Development of selectable marker free, insect resistant, transgenic mustard (Brassica juncea) plants using Cre/loxmediated recombination

Abstract

BackgroundAntibiotic/ herbicide resistant marker genes have been proven to be very useful in plant transformation for the initial selection of desired transgenic events. However, presence of these genes in the genetically modified crops may render the crop less acceptable to the consumers. Among several different approaches, the effectiveness of Cre/lox mediated recombination strategy for selectable marker gene (SMG) elimination has previously been demonstrated by different groups in several plants including Brassica. In the present study exploiting Cre/lox mediated recombination strategy, attempt has been made for selectable marker gene elimination from Allium sativum leaf agglutinin (ASAL) expressing Brassica plants with hemipteran insect resistant phenotype.ResultsAllium sativum leaf agglutinin (ASAL) linked with lox flanked hygromycin resistant (hpt) gene was introduced in mustard. Cre recombinase gene cassette was also integrated in separate event. A Cre/lox mediated recombination using crossing strategy was adopted to remove the hpt gene from the subsequent generation of selected hybrid events. Reciprocal crosses were made between T1ASAL-lox-hpt-lox and cre-bar plants. Marker gene elimination was confirmed in the resulting F1 hybrid progenies by PCR analysis, using hpt, cre and ASAL specific primers followed by Southern hybridization. In marker free plants, expression of ASAL was also confirmed by western blotting and ELISA analysis. Retention of functionality of expressed ASAL was investigated by agglutination assay using rabbit erythrocytes. Expressed ASAL was also found to be thermo-sensitive. In planta insect bioassay on F1 hybrid progenies exhibited detrimental effect on the performance of devastating target pest, Lipaphis erysimi. The F1 hybrid hpt negative, ASAL positive plants were allowed to self- fertilize to obtain F2 progeny plants. In some of these plants cre gene was found to be segregated out of the ASAL gene by genetic segregation yielding completely marker free plants.ConclusionsThe present study establishes the efficient expression of the newly introduced insect resistant ASAL gene even after Cre/lox mediated recombination resulting in elimination of selectable marker gene.

Strong activity of FLPe recombinase in rice plants does not correlate with the transmission of the recombined locus to the progeny

“…Consistent with its activity in the germline, the ZmUBI1:Cre construct successfully generated stable marker-free lines of rice and wheat (Moore and Srivastava 2006;Srivastava et al 1999). Similarly, other promoters, including the 35S promoter, heat-inducible, and chemical-inducible promoters, have also been successful in generating stable marker-free lines when the Cre-lox system was used for marker excision (Bala et al 2013;Dale and Ow 1991;Nandy and Srivastava 2012;Russell et al 1992;Sreekala et al 2005;Zhang et al 2003). Thus, gametophytic promoters have mostly been used for pollen-specific excision of the transgene for biocontainment applications (Luo et al 2007;Mlynárová et al 2006).…”

Development of marker-free transgenic pigeon pea (Cajanus cajan) expressing a pod borer insecticidal protein

“…Extensive research has been done to develop insect-resistant pigeon pea, but until now, no marker-free plant has been generated. Thus, in the current experiment, an attempt was undertaken to develop a marker-free pigeon pea plant using Cre/ lox technology with insecticidal activity 31 , 32 , 33 , 34 . To perform this, we also developed a cre line with a pHC construct and crossed this line with the cry1Ab line.…”

Targeted Genome Engineering and Its Application in Trait Improvement of Crop Plants

“…For example, by using Cre/LoxP-mediated recombination, the selectable maker gene, HPT, has been successfully eliminated from transgenic mustard plants with insect resistance [22]. Elimination of selectable marker genes has also been reported in rice [23], potato [24], tomato [25] and so on.…”