Skip to main content

Somatic embryogenesis and plant regeneration of tropical maize genotypes


In Latin America and sub-Saharan Africa, tropical maize (Zea mays L.) is a major crop for human consumption. To cope with the increasing population and changing environment, there is a need for improving tropical maize germplasm. As part of a biotechnological approach, efficient in vitro regeneration of two tropical maize inbred lines (CML216 and CML244) was established. A number of parameters were optimized, such as age of the immature embryos, plant media and growth regulator concentration. After 6 weeks of culture, somatic embryos that had already reached the coleoptilar stage produced shoots after light induction and developed into fertile plants after acclimation in the soil. The callus induction frequencies and somatic embryo-derived plantlet formation were higher when cultured with the Linsmaier and Skoog medium than those with the Chu’s N6 basal medium. Regeneration of tropical maize shoots depended on the 2,4-dichlorophenoxyacetic acid (2,4-D) concentration at the callus initiation stage from immature embryos. The recalcitrance of the tropical maize inbred line TL26 to in vitro regeneration was overcome in a single-cross hybrid with the CML216 and CML244 genotypes. Remarkably, tropical maize somatic embryos were formed at the abaxial side of the scutellum facing the medium, probably from the axis of the immature embryos, as shown by histological sections. Upon co-cultivation, agrobacteria transiently expressed their intronless β-glucuronidase-encoding gene at the embryogenic tissue, but not with an intron-containing gene, suggesting that virulence genes are induced in Agrobacterium, but that subsequent steps in the T-DNA transfer are inhibited.

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

Fig. 1
Fig. 2
Fig. 3



2,4-Dichlorophenoxyacetic acid

bar :

Phosphinothricin acetyltransferase-encoding gene


Callus induction medium or resting medium


Callus maturation medium


Embryo suspension medium/infection medium




Linsmaier and Skoog


2-(N-morpholino)ethanesulphonic acid


Murashige and Skoog


Chu’s N6 basal medium


Cauliflower mosaic virus 35S promoter


Piperazine-N,N-bis(2-ethanesulphonic acid)


Regenerated shoot from somatic embryo


Progeny of R0 shoot


Shoot induction medium or regeneration medium


Transfer DNA


Cauliflower mosaic virus 35S terminator


Nopaline synthase terminator


Maize long ubiquitin promoter

uidA :

β-d-glucuronidase gene from Escherichia coli


5-Bromo-4-chloro-3-indolyl-β-d-glucuronic acid


Yeast Extract Peptone


  • Al-Abed D, Rudrabhatla S, Talla R, Goldman S (2006) Split-seed: a new tool for maize researchers. Planta 223:1355–1360

    Article  CAS  PubMed  Google Scholar 

  • Anami S, De Block M, Machuka J, Van Lijsebettens M (2009) Molecular improvement of tropical maize for drought stress tolerance in sub-Saharan Africa. Crit Rev Plant Sci 28:16–35

    Article  CAS  Google Scholar 

  • Armstrong CL, Phillips RL (1988) Genetic and cytogenetic variation in plants regenerated from organogenic and friable, embryogenic tissue cultures of maize. Crop Sci 28:363–369

    Article  Google Scholar 

  • Bancroft JD, Stevens A (1990) Theory and practice of histological techniques, 3rd edn. Churchill Livingstone, New York

    Google Scholar 

  • Bohorova NE, Luna B, Brito RM, Huerta LD, Hoisington DA (1995) Regeneration potential of tropical, subtropical, midaltitude, and highland maize inbreds. Maydica 40:275–281

    Google Scholar 

  • Bohorova N, Zhang W, Julstrum P, McLean S, Luna B, Brito RM, Diaz L, Ramos ME, Estanol P, Pacheco M, Salgado M, Hoisington D (1999) Production of transgenic tropical maize with cryIAb and cryIAc genes via microprojectile bombardment of immature embryos. Theor Appl Genet 99:437–444

    Article  CAS  Google Scholar 

  • Bohorova N, Frutos R, Royer M, Estañol P, Pacheco M, Rascón Q, McLean S, Hoisington D (2001) Novel synthetic Bacillus thuringiensis cry1B gene and cry1B-cry1Ab translational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in transgenic tropical maize. Theor Appl Genet 103:817–826

