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Stable Agrobacterium-mediated genetic transformation of London plane tree (Platanus acerifolia Willd.)

  • Genetic Transformation and Hybridization
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Abstract

London plane tree (Platanus acerifolia Willd.) is an important tree in urban landscaping but it suffers from a number of negative traits which genetic engineering could be used to address. As with many woody species, P. acerifolia has appeared recalcitrant to genetic transformation. However, the recent development of a method for regenerating shoots from P. acerifolia leaf explants suggests that such material could be a target for gene-transfer. Using an Agrobacterium tumefaciens strain in which the T-DNA carries the histochemically detected reporter gene β-glucuronidase (GUS), we have followed the transfer of genes from Agrobacterium to leaf explants of Platanus acerifolia. Using this system, we have identified a set of inoculation and co-cultivation conditions (notably: the pre-treatment of leaf explants with 0.4 M mannitol, an inoculation period of 10 min, a bacterial OD600 of 0.8–1.0 and a co-cultivation period of 5 days) that permit a good frequency and reliability of transient gene-transfer. Optimum levels of antibiotics for bacterial elimination and kanamycin-resistant shoot regeneration were also established. By applying these parameters, we recovered eight independent stably transformed shoots that were kanamycin-resistant and contained the nptII T-DNA gene, as confirmed by PCR analysis. Furthermore, Southern blot analysis confirmed that, in at least five of these lines, the transgene was associated with high molecular weight DNA, so indicating integration into the plant genome.

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Abbreviations

AS:

Acetosyringone

BA:

6-Benzylaminopurine

Cx:

Cefotaxime

GUS :

β-Glucuronidase

IBA:

Indole-3-butyric acid

Kan:

Kanamycin

LB:

Luria-Bertani (medium)

MS:

Murashige and Skoog (1962)

NAA:

Naphthalene acetic acid

nptII:

Neomycin phosphotransferase

Tm:

Timentin

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Acknowledgements

This research was partially supported by a grant from the National Natural Science Foundation of China (30371015) and Ministry of Education Foundation of China (NCET-04-0733) to Bao MZ. Liu GF initiated the transformation work. Fang F made the experiments on high osmotic treatment and obtained some Kan-resistant lines. Li ZN optimized the transformation system and analysed the transgenic plants. The kindness of Dr. Lin Yongjun, Jin Shuangxia and professor Zhang Xianlong in National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University for technical support are appreciated. We thank Dr. Alex C. McCormac for critical editing of the manuscript and all the colleagues in our laboratory for technical assistance.

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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, P.R. China

    Z. N. Li, F. Fang, G. F. Liu & M. Z. Bao

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  1. Z. N. Li
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  2. F. Fang
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  3. G. F. Liu
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  4. M. Z. Bao
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Correspondence to M. Z. Bao.

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Communicated by S. A. Merkle

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Li, Z.N., Fang, F., Liu, G.F. et al. Stable Agrobacterium-mediated genetic transformation of London plane tree (Platanus acerifolia Willd.). Plant Cell Rep 26, 641–650 (2007). https://doi.org/10.1007/s00299-006-0271-x

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  • DOI: https://doi.org/10.1007/s00299-006-0271-x

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