Details

Autor(en) / Beteiligte

• Gao, Xiaoqing
• Zhou, Jie
• Li, Jun
• Zou, Xiaowei
• Zhao, Jianhua
• Li, Qingliang
• Xia, Ran
• Yang, Ruifang
• Wang, Dekai
• Zuo, Zhaoxue
• Tu, Jumin
• Tao, Yuezhi
• Chen, Xiaoyun
• Xie, Qi
• Zhu, Zengrong
• Qu, Shaohong

Titel

Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy

Ist Teil von

• Plant physiology (Bethesda), 2015-01, Vol.167 (1), p.11-24

Ort / Verlag

United States: American Society of Plant Biologists

Erscheinungsjahr

2015

Link zum Volltext

Quelle

EZB Electronic Journals Library

Beschreibungen/Notizen

• Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays)Activator/Dissociation (Ds)transposable element can be routinely used to generate marker-free transgenic plants. ADs-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned theBacillus thuringiensis(Bt)δ-endotoxin gene into theDselements and transformed transposon vectors into rice (Oryza sativa) cultivars viaAgrobacterium tumefaciens.PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determineDs(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detectBtexpression levels, and bioassays to confirm resistance against the striped stem borerChilo suppressalis.Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops.