Efficient transposition of the Tnt1 tobacco retrotransposon in the model legume Medicago truncatula

Abstract

SummaryThe tobacco element, Tnt1, is one of the few active retrotransposons in plants. Its transposition is activated during protoplast culture in tobacco and tissue culture in the heterologous host Arabidopsis thaliana. Here, we report its transposition in the R108 line of Medicago truncatula during the early steps of the in vitro transformation±regeneration process. Two hundred and twenty-®ve primary transformants containing Tnt1 were obtained. Among them, 11.2% contained only transposed copies of the element, indicating that Tnt1 transposed very early and ef®ciently during the in vitro transformation process, possibly even before the T-DNA integration. The average number of insertions per transgenic line was estimated to be about 15. These insertions were stable in the progeny and could be separated by segregation. Inspection of the sequences anking the insertion sites revealed that Tnt1 had no insertion site speci®city and often inserted in genes (one out of three insertions). Thus, our work demonstrates the functioning of an ef®cient transposable element in leguminous plants. These results indicate that Tnt1 can be used as a powerful tool for insertion mutagenesis in M. truncatula.

Successful Gene Tagging in Lettuce Using the Tnt1 Retrotransposon from Tobacco

“…Furthermore, several different single-copy insert plants were easily obtained after only three backcrosses and one self-pollination, starting with a plant containing 28 copies of Tnt1, demonstrating the feasibility of separating a given Tnt1 insertion from the others even from a genotype containing large numbers of transposed copies. No new transposition events were observed in any of the progenies studied, as already observed in Arabidopsis and Medicago (Lucas et al, 1995;d'Erfurth et al, 2003). Thus, the insertions of Tnt1 are stable and the element does not transpose at a detectable frequency in the germ cells of lettuce under standard growth conditions in the greenhouse.…”

“…Analysis of 25 different lettuce Tnt1 flanking sequences showed that the element was inserted close to transcribed regions in one-half of the sequences analyzed. This Tnt1 preference to transpose into coding regions was also observed in Medicago and Arabidopsis (Courtial et al, 2001;d'Erfurth et al, 2003;Tadege et al, 2005) and also in the two other retroelements Tto1 and Tos17 (Okamoto and Hirochika, 2000;Yamazaki et al, 2001;Miyao et al, 2003), even if some bias and insertion site specificity have been shown for Tos17. Thus, retrotransposon elements such as Tnt1 have great potential as tools for insertional mutagenesis in crop plants with large genomes like lettuce.…”

“…For Medicago, genes are believed to be clustered in the euchromatic regions, representing only 25% of the complete genome (Kulikova et al, 2001). As a result, transcribed regions are expected to represent less than 15% of the Medicago genome (d'Erfurth et al, 2003). Accordingly, in the lettuce genome, which is 5 times larger than Medicago, we would expect to find an even lower percentage of transcribed sequences than in Medicago.…”

“…The number of transposed copies per plant was particularly high and difficult to estimate accurately on the T 0 Southern blots. Whereas in Arabidopsis the number of transposed Tnt1 copies fluctuated between 0 and 26 (Courtial et al, 2001) and in Medicago between four and more than 30 (d'Erfurth et al, 2003), the number of Tnt1 insertions The number of Tnt1 inserts in each plant was determined by Southern blot (Fig. 3).…”

“…Retrotransposons have already been used successfully as mutagens in plants: Tos17, an endogenous retrotransposon of rice, is used for gene tagging in rice (Yamazaki et al, 2001). Moreover, it was shown that Tto1 and Tnt1 transpose in Arabidopsis (Okamoto and Hirochika, 2000;Courtial et al, 2001), and Tnt1 from tobacco (Nicotiana tabacum) is used in Medicago truncatula (d'Erfurth et al, 2003;Tadege et al, 2005). These studies revealed another important advantage of the use of retrotransposons over more traditional DNAtype elements: They can be highly mutagenic because some of them prefer to transpose into gene-rich regions.…”