Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 14, Issue 2, Pages 2515-2528Publisher
MDPI
DOI: 10.3390/ijms14022515
Keywords
regeneration; transformation; leaf age; poplar; Agrobacterium
Funding
- State Forestry Administration 948 Program of China [2008429]
- Heilongjiang Provincial Program for Distinguished Young Scholars [JC200609]
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Poplar is a model organism for high in vitro regeneration in woody plants. We have chosen a hybrid poplar Populus davidiana Dode x 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 x 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 (similar to 30%) than older leaves (10%).
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