biologia plantarum

Biologia plantarum 60:767-773, 2016 | DOI: 10.1007/s10535-016-0653-z

A novel double T-DNA system for producing stack and marker-free transgenic plants

X. J. Wang¹, Y. Y. Su¹, Y. F. Dong¹, Q. L. Tang¹, Z. X. Wang^1,*

¹ Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, P.R. China

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.

Keywords: Agrobacterium tumefaciens; isocaudamer technique; multi-gene stack; Nicotiana tabacum

Subjects: marker-free transgenic plants; isodaumer technique; multi-gene stack; GUS staining; tobacco

Received: July 27, 2015; Revised: March 7, 2016; Accepted: March 10, 2016; Published: December 1, 2016  Show citation