Study on factors influencing transformation efficiency in Pinus massoniana using Agrobacterium tumefaciens
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Genetic manipulation of gymnosperms using Agrobacterium—mediated system, a powerful field in forest biotechnology, has a potential to accelerate forest tree improvements with desired attributes. However, most conifers such as pine have been considered recalcitrant to genetic transformation. The present study reports the successful production of transgenic Pinus massoniana from zygotic embryos using Agrobacterium tumefaciens harboring the pBI121:CslA2 binary vector. Various independent parameters were tested for their effects on transformation efficiency in P. massoniana. According to our results, combination of Agrobacterium density at OD600 of 0.5, cold treatment of Agrobacterium suspension at 4 °C for approximately 5 h, inoculation period of 5 h, and the addition of 100 µM acetosyringone in co-cultivation medium significantly enhanced the transformation efficiency. The stable integration of CslA2 gene into the genome of putative transgenic plants was confirmed by polymerase chain reaction and Southern blot hybridization, and the expression levels were determined using quantitative reverse transcription-PCR. Our optimized transformation procedure could provide an opportunity for transferring economically important genes into P. massoniana and other conifer species as well.
KeywordsEfficient transformation system Pinus massoniana Agrobacterium tumefaciens Molecular analysis
2,4-Dichlorophenoxy acetic acid
Naphthalene acetic acid
Optical density at 600nm
This study was supported by “The Thirteenth 5-year Plan” National Key Research Project “Pinus massoniana sustainable management technology research on Pinus massoniana”,and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
SM and KJ designed the experiments. SM conducted the experiments and collated the results. KM performed the statistical analysis and prepared the graphs. Drafting of manuscript and reading and final approval of the version to be published; SM, KM and KJ.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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