Generation of Stable Transgenic Rice (Oryza sativa L.) by Agrobacterium‐Mediated Transformation

Yi Zhang1, Jun Li1, Caixia Gao2

1 University of Chinese Academy of Sciences, Beijing, China, 2 State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20004
Online Posting Date:  June, 2016
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Rice (Oryza sativa L.) is the staple food for more than half of the world's population, and has also become an important model monocot. As a result, numerous genetic transformation protocols have been developed to improve and better understand this particular agronomic plant. Here we introduce a convenient transformation method using Agrobacterium. The explants used are embryogenic calli derived from mature seeds, which are easily obtained and can be used all year round. After selection and regeneration, transformants are obtained from resistant calli cultured on the regeneration medium. This protocol has been used to generate transgenic rice (Oryza sativa L.) in as little as 4 months. © 2016 by John Wiley & Sons, Inc.

Keywords: Agrobacterium‐mediated transformation; calli; regeneration; rice

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Table of Contents

  • Introduction
  • Basic Protocol 1: Agrobacterium–Mediated Transformation of Rice Calli
  • Support Protocol 1: Preparation of Agrobacterium Harboring Constructs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Agrobacterium–Mediated Transformation of Rice Calli

  • Rice seeds
  • 2% (v/v) sodium hypochloride
  • Tween 20
  • M1 calli induction medium (see recipe)
  • Agrobacterium tumefaciens strain harboring constructs (see the protocol 2Support Protocol)
  • LB medium (see recipe)
  • AAM medium (see recipe)
  • Sterile water containing 200 mg/liter carbenicillin or cefotaxime
  • M2 selection medium (see recipe)
  • M5 regeneration medium (see recipe)
  • M6 rooting medium (see recipe)
  • Shaker: setting 250 rpm/min; 28°C (used for Agrobacterium multiplication and activation)
  • 50‐ml tubes
  • Sterile filter papers (9‐cm diameter)
  • 9‐cm diameter plastic petri dishes
  • Parafilm
  • Sterile glass containers (18‐cm in length)
  • 16 × 16‐cm plastic film
  • Growth chamber: settings 28°C/24 hr dark (used for calli induction, subculture and selection)
  • Forceps
  • Stereoscopic microscope
  • 100‐ml conical flasks
  • Sterile glass petri dishes (6‐ and 9‐cm in diameter)
  • Micropore medical sealing tape
  • Growth chamber: settings 25°C/24 hr dark (used for co‐cultivation)
  • Growth chamber: settings 28°C/24 hr light (used for calli regeneration and plantlet growth)

Support Protocol 1: Preparation of Agrobacterium Harboring Constructs

  • Competent Agrobacterium tumefaciens cells (TransGen Biotech)
  • The construct: a binary plasmid with the gene of interest
  • Ice
  • Liquid nitrogen
  • LB liquid and solid media
  • Appropriate selective antibiotic
  • 40% glycerin
  • 1.5‐ml microcentrifuge tubes
  • Shaking incubator
  • Centrifuge
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Literature Cited

Literature Cited
  Christou, P., Ford, T.L., and Kofron, M. 1991. Production of transgenic rice (Oryza sativa L.) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Bio. Technol. 9:957‐962. doi: 10.1038/nbt1091‐957.
  Chu, C.C. 1978. The N6 medium and its application to anther culture of cereal crops. In Proceedings of the Symposium on Plant Tissue Cultures. pp. 43‐50. Science Press, Beijng, China.
  Datta, S.K., Peterhans, A., Datta, K., and Potrykus, I. 1990. Genetically engineered fertile indica‐rice recovered from protoplasts. Bio. Technol. 8:736‐740. doi: 10.1038/nbt0890‐736.
  Gamborg, O.L., Miller, R.A., and Ojima, K. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50:151‐158. doi: 10.1016/0014‐4827(68)90403‐5.
  Gao, S.J., Chen, R.K., and Ma, H.M. 2004. Factors influencing the regeneration frequency of mature embryo‐derived callus in Hsien Rice Cultivars. Acta Bot. Sinica 30:1254‐1258.
  Hiei, Y. and Komari, T. 2008. Agrobacterium‐mediated transformation of rice using immature embryos or calli induced from mature seed. Nat. Protoc. 3:824‐834. doi: 10.1038/nprot.2008.46.
  Hiei, Y., Ohta, S., Komari, T., and Kumashiro, 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.
  Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant 15:473‐497. doi: 10.1111/j.1399‐3054.1962.tb08052.x.
  Shimamoto, K., Terada, R., Izawa, T., and Fujimoto, H. 1989. Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 338:274‐276. doi: 10.1038/338274a0.
  Toriyama, K. and Hinata, K. 1985. Cell suspension and protoplast culture in rice. Plant Sci. 41:179‐183. doi: 10.1016/0168‐9452(85)90086‐X.
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