Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy

Abstract

(S.Q.).Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) d-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops.

An Efficient Gene Excision System in Maize

“…In a transgenic plant with independent insertions of each of these constructs, the selectable marker can be segregated genetically ( Hare and Chua, 2002 ; Puchta, 2003 ; Darbani et al., 2007 ; Ling et al., 2016 ). Alternatively, SMGs can be removed by excision via homologous recombination ( Puchta, 2000 ; Zubko et al., 2000 ), elimination by transposition ( Maeser and Kahmann, 1991 ; Gao et al., 2015 ) or, by the use of recombinases to excise unwanted DNA. Several recombination systems have been used to excise SMGs, including Cre/lox from bacteriophage P1 ( Hoess et al., 1982 ; Hoess and Abremski, 1985 ), Flp/frt from Saccharomyces cerevisiae ( Cox, 1983 ; Senecoff et al., 1985 ), R/RS from Zygosaccharomyces rouxii ( Araki et al., 1985 ), and Gin / gix from bacteriophage ( Klippel et al., 1988 ).…”

Low Long Terminal Repeat (LTR)-Retrotransposon Expression in Leaves of the Marine Phanerogam Posidonia Oceanica L.

“…The analyzed REs resulted generally lowly transcribed. The low transcript abundance of LTR-REs in plants has often been reported [51,[53][54][55][56]. In certain species, increased expression in plants exposed to biotic or abiotic stresses was reported; however, the global level of expression remained low even during stressful treatments [57][58][59][60][61][62].…”

Construction of Marker-Free Genetically Modified Maize Using a Heat-Inducible Auto-Excision Vector

“…In the first approach, the selectable marker genes and the gene of interest are introduced at different loci of the plant genome by co-transformation (the mechanism is shown in Figure 1(a1–a4), after which the selectable marker gene is segregated out by crossing sexually [30]. The second method entails the elimination of selectable markers by transposition [31,32]. To eliminate the selectable marker using transposon-mediated transgene reintegration is an advantageous strategy for marker gene removal (the mechanism is shown in Figure 1(b1,b2), because it allows intact transgene insertion with defined boundaries and requires only a few primary transformants [33].…”