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An efficient system to produce transgenic plants via cyclic leave-originated secondary somatic embryogenesis in Rosa rugosa

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Abstract

An efficient and reproducible Agrobacterium-mediated transformation system via repetitive secondary somatic embryogenesis was developed for Rosa rugosa ‘Bao white’. Somatic embryogenesis was induced from in vitro-derived unexpanded leaflet explants on MS medium supplemented with 4.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 mg/L Kinetin and 30 g/L glucose. Secondary somatic embryos were successfully proliferated via cyclic secondary somatic embryogenesis on MS medium containing 1.0 mg/L 2,4-D, 0.01 mg/L 6-benzyladenine and 45 g/L glucose under light intensity of 500–1,000 lux. The highest germination rate (86.33 %) of somatic embryos was observed on 1/2-strength MS medium containing 1.0 mg/L BA. Relying on the repetitive secondary somatic embryogenesis and A. tumefaciens strain EHA105 harboring the binary vector pBI121, a stable and effective Agrobacterium-mediated transformation pattern was developed. The presented transformation protocol, in which somatic embryo clumps at globular stage (0.02–0.04 g) were infected by Agrobacterium for 60 min and co-cultivated for 2 days, and then selected under a procedure of 3 steps, were confirmed to be optional by GUS histochemical assay and Southern blot analysis. The procedure described here will be very useful for the introgression of desired genes into R. rugosa ‘Bao white’ and the molecular analysis of gene function.

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Abbreviations

MS:

Murashige and Skoog (1976)

2,4-D:

2,4-Dichlorophenoxyacetic acid

TDZ:

Thidiazuron

BA:

6-Benzyladenine

NAA:

a-Naphthalene acetic acid

KT:

Kinetin

ABA:

Gibberellin

PGR:

Plant growth regulator

nptII :

Neomycin phosphotransferase

GUS:

β-Glucuronidase

Kan:

Kanamycin

Cef:

Cefotaxime

AS:

Acetosyringone

X-Gluc:

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

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (No. 31171985) and the National Science and Technology Ministry of China (No. 2011AA100208). We thank all the colleagues in our lab for constructive discussion and technical support. We are also grateful to Dr. Alex C. McCormac for critical editing of the manuscript.

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Authors and Affiliations

  1. Key Laboratory of Horticultural Plant Biology, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    Wen Xing, Ying Bao, Ping Luo, Manzhu Bao & Guogui Ning

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  1. Wen Xing
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  2. Ying Bao
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  3. Ping Luo
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  4. Manzhu Bao
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  5. Guogui Ning
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Correspondence to Guogui Ning.

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Communicated by S. Werbrouck.

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Xing, W., Bao, Y., Luo, P. et al. An efficient system to produce transgenic plants via cyclic leave-originated secondary somatic embryogenesis in Rosa rugosa . Acta Physiol Plant 36, 2013–2023 (2014). https://doi.org/10.1007/s11738-014-1578-9

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  • DOI: https://doi.org/10.1007/s11738-014-1578-9

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