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JXB Advance Access published online on October 10, 2006
Journal of Experimental Botany, doi:10.1093/jxb/erl145
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© 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Received March 12, 2006
Accepted July 25, 2006

RESEARCH PAPER

Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment

Qin Yao 1, Ling Cong 1, Jun Li Chang 1, Ke Xiu Li 2, Guang Xiao Yang 1, Peter Shewry 2, and Guang Yuan He 1 *

1 China-UK HUST-RRes Genetic Engineering and Genomics Joint Laboratory, Huazhong University of Science and Technology (HUST), 430074 Luoyu Road 1037, Wuhan, Hubei, People's Republic of China
2 Rothamsted Research (RRes), Harpenden, Hertfordshire AL5 2JQ, UK

* To whom correspondence should be addressed.
Guang Yuan He, E-mail: guangyuan.he{at}bbsrc.ac.uk


  Abstract

Two groups of linear gene constructs (gus and bar, and 1Ax1 and bar) lacking vector backbone sequences were independently transferred into the elite wheat (Triticum aestivum L.) variety EM12, and genetically stable transgenic plants with low copy number transgene integration were recovered. Co-transformation experiments were carried out in parallel using either circular whole plasmid(s) or linear gene cassettes which were purified from the same plasmid by restrictive digestion, each cassette consisting of a promoter, an open reading frame, and a terminator. Six transgenic wheat lines transformed with 1Ax1 plus bar gene cassettes, five lines with gus plus bar gene cassettes, three lines with p1Ax1 plus pAHC20, and two lines with pAHC25 were regenerated with transformation frequencies of 0.6, 0.5, 0.3, and 0.2%, respectively. Southern blotting analysis showed that there were 1-4 hybridizing bands in transgenic lines carrying gene cassettes, of which most lines displayed single-copy transgene insertion. Expression analyses showed that 50.5% of the T1 lines carrying gus plus bar gene cassettes have the expression signals of two genes. SDS-PAGE analysis of the T1 generation revealed that 71% of herbicide-resistant plants carrying 1Ax1 plus bar gene cassettes expressed the high molecular weight subunit 1Ax1 in the endosperm. Gene cassettes were transmitted and segregated in the subsequent generations, in simple Mendelian ratios. In addition, reverse tanscription-polymerase chain reaction (RT-PCR) results confirmed that 1Ax1 gene cassettes were expressed specifically in the endosperm of the transgenic wheat plant. It is proposed that gene transfer using multiple gene cassettes offers an efficient and rapid method to obtain the single-copy transgenic wheat.

Keywords: Gene cassette; integration pattern; particle bombardment; vector backbone sequence; wheat.
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