeira da Silva JA (2012a) Bud length, plating density, and incubation time on microspore embryogenesis in Brassica napus. Int J Vegetable Sci 18:346–357

    Article  Google Scholar 

  • Ahmadi B, Alizadeh K, Teixeira da Silva JA (2012b) Enhanced regeneration of haploid plantlets from microspores of Brassica napus L. using bleomycin, PCIB, and phytohormones. Plant Cell Tiss Org Cult 109:525–533

    Article  CAS  Google Scholar 

  • Akula A, Akula C, Bateson MF (2000) Betaine a novel candidate for rapid induction of somatic embryogenesis in tea (Camellia sinensis (L.) O. Kuntze). Plant Growth Regul 30(3):241–246

    Article  CAS  Google Scholar 

  • Babbar BS, Agarwal PK, Sahay S, Bhojwani SS (2004) Isolate microspore culture of Brassica: an experimental tool for developmental studies and crop improvement. Indian J Biotech 3:185–202

    Google Scholar 

  • Barro F, Fernández-Escobar J, De la Vega M, Martín A (2002) Modification of glucosinolate and erucic acid contents in doubled haploid lines of Brassica carinata by UV treatment of isolated microspores. Euphytica 129:1–6

    Article  Google Scholar 

  • Bialecka B, Kepczynsky J (2003) Regulation of α-amylase activity in Amaranthus caudatus seeds by methyl jasmonate, gibberellin A3, benzyladenine and ethylene. Plant Growth Regul 39:51–56

    Article  CAS  Google Scholar 

  • Blázquez S, Piqueras A, Serna MD, Casas JL, Fernández JA (2004) Somatic embryogenesis in saffron: optimization through temporary immersion and polyamine metabolism. Acta Hort 650:269–276

    Google Scholar 

  • Carimi F, Zottini M, Formentin E, Terzi M, Schiavo FL (2003) Cytokinins: new apoptotic inducers in plants. Planta 216:413–421

    CAS  PubMed  Google Scholar 

  • Catinot J, Buchala A, Abou-Mansour E, Métraux JP (2008) Salicylic acid production in response to biotic and abiotic stress depends on isochorismate in Nicotiana benthamiana. FEBS Lett 582:473–478

    Article  CAS  PubMed  Google Scholar 

  • Choudhary K, Singh M, Rathore MS, Shekhawat NS (2009) Somatic embryo formation and in vitro plant regeneration in moth bean [Vigna aconitifolia (Jacq.) Marechal]: a recalcitrant legume grain. Plant Biotech Rep 3:205–211

    Article  Google Scholar 

  • Coventry J, Kott LS (1998) Doubled haploid technology for spring and winter Brassica napus (revised ed.). OAC Publication, University of Guelph, Ontario, Canada. Technol Bull., 42 pp

  • Fang JY, Wetten A, Hadley P (2004) Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos for long-term germplasm storage. Plant Sci 166:669–675

    Article  CAS  Google Scholar 

  • Ferrie AMR, Caswell KL (2011) Isolated microspore culture technique and recent progress for haploid and doubled haploid plant production. Plant Cell Tiss Org Cult 104:301–309

    Article  Google Scholar 

  • Ferrie AMR, Keller WA (2007) Optimization of methods for using polyethylene glycol as a non-permeating osmoticum for the induction of microspore embryogenesis in Brassicaceae. In Vitro Cell Dev Biol-Plant 43:348–355

    Article  CAS  Google Scholar 

  • Ferrie AMR, Taylor DC, MacKenzie SL, Keller WA (1999) Microspore embryogenesis of high sn-2 erucic acid Brassica oleracea germplasm. Plant Cell Tiss Org Cult 57:79–84

    Article  CAS  Google Scholar 

  • Gamborg OL, Miller RA, Ojima L (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158

    Article  CAS  PubMed  Google Scholar 

  • Ghelis T, Bolbach G, Clodic G, Habricot Y, Miginiac E, Sotta B, Jeannette E (2008) Protein tyrosine kinases and protein tyrosine phosphatases are involved in abscisic acid-dependent processes in Arabidopsis seeds and suspension cells. Plant Physiol 148:1668–1680

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hao L, Zhou L, Xu X, Cao J, Xi T (2006) The role of salicylic acid and carrot somatic embryogenic callus extracts in somatic embryogenesis of naked oat (Avena nuda). Plant Cell Tiss Org Cult 85(1):109–113

    Article  CAS  Google Scholar 

  • He Y, Fukushige H, Hildebrand DF, Gan S (2002) Evidence supporting a role of jasmonic acid in Arabidopsis leaf senescence. Plant Physiol 128(3):876–884

