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Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat

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Abstract

Immature embryos have been used frequently as target tissues in the genetical transformation of wheat. However, obtaining a large number of high quality immature embryos throughout the year is a laborious and delicate process, because of the need to cultivate the plants under controlled conditions. To circumvent this, we have employed mature embryos rather than immature ones as starter explants for Agrobacterium-mediated transformation of an elite wheat (Triticum aestivum L.) cultivar EM12. The neomycin phosphotransferase ІІ (npt ІІ) and β-glucuronidase (gus) genes were used as selectable and screenable marker genes, respectively, to assess and optimize the performance of T-DNA delivery. With the aid of an orthogonal design, the effect of four factors in combination on transfer DNA (T-DNA) delivery was studied. These factors were preculture duration, different kinds of inoculation, length of inoculation and co-culture condition. Optimal conditions for T-DNA delivery were obtained for mature embryos precultured for 14 days, followed by immersing in inoculation suspension with full strength Murashige and Skoog (MS) salts in darkness at 23–25°C for 3 h, and then co-culturing with Agrobacterium under desiccating condition in the dark at 23–24°C for 2–3 days. Complete analysis of transgene insertion demonstrated that the optimized method for Agrobacterium-mediated transformation of mature embryos of wheat was efficient and practicable.

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Abbreviations

ANOVA:

Analysis of variance

AS:

Acetosyringone

GUS:

β-glucuronidase

MS:

Murashige and Skoog

Picloram:

4-amino-3, 5, 6-trichloropicolinic acid

2, 4-D:

2, 4-dichlorophenoxyacetic acid

T-DNA:

Transfer DNA

X-Gluc:

5-bromo-4-chloro-3-indolyl β-d-glucuronide

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Acknowledgments

The study was supported by National Basic Research Program of China 2002 CB111301-”Functional genomics and molecular improvement of quality related traits in important agricultural crops.”

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Authors and Affiliations

  1. China-UK HUST-RRes Genetic Engineering and Genomics Joint Laboratory, Huazhong University of Science and Technology (HUST), Luoyu Road 1037, Wuhan, Hubei, 430074, P.R. China

    Liping Ding, Shengchun Li, Jianming Gao, Yuesheng Wang, Guangxiao Yang & Guangyuan He

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  1. Liping Ding
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  2. Shengchun Li
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Correspondence to Guangyuan He.

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Ding, L., Li, S., Gao, J. et al. Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat. Mol Biol Rep 36, 29–36 (2009). https://doi.org/10.1007/s11033-007-9148-5

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