Abstract
Key message
Using hypocotyls as explants, an in vitro adventitious shoot regeneration system of Manchurian ash was established, and it has been successfully applied in Agrobacterium-mediated genetic transformation.
Abstract
Manchurian ash, Fraxinus mandshurica Rupr., is an important tree species for landscaping, timber, and afforestation. However, a genetic transformation system of this tree species has not been reported thus far, which impedes its breeding and investigation (e.g., functional gene assays). In this study, we present an ultrasound-aided Agrobacterium-mediated genetic transformation system for this tree species using hypocotyls as explants. The optimum medium for bud induction was WPM (Woody Plant Medium) + 1.0 mg L−1 TDZ (thidiazuron) + 30 g L−1 sucrose. Sonication for 90 s and vacuum treatment for 10 min doubled the number of adventitious buds from 33.23 to 77.67 buds per hypocotyl. The optimum medium for shoot elongation was WPM supplemented with 0.025 mg L−1 TDZ and 1.0 mg L−1 gibberellic acid (GA3), which resulted in a survival rate of 70.97%. Our results also showed that lower light intensity culture can prevent tissue browning, which is of vital concern for the in vitro propagation of this tree species. Using this regeneration system, precultured hypocotyl explants were transformed with the aid of 90 s sonication plus 10 min vacuum infiltration. After performing a screening culture at 30 mg L−1 kanamycin, 16 transgenic plant lines were obtained, for a transformation rate of 7.31%.
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Funding
This study was co-funded by the China National Key R&D Program during the 14th Five-year Plan Period (2021YFD220030301), Heilongjiang Provincial R&D Project on Provincial Applied Technology (GA19B201), and Fundamental Research Funds for the Central Universities (2572020DR09). The funders had no role in the study design, analysis, or preparation of the manuscript.
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Qi, F., Tang, M., Wang, W. et al. In vitro adventitious shoot regeneration system for Agrobacterium-mediated genetic transformation of Fraxinus mandshurica Rupr.. Trees 36, 1387–1399 (2022). https://doi.org/10.1007/s00468-022-02302-3
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DOI: https://doi.org/10.1007/s00468-022-02302-3