    Article  CAS  Google Scholar 

  • Carvalho CHS, Bohorova N, Bordallo PN, Abreu LL, Valicente FH, Bressan W, Paiva E (1997) Type II callus production and plant regeneration in tropical maize genotypes. Plant Cell Rep 17:73–76

    Article  CAS  Google Scholar 

  • Cheng M, Lowe BA, Spencer TM, Ye X, Armstrong CL (2004) Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. In Vitro Cell Dev Biol Plant 40:31–45

    Article  Google Scholar 

  • Chu CC, Wang CC, Sun CS, Chen H, Yin KC, Chu CY, Bi FY (1975) Establishment of an efficient medium for anther culture of rice through comparative experiments on nitrogen sources. Sci Sin 18:659–668

    Google Scholar 

  • D’Halluin K, Vanderstraeten C, Stals E, Cornelissen M, Ruiter R (2007) Homologous recombination: a basis for targeted genome optimization in crop species such as maize. Plant Biotechnol J 6:93–102

    PubMed  Google Scholar 

  • Endo M, Ishikawa Y, Osakabe K, Nakayama S, Kaya H, Araki T, Shibahara K, Abe K, Ichikawa H, Valentine L, Hohn B, Toki S (2006) Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants. EMBO J 25:5579–5590

    Article  CAS  PubMed  Google Scholar 

  • Escudero J, Neuhaus G, Schläppi M, Hohn B (1996) T-DNA transfer in meristematic cells of maize provided with intracellular Agrobacterium. Plant J 10:355–360

    Article  CAS  Google Scholar 

  • Fluminhan A, De Aguiar-Perecin MLR (1998) Embryogenic response and mitotic instability in callus cultures derived from maize inbred lines differing in heterochromatic knob content of chromosomes. Ann Bot 82:569–576

    Article  Google Scholar 

  • Frame BR, Shou H, Chikwamba RK, Zhang Z, Xiang C, Fonger TM, Pegg SEK, Li B, Nettleton DS, Pei D, Wang K (2002) Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiol 129:13–22

    Article  CAS  PubMed  Google Scholar 

  • Frame BR, McMurray JM, Fonger TM, Main ML, Taylor KW, Torney FJ, Paz MM, Wang K (2006) Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts. Plant Cell Rep 25:1024–1034

    Article  CAS  PubMed  Google Scholar 

  • Golovkin MV, Ábrahám M, Mórocz S, Bottka S, Fehér A, Dudits D (1993) Production of transgenic maize plants by direct DNA uptake into embryogenic protoplasts. Plant Sci 90:41–52

    Article  CAS  Google Scholar 

  • Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O’Brien JV, Chambers SA, Adams WR Jr, Willetts NG, Rice TB, Mackey CJ, Krueger RW, Kausch AP, Lemaux PG (1990) Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2:603–618

    Article  CAS  PubMed  Google Scholar 

  • Hodges TK, Kamo KK, Imbrie CW, Becwar MR (1986) Genotype specificity of somatic embryogenesis and regeneration in maize. BioTechnology 4:219–223

    Google Scholar 

  • Hood EE, Helmer GL, Fraley RT, Chilton M-D (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168:1291–1301

    CAS  PubMed  Google Scholar 

  • Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750

    Article  CAS  PubMed  Google Scholar 

  • Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405

    Article  CAS  Google Scholar 

  • Karimi M, Depicker A, Hilson P (2007) Recombinational cloning with plant Gateway vectors. Plant Physiol 145:1144–1154

    Article  CAS  PubMed  Google Scholar 

  • Kay R, Chan A, Daly M, McPherson J (1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236:1299–1302

    Article  CAS  PubMed  Google Scholar 

  • Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18:100–127

    Article  CAS  Google Scholar 

  • Lu C, Vasil IK, Ozias-Akins P (1982) Somatic embryogenesis in Zea mays L. Theor Appl Genet 62:109–112

    Article  Google Scholar 

  • Mondal KC, Banerjee D, Jana M, Pati BR (2001) Colorimetric assay method for determination of the tannin acyl hydrolase (EC activity. Anal Biochem 295:168–171

