Abstract
Both linked and un-linked co-transformation can be used to overcome limitations of methods, such as re-transformation or sexual crossing of transgenic plants, to enable transfer of multiple genes to a single plant. Un-linked co-transformation can also facilitate the production of selectable marker-free transgenic plants. In this study, transgenic white clover plants were generated by Agrobacterium-mediated linked co-transformation using a single T-DNA of 9803 bp expressing: an isopentenyl transferase (IPT) gene for delayed leaf senescence under the control of an organ specific MYB32 promoter from Arabidopsis, a white clover nodule enhanced malate dehydrogenase (neMDH) gene for aluminium tolerance controlled by the endogenous Phosphate Transporter 1 (PT1) promoter, and the coat protein gene from Alfalfa Mosaic Virus (CP-AMV) controlled by the 35S promoter from Cauliflower Mosaic Virus. The selectable marker gene encoding hygromycin phosphotransferase (hph) was borne on a separate T-DNA. Forty independent transgenic events carrying the triple stack were generated, with estimated co-transformation efficiencies of 0.22 to 0.23%. Forty three percent of the events generated had a single insertion, while two events were selectable marker-free. Transcript abundance studies of the three transgenes of interest demonstrated the transcriptional competence of the inserted T-DNA. This study illustrates the feasibility of transferring multiple genes in a large single T-DNA into white clover by Agrobacterium-mediated co-transformation. Furthermore, observations of consistently delayed leaf senescence, statistically significant increases in TrneMDH transcript, and presence of CP-AMV transcript, support further analysis of these events for delayed leaf senescence under drought conditions, aluminium tolerance, and resistance to AMV.
Key message
The generation of marker-free white clover plants with three transgenes in a single T-DNA by cotransformation in one step is reported here.
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Change history
21 October 2020
In the original article, the order of transgene names in the column headings of Table 3 was incorrect. The correct Table 3 is printed below. The error does not change the outcome of the study. We apologize for any inconvenience.
Abbreviations
- Atmyb32 :
-
Arabidopsis thaliana MYB 32
- CaMV35S :
-
Cauliflower mosaic virus 35S
- CP-AMV:
-
Coat protein Gene from Alfalfa Mosaic Virus
- ddPCR:
-
Droplet digital PCR
- EF1alpha :
-
Eukaryotic elongation factor alpha 1
- GFP:
-
Green fluorescent protein
- GOI:
-
Gene of interest
- hph :
-
Hygromycin phosphotransferase
- IPT :
-
Isopentenyl transferase
- RIM:
-
Root-inducing media
- RM:
-
Regeneration media
- PDH :
-
Pyruvate dehydrogenase
- qPCR:
-
Quantitative PCR
- T-DNA:
-
Transfer DNA
- TrneMDH :
-
Trifolium repens nodule-enhanced malate dehydrogenase
- TrPt1 :
-
Trifolium repens phosphate transporter gene
- tuGFP:
-
Turbo green fluorescent protein
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Acknowledgements
We thank Dr. Daniel Isenegger for critical reading of the manuscript.
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This project was funded by Agriculture Victoria, and Dairy Australia.
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Communicated by Sergio J. Ochatt.
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Narancio, R., Ding, YL., Lin, YH. et al. Application of linked and unlinked co-transformation to generate triple stack, marker-free, transgenic white clover (Trifolium repens L.). Plant Cell Tiss Organ Cult 142, 635–646 (2020). https://doi.org/10.1007/s11240-020-01891-6
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DOI: https://doi.org/10.1007/s11240-020-01891-6