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Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 133, Issue 3, pp 437–445 | Cite as

Study on factors influencing transformation efficiency in Pinus massoniana using Agrobacterium tumefaciens

  • Samaneh Sadat Maleki
  • Kourosh Mohammadi
  • Kong Shu Ji
Research Note
  • 335 Downloads

Abstract

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.

Keywords

Efficient transformation system Pinus massoniana Agrobacterium tumefaciens Molecular analysis 

Abbreviations

2,4-D

2,4-Dichlorophenoxy acetic acid

6-BA

Benzylaminopurine

NAA

Naphthalene acetic acid

AS

Acetosyringone

OD

Optical density at 600nm

NPTII

Neomycin phosphotransferase

Notes

Acknowledgements

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).

Author contributions

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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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Co-Innovation Center for Sustainable Forestry in Southern China, The key Forest Genetics & Biotechnology, Ministry of EducationNanjing Forestry UniversityNanjingChina

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