    Article  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Negrotto D, Jolley M, Beer S, Wenck AR, Hansen G (2000) The use of phoshomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Rep 19:798–803

    Article  CAS  Google Scholar 

  • O’Connor-Sánchez A, Cabrera-Ponce JL, Valdez-Melara M, Téllez-Rodríguez P, Pons-Hernández JL, Herrera-Estrella L (2002) Transgenic maize plants of tropical and subtropical genotypes obtained from calluses containing organogenic and embryogenic-like structures derived from shoot tips. Plant Cell Rep 21:302–312

    Article  Google Scholar 

  • Oduor RO, Njagi ENM, Ndung’u S, Machuka JS (2006) In vitro regeneration of dryland Kenyan maize genotypes through somatic embryogenesis. Int J Bot 2:146–151

    Article  Google Scholar 

  • Prioli LM, da Silva WJ (1989) Somatic embryogenesis and plant regeneration capacity in tropical maize inbreds. Rev Bras Genét 12:553–566

    Google Scholar 

  • Sairam RV, Parani M, Franklin G, Lifeng Z, Smith B, MacDougall J, Wilber C, Sheikhi H, Kashikar N, Meeker K, Al-Abed D, Berry K, Vierling R, Goldman SL (2003) Shoot meristem: an ideal explant for Zea mays L. transformation. Genome 46:323–329

    Article  CAS  PubMed  Google Scholar 

  • Schläppi M, Hohn B (1992) Competence of immature maize embryos for Agrobacterium-mediated gene transfer. Plant Cell 4:7–16

    Article  PubMed  Google Scholar 

  • Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S, Liang C, Zhang J, Fulton L, Graves TA, Minx P, Reily AD, Courtney L, Kruchowski SS, Tomlinson C, Strong C, Delehaunty K, Fronick C, Courtney B, Rock SM, Belter E, Du F, Kim K, Abbott RM, Cotton M, Levy A, Marchetto P, Ochoa K, Jackson SM, Gillam B, Chen W, Yan L, Higginbotham J, Cardenas M, Waligorski J, Applebaum E, Phelps L, Falcone J, Kanchi K, Thane T, Scimone A, Thane N, Henke J, Wang T, Ruppert J, Shah N, Rotter K, Hodges J, Ingenthron E, Cordes M, Kohlberg S, Sgro J, Delgado B, Mead K, Chinwalla A, Leonard S, Crouse K, Collura K, Kudrna D, Currie J, He R, Angelova A, Rajasekar S, Mueller T, Lomeli R, Scara G, Ko A, Delaney K, Wissotski M, Lopez G, Campos D, Braidotti M, Ashley E, Golser W, Kim H, Lee S, Lin JK, Dujmic Z, Kim W, Talag J, Zuccolo A, Fan C, Sebastian A, Kramer M, Spiegel L, Nascimento L, Zutavern T, Miller B, Ambroise C, Muller S, Spooner W, Narechania A, Ren L, Wei S, Kumari S, Faga B, Levy MJ, McMahan L, Van Buren P, Vaughn MW, Ying K, Yeh C-T, Emrich SJ, Jia Y, Kalyanaraman A, Hsia A-P, Barbazuk WB, Baucom RS, Brutnell TP, Carpita NC, Chaparro C, Chia J-M, Deragon J-M, Estill JC, Fu Y, Jeddeloh JA, Han Y, Lee H, Li P, Lisch DR, Liu S, Liu Z, Nagel DH, McCann MC, SanMiguel P, Myers AM, Nettleton D, Nguyen J, Penning BW, Ponnala L, Schneider KL, Schwartz DC, Sharma A, Soderlund C, Springer NM, Sun Q, Wang H, Waterman M, Westerman R, Wolfgruber TK, Yang L, Yu Y, Zhang L, Zhou S, Zhu Q, Bennetzen JL, Dawe RK, Jiang J, Jiang N, Presting GG, Wessler SR, Aluru S, Martienssen RA, Clifton SW, McCombie WR, Wing RA, Wilson RK (2009) The B73 maize genome: complexity, diversity, and dynamics. Science 326:1112–1115

