Plant Cell, Tissue and Organ Culture

, Volume 83, Issue 2, pp 187–200

Successful Agrobacterium-Mediated Genetic Transformation of Maize Elite Inbred lines



An efficient transformation system was developed for maize (Zea mays L.) elite inbred lines using Agrobacterium-mediated gene transfer by identifying important factors that affected transformation efficiency. The hypervirulent Agrobacterium tumefaciens strain EHA105 proved to be better than octopine LBA4404 and nopaline GV3101. Improved transformation efficiencies were obtained when immature embryos were inocubated with Agrobacterium suspension cells (A600 = 0.8) for 20 min in the presence of 0.1% (v/v) of a surfactant (Tween20) in the infection medium. Optimized cocultivation was performed in the acidic medium (pH5.4) at 22 °C in the dark for 3 days. Using the optimized system, we obtained 42 morphologically normal, independent transgenic plants in four maize elite inbred lines representing different genetic backgrounds. Most of them (about 85%) are fertile. The transformation frequency (the number of independent, PCR-positive transgenic plants per 100 embryos infected) ranged from 2.35 to 5.26%. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to three copies of the transgene were integrated into the maize nuclear genome. About 70% of the transgenic plants received a single insertion of the transgenes based on Southern analysis of 10 transformed events. T1 plants were analyzed and transmission of transgenes to the T1 generation in a Mendelian fashion was verified. This system should facilitate the introduction of agronomically important genes into commercial genotypes.

Key words:

Agrobacterium tumefaciens genetic transformation immature embryos transgenic plants Zea mays 





phosphinothricin acetyltransferase gene


2,4-dichloro phenoxyactic acid




indole-3-butyric acid


2-(N-morpholino) ethanesulfonic acid


polymerase chain reaction




β-glucuronidase gene from Escherichiacoli




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  1. Alt-Moerbe, J, Neddemann, P, Lintig, J, Weiler, EW, Schroder, J 1988Temperature sensitive step in Ti plasmid vir region induction and correlation with cytokinin secretion by AgrobacteriumMol. Gen. Genet.21318CrossRefGoogle Scholar
  2. Amoah, BK, Wu, H, Sparks, C, Jones, D 2001Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissueJ. Exp. Bot.5111351142CrossRefGoogle Scholar
  3. Birch, RG 1997Plant transformation: problems and strategies for practical applicationAnnu. Rev. Plant Physiol. Plant. Mol. Biol.48297326CrossRefPubMedGoogle Scholar
  4. Chan, MT, Lee, TM, Chang, HH 1992Transformation of indica rice (Oryza sativa L.) mediated by Agrobacterium tumefaciensPlant Cell Physiol.33577583Google Scholar
  5. Cheng, M, Fry, JE, Pang, SZ, Zhou, HP, Hironaka, CM, Duncan, DR, Conner, TW, Wan, YC 1997Genetic transformation of wheat mediated by Agrobacterium tumefaciensPlant Physiol.115971980PubMedGoogle Scholar
  6. Chilton, MD, Currier, TC, Farrand, SK, Bendich, AJ, Gordon, MP, Nester, EW 1974Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumorsProc. Natl. Acad. Sci. USA7136723676PubMedGoogle Scholar
  7. Chu, CC, Wang, CC, Sun, CS, Hus, C, Yin, KC, Chu, CY, Bi, FY 1975Establishment of an efficient medium for another culture of rice through comparative experiments on the nitrogen sourcesSci. Sin.18659668Google Scholar
  8. Curtis, IS, Nam, HG 2001Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method – plant development and surfactant are important in optimizing transformation efficiencyTransgenic Res.10363371CrossRefPubMedGoogle Scholar
  9. Dellaporta, SL, Wood, J, Hicks, JB 1983A plant DNA miniprepration: version IIPlant Mol. Biol. Rep.4921Google Scholar
  10. D’Halluin, K, Bonne, E, Bossut, M, Beuckeleer, MD, Leemans, J 1992Transgenic maize plants by tissue electroporationPlant Cell414951505CrossRefPubMedGoogle Scholar
  11. Dillen, W, DeClercq, J, Kapila, J, Zambre, M, Van Montagu, M, Angenon, G 1997The effect of temperature on Agrobacterium tumefaciens-mediated gene transfer to plantsPlant J.1214591463CrossRefGoogle Scholar
  12. Frame, BR, Drayton, PR, BagNall, SV, Lewnau, CJ, Bullock, WP, Wilson, HM, Dunwell, JM, Thompson, JA, Wang, K 1994Production of fertile transgenic maize plants by silicon carbide whisker-mediated transformationPlant J.6941948CrossRefGoogle Scholar
  13. Frame, BR, Shou, H, Chikwamba, RK, Zhang, Z, Xiang, C, Fonger, TM, Pegg, SEK, Li, B, Nettleton, DS, Pei, D, Wang, K 2002Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector systemPlant Physiol.1291322CrossRefPubMedGoogle Scholar
  14. Fullner, KJ, Lara, JC, Nester, EW 1996Pilus assembly by Agrobacterium T-DNA transfer genesScience27311071109PubMedGoogle Scholar
  15. Fullner, KJ, Nester, EW 1996Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciensJ.␣Bacteriol.17814981504PubMedGoogle Scholar
  16. Gamborg, OL, Miller, RA, Ojima, K 1968Nutrient requirements of suspension cultures of soybean root cellsExp. Cell Res.50151158CrossRefPubMedGoogle Scholar
  17. Gelvin, SB 2000Agrobacterium and plant genes: involved in T-DNA transfer and integrationAnnu. Rev. Plant Physiol. Plant Mol. Biol.51223256CrossRefPubMedGoogle Scholar
  18. Gelvin, SB 2003Agrobacterium-mediated plant transformation: the biology behind the “gene-jocking” toolMicrobiol. Mol. Biol. Rev.671637CrossRefPubMedGoogle Scholar
  19. Golovkein, MV, Abraham, M, Morocz, S, Bottka, S, Feher, A, Dudits, D 1993Productions of transgenic maize plants by direct DNA uptake into embryogenic protoplastsPlant Sci.904152CrossRefGoogle Scholar
  20. 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 1990Transformation of maize cells and regeneration of fertile transgenic plantsPlant Cell2603618CrossRefPubMedGoogle Scholar
  21. Gutlitz, RGH, Lamb, PW, Matthsse, AG 1987Involvement of carrot cell surface proteins in attachment of Agrobacterium tumefaciensPlant Physiol.83564575Google Scholar
  22. Hiei, Y, Komari, T, Kubo, T 1997Transformation of rice mediated by Agrobacterium tumefaciensPlant Mol. Biol.35205218CrossRefPubMedGoogle Scholar
