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Improvement of Agrobacterium-mediated transformation and rooting of black cherry

  • Biotechnology/Genetic Transformation/Functional Genomics
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Abstract

An improved protocol for Agrobacterium-mediated transformation of an elite, mature black cherry genotype was developed. To increase transformation efficiency, vacuum infiltration, sonication, and a combination of the two treatments were applied during the cocultivation of leaf explants with Agrobacterium tumefaciens strain EHA105 harboring a PsAGAMOUS RNAi plasmid (pART27-PsAGRNAi). The effects of Agrobacterium culture density and cocultivation duration on transformation efficiency were examined using EHA105 harboring either pBI121-MDL4 or pBI121-PsTFL1. In addition, the effect of the binary vector on transformation efficiency was also studied. Fifteen-minute vacuum infiltration without sonication produced the highest transformation efficiency (21.7%) in experiments using pART27-PsAGRNAi. OD600 values of 1.0 and 1.5 resulted in a transformation efficiency of 5% when pBI121-PsTFL1 was used for transformation. Transformation efficiency of 5% was also obtained from 3-d cocultivation using construct pBI121-MDL4 whereas no shoots regenerated after 4-d cocultivation. The binary vectors used also impacted transformation efficiency. PCR and quantitative-PCR analyses were used to confirm the integration of transgenes and determine the copy number of the selectable marker gene, neomycin phosphotransferase II, in 18 putative transgenic lines. Rooting of transgenic black cherry shoots was achieved at a frequency of 30% using half-strength Murashige and Skoog medium supplemented with 2% sucrose, 5 μM naphthaleneacetic acid, 0.01 μM kinetin, and 0.793 mM phloroglucinol, and the resulting transgenic plants were successfully acclimatized.

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Acknowledgments

This work was supported financially by a Fred M. van Eck scholarship for Purdue University to Ying Wang. The authors gratefully acknowledge Drs. Sadanand Dhekney, Lining Tian, and Charles Maynard for their constructive review and suggestions for the improvement of this manuscript. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that also may be suitable.

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Authors and Affiliations

  1. Department of Forestry and Natural Resources, Hardwood Tree Improvement and Regeneration Center (HTIRC), Purdue University, 715 West State St, West Lafayette, IN, 47907, USA

    Ying Wang

  2. USDA Forest Service, Northern Research Station, HTIRC, 715 West State St, West Lafayette, IN, 47907, USA

    Paula M. Pijut

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  1. Ying Wang
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  2. Paula M. Pijut
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Correspondence to Paula M. Pijut.

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Editor: Harold Trick

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Wang, Y., Pijut, P.M. Improvement of Agrobacterium-mediated transformation and rooting of black cherry. In Vitro Cell.Dev.Biol.-Plant 50, 307–316 (2014). https://doi.org/10.1007/s11627-014-9608-2

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  • DOI: https://doi.org/10.1007/s11627-014-9608-2

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