    Article  CAS  PubMed  Google Scholar 

  • Shepherd DN, Mangwende T, Martin DP, Bezuidenhout M, Kloppers FJ, Carolissen CH, Monjane AL, Rybicki EP, Thomson JA (2007) Maize streak virus-resistant transgenic maize: a first for Africa. Plant Biotechnol J 5:759–767

    Article  CAS  PubMed  Google Scholar 

  • Shohael AM, Akanda MAL, Parvez S, Mahfuja S, Alam MF, Islam R, Joarder N (2003) Somatic embryogenesis and plant regeneration from immature embryo derived callus of inbred maize (Zea mays L.). Biotechnology 2:154–161

    Article  Google Scholar 

  • Strable J, Scanlon MJ (2009) Maize (Zea mays): a model organism for basic and applied research in plant biology. Cold Spring Harb Protoc 2009:pdb.emo132

  • Tenea GN, Spantzel J, Lee L-Y, Zhu Y, Lin K, Johnson SJ, Gelvin SB (2009) Overexpression of several Arabidopsis histone genes increases Agrobacterium-mediated transformation and transgene expression in plants. Plant Cell 21:3350–3367

    Article  CAS  PubMed  Google Scholar 

  • Tomes DT, Smith OS (1985) The effect of parental genotype on initiation of embryogenic callus from elite maize (Zea mays L.) germplasm. Theor Appl Genet 70:505–509

    Article  Google Scholar 

  • Valdez-Ortiz A, Medina-Godoy S, Valverde ME, Paredes-López O (2007) A transgenic tropical maize line generated by the direct transformation of the embryo-scutellum by A. tumefaciens. Plant Cell Tiss Organ Cult 91:201–214

    Article  Google Scholar 

  • Vielle-Calzada J-P, Martínez de la Vega O, Hernández-Guzmán G, Ibarra-Laclette E, Alvarez-Mejía C, Vega-Arreguín JC, Jiménez-Moraíta B, Fernández-Cortés A, Corona-Armenta G, Herrera-Estrella L, Herrera-Estrella A (2009) The Palomero genome suggests metal effects on domestication. Science 326:1078

    Article  CAS  PubMed  Google Scholar 

  • Williams ME, Hepburn AG, Widholm JM (1990) Somaclonal variation in a maize inbred line is not associated with changes in the number or location of Ac-homologous sequences. Theor Appl Genet 81:272–276

    Article  Google Scholar 

  • Xu Y, Skinner DJ, Wu H, Palacios-Rojas N, Araus JL, Yan J, Gao S, Warburton ML, Crouch JH (2009) Advances in maize genomics and their value for enhancing genetic gains from breeding. Int J Plant Genomics 2009:957602

    PubMed  Google Scholar 

  • Yu X, Li Y, Wu D (2004) Enzymatic synthesis of gallic acid esters using microencapsulated tannase: effect of organic solvents and enzyme specificity. J Mol Catal B Enzym 30:69–73

    Article  CAS  Google Scholar 

  • Zhao ZY, Gu W, Cai T, Tagliani LA, Hondred D, Bond D, Krell S, Rudert ML, Bruce WB, Pierce DA (1998) Molecular analysis of T0 plants transformed by Agrobacterium and comparison of Agrobacterium-mediated transformation with bombardment transformation in maize. Maize Genet Coop Newslett 72:34–37

    Google Scholar 

  • Zheng Y, He X-W, Ying Y-H, Lu J-F, Gelvin SB, Shou HX (2009) Expression of the Arabidopsis thaliana histone gene AtHTA1 enhances rice transformation efficiency. Mol Plant 24:832–837

    Article  Google Scholar 

Download references


We thank Dr. Martine De Cock for help in preparing the manuscript. S.E.A., A.J.M., and C.T. are indebted to the Vlaamse Interuniversitaire Raad, The Rockefeller Foundation, and the United States Agency for International Development for predoctoral fellowships, respectively.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Mieke Van Lijsebettens.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Anami, S.E., Mgutu, A.J., Taracha, C. et al. Somatic embryogenesis and plant regeneration of tropical maize genotypes. Plant Cell Tiss Organ Cult 102, 285–295 (2010).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: