Co-culturing on dry filter paper significantly increased the efficiency of Agrobacterium-mediated transformations of maize immature embryos

  • Xueqing DuanEmail author
  • Liru Zheng
  • Jinhao Sun
  • Wenbo Liu
  • Wenqian Wang
  • Hailong An
Research Article


In the Agrobacterium tumefaciens-mediated transformations of maize immature embryos (IEs), the common co-culturing media used are MS or N6-based (MC). Here, we used a novel co-culturing method in which maize ‘Qi319’ IEs inoculated with Agrobacterium-harboring target vector were placed on dry filter paper (DC) in a petri dish. To compare the effects of the DC and MC co-culturing methods on transformation efficiency, we designed three experiments: (1) A. tumefaciens strain AGL1 independently carrying two plasmids, pXQD12 and pXQD70; (2) two A. tumefaciens strains, AGL1 and EHA105, carrying pXQD12; and (3) strains AGL1 and EHA105 each independently inoculated with pXQD12 and pXQD70 for different infiltration periods, 5, 10, 15, 20 and 25 min. We used A. tumefaciens to inoculate IEs derived from maize ears 9–15 d after pollination, and then IEs were placed in petri dishes for co-culturing. The DC treatment significantly increased the percentage of IEs expressing green fluorescence protein (%GFP), indicating positive transformants. DC-treated IEs had ~ 3 to 4 times the %GFP compared with MC-treated IEs at 8 d after inoculation (3 d co-culture and 5 d restoration). The average regeneration frequency (%GFP positive regenerated calli of infected IEs) and stable transformation frequency (%GFP positive T0 plants of infected IEs) significantly increased with the DC treatment. Thus, the DC method may be used to develop a more efficient Agrobacterium-mediated transformation method for maize IEs.


Dry filter paper Co-culturing Transformation efficiency Agrobacterium Maize 



This work was supported by the Ministry of Agriculture of China (Grant Number 2016ZX08009002), the National Natural Science Foundation of China (Grant Number 31301080) and the foundation of the Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations (Grant Number lzujbky-2016-bt05). We thank also LetPub ( and Liwen Bianji ( for their linguistic assistance during the preparation of this manuscript.

Authors contribution

XD contributed all reagents and materials used in the experiments. XD, LZ, JS, WL, WW and HA designed and performed the experiments. HA and XD analyzed the data and wrote the paper.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12298_2018_641_MOESM1_ESM.docx (162 kb)
Supplementary material 1 (DOCX 161 kb)


