One of the limitations to conducting maize Agrobacterium-mediated transformation using explants of immature zygotic embryos routinely is the availability of the explants. To produce immature embryos routinely and continuously requires a well-equipped greenhouse and laborious artificial pollination. To overcome this limitation, an Agrobacterium-mediated transformation system using explants of type II embryogenic calli was developed. Once the type II embryogenic calli are produced, they can be subcultured and/or proliferated conveniently. The objectives of this study were to demonstrate a stable Agrobacterium-mediated transformation of maize using explants of type II embryonic calli and to evaluate the efficiency of the protocol in order to develop herbicide-resistant maize. The type II embryogenic calli were inoculated with Agrobacterium tumefaciens strain C58C1 carrying binary vector pTF102, and then were subsequently cultured on the following media: co-cultivation medium for 1 day, delay medium for 7 days, selection medium for 4 × 14 days, regeneration medium, and finally on germination medium. The T-DNA of the vector carried two cassettes (Ubi promoter-EPSPs ORF-nos and 35S promoter–bar ORF-nos). The EPSPs conferred resistance to glyphosate and bar conferred resistance to phosphinothricin. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to phosphinothricin indicated by the growth of putative transgenic calli on selection medium amended with 4 mg l−1 phosphinothricin, northern blot analysis of bar gene, and leaf painting assay for detection of bar gene-based herbicide resistance. Northern blot analysis and leaf painting assay confirmed the expression of bar transgenes in the R1 generation. The average transformation efficiency was 0.60%. Based on northern blot analysis and leaf painting assay, line 31 was selected as an elite line of maize resistant to herbicide.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
Armstrong CL, Green CE (1985) Establishment and maintenance of friable, embryogenic maize callus and the involvement of l-proline. Planta 164:207–214
Armstrong CL, Green CE, Phillips RL (1991) Development and availability of germplasm with high type II culture formation response. Maize Genet Coop Newslett 65:92–93
Cho MA, Park YO, Kim JS, Park KJ, Min HK, Liu JR, Choi PS (2005) Yellowish friable embryogenic callus (YFEC) production and plant regeneration from immature embryo cultures of domestic maize cultivars and genotypes (Zea may L.). Korean J Plant Biotech 32:1–5
Choi HW, Lemaux PG, Cho MJ (2000) Increased chromosomal variation in transgenic versus nontransgenic barley (Hordeum vulgare L.) plants. Crop Sci 40:524–533
Danilova SA, Dolgikh YI (2005) Optimization of agrobacterial (Agrobacterium tumefaciens) transformation of maize embryogenic callus. Russ J Plant Phys 52:535–541
Dennehey BK, Peterson WL, Ford-Santino C, Pajeau M, Armstrong CL (1994) Comparison of selective agents for use with the selectable marker gene bar in maize transformation. Plant Cell Tissue Organ Cult 36:1–7
Frame BR, Zhang H, Cocciolone S, Sidorenko L, Dietrich C, Pegg S, Zhen S, Schnable P, Wang K (2000) Production of transgenic maize from bombarded Type II callus: effect of gold particle size and callus morphology on transformation efficiency. In Vitro Cell Dev Biol-Plant 36:21–29
Frame BR, Shou H, Chikwamba RK, Zhang Z, Xiang C, Fonger TM, Ellen S, Pegg K, 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
Fromm ME, Morrish F, Armstrong CL, William R, Thomas J, Klein T (1990) Inheritance and expression of chimeric genes in the progeny of transgenic maize plants. Biotechnology 8:833–839
Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994
Hinchee MAW, Connor-Ward DW, Newell CA, McDonnell RE, Sato SJ, Gasser CS, Fischhoff DA, Re DB, Fraley RT, Horsch RB (1988) Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. Biotechnology 6:915–922
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
Jorgensen RA (1995) Cosuppression, flower color patterns, and metastable gene expression states. Science 268:686–691
Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–390
Larkin PJ, Scowcroft WR (1981) Somaclonal variation—a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60:197–214
Negrotto D, Beer MJS, Wench AR, Hansen G (2000) The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Rep 19:798–803
Olhoft PM, Flagel LE, Donovan CM, Somers DA (2003) Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method. Planta 216:723–735
Pescitelli SM, Sukhapinda K (1995) Stable transformation via electroporation into maize Type II callus and regeneration of fertile transgenic plants. Plant Cell Rep 14:712–716
Sidorov V, Gilbertson L, Addae P, Duncan D (2006) Agrobacterium-mediated transformation of seedling-derived maize callus. Plant Cell Rep 25:320–328
Utomo SD (2004) Transformasi genetik lima varietas kedelai menggunakan Agrobacterium. J Agrotropika IX:95–101
Utomo SD (2005) Pengaruh L-Sistein terhadap efisiensi transformasi genetik jagung (Zea mays) menggunakan Agrobacterium. Bul Agronomi XXXIII:7–16
Vega JM, Yu W, Kennon AR, Chen X, Zhang ZJ (2008) Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors. Plant Cell Rep 27:297–305
Zhao ZY, Gu W, Cai T, Tagliani LA, Hondred DA, 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
Zhao ZY, Gu W, Cai T, Tagliani LA, Hondred DA, Bond D, Krell S, Rudert ML, Pierce DA (2001) High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Mol Breed 8:323–333
This work was supported by a grant from the Biogreen 21 (20050301034466, 20080401034048). We thank Dr. Kan Wang from the Iowa State University (ISU) for providing binary expression vector (pTF102).
Rights and permissions
About this article
Cite this article
Kim, H.A., Utomo, S.D., Kwon, S.Y. et al. The development of herbicide-resistant maize: stable Agrobacterium-mediated transformation of maize using explants of type II embryogenic calli. Plant Biotechnol Rep 3, 277–283 (2009). https://doi.org/10.1007/s11816-009-0099-2
- Genetic transformation
- Type II embryogenic calli
- Zea mays L.