Genetic Transformation Using Maize Immature Zygotic Embryos

  • Bronwyn Frame
  • Marcy Main
  • Rosemarie Schick
  • Kan Wang
Part of the Methods in Molecular Biology book series (MIMB, volume 710)


Epidermal and subepidermal cells in the abaxial, basal region of the maize (Zea mays L.) immature zygotic embryo (IZE) scutellum can be induced by exogenous auxin to proliferate and undergo somatic embryogenesis. Successful genetic transformation of IZEs depends not only on optimizing transformation parameters for these totipotent cells, but also on achieving high embryogenic callus induction frequency (ECIF) in a population of targeted explants. In maize, ECIF is strongly influenced by genotype, the tissue culture media used, and the interaction of these two factors. Altering tissue culture media components to increase ECIF and/or transformation frequency (TF) has been one approach used to expand the range of maize genotypes amenable to genetic transformation using the IZE. This chapter outlines such an approach – an Agrobacterium-mediated transformation protocol is used for direct-targeting IZEs of the hybrid Hi Type II and inbred B104 lines. Two different media regimes are used for successful culture and transformation of two distinct genotypes.

Key words

Agrobacterium tumefaciens B104 Callus induction frequency Embryogenic callus Genetic transformation Hi II Immature zygotic embryo Maize 



Our thanks to Jennifer McMurray and Tina Paque for their contributions in the laboratory and greenhouse, and to Dr. Arnel Hallauer for providing the original B104 seed. This work is ­supported partially by the National Science Foundation (DBI #0110023), the Iowa State University Agricultural Experiment Station, the Office of Biotechnology, the Plant Science Institute, and the Baker Endowment Advisory Council for Excellence in Agronomy at Iowa State University.


