Osmotic shock improves Tnt1 transposition frequency in Medicago truncatula cv Jemalong during in vitro regeneration.
Insertion mutant collections are powerful tools for genetic studies in plants. Although large-scale insertional mutagenesis using T-DNA is not feasible in legumes, the Tnt1 tobacco retrotransposon can be used as a very efficient mutagen in the Medicago truncatula R108 genotype. In this article, we show that Tnt1 can also be exploited to create insertional mutants via transformation and/or regeneration in the reference cultivar Jemalong. Tnt1 insertional mutagenesis in Jemalong following Agrobacterium tumefaciens-mediated transformation was found to be very efficient, with an average of greater than 15 insertions/line. In contrast, regeneration using low-copy transgenic starter lines resulted in a highly variable rate of new Tnt1 insertions. With the goal of increasing the number of additional Tnt1 insertions during regeneration of starter lines, we have compared the insertion frequencies for a number of different regeneration protocols. In addition, we have been able to show that sucrose-mediated osmotic shock preceding regeneration significantly increases the transposition frequency. Under optimal conditions, 95% of the regenerated Jemalong plants possess new insertions.
Iantcheva
Anelia
A
AgroBioInstitute, bul. Dragan Tzankov 8, 1164 Sofia, Bulgaria. aneliaiancheva@abi.bg
Chabaud
Mireille
M
Cosson
Viviane
V
Barascud
Marielle
M
Schutz
Bernadette
B
Primard-Brisset
Catherine
C
Durand
Patricia
P
Barker
David G
DG
Vlahova
Mariana
M
Ratet
Pascal
P
D016960:Agrobacterium tumefaciens / Q000235:genetics; D003470:Culture Media; D018744:DNA, Plant / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D046913:Medicago truncatula / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional / Q000379:methods*; D009997:Osmotic Pressure; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D012038:Regeneration; D018626:Retroelements; D013395:Sucrose / Q000494:pharmacology; D014170:Transformation, Genetic
D003470:Culture Media; D018744:DNA, Plant; D018626:Retroelements; D013395:Sucrose
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