Abstract
This study aimed to develop a new vector system to remove selection genes and to introduce two or more genes of interest into plants in order to express them in a coordinated manner. A multigene expression vector was established based on pCamBIA2300 using a selectable marker gene (SMG)-free system based on the combination of the isocaudamer technique and double T-DNA. The vector DT7 containing seven target genes was constructed and introduced into tobacco using Agrobacterium-mediated transformation. Twenty-one of 27 positive transgenic plants contained both T-DNA regions. The co-transformation frequency was 77.8 %. The frequency of unlinked integration of two intact T-DNAs was 22.22 % (6/27). The frequency of removal of SMG from transgenic T1 plants was 19.10 %. These results suggest that this vector system was functional and effective for multigene expression and SMG-free transgenic plant cultivation. At least seven target genes can be co-expressed using this system. Overall, these findings provide a new and highly effective platform for multigene and marker-free transgenic plant production.
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Abbreviations
- cn:
-
copy number
- CF:
-
co-transformation frequency
- CT:
-
cycle threshold
- GOI:
-
gene of interest
- GUS:
-
β-glucuronidase
- KanR :
-
kanamycin resistant
- KanS :
-
kanamycin sensitive
- LB:
-
left border
- MCS:
-
multi cloning site
- MS:
-
Murashige and Skoog
- Nos P:
-
nopaline synthase promoter
- Nos T:
-
Nos terminator
- PPTR :
-
phosphinothricin (PPT)-resistant plant
- PPTS :
-
PPT-susceptible
- RB:
-
right border
- SMG:
-
selectable marker gene
- YEB:
-
yeast extract broth
- ZFNs:
-
zinc finger nucleases
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Acknowledgments: This study was supported by the Major Project of China on New Varieties of GMO Cultivation (grant No. 2014ZX08010-003) and the Special Fund for Agro-scientific Research in the Public Interest of the People's Republic of China (grant No. 201403075).
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Wang, X.J., Su, Y.Y., Dong, Y.F. et al. A novel double T-DNA system for producing stack and marker-free transgenic plants. Biol Plant 60, 767–773 (2016). https://doi.org/10.1007/s10535-016-0653-z
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DOI: https://doi.org/10.1007/s10535-016-0653-z