  23. Hiei, Y, Komari, T, Ishida, Y, Saito, H 2000Development of Agrobacterium-mediated transformation method for monocotyledonous plantsBreed. Res.2205213Google Scholar
  24. Hoekema, A, Hirsch, PR, Hooykaas, PJJ, Schilperoort, RA 1983A binary plant vector strategy based on separation of the vir and T-region of the Agrobacterium tumefaciens Ti plasmidNature303179180CrossRefGoogle Scholar
  25. Höfgen, R, Willmitzer, L 1988Storage of competent cells for Agrobacterium tumefaciensNucleic Acids Res.169877 PubMedGoogle Scholar
  26. Hood, EE, Gelvin, SB, Melchers, LS, Hoekema, A 1993New Agrobacterium helper plasmids for gene transfer to plantsTransgenic Res.2208218CrossRefGoogle Scholar
  27. Ishida, Y, Satto, H, Ohta, S, Hiei, Y, Komari, T, Kumashiro, T 1996High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciensNat. Biotechnol.14745750CrossRefPubMedGoogle Scholar
  28. Jefferson, RA 1987Assaying chimeric in plants: the GUS gene fusion systemPlant Mol. Biol. Rep.5387405Google Scholar
  29. Ko, T-S, Lee, S, Krasnyanski, S, Korban, SS 2003Two critical factors are required for efficient transformation of multiple soybean cultivars: Agrobacterium strain and orientation of immature cotyledonary explantTheor. Appl. Genet.107439447CrossRefPubMedGoogle Scholar
  30. Koncz, C, Schell, J 1986The promoter of the TL-DNA gene 5 controls the tissue-specific expression of the chimeric genes carried by a novel type of Agrobacterium binary vectorMol. Gen. Genet.204383396CrossRefGoogle Scholar
  31. Kondo, T, Hasegawa, H, Suzuki, M 2000Transformation and regeneration of garlic (Allium sativum L.) by Agrobacterium-mediated gene transferPlant Cell Rep.19989993CrossRefGoogle Scholar
  32. Mondal, TK, Bhattacharya, A, Ahuja, PS, Chand, PK 2001Transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] plants obtained by Agrobacterium-mediated transformation of somatic embryosPlant Cell Rep.20712720CrossRefGoogle Scholar
  33. Negrotto, D, Jolley, M, Beer, S, Wenck, AR, Hansen, G 2000The use of phosphomanose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformationPlant Cell Rep.19798803CrossRefGoogle Scholar
  34. Petolino, JF, Hopkins, NL, Kosegi, BD, Skokut, M 2000Whisker-mediated transformation of embryogenic callus of maizePlant Cell Rep.19781786CrossRefGoogle Scholar
  35. Rashid, H, Yokoi, S, Toriyama, K, Hinata, K 1996Transgenic plant production mediated by Agrobacterium in Indica ricePlant Cell Rep.15727730CrossRefGoogle Scholar
  36. Sambrook, J, Fritsch, EF, Maniatis, T 1989Molecular Cloning: A Laboratory Manual, 2nd ednCold Spring Harbor Laboratory PressCold Spring Harbor, NYGoogle Scholar
  37. Stachel, SE, Nester, EW, Zambryski, PC 1986A plant cell factor induces Agrobacterium tumefaciens vir gene expressionProc. Nat.l Acad. Sci. USA83379383Google Scholar
  38. Sunilkumar, G, Rathore, KS 2001Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regenerationMol. Breed.83752CrossRefGoogle Scholar
  39. Suzuki, S, Nakano, M 2002Agrobacterium-mediated production of transgenic plants of Muscariarmeniacum Leichtl. ex BakPlant. Cell. Rep.20835841CrossRefGoogle Scholar
  40. Wan, Y, Widholm, JM, Lemaux, PG 1995Type-I callus as a bombardment target for generating fertile transgenic maize (Zea mays L.)Planta196714CrossRefGoogle Scholar
  41. Wang, H, Qi, M, Cutler, AJ 1993A simple method of preparing plant samples for PCRNucleic Acids Res.2141534154PubMedGoogle Scholar
  42. Wu, H, Sparks, C, Amoah, B, Jones, D 2003Factors influencing successful Agrobacterium-mediated genetic transformation of wheatPlant Cell Rep.21659668PubMedGoogle Scholar
  43. Yu, H, Yang, SH, Goh, CJ 2001Agrobacterium-mediated transformation of a Dendrobium orchid with the class 1 knox gene DOH1Plant Cell Rep.20301305CrossRefGoogle Scholar
  44. Zhao, Z, Gu, W, Cai, T, Tagliani, L, Hondred, D, Bond, D, Schroeder, S, Rudert, M, Pierce, D 2001High throughput genetic transformation mediated by Agrobacteriumtumefaciens in maizeMol. Breed.8323333CrossRefGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  1. 1.Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological SciencesThe Chinese Academy of SciencesShanghaiP.R. China

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