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A non-antibiotic selection strategy uses the phosphomannose-isomerase (PMI) gene and green fluorescent protein (GFP) gene for Agrobacterium-mediated transformation of Prunus domestica L. leaf explants

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Abstract

We developed an efficient system for agrobacterial transformation of plum (Prunus domestica L.) leaf explants using the PMI/mannose and GFP selection system. The cultivar ‘Startovaya’ was transformed using Agrobacterium tumefaciens strain CBE21 carrying the vector pNOV35SGFP. Leaf explants were placed onto a nutrient medium containing various concentrations and combinations of mannose and sucrose to develop an efficient selection system. Nine independent transgenic lines of plum plants were obtained on a regeneration medium containing 20 g/L sucrose and 15 g/L mannose. The highest transformation frequency (1.40 %) was produced using a delayed selection strategy. Starting from the 1st days after transformation and ending by regeneration of shoots from the transgenic callus, selection of transgenic cells was monitored by GFP fluorescence that allowed avoiding formation of escapes. Integration of the manA and gfp transgenes was confirmed by PCR and Southern blotting. The described transformation protocol using a positive PMI/mannose system is an alternative selection system for production of transgenic plum plants without genes of antibiotic and herbicide resistance, and the use of leaf explants enables retention of cultivar traits of plum plants.

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Acknowledgments

The work was supported by the Russian Science Foundation, Grant No 14-50-00079.

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Author notes

  1. Roman Mikhailov

    Present address: LLC “Antherix”, Institutskaya 7, Pushchino, Moscow Region, Russia, 142290

Authors and Affiliations

  1. Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Science Ave 6, Pushchino, Moscow Region, Russia, 142290

    Tatiana Sidorova, Roman Mikhailov, Alexander Pushin, Dmitry Miroshnichenko & Sergey Dolgov

  2. Nikitsky Botanical Gardens, Nikita 52, Yalta, Russia, 298648

    Tatiana Sidorova, Alexander Pushin & Sergey Dolgov

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  1. Tatiana Sidorova
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  2. Roman Mikhailov
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Correspondence to Tatiana Sidorova.

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T. Sidorova and R. Mikhailov have contributed equally to this work.

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11240_2016_1100_MOESM1_ESM.docx

Supplementary material 1 Supplementary Fig. 1 Intensity of GFP fluorescence in leaves of nine independent transgenic plum lines (left) compared to control (right). Leaves were taken from 5-year-old plum trees growing in greenhouse. a leaves under ultraviolet light (GFP plant filter); b leaves under daylight (DOCX 1444 KB)

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Sidorova, T., Mikhailov, R., Pushin, A. et al. A non-antibiotic selection strategy uses the phosphomannose-isomerase (PMI) gene and green fluorescent protein (GFP) gene for Agrobacterium-mediated transformation of Prunus domestica L. leaf explants. Plant Cell Tiss Organ Cult 128, 197–209 (2017). https://doi.org/10.1007/s11240-016-1100-8

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