  1. 1.
    Green CE, Phillips RL (1975) Plant regeneration from tissue cultures of maize. Crop Sci 15:417–421CrossRefGoogle Scholar
  2. 2.
    Vasil V, Vasil IK, Lu C (1984) Somatic embryogenesis in long-term callus cultures of Zea mays L (Gramineae). Am J Bot 71:158–161CrossRefGoogle Scholar
  3. 3.
    Fransz PF, Schel JHN (1990) Cyto-differentiation during the development of friable embryogenic callus in maize (Zea mays). Can J Bot 69:26–33CrossRefGoogle Scholar
  4. 4.
    Armstrong CL, Green CE (1985) Establishment and maintenance of friable, embryogenic maize callus and the involvement of L-proline. Planta 164:207–214CrossRefGoogle Scholar
  5. 5.
    Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O’Brien JV, Chambers SA, Whitney J, Adams R, 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 2:603–618PubMedCrossRefGoogle Scholar
  6. 6.
    Fromm ME, Morrish F, Armstrong C, Williams R, Thomas J, Klein TM (1990) Inheritance and expression of chimeric genes in the progeny of transgenic maize plants. Bio/Technology 8:833–839PubMedCrossRefGoogle Scholar
  7. 7.
    D’Halluin K, Bonne E, Bossut M, De Beuckeleer M, Leemans J (1992) Transgenic maize plants by tissue electroporation. Plant Cell 4:1495–1505PubMedCrossRefGoogle Scholar
  8. 8.
    Koziel MG, Beland GL, Bowman C, Carozzi NB, Crenshaw R, Crossland L, Dawson J, Desai N, Hill M, Kadwell S, Launis K, Lewis K, Maddox D, McPherson K, Meghji MR, Merlin E, Rhodes R, Warren GW, Wright M, Evola SV (1993) Field performance of elite transgenic maize plants expressing and insecticidal protein derived from Bacillus thuringiensis. Bio/Technology 11:194–200CrossRefGoogle Scholar
  9. 9.
    Songstad DD, Armstrong CL, Petersen WL, Hairston B, Hinchee MAW (1996) Production of transgenic maize plants and progeny by bombardment of Hi-II immature embryos. In Vitro Cell Dev Biol Plant 32:179–183CrossRefGoogle Scholar
  10. 10.
    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 14:745–750PubMedCrossRefGoogle Scholar
  11. 11.
    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 Gen Coop Newsl 72:34–37Google Scholar
  12. 12.
    Frame BR, Shou H, Chikwamba RK, Zhang Z, Xiang C, Fonger TM, Pegg SE, Li B, Nettleton DS, Pei D, Wang K (2002) Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiol 129:13–22PubMedCrossRefGoogle Scholar
  13. 13.
    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 25:1024–1034PubMedCrossRefGoogle Scholar
  14. 14.
    Zhao ZY, Gu W, Cai T, Tagliani LA, Hondred D, Bond D, Schroeder S, Rudert M, Pierce DA (2001) High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Mol Breed 8:323–333CrossRefGoogle Scholar
  15. 15.
    Brettschneider R, Becker D, Lorz H (1997) Efficient transformation of scutellar tissue of immature maize embryos. Theor Appl Genet 94:737–748CrossRefGoogle Scholar
  16. 16.
    Ishida Y, Saito H, Hiei Y, Komari T (2003) Improved protocol for transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Plant Biotech 20:57–66CrossRefGoogle Scholar
  17. 17.
    Lee BK, Kennon AR, Chen X, Jung TW, Ahn BO, Lee JY, Zhang Z (2007) Recovery of transgenic events from two highly recalcitrant maize (Zea mays L.) genotypes using Agrobacterium-mediated standard-binary-vector transformation. Maydica 52:457–469Google Scholar
  18. 18.
    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
  19. 19.
    Duncan DR, Williams ME, Zehr BE, Widholm JM (1985) The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes. Planta 165:322–332CrossRefGoogle Scholar
  20. 20.
    Hodges TK, Kamo KK, Imbrie CW, Becwar MR (1986) Genotype specificity of somatic embryogenesis and regeneration in maize. Bio/Technology 4:219–223Google Scholar
  21. 21.
    Vain P, Yean H, Flament P (1989) Enhancement of production and regeneration of embryogenic type II callus in Zea mays by AgNO3. Plant Cell Tiss Org Cult 18:143–151CrossRefGoogle Scholar
  22. 22.
    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
  23. 23.
    Lu C, Vasil V, Vasil IK (1983) Improved efficiency of somatic embryogenesis and plant regeneration from tissue cultures of maize (Zea mays L.). Theor Appl Genet 66:285–289CrossRefGoogle Scholar
  24. 24.
    Vain P, McMullen MD, Finer JJ (1993) Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize. Plant Cell Rep 12:84–88CrossRefGoogle Scholar
  25. 25.
    Armstrong CL, Green CE, Phillips RL (1991) Development and availability of germplasm with high Type II culture formation response. Maize Genet Coop Newsl 65:92–93Google Scholar
  26. 26.
    Vega JM, Yu W, Kennon AR, Chen X, Zhang Z (2008) Improvement of Agrobacterium-mediated transformation in Hi II maize (Zea mays) using standard binary vectors. Plant Cell Rep 27:297–305PubMedCrossRefGoogle Scholar
  27. 27.
    Lupotto E, Conti E, Reali A, Lanzanova C, Baldoni E, Allegri L (2004) Improving in vitro culture and regeneration conditions for Agrobacterium-mediated maize transformation. Maydica 49:21–29Google Scholar
  28. 28.
    Huang X, Wei Z (2005) Successful Agrobacterium-mediated genetic transformation of maize elite inbred lines. Plant Cell, Tiss Org Cult 83:187–200CrossRefGoogle Scholar
  29. 29.
    Ishida Y, Hiei Y, Komari T (2007) Agrobacterium-mediated transformation of maize. Nat Protoc 2:1614–1621PubMedCrossRefGoogle Scholar
  30. 30.
    Hallauer R, Lamkey KR, White PR (1997) Registration of five inbred lines of maize: B102, B103, B104, B105 and B106. Crop Sci 37:1405–1406CrossRefGoogle Scholar
  31. 31.
    Paz MM, Shou H, Guo Z, Zhang Z, Banerjee AK, Wang K (2004) Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explants. Euphytica 136:167–179CrossRefGoogle Scholar
  32. 32.
    Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994PubMedCrossRefGoogle Scholar
  33. 33.
    Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168:1291–1301PubMedGoogle Scholar
  34. 34.
    White J, Chang S, Bibb MJ, Bibb MJ (1990) A cassette containing the bar gene of Streptomyces hygroscopicus: a selectable marker for plant transformation. Nucleic Acids Res 18:1062PubMedCrossRefGoogle Scholar
  35. 35.
    Carrington JC, Freed DD (1990) Cap-independent enhancement of translation by a plant potyvirus 5′ nontranslated region. J Virol 64:1590–1597PubMedGoogle Scholar
  36. 36.
    Mason HS, DeWald DB, Mullet JE (1993) Identification of a methyl jasmonate-­responsive domain in the soybean vspB ­promoter. Plant Cell 5:241–251PubMedCrossRefGoogle Scholar
  37. 37.
    An G, Ebert PR, Mitra A, Ha SB (1988) Binary vectors. In: Gelvin SB, Schilperoort RA (eds) Plant molecular biology manual. Kluwer Academic, Dordrecht, pp 1–19Google Scholar
  38. 38.
    Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bi FY (1975) Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen source. Sci Sin 18:659–668Google Scholar
  39. 39.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  40. 40.
    McCain JW, Kamo KK, Hodges TK (1988) Characterization of somatic embryo devel­opment and plant regeneration from friable maize callus cultures. Bot Gaz 149:16–20CrossRefGoogle Scholar

Copyright information

© Humana Press 2011

Authors and Affiliations

  • Bronwyn Frame
    • 1
  • Marcy Main
    • 2
  • Rosemarie Schick
    • 2
  • Kan Wang
    • 2
  1. 1.Plant Science Institute, Department of Agronomy, Center for Plant TransformationIowa State UniversityAmesUSA
  2. 2.Department of Agronomy, Center for Plant Transformation, Plant Science InstituteIowa State UniversityAmesUSA

Personalised recommendations