Addition–deletion mutations in transgenic Arabidopsis thaliana generated by the seed co-cultivation method

Abstract

We have characterized three mutant alleles of the CER2 gene of Arabidopsis thaliana by sequencing polymerase chain reaction products of this gene generated with template DNA isolated from lines MK1, BRL7, and BRL17. Sequence analysis of the amplification product from line BRL17 revealed a 17-bp deletion in the CER2 gene. The CER2 gene of BRL7 differs from the wild-type sequence by a 2-base substitution and 2-base insertion. As both of these lines were isolated from the transformant populations generated by Dr. K. Feldmann and his collaborators, we suggest that these small rearrangements may be caused by unsuccessful T-DNA insertions. Comparative sequence analysis of the sequence of line MK1 and the wild type revealed a single nucleotide substitution located 360 bp downstream from the intron-exon junction that changes a tryptophan triplet TGG to TGA; i.e., an opal non-sense mutation. In accordance with these observations, the cer phenotype of line MK1 was complemented by transformation with a fusion construct of the CaMV 35S promoter and the CER2 structural gene. Comparative analysis of the deduced amino acid sequences encoded by the CER2 gene from Arabidopsis and by the glossy2 gene from Zea mays revealed a significant similarity between them. This suggests that these gene products may have a similar biochemical function.

Over‐expression of the Arabidopsis AtMYB41 gene alters cell expansion and leaf surface permeability

“…, 2004) and LCR (Wellesen et al. , 2001), which are involved in biosynthesis of cutin, CER2 (Negruk et al. , 1996; St‐Pierre et al.…”

A chromosomal paracentric inversion associated with T‐DNA integration in Arabidopsis

“…The breakpoints can be ‘tagged’ by the T‐DNA as reported here but breakpoints without T‐DNA integration have also been noted ( Nacry et al . 1998 ), and could result from abortive T‐DNA integration as proposed for other untagged mutations ( Negruk et al . 1996 ).…”

Transformation-induced Mutations in Transgenic Plants: Analysis and Biosafety Implications

“…Agrobacterium-mediated transformation methods that do not involve tissue culture have been used to create large populations of T-DNA containing plants (Feldman, 1991;McElver et al, 2001). When such T-DNA tagging populations are screened for mutations, a large proportion of the identified mutant phenotypes are not linked to a T-DNA insertion event (Forsthoefel et al, 1992;McNevin et al, 1993;Negruk et al, 1996;Budziszewski et al, 2001). In one experiment, only 1/3 of the mutant phenotypes identified in the T-DNA tagging population co-segregated with a T-DNA (Budziszewski et al, 2001).…”

“…A few such untagged mutations have been characterised by DNA sequence analysis. For example, two untagged Cer2 mutant alleles have been isolated from a transformed A. thaliana population and sequenced (Negruk et al, 1996). One of the two alleles had a 17 bp deletion and the other had a 2 bp substitution and a 2 bp insertion.…”