  1. Al-Abed D, Rudrabhatla S, Talla R, Goldman S (2006) Split-seed: a new tool for maize researchers. Planta 223:1355–1360CrossRefGoogle Scholar
  2. Alves SC, Worland B, Thole V, Snape JW, Bevan MW, Vain P (2009) A protocol for Agrobacterium-mediated transformation of Brachypodium distachyon community standard line Bd21. Nat Protoc 4:638–649CrossRefGoogle Scholar
  3. Arencibia AD, Carmona E, Teallez P, Chan MT, Yu SM, Trujillo LE, Oramas P (1998) An efficient protocol for sugarcane (Saccharum spp. L.) transformation mediated by Agrobacterium tumefaciens. Transgenic Res 7:213–222CrossRefGoogle Scholar
  4. Cao SL, Masilamany P, Wb Li, Pauls KP (2014) Agrobacterium tumefaciens-mediated transformation of corn (Zea mays L.) multiple shoots. Biotechnol Biotechnol Equip 28:208–216CrossRefGoogle Scholar
  5. Ceasar SA, Ignacimuthu S (2011) Agrobacterium-mediated transformation of finger millet (Eleusine coracana (L.) Gaertn.) using shoot apex explants. Plant Cell Rep 30:1759–1770CrossRefGoogle Scholar
  6. Ceasar SA, Baker A, Ignacimuthu S (2017) Functional characterization of the PHT1 family transporters of foxtail millet with development of a novel Agrobacterium-mediated transformation procedure. Sci Rep 7:14064CrossRefGoogle Scholar
  7. Cheng M, Hu T, Lagton J, Liu C, Fry J (2003) Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat. Vitro Cell Dev Biol Plant 39:595–604CrossRefGoogle Scholar
  8. Cheng M, Lowe BA, Spencer M, Ye X, Armstrong CL (2004) Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. Vitro Cell Dev Biol Plant 40:31–45CrossRefGoogle Scholar
  9. Cho MJ, Wu E, Kwan J, Yu M, Banh J, Linn W, Anand A, Li Z, TeRonde S, Register JC, Jones TJ, Zhao ZY (2014) Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line. Plant Cell Rep 33:1767–1777CrossRefGoogle Scholar
  10. Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5:213–218CrossRefGoogle Scholar
  11. D’Halluin K, Bonne E, Bossut M, Beuckeleer MD, Leemans J (1992) Transgenic maize plants by tissue electroporation. Plant Cell 4:1495–1505CrossRefGoogle Scholar
  12. Di H, Lu CH, Wang ZH, Liu ZJ (2008) Study on transformation of bar gene into maize (Zea mays L.) by Agrobacterium tumefaciens mediated. Dongbei Nongye Daxue Xuebao (J Northeast Agric Univ) 39:150–154Google Scholar
  13. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
  14. Frame BR, Drayton PR, Bagnall SV, Lewnau CJ, Bullock WP, Wilson HM, Dunwell JM, Thompson JA, Wang K (1994) Production of fertile transgenic maize plants by silicon carbide whisker-mediated transformation. Plant J 6:941–948CrossRefGoogle Scholar
  15. 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 129:13–22CrossRefGoogle 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 25:1024–1034CrossRefGoogle Scholar
  17. Frame B, Main M, Schick R, Wang K (2011) Genetic transformation using maize immature zygotic embryos. Methods Mol Biol 710:327–341CrossRefGoogle Scholar
  18. Fromm ME, Taylor LP, Walbot V (1986) Stable transformation of maize after gene transfer by electroporation. Nature 319:791–793CrossRefGoogle Scholar
  19. 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. Nat Biotechnol 8:833–839CrossRefGoogle Scholar
  20. Golovkin MV, Abraham M, Morocz S, Bottka S, Feher A, Dudits D (1993) Production of transgenic maize plants by direct DNA uptake into embryogenic protoplasts. Plant Sci 90:41–52CrossRefGoogle Scholar
  21. Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O’Brien JV, Chambers SA, Adams WR, 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 2:603–618CrossRefGoogle Scholar
  22. Hiei Y, Ishida Y, Kasaoka K, Komari T (2006) Improved frequency of transformation in rice and maize by treatment of immature embryos with centrifugation and heat prior to infection with Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 87:233–243CrossRefGoogle Scholar
  23. Hood E, Jen G, Kayes L, Kramer J, Fraley RT, Chilton M (1984) Restriction endonuclease map of pTiBo542, a potential Ti plasmid vector for genetic engineering of plants. Biotechnology 2:702–708Google Scholar
  24. 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 14:745–750CrossRefGoogle Scholar
  25. Ishida Y, Saito H, Hiei Y, Komari T (2003) Improved protocol for transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Plant Biotechnol 20:57–66CrossRefGoogle Scholar
  26. Ishida Y, Hiei Y, Komari T (2007) Agrobacterium-mediated transformation of maize. Nat Protoc 2:1614–1621CrossRefGoogle Scholar
  27. Joyce P, Kuwahata M, Turner N, Lakshmanan P (2010) Selection system and co-cultivation medium are important determinants of Agrobacterium-mediated transformation of sugarcane. Plant Cell Rep 29:173–183CrossRefGoogle Scholar
  28. Kant P, Gulati A, Harris L, Gleddie S, Singh J (2012) Transgenic corn plants with modified ribosomal protein L3 show decreased ear rot disease after inoculation with Fusarium graminearum. Aust J Crop Sci 6:1598–1605Google Scholar
