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Factors affecting Agrobacterium-mediated genetic transformation of embryogenic callus of Parthenocissus tricuspidata Planch

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Abstract

A protocol for Agrobacterium-mediated transformation was developed for embryogenic callus of an excellent climber species, Parthenocissus tricuspidata. A. tumefaciens strain EHA105 or C58 harboring the pCAMBIA2301 binary vector with the neomycin phosphotransferase (nptII) and β-glucuronidase (uidA) gene was used. Factors affecting the transformation efficiency, including the Agrobacterium strains, co-cultivation time, Agrobacterium concentration, and infection time, were evaluated. Strain EHA105 proved to be significantly better than C58, and 4 days of co-culture was critical for transformation. An Agrobacterium suspension at a concentration of 0.5–0.7 × 108 cells ml−1 (OD600 = 0.5–0.7) and an infection time of 40 min was optimal for transformation. By applying these optimized parameters, we recovered six independent transformed shoots that were kanamycin-resistant and contained the nptII gene, as verified by polymerase chain reaction (PCR) analysis. Southern blot analysis confirmed that T-DNA was stably integrated into the genome of three out of six PCR-positive lines. Furthermore, histochemical GUS assay revealed the expression of the uidA gene in kanamycin-resistant calli, somatic embryos, and leaves of transgenic plants.

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Abbreviations

AS:

Acetosyringone

BA:

6-benzylaminopurine

Cx:

Cefotaxime

GUS:

β-glucuronidase

2,4-D:

2,4-dichlorophenoxyacetic acid

Kan:

Kanamycin

LB:

Luria–Bertani (medium)

MS:

Murashige and Skoog (1962)

NAA:

Naphthaleneacetic acid

nptII :

Neomycin phosphotransferase

CH:

Casein hydrolysate

PPF:

Photosynthetic photon flux

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Acknowledgments

This research is supported by the 863 Project (2001AA244049) of the Ministry of Science and Technology of the People’s Republic of China. The authors gratefully acknowledge all colleagues of our laboratory for the constructive discussion and technical support. We also especially thank the two anonymous reviewers for their helpful suggestions and revisions to the manuscript.

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  1. Yongxue Yang

    Present address: Oil Crops Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 430062, Wuhan, People’s Republic of China

Authors and Affiliations

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

    Yongxue Yang, Manzhu Bao & Guofeng Liu

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  1. Yongxue Yang
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  2. Manzhu Bao
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  3. Guofeng Liu
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Correspondence to Guofeng Liu.

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Yang, Y., Bao, M. & Liu, G. Factors affecting Agrobacterium-mediated genetic transformation of embryogenic callus of Parthenocissus tricuspidata Planch. Plant Cell Tiss Organ Cult 102, 373–380 (2010). https://doi.org/10.1007/s11240-010-9742-4

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