GBSS T-DNA inserts giving partial complementation of the amylose-free potato mutant can also cause co-suppression of the endogenous GBSS gene in a wild-type background

Abstract

The wild-type gene encoding granule-bound starch synthase (GBSS) is capable of both complementing the amylose-free (amf) potato mutant and inhibiting the endogenous GBSS gene expression in wild-type potato. Co-suppression of the endogenous GBSS gene, easily visualised by staining the starch with iodine, occurred when the full-size GBSS sequence (genomic), GBSS cDNA or even the mutant amf allele were introduced into the wild-type potato. Conversely, introduction of the GBSS promoter sequence alone, did not result in co-suppression in the 80 analysed transformants. Neither the orientation of the GBSS gene with respect to kanamycin resistance nor the presence of an enhancer influenced the frequency of plants showing a co-suppression phenotype. After crossing a partially complemented amf mutant with a homozygous wild-type plant, the F1 offspring segregated into plant phenotypes with normal and decreased expression of the GBSS gene. This decreased expression correlated with the presence of a linked block of five T-DNA inserts which was previously shown to be correlated with partial complementation of the amf mutant. This crossing experiment indicates that co-suppression can cause inhibition of gene expression of both inserted and endogenous wild-type GBSS genes. The frequency of partially complemented amf plants was equal to the frequency of co-suppressed wild types when a construct, with an enhancer in front of the GBSS promoter, was used (pWAM 101E). This might suggest that partial complementation of the amf genotype caused by unstable expression of the transgene can be overcome by inserting an enhancer in front of the GBSS promoter.

Improved Cassava Starch by Antisense Inhibition of Granule-bound Starch Synthase I

“…Also in potato it was reported that the values of mRNA in antisense and co-suppression transformants varied, and was different within one tuber and different tubers from the same clone. The only relation which could be found was that in all antisense plants the level of mRNA was always lower than in the controls (Kuipers et al 1995;Flipse et al 1996). This was also observed in cassava.…”

Silencing of waxy genes in rice containing Wx transgenes

“…Since our Wx transgene has homology with the silenced endogenous genes in both the promoter and the coding sequence it was not possible to distinguish which eect caused silencing. In the case of cosuppression of the GBSS gene in potato, it has been shown that the coding region is sucient to cause cosuppression (Flipse et al 1996). Our observation that no silencing of the transgene occurs in the mutant wx rice in which no Wx mRNA was detected might indicate the involvement of RNA in gene silencing; this is a characteristic of post-transcriptional silencing.…”

“…Many mutations of the locus are available in rice and it is one of the best-studied loci in rice (Sano 1984;Okagaki and Wessler 1988;Hirano and Sano 1991;Wang et al 1995). In potato, cosuppression of the GBSS gene occurs when either the genomic GBSS gene or the cDNA fused to the CaMV35S promoter is introduced into wild-type plants (Flipse et al 1996). It was also shown that multicopy GBSS transgenes were not only partially suppressed due to cis-interaction among them, but also caused cosuppression by acting on the wild-type gene (Flipse et al 1994;1996).…”

Plant Gene Silencing

“…Silencing of GBSSI in potato has been achieved by introduction of either antisense or sense copies of GBSSI cDNA or genomic DNA (Visser et al, 1991a(Visser et al, , b, 1996Hofvander et al, 1992;Kuipers et al, 1992Kuipers et al, , 1994aKuipers et al, , b, 1995Kuipers et al, , 1997Kull et al, 1995;Flipse et al, 1996c). Down-regulation of GBSSI was most efficiently attained with antisense GBSSI T-DNA insertions: 50-80% of the primary antisense transformants exhibited a decrease in amylose content, compared with ca.…”