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Agrobacterium-mediated transformation of black cherry for flowering control and insect resistance

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Abstract

Black cherry is one of the most valuable hardwood species for cabinetry, furniture, and veneer. The goal of this study was to develop transgenic black cherry plants with reproductive sterility and enhanced insect resistance. Black cherry TERMINAL FLOWER 1 (PsTFL1) was overexpressed under the control of the CaMV 35S promoter in black cherry via Agrobacterium-mediated transformation, as a strategy for gene containment. PsTFL1 is a homolog of Arabidopsis TFL1 which is known to play a key role in regulating flowering time by counteracting with FLOWERING LOCUS T and repressing the transcription of the floral-related genes. The elevated expression level of PsTFL1 was proven to be able to significantly delay flowering and cause abnormal floral structure which led to sterility in Arabidopsis. Therefore, the overexpression of PsTFL1 was expected to induce the similar phenotype in black cherry to achieve reproductive sterility. To enhance insect resistance in black cherry, the black cherry endogenous genes encoding prunasin hydrolase isoform 3 (PH3) and mandelonitrile lyase isoform 4 (MDL4) were inserted into black cherry under the control of the phloem-specific promoter rolC or the CaMV 35S promoter. The two enzymes catalyze the hydrolysis of prunasin to mandelonitrile and the dissociation of mandelonitrile to hydrogen cyanide (HCN), respectively, and the overexpression of PH3 or MDL4 might accelerate the release of toxic HCN and lead to an effective protection from cambial-mining insects. Three independent transgenic lines of 35S::PsTFL1, three of rolC::MDL4-FLAG, two of rolC::PH3-FLAG, and eight of 35S::MDL4-FLAG were obtained. The integration of transgenes and the copy number of neomycin phosphotransferase were examined by polymerase chain reaction (PCR) and quantitative PCR (qPCR) analysis. The mRNA levels of PsTFL1, MDL4, and PH3 were examined by real-time qPCR and were compared to the wild-type. The expression level of PsTFL1 in the three 35S::PsTFL1 lines were 3.7–5.8-times higher than that of the wild-type. However, the mRNA level of MDL4 in the 35S::MDL4-FLAG lines and rolC::MDL4-FLAG lines, and the mRNA level of PH3 in the rolC::PH3-FLAG lines showed no significant change indicating that transgene silencing was induced. Western blot analysis was carried out using anti-FLAG antibody to detect the FLAG-tagged PH3 in the transgenic line that had slightly increased mRNA level of PH3, but no signal was detected.

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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 Dr. Marina Kalyaeva and Mary Welter 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. Northern Research Station, HTIRC, USDA Forest Service, 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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Wang, Y., Pijut, P.M. Agrobacterium-mediated transformation of black cherry for flowering control and insect resistance. Plant Cell Tiss Organ Cult 119, 107–116 (2014). https://doi.org/10.1007/s11240-014-0517-1

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  • DOI: https://doi.org/10.1007/s11240-014-0517-1

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