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hoseini M, Ghadimzadeh M, Ahmadi B, Teixeira da Silva JA (2013) Effects of ascorbic acid, alpha-tocopherol and glutathione on microspore embryogenesis in Brassica napus L. In Vitro Cell Dev Biol-Plant in press

  • Hutchinson MJ, Saxena PK (1996) Acetylsalicylic acid enhances and synchronizes thidiazuron induced somatic embryogenesis in geranium (Pelargonium × hortorum Baily) tissue cultures. Plant Cell Rep 15:512–515

    Article  CAS  PubMed  Google Scholar 

  • Jacquard C, Mazeyrat-Gourbeyre F, Devaux P, Boutilier K, Baillieul F, Clément C (2009) Microspore embryogenesis in barely: anther pre-treatment stimulates plant defense gene expression. Planta 229:393–402

    Article  CAS  PubMed  Google Scholar 

  • Kott LS, Beversdorf WD (1990) Enhanced plant regeneration fro microspore-derived embryos of Brassica napus by chilling, partial desiccation and age selection. Plant Cell Tiss Org Cult 23:187–192

    Google Scholar 

  • Lichter R (1982) Induction of haploid plants from isolated pollens of Brassica napus. Z Pflanzenphysiol 105:427–434

    Article  Google Scholar 

  • Liu S, Wang H, Zhang J, Fitt BDL, Xu Z, Evans N, Liu Y, Yang W, Guo X (2005) In vitro mutation and selection of doubled-haploid Brassica napus lines with improved resistance to Sclerotinia sclerotiorum. Plant Cell Rep 24:133–144

    Article  CAS  PubMed  Google Scholar 

  • Luo JP, Jiang ST, Pan LJ (2001) Enhanced somatic embryogenesis by salicylic acid of Astragalus adsurgens Pall: relationship with H2O2 production and H2O2-metabolizing enzyme activities. Plant Sci 161:125–132

    Article  CAS  Google Scholar 

  • Malabadi RB, Teixeira da Silva JA, Nataraja K (2008) Salicylic acid induces somatic embryogenesis from nature trees of Pinus roxburghii (Chir pine) using TCL technology. Tree Forestry Sci Biotechnol 2(1):34–39

    Google Scholar 

  • Maraschin SDF, de Priester W, Spaink HP, Wang M (2005) Androgenic switch: an example of plant embryogenesis from the male gametophyte perspective. J Exp Bot 56:1711–1726

    Article  CAS  PubMed  Google Scholar 

  • Maraschin SDF, Caspers M, Potokina E, Wulfert F, Graner A, Spaink HP, Wang M (2006) cDNA array analysis of stress-induced gene expression in barley androgenesis. Physiol Plant 127:535–550

    Article  CAS  Google Scholar 

  • Mulgund GS, Meti NT, Malabadi RB, Nataraja K, Kumar SV (2012) Role of salicylic acid on conifer somatic embryogenesis. Res Biotechnol 3(2):57–61

    Google Scholar 

  • Nojavan-Asghari M, Ishizava K (1998) Inhibitory effects of methyl jasmonate on the germination and ethylene production and cocklebur seeds. J Plant Growth Regul 17:13–18

    Article  CAS  Google Scholar 

  • Prem D, Gupta K, Agnihotri A (2005) Effect of various exogenous and endogenous factors on microspore embryogenesis in Indian mustard (Brassica juncea L. Czern and Coss). In Vitro Cell Dev Biol-Plant 41:266–273

    Article  Google Scholar 

  • Preston CA, Betts H, Baldwin IT (2002) Methyl jasmonate as an allelopathic agent: sagebrush inhibits germination of a neighboring tobacco, Nicotiana attenuata. J Chem Ecol 28:2343–2369

    Article  CAS  PubMed  Google Scholar 

  • Rai MK, Jaiswal VS, Jaiswal U (2009) Effect of selected amino acids and polyethylene glycol on maturation and germination of somatic embryos of guava (Psidium guajava L.). Sci Hort 121:233–236

    Article  CAS  Google Scholar 

  • Reynolds TL (2000) Effects of calcium on embryogenic induction and the accumulation of abscisic acid, and an early cystein-labeled metallothionein gene in androgenic microspores of Triticum aestivum. Plant Sci 150:201–207

    Article  CAS  Google Scholar 

  • Reynolds TL, Crawford RL (1996) Changes in abundance of an abscisic acid-responsive, early cystein-labeled metalothionein transcript during pollen embryogenesis in bread wheat (Triticum aestivum). Plant Mol Biol 32:823–829

