Feasibility of the seed specific cruciferin C promoter in the self excision Cre/loxP strategy focused on generation of marker-free transgenic plants

Abstract

This work is focused on the generation of selectable marker-free transgenic tobacco plants using the self excision Cre/loxP system that is controlled by a strong seed specific Arabidopsis cruciferin C (CRUC) promoter. It involves Agrobacterium-mediated transformation using a binary vector containing the gus reporter gene and one pair of the loxP sites flanking the cre recombinase and selectable nptII marker genes (floxed DNA). Surprisingly, an ectopic activation of CRUC resulting in partial excision of floxed DNA was observed during regeneration of transformed cells already in calli. The regenerated T(0) plants were chimeric, but no ongoing ectopic expression was observed in these one-year-long invitro maintained plants. The process of the nptII removal was expected in the seeds; however, none of the analysed T(0) transgenic lines generated whole progeny sensitive to kanamycin. Detailed analyses of progeny of selected T(0)-30 line showed that 10.2% GUS positive plants had completely removed nptII gene while the remaining 86.4% were still chimeras. Repeated activation of the cre gene in T(2) seeds resulted in increased rate of marker-free plants, whereas four out of ten analysed chimeric T(1) plants generated completely marker-free progenies. This work points out the feasibility as well as limits of the CRUC promoter in the Cre/loxP strategy.

Generation of marker‐free transgenic hexaploid wheat via an Agrobacterium‐mediated co‐transformation strategy in commercial Chinese wheat varieties

“…It is considered important to generate marker‐free varieties where the marker gene used for the generation of positive transgenic plants has been eliminated. Marker‐free transgenic plants can be produced by FLP/FRT site‐specific recombination (Nandy and Srivastava, ; Nanto and Ebinuma, ), Cre/lox site‐specific recombination (Li et al ., ; Mészáros et al ., ; Mlynarova and Nap, ; Moravcikova et al ., ), multi‐auto‐transformation (Khan et al ., ), co‐transformation (Miller et al ., ) and using a marker‐free binary vector (McCormac et al ., ). Among these methods, co‐transformation is the most efficient and simple technique (Tuteja et al ., ).…”

Strategies for developing marker-free transgenic plants

“…Application of heat shock inducible Cre/lox systems in tobacco and potato resulted in 30 ~ 80% and 4.7% excision rates, respectively [36,37]. In recent publications it was shown that the Cre recombinase in this self-excision unit can be activated by developmentally regulated promoters in different tissues such as microspores, inflorescences and seeds [38][39][40].…”

Elimination of Transgenic Sequences in Plants by Cre Gene Expression

“…During the last few years a number of promoters active in different stages of plant development, namely in germline Van Ex et al, 2009), embryo , microspore (Mlynarova et al, 2006;Luo et al, 2007), floral (Bai et al, 2008) and seed (Odell et al, 1994;Moravčíková, et al, 2008;Kopertekh et al, 2010) tissues have been tested to control cre expression. High efficiency of such promoters in Arabidopsis , tobacco (Mlynarova et al, 2006), rice (Bai et al, 2008), soybean (Lie et al, 2007) and oilseed rape (Kopertekh et al, 2009) makes this approach universal for model and agronomically important species.…”

“…The essential feature of conditional Cre systems is a careful regulation with respect to time and tissue. Background Cre activation was observed for heat-shock inducible (Hoff et al, 2001;Wang et al, 2005) and some seed-specific promoters (Odell et al, 1994;Moravčíková, et al, 2008), resulting in reduced efficiency of the systems.…”