Maize (Zea mays) Hi‐II Transformation via Agrobacterium‐Mediated T‐DNA Transfer

Hyeyoung Lee1, Zhanyuan J. Zhang1

1 University of Missouri, Plant Transformation Core Facility, Division of Plant Sciences, Columbia, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20016
Online Posting Date:  May, 2016
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Genetic transformation of maize via Agrobacterium tumefaciens is still more art than science, with different researchers achieving substantially different transformation results. This article describes our advanced Agrobacterium‐mediated transformation system in Hi‐II maize. The system utilizes simple binary vectors and immature embryos for the transformation, employing the bar gene as a plant selectable marker in combination with bialaphos for subsequent culture selection. The transformation process is efficient and highly reproducible. Certain inbred maize lines can also be transformed with some modification of the system. © 2016 by John Wiley & Sons, Inc.

Keywords: Agrobacterium tumefaciens; binary vectors; Hi‐II; maize

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Table of Contents

  • Introduction
  • Basic Protocol 1: Stable Transformation of Maize
  • Support Protocol 1: Greenhouse Maize Plant Management
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: Stable Transformation of Maize

  • A. tumefaciens (also called Rhizobium radiobacter) strain AGL1 carrying a simple binary vector (e.g., pZY102; ATCC,
  • ABC agar plates containing appropriate antibiotics (see recipe)
  • YEP agar plates containing appropriate antibiotics (see recipe)
  • PHI‐A (inoculation medium; see recipe)
  • Ears of maize Hi‐II A × B ( protocol 2Support Protocol)
  • 30% (v/v) commercial bleach
  • Tween 20
  • PHI‐B (co‐cultivation medium plates; see recipe)
  • PHI‐C (resting medium plates; see recipe)
  • PHI‐D1 (selection medium I plates; see recipe)
  • PHI‐D2 (selection medium II plates; see recipe)
  • PHI‐E (maturation medium plates; see recipe)
  • PHI‐F (regeneration medium; see recipe)
  • Soil mixture (e.g., Promix BX)
  • Dark culture incubators with temperature control and air circulation (Percival Scientific; one at 20°C for co‐cultivation and the other at 28°C for callus culture)
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Inoculating loop
  • Spectrophotometer and cuvettes
  • Shaker
  • 2‐ml microcentrifuge tubes
  • Forceps, sterile
  • 1‐liter wide‐mouth bottle, sterile
  • 150 × 15–mm and 100 × 15–mm Petri dishes
  • #11 razor blades, sterile
  • Microspatula, sterile
  • Dissecting microscope such as Olympus stereo zoom microscope SZ40 MODEL LMS‐225R with 5‐40× amplification and built‐in light source (Leeds Precision Instruments)
  • 3 M porous tape
  • 25 × 150–mm test tubes
  • Small plastic planting pots
  • Growth chamber: settings 25°C, 16 hr/light, 8 hr/dark
  • Light incubator or culture room: settings 24°C 18 hr/light, 6 hr/dark
  • Additional reagents and equipment for maintenance and pollination of maize ( protocol 2Support Protocol)
NOTE: Autoclave and discard all contaminated Agrobacterium and maize cultures promptly.

Support Protocol 1: Greenhouse Maize Plant Management

  • Soil mixture (e.g., Promix BX)
  • Osmocote 18‐6‐12 or 19‐6‐12 fertilizer (Hummert International, cat. no. 07‐6300‐1 or 07‐6330‐1)
  • Source of water (avoid high concentrations of organic or inorganic compounds)
  • Maize Hi‐II immature embryos derived from the self‐pollinated ears (F2) of the F1 cross between Hi‐II A and Hi‐II B or between A188 and B73 are used as starting material seeds of Hi‐A (or A188) and Hi‐II B (or B73) can be requested from Maize Genetic Stock Center (
  • Iron sulfate (Hummert International, cat. no. 07‐0851‐1)
  • Peters 20‐20‐20 fertilizer (Hummert International, cat. no. 07‐5400‐1)
  • Greenhouse module with maximal light exposure and intensity using combined daylight and supplemental lighting from metal halide and high pressure sodium light bulbs (Hummert International)
  • 3‐gallon pots (Hummert International)
  • Measuring spoon (approximately 1 oz.; Hummert International, cat. no. 060092‐1)
  • Tags for labeling pots (Hummert International)
  • Jiffy Pots (Hummert International)
  • Humidomes (Hummert International, cat no. 143850‐1))
  • Shoot bags (Lawson Bags, cat. no. 217;
  • Tassel bags (Lawson Bags, cat. no. 402;
  • Stapling pliars (Hummert International, cat no. 51‐3800‐1))
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Literature Cited

