Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene: increase of the antisense effect during tuber growth

Abstract

Transgenic plants of a tetraploid potato cultivar were obtained in which the amylose content of tuber starch was reduced via antisense RNA-mediated inhibition of the expression of the gene encoding granule-bound starch synthase (GBSS). GBSS is one of the key enzymes in the biosynthesis of starch and catalyses the formation of amylose. The antisense GBSS genes, based on the full-length GBSS cDNA driven by the 35S CaMV promoter or the potato GBSS promoter, were introduced into the potato genome by Agrobacterium tumefaciens-mediated transformation. Expression of each of these genes resulted in the complete inhibition of GBSS gene expression, and thus in the production of amylose-free tuber starch, in mature field-grown plants originating from rooted in vitro plantlets of 4 out of 66 transgenic clones. Clones in which the GBSS gene expression was incompletely inhibited showed an increase of the extent of inhibition during tuber growth. This is likely to be due to the increase of starch granule size during tuber growth and the specific distribution pattern of starch components in granules of clones with reduced GBSS activity. Expression of the antisense GBSS gene from the GBSS promoter resulted in a higher stability of inhibition in tubers of field-grown plants as compared to expression from the 35S CaMV promoter. Field analysis of the transgenic clones indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch and sugar content of transgenic tubers, the expression level of other genes involved in starch and tuber metabolism and agronomic characteristics such as yield and dry matter content.

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.…”

Multiple allelism as a control mechanism in metabolic pathways: GBSSI allelic composition affects the activity of granule-bound starch synthase I and starch composition in potato

“…Northern analysis RNA was isolated according to Kuipers et al (1994b), fractionated by electrophoresis, blotted and hybridised to the cDNA for potato GBSSI, as described by Sambrook et al (1989). A 24S rRNA probe was used as a standard (Landsmann et al 1985).…”

Molecular characterization and biotechnological improvement of the processing quality of potatoes

“…Expression of the antisense GBSS gene using the GBSS promoter resulted in a higher stability for the amylosefree trait than with the 35S promoter (Visser et al 1991). Field analyses of these transgenic plants indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch or sugar content of tubers (Kuipers et al 1994). The amylosefree trait in transgenic plants showed a dominant Mendelian inheritance, so the genetically modified amylose-free potato clones have use as either male or female progenitors in a breeding program (Heeres et al 1997).…”

“…Candidate genes located in the QTL regions affecting the dry matter content include two starch phosphorylase genes on chromosome I, a Rubisco gene on chromosome II, a starch synthase gene on chromosome III, a starch phosphorylase gene and a debranching enzyme gene on chromosome IX, a Rubisco activase gene and an apoplast invertase gene on chromosome X, a sucrose transporter gene on chromosome XI, and a sucrose synthase gene on chromosome XII (Chen et al 2001). Transgenic evidence for the involvement in starch dry matter has been available for several of these enzymes, including sucrose synthase (Zrenner et al 1995), starch synthase (Kuipers et al 1994), and glucan branching enzyme (Kortstee et al 1996). Some of the genes have already converted to molecular markers for markerassisted selection of dry matter content of potato tubers.…”

“…Transgenic potato plants with amylose-free tubers were successfully created through inhibiting granulebound starch synthase (GBSS) using an antisense technology (Visser et al 1991;Kuipers et al 1994). Expression of the antisense GBSS gene using the GBSS promoter resulted in a higher stability for the amylosefree trait than with the 35S promoter (Visser et al 1991).…”