Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 102, Issue 3, pp 285–295 | Cite as

Somatic embryogenesis and plant regeneration of tropical maize genotypes

  • Sylvester Elikana Anami
  • Allan Jalemba Mgutu
  • Catherine Taracha
  • Griet Coussens
  • Mansour Karimi
  • Pierre Hilson
  • Mieke Van Lijsebettens
  • Jesse Machuka
Original Paper


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.


Tropical maize Callus induction Somatic embryogenesis 



2,4-Dichlorophenoxyacetic acid


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


β-d-glucuronidase gene from Escherichia coli


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


Yeast Extract Peptone


  1. Al-Abed D, Rudrabhatla S, Talla R, Goldman S (2006) Split-seed: a new tool for maize researchers. Planta 223:1355–1360CrossRefPubMedGoogle Scholar
  2. 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 28:16–35CrossRefGoogle Scholar
  3. Armstrong CL, Phillips RL (1988) Genetic and cytogenetic variation in plants regenerated from organogenic and friable, embryogenic tissue cultures of maize. Crop Sci 28:363–369CrossRefGoogle Scholar
  4. Bancroft JD, Stevens A (1990) Theory and practice of histological techniques, 3rd edn. Churchill Livingstone, New YorkGoogle Scholar
  5. Bohorova NE, Luna B, Brito RM, Huerta LD, Hoisington DA (1995) Regeneration potential of tropical, subtropical, midaltitude, and highland maize inbreds. Maydica 40:275–281Google Scholar
  6. 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 99:437–444CrossRefGoogle Scholar
  7. 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 103:817–826CrossRefGoogle Scholar
  8. 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 17:73–76CrossRefGoogle Scholar
  9. 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 40:31–45CrossRefGoogle Scholar
  10. 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 18:659–668Google Scholar
  11. 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 6:93–102PubMedGoogle Scholar
  12. 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 25:5579–5590CrossRefPubMedGoogle Scholar
  13. Escudero J, Neuhaus G, Schläppi M, Hohn B (1996) T-DNA transfer in meristematic cells of maize provided with intracellular Agrobacterium. Plant J 10:355–360CrossRefGoogle Scholar
  14. 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 82:569–576CrossRefGoogle Scholar
  15. 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 129:13–22CrossRefPubMedGoogle Scholar
  16. 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 25:1024–1034CrossRefPubMedGoogle Scholar
  17. 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 90:41–52CrossRefGoogle Scholar
  18. 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 2:603–618CrossRefPubMedGoogle Scholar
  19. Hodges TK, Kamo KK, Imbrie CW, Becwar MR (1986) Genotype specificity of somatic embryogenesis and regeneration in maize. BioTechnology 4:219–223Google Scholar
  20. 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 168:1291–1301PubMedGoogle Scholar
  21. 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 14:745–750CrossRefPubMedGoogle Scholar
  22. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405CrossRefGoogle Scholar
  23. Karimi M, Depicker A, Hilson P (2007) Recombinational cloning with plant Gateway vectors. Plant Physiol 145:1144–1154CrossRefPubMedGoogle Scholar
  24. Kay R, Chan A, Daly M, McPherson J (1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236:1299–1302CrossRefPubMedGoogle Scholar
  25. Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18:100–127CrossRefGoogle Scholar
  26. Lu C, Vasil IK, Ozias-Akins P (1982) Somatic embryogenesis in Zea mays L. Theor Appl Genet 62:109–112CrossRefGoogle Scholar
  27. Mondal KC, Banerjee D, Jana M, Pati BR (2001) Colorimetric assay method for determination of the tannin acyl hydrolase (EC activity. Anal Biochem 295:168–171CrossRefPubMedGoogle Scholar
  28. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  29. 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 19:798–803CrossRefGoogle Scholar
  30. 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 21:302–312CrossRefGoogle Scholar
  31. Oduor RO, Njagi ENM, Ndung’u S, Machuka JS (2006) In vitro regeneration of dryland Kenyan maize genotypes through somatic embryogenesis. Int J Bot 2:146–151CrossRefGoogle Scholar
  32. Prioli LM, da Silva WJ (1989) Somatic embryogenesis and plant regeneration capacity in tropical maize inbreds. Rev Bras Genét 12:553–566Google Scholar
  33. 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 46:323–329CrossRefPubMedGoogle Scholar
  34. Schläppi M, Hohn B (1992) Competence of immature maize embryos for Agrobacterium-mediated gene transfer. Plant Cell 4:7–16CrossRefPubMedGoogle Scholar
  35. 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 326:1112–1115CrossRefPubMedGoogle Scholar
  36. 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 5:759–767CrossRefPubMedGoogle Scholar
  37. 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 2:154–161CrossRefGoogle Scholar
  38. 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.emo132Google Scholar
  39. 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 21:3350–3367CrossRefPubMedGoogle Scholar
  40. 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 70:505–509CrossRefGoogle Scholar
  41. 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 91:201–214CrossRefGoogle Scholar
  42. 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 326:1078CrossRefPubMedGoogle Scholar
  43. 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 81:272–276CrossRefGoogle Scholar
  44. 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 2009:957602PubMedGoogle Scholar
  45. 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 30:69–73CrossRefGoogle Scholar
  46. 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 72:34–37Google Scholar
  47. 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 24:832–837CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Sylvester Elikana Anami
    • 1
    • 2
    • 3
  • Allan Jalemba Mgutu
    • 1
  • Catherine Taracha
    • 4
  • Griet Coussens
    • 2
    • 3
  • Mansour Karimi
    • 2
    • 3
  • Pierre Hilson
    • 2
    • 3
  • Mieke Van Lijsebettens
    • 2
    • 3
  • Jesse Machuka
    • 1
  1. 1.Plant Transformation Facility, Department of Biochemistry and BiotechnologyKenyatta UniversityNairobiKenya
  2. 2.Department of Plant Systems BiologyVIBGentBelgium
  3. 3.Department of Plant Biotechnology and GeneticsGhent UniversityGentBelgium
  4. 4.Kenya Agricultural Research InstituteNairobiKenya

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