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Efficient Agrobacterium-Mediated Transformation of Hybrid Poplar Populus davidiana Dode × Populus bollena Lauche
Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field (SAVER), Ministry of Education, Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University, Harbin Hexing Road, Harbin 150040, China
Institute of Microbiology, Heilongjiang Academy of Sciences, Harbin 150010, China
Asian Natural Environmental Science Center, University of Tokyo, Nishitokyo-shi, Tokyo 188-0002, Japan
* Author to whom correspondence should be addressed.
Received: 7 November 2012; in revised form: 29 December 2012 / Accepted: 17 January 2013 / Published: 25 January 2013
Abstract: Poplar is a model organism for high in vitro regeneration in woody plants. We have chosen a hybrid poplar Populus davidiana Dode × Populus bollena Lauche. By optimizing the Murashige and Skoog medium with (0.3 mg/L) 6-benzylaminopurine and (0.08 mg/L) naphthaleneacetic acid, we have achieved the highest frequency (90%) for shoot regeneration from poplar leaves. It was also important to improve the transformation efficiency of poplar for genetic breeding and other applications. In this study, we found a significant improvement of the transformation frequency by controlling the leaf age. Transformation efficiency was enhanced by optimizing the Agrobacterium concentration (OD600 = 0.8–1.0) and an infection time (20–30 min). According to transmission electron microscopy observations, there were more Agrobacterium invasions in the 30-day-old leaf explants than in 60-day-old and 90-day-old explants. Using the green fluorescent protein (GFP) marker, the expression of MD–GFP fusion proteins in the leaf, shoot, and root of hybrid poplar P. davidiana Dode × P. bollena Lauche was visualized for confirmation of transgene integration. Southern and Northern blot analysis also showed the integration of T-DNA into the genome and gene expression of transgenic plants. Our results suggest that younger leaves had higher transformation efficiency (~30%) than older leaves (10%).
Keywords: regeneration; transformation; leaf age; poplar; Agrobacterium
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Han, X.; Ma, S.; Kong, X.; Takano, T.; Liu, S. Efficient Agrobacterium-Mediated Transformation of Hybrid Poplar Populus davidiana Dode × Populus bollena Lauche. Int. J. Mol. Sci. 2013, 14, 2515-2528.
Han X, Ma S, Kong X, Takano T, Liu S. Efficient Agrobacterium-Mediated Transformation of Hybrid Poplar Populus davidiana Dode × Populus bollena Lauche. International Journal of Molecular Sciences. 2013; 14(2):2515-2528.
Han, Xue; Ma, Shurong; Kong, Xianghui; Takano, Tetsuo; Liu, Shenkui. 2013. "Efficient Agrobacterium-Mediated Transformation of Hybrid Poplar Populus davidiana Dode × Populus bollena Lauche." Int. J. Mol. Sci. 14, no. 2: 2515-2528.