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Gene Manipulation of a Heavy Metal Hyperaccumulator Species Thlaspi caerulescens L. via Agrobacterium-mediated Transformation

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Abstract

Thlaspi caerulescens L. is well known as a Zn/Cd hyperaccumulator. The genetic manipulation of T. caerulescens through transgenic technology can modify plant features for use in phytoremediation. Here, we describe the efficient transformation of T. caerulescens using Agrobacterium tumefaciens strain EHA105 harboring a binary vector pBI121 with the nptII gene as a selectable marker, the gus gene as a reporter and a foreign catalase gene. Based on the optimal concentration of growth regulators, the shoot cluster regeneration system via callus phase provided the basis of the genetic transformation in T. caerulescens. The key variables in transformation were examined, such as co-cultivation period and bacterial suspension density. Optimizing factors for T-DNA delivery resulted in kanamycin-resistant transgenic shoots with transformation efficiency more than 20%, proven by histochemical GUS assay and PCR analysis. Southern analysis of nptII and RT-PCR of catalase gene demonstrated that the foreign genes were integrated in the genome of transformed plantlets. Moreover, the activity of catalase enzyme in transgenic plants was obviously higher than in wild-type plants. This method offers new prospects for the genetic engineering of this important hyperaccumulator species.

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Abbreviations

KT:

Kinetin

BA:

Benzylaminopurine

NAA:

α-Naphthaleneacetic acid

AS:

Acetosyringone

LH:

Lactalbumin hydrolysate

GA3 :

Gibberellin

BCM:

Basic culture medium

RCM:

Regeneration culture medium

PCM:

Pre-culture medium

CCM:

Co-cultivation medium

SCM-Cb:

Selection culture medium with carbenicillin

SCM-Km:

Selection culture medium with carbenicillin and kanamycin

SCM-GA3 :

Selection culture medium with GA3

RM-Km:

Rooting medium with kanamycin

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Acknowledgments

The research was supported by the National High Technology Planning Program of China (Grant nos. 2006AA10Z407 and 2007AA021404) and China National Natural Sciences Foundation (Grant no. 30570146).

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Authors and Affiliations

  1. Department of Biology, Graduate University of Chinese Academy of Sciences, Yuquan Rd 19A, Beijing, 100049, China

    Zi Qiu Guan, Tuan Yao Chai, Jin Xu, Wei Wei, Lu Han & Lin Cong

  2. Department of Biological Engineering, School of Chemical and Environmental Engineering, China University of Mining and Technology (Beijing), Xueyuan Rd 11, Beijing, 100083, China

    Yu Xiu Zhang

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  1. Zi Qiu Guan
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Correspondence to Tuan Yao Chai.

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Guan, Z.Q., Chai, T.Y., Zhang, Y.X. et al. Gene Manipulation of a Heavy Metal Hyperaccumulator Species Thlaspi caerulescens L. via Agrobacterium-mediated Transformation. Mol Biotechnol 40, 77–86 (2008). https://doi.org/10.1007/s12033-008-9065-4

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