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

Gào, Xīn; Zhou, Jie; Li, Jun; Zou, Xiaowei; Zhao, Jinyan; Li, Qingliang; R, Xia; Yang, Ruifang; Wang, Dekai; Zuo, Zhaoxue; Tu, J. C.; Tao, Yuezhi; Chen, Xiaoyun; Xie, Qi; Zhu, Zhiliang; Qu, Shaohong

doi:10.1104/pp.114.246173

Cited by 20 publications

(26 citation statements)

References 39 publications

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“…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 ).…”

Section: Introductionmentioning

confidence: 99%

An Efficient Gene Excision System in Maize

et al. 2020

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Use of the morphogenic genes Baby Boom ( Bbm ) and Wuschel2 ( Wus2 ), along with new ternary constructs, has increased the genotype range and the type of explants that can be used for maize transformation. Further optimizing the expression pattern for Bbm / Wus2 has resulted in rapid maize transformation methods that are faster and applicable to a broader range of inbreds. However, expression of Bbm / Wus2 can compromise the quality of regenerated plants, leading to sterility. We reasoned excising morphogenic genes after transformation but before regeneration would increase production of fertile T0 plants. We developed a method that uses an inducible site-specific recombinase ( Cre ) to excise morphogenic genes. The use of developmentally regulated promoters, such as Ole , Glb1 , End2 , and Ltp2 , to drive Cre enabled excision of morphogenic genes in early embryo development and produced excised events at a rate of 25–100%. A different strategy utilizing an excision-activated selectable marker produced excised events at a rate of 53–68%; however, the transformation frequency was lower (13–50%). The use of inducible heat shock promoters (e.g. Hsp17.7 , Hsp26 ) to express Cre, along with improvements in tissue culture conditions and construct design, resulted in high frequencies of T0 transformation (29–69%), excision (50–97%), usable quality events (4–15%), and few escapes (non-transgenic; 14–17%) in three elite maize inbreds. Transgenic events produced by this method are free of morphogenic and marker genes.

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Section: Introductionmentioning

confidence: 99%

An Efficient Gene Excision System in Maize

et al. 2020

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“…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].…”

Section: Discussionmentioning

confidence: 99%

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

Vangelisti

Mascagni

Usai

et al. 2020

Life

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Seagrasses as Posidonia oceanica reproduce mostly by vegetative propagation, which can reduce genetic variability within populations. Since, in clonally propagated species, insurgence of genetic variability can be determined by the activity of transposable elements, we have estimated the activity of such repeat elements by measuring their expression level in the leaves of plants from a Mediterranean site, for which Illumina complementary DNA (cDNA) sequence reads (produced from RNAs isolated by leaves of plants from deep and shallow meadows) were publicly available. Firstly, we produced a collection of retrotransposon-related sequences and then mapped Illumina cDNA reads onto these sequences. With this approach, it was evident that Posidonia retrotransposons are, in general, barely expressed; only nine elements resulted transcribed at levels comparable with those of reference genes encoding tubulins and actins. Differences in transcript abundance were observed according to the superfamily and the lineage to which the retrotransposons belonged. Only small differences were observed between retrotransposon expression levels in leaves of shallow and deep Posidonia meadow stands, whereas one TAR/Tork element resulted differentially expressed in deep plants exposed to heat. It can be concluded that, in P. oceanica, the contribution of retrotransposon activity to genetic variability is reduced, although the nine specific active elements could actually produce new structural variations.

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“…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].…”

Section: Introductionmentioning

confidence: 99%

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

Jin

Guo

et al. 2019

Genes

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Gene modification is a promising tool for plant breeding, and gradual application from the laboratory to the field. Selectable marker genes (SMG) are required in the transformation process to simplify the identification of transgenic plants; however, it is more desirable to obtain transgenic plants without selection markers. Transgene integration mediated by site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. Here, we present an auto-elimination vector system that uses a heat-inducible Cre to eliminate the selectable marker from transgenic maize, without the need for repeated transformation or sexual crossing. The vector combines an inducible site-specific recombinase (hsp70::Cre) that allows for the precise elimination of the selectable marker gene egfp upon heating. This marker gene is used for the initial positive selection of transgenic tissue. The egfp also functions as a visual marker to demonstrate the effectiveness of the heat-inducible Cre. A second marker gene for anthocyanin pigmentation (Rsc) is located outside of the region eliminated by Cre and is used for the identification of transgenic offspring in future generations. Using the heat-inducible auto-excision vector, marker-free transgenic maize plants were obtained in a precisely controlled genetic modification process. Genetic and molecular analyses indicated that the inducible auto-excision system was tightly controlled, with highly efficient DNA excision, and provided a highly reliable method to generate marker-free transgenic maize.

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

Cited by 20 publications

References 39 publications

An Efficient Gene Excision System in Maize

An Efficient Gene Excision System in Maize

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

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

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