  29. Khanna H, Becker D, Kleidon J, Dale J (2004) Centrifugation assisted Agrobacterium tumefaciens-mediated transformation (CAAT) of embryogenic cell suspensions of banana (Musa spp. Cavendish AAA and Lady finger AAB). Mol Breed 14:239–252CrossRefGoogle Scholar
  30. Komari T (1990) Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542. Plant Cell Rep 9:303–306CrossRefGoogle Scholar
  31. 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 an insecticidal protein derived from Bacillus thuringiensis. Nat Biotechnol 11:194–200CrossRefGoogle Scholar
  32. Li Z, TeRonde S, Meyer S, Arling ML, Register JC, Zhao ZY, Jones TJ, Anand A (2015) Effect of Agrobacterium strain and plasmid copy number on transformation frequency, event quality and usable event quality in an elite maize cultivar. Plant Cell Rep 34:745–754CrossRefGoogle Scholar
  33. Nahampun HN, Arredondo DL, Xu X, Estrella LH, Wang K (2016) Assessment of ptxD gene as an alternative selectable marker for Agrobacterium-mediated maize transformation. Plant Cell Rep 35:1121–1132CrossRefGoogle Scholar
  34. Oltmanns H, Frame B, Lee LY, Johnson S, Li B, Wang K, Gelvin SB (2010) Generation of backbone-free, low transgene copy plants by launching T-DNA from the Agrobacterium chromosome. Plant Physiol 152:1158–1166CrossRefGoogle Scholar
  35. Petti C, Wendt T, Meade C, Mullins E (2009) Evidence of genotype dependency within Agrobacterium tumefaciens in relation to the integration of vector backbone sequence in transgenic Phytophthora infestans-tolerant potato. J Biosci Bioeng 107:301–306CrossRefGoogle Scholar
  36. Qu S, Desai A, Wing R, Sundaresan V (2008) A versatile transposon-based activation tag vector system for functional genomics in cereals and other monocot plants. Plant Physiol 146:189–199CrossRefGoogle Scholar
  37. Que Q, Elumalai S, Li X, Zhong H, Nalapalli S, Schweiner M, Fei X, Nuccio M, Kelliher T, Gu W, Chen Z, Chilton MM (2014) Maize transformation technology development for commercial event generation. Front Plant Sci 5:1–19CrossRefGoogle Scholar
  38. Rhodes CA, Pierce DA, Mettler IJ, Mascarenhas D, Detmer JJ (1988a) Genetically transformed maize plants from protoplasts. Science 240:204–207CrossRefGoogle Scholar
  39. Rhodes CA, Lowe KS, Ruby KL (1988b) Plant regeneration from protoplasts isolated from embryogenic maize cell cultures. Biotechnology 6:56–60Google Scholar
  40. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  41. Shou H, Frame BR, Whitham SA, Wang K (2004) Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation. Mol Breed 13:201–208CrossRefGoogle Scholar
  42. Sidorov V, Duncan D (2009) Agrobacterium-mediated maize transformation: immature embryos versus callus. Methods Mol Biol 526:47–58CrossRefGoogle Scholar
  43. Sidorov V, Gilbertson L, Addae P, Duncan D (2006) Agrobacterium-mediated transformation of seedling-derived maize callus. Plant Cell Rep 25:320–328CrossRefGoogle Scholar
  44. Simon E (1978) Membranes in dry and imbibed seeds. In: Crow J, Clegg J (eds) Dry biological systems. London Academic Press, LondonGoogle Scholar
  45. Sun CB, Guo J, Tao R, Li HH, Xing SC, Yuan Y (2012) Study on genetic transformation system of maize mediated by Agrobacterium tumefaciens. Chin Agric Sci Bull 28:71–75Google Scholar
  46. Trifonova A, Madsen S, Olesen A (2001) Agrobacterium-mediated transgene delivery and integration into barley under a range of in vitro culture conditions. Plant Sci 161:871–880CrossRefGoogle Scholar
  47. Wang HW, Liang YH, Shi ZS, Zhang SH, Ge YX (2011) Study on Agrobacterium tumefaciens mediated transformation of maize immature embryos. J Maize Sci 19:73–75Google Scholar
  48. Wu H, Sparks C, Amoah B, Jones HD (2003) Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Rep 21:659–668Google Scholar
  49. Wu E, Lenderts B, Glassman K, Berezowska-Kaniewska M, Christensen H, Asmus T, Zhen S, Chu U, Cho MJ, Zhao ZY (2014) Optimized Agrobacterium-mediated sorghum transformation protocol and molecular data of transgenic sorghum plants. Vitro Cell Dev Biol Plant 50:9–18CrossRefGoogle Scholar
  50. Yin K, Gao C, Qiu JL (2017) Progress and prospects in plant genome editing. Nat Plants 3:17107CrossRefGoogle Scholar
  51. 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 Newsl 72:34–37Google Scholar
  52. Zhao ZY, Gu W, Cai T, Tagliani L, Hondred D, Bond D, Schroeder S, Rudert M, Pierce D (2002) High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Mol Breed 8:323–333CrossRefGoogle Scholar
  53. Zhuang ZY, Wang HN, Zhang JW, Wen RT, Li YS, Zheng Q (2010) Optimization of genetic transformation system for maize immature embryo calli mediated by Agrobacterium tumefaciens. J Gansu Agric Univ 45:49–54Google Scholar

Copyright information

© Prof. H.S. Srivastava Foundation for Science and Society 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Crop Biology, College of Life SciencesShandong Agricultural UniversityTai’anPeople’s Republic of China

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