Literature Cited
  Chu, C.C., Wang, C.C., Sun, C.S., Hsu, C., Yin, K.C., Chu, C.Y., and Bi, F.Y. 1975 Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen source. Sci. Sin. 18:659‐668.
  Frame, B.R., McMurray, J.M., Fonger, T.M., Main, M.L., Taylor, K.W., Torney, F.J., Paz, M.M., and Wang, K. 2006. Improved Agrobacterium‐mediated transformation of three maize inbred lines using MS salts. Plant Cell Rep. 25:1024‐1034. doi: 10.1007/s00299‐006‐0145‐2.
  Frame, B.R., Shou, H., Chikwamba, R.K., Zhang, Z., Xiang, C., Fonger, T.M., Pegg, S.E.K., Li, B., Nettleton, D.S., Pei, D., and Wang, K. 2002. Agrobacterium tumefaciens‐mediated transformation of maize embryos using a standard binary vector system. Plant Physiol. 129:13‐22. doi: 10.1104/pp.000653.
  Gould, J., Devey, M., Hasegawa, O., Ulian, E.C., Peterson, G., and Smith, R.H. 1991. Transformation of Zea mays L. using Agrobacterium tumefaciens and the shoot apex. Plant Physiol. 95:426‐434. doi: 0032‐0889/91/95/0426/09/$01.00/0.
  Hiei, Y., Ohta, S., and Komari, T. 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T‐DNA. Plant J. 6:271‐282. doi: 10.1046/j.1365‐313X.1994.6020271.x.
  Huang, X.Q. and Wei, Z.M. 2004. High‐frequency plant regeneration through callus initiation from mature embryos of maize (Zea mays L.). Plant Cell Rep. 22:793‐800. doi: 10.1007/s00299‐003‐0748‐9.
  Huang, X. and Wei, Z. 2005. Successful Agrobacterium‐mediated genetic transformation of maize elite inbred lines. Plant Cell Tissue Organ. Cult. 83:187‐200. doi: 10.1007/s11240‐005‐5772‐8.
  Ishida, Y., Saito, H., Ohta, S., Hiel, Y., Komari, T., and Kumashiro, T. 1996. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat. Biotechnol. 14:745‐750. doi: 10.1038/nbt0696‐745.
  Lee, B.K., Kennon, A.R., Chen, X., Jung, T.W., Ahn, B.O., Lee, Z.Y., and Zhang, Z. 2007. Recovery of transgenic events from two highly recalcitrant maize (Zea mays L.) genotypes using Agrobacterium‐mediated standard–binary‐vector transformation. Maydica 52:457‐469.
  Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:473‐497. doi: 10.1111/j.1399‐3054.1962.tb08052.x.
  Sidorov, V., Gilbertson, L., Addae, P., and Duncan, D. 2006. Agrobacterium‐mediated transformation of seedling‐derived maize callus. Plant Cell Rep. 25:320‐328. doi: 10.1007/s00299‐005‐0058‐5.
  Vega, J.M., Yu, W., Kennon, A.R., Chen, X., and Zhang, Z. 2008. Improvement of Agrobacterium‐mediated transformation in Hi‐II maize (Zea mays) using standard binary vectors. Plant Cell Rep. 27:297‐305. doi: 10.1007/s00299‐007‐0463‐z.
  Zhang, W., Subbarao, S., Addae, P., Shen, A., Armstrong, C., Peschke, V., and Gilbertson, L. 2003. Cre/lox mediated marker gene excision in transgenic maize (Zea may L.) plants. Theor. Appl. Genet. 107:1157‐1168. doi: 10.1007/s00122‐003‐1368‐z.
  Zhao, Z.Y., Gu, W., and Cai, T. 1999. Methods for Agrobacterium‐mediated transformation. United States Patent No. 5981840.
  Zhao, Z.Y., Gu, W., Cai, T., and Pierce, D.A. 2004. Methods for Agrobacterium‐mediated transformation. United States Patent No. 963096. Pioneer Hi‐bred International, Inc. Des Moines, Ia.
  Zhao, Z.Y., Gu, W., Cai, T., Tagliani, L., Hondred, D., Bond, D., Schroeder, S., Rudert, M., and Pierce, D. 2002. High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Mol. Breed. 8:323‐333. doi: 10.1023/A:1015243600325.
Key References
  Ishida et al., 1996. See above.
  A ground‐breaking work establishing that maize could be transformed by Agrobacterium‐mediated transformation.
  Zhao et al., 1999. See above.
  A milestone work establishing a basic protocol for subsequent improvement of maize transformation
  Frame et al., 2002. See above.
  A milestone work making it possible for the public to transform maize Hi‐II using standard binary vectors
  Vega et al., 2008. See above.
  A milestone work making maize transformation more efficient by combined use of low‐salt medium with antioxidants during the infection stage, using a standard binary vector system.
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