    Article  CAS  PubMed  Google Scholar 

  • Rodríguez-Serrano M, Bárány I, Prem D, Coronado MJ, Risueño MC, Testilano PS (2012) NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley. J Exp Bot 63:2007–2024

    Article  PubMed  Google Scholar 

  • Roustan JP, Latche A, Fallot J (1989) Effect of salicylic acid and acetylsalcylicon ethylene production and somatic embryogenesis in carrot (Daucus carota L.) cell suspensions. C R Acad Sci 308:395–399

    CAS  Google Scholar 

  • Ruduś I, Kępczyński J, Kępczyńska E (2001) The influence of the jasmonates and abscisic acid on callus growth and somatic embryogenesis in Medicago sativa L. tissue culture. Acta Physiol Plant 23(1):103–107

    Article  Google Scholar 

  • Sakhanokho HF, Rajasekaran K, Kelley RY (2009) Somatic embryogenesis in Hedychium bousigonianum. Hort Sci 44(5):1487–1490

    Google Scholar 

  • Santra M, Ankrah N, Santra DK, Kidwell KK (2012) An improved wheat microspore culture technique for the production of doubled haploid plants. Crop Sci 52(5):2314–2320

    Article  Google Scholar 

  • Shariatpanahi ME, Bal U, Heberle-Bors E, Touraev A (2006) Stresses applied for the re-programming of plant microspores towards in vitro embryogenesis. Physiol Plant 127:519–534

    Article  CAS  Google Scholar 

  • Sharma P, Pandey S, Bhattacharya A, Nagar PK, Ahuja PS (2004) ABA associated biochemical changes during somatic embryo development in Camellia sinensis (L.) O. Kuntze. J Plant Physiol 161:1269–1276

    Article  CAS  PubMed  Google Scholar 

  • Takahashi K, Fujino K, Kikuta Y, Koda Y (1994) Expansion of potato cells in response to jasmonic acid. Plant Sci 100:3–8

    Article  CAS  Google Scholar 

  • Teixeira da Silva JA (2012) Impact of methyl jasmonate on PLB formation of hybrid Cymbidium (Orchidaceae). J Plant Develop 19:47–52

    Google Scholar 

  • Teixeira da Silva JA (2013) Jasmonic acid, but not salicylic acid, improves PLB formation of hybrid Cymbidium. Plant Tiss Cult Biotech 22(2):187–192

    Google Scholar 

  • Tsuwamoto R, Fukuoka H, Takahata Y (2007) Identification and characterization of genes expressed in early embryogenesis from microspores of Brassica napus. Planta 225:641–652

    Article  CAS  PubMed  Google Scholar 

  • Van Bergen S, Kottenhagen MJ, van der Meulen RM, Wang M (1999) Effects of ABA during the pretreatment of barley anthers on androgenesis of Hordeum vulgare L. cultivras Igri and Digger. J Plant Growth Regul 18:135-143

    Google Scholar 

  • Von Arnold S, Sabala I, Bozhkov P, Dyachok J, Filonova L (2002) Developmental pathway of somatic embryogenesis. Plant Cell Tiss Org Cult 69:233–249

    Article  Google Scholar 

  • Wang M, Hoekstra S, van Bergen S, Lamers GEM, Oppedijk BJ, van der Heijden MW, de Priester W, Schilperoort RA (1999) Apoptosis in developing anthers and the role of ABA in this process during androgenesis in Hordeum vulgare L. Plant Mol Biol 39:489–501

    Article  CAS  PubMed  Google Scholar 

  • Zhang GQ, Zhang DQ, Tang GX, He Y, Zhou WJ (2006) Development from microspore derived embryos in oilseed rape as affected by chilling, desiccation and cotyledon excision. Biol Plant 150:180–186

    Article  Google Scholar 

  • Zur I, Dubas E, Golemiec E, Szechynska-Hebda M, Janowiak F, Wedzony M (2008) Stress-induced changes important for effective androgenic induction in isolated microspore culture of triticale (× Triticosecale Wittm.). Plant Cell Tiss Org Cult 94(3):319–328

    Article  CAS  Google Scholar 

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Ahmadi, B., Shariatpanahi, M.E. & Teixeira da Silva, J.A. Efficient induction of microspore embryogenesis using abscisic acid, jasmonic acid and salicylic acid in Brassica napus L. Plant Cell Tiss Organ Cult 116, 343–351 (2014).

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