Expression of a wild-type GBSS gene introduced into an amylose-free potato mutant by Agrobacterium tumefaciens and the inheritance of the inserts at the microsporic level

Abstract

Granule-bound starch synthase (GBSS) catalyses the synthesis of amylose in starch granules. Transformation of a diploid amylose-free (amf) potato mutant with the gene encoding GBSS leads to the restoration of amylose synthesis. Transformants were obtained which had wild-type levels of both GBSS activity and amylose content. It proved to be difficult to increase the amylose content above that of the wild-type potato by the introduction of additional copies of the wild-type GBSS gene. Staining of starch with iodine was suitable for investigating the degree of expression of the inserted GBSS gene in transgenic amf plants. Of the 19 investigated transformants, four had only red-staining starch in tubers indicating that no complementation of the amf mutation had occured. Fifteen complemented transformants had only blue-staining starch in tubers or tubers of different staining categories (blue, mixed and red), caused either by full or partial expression of the inserted gene. Complementation was also found in the microspores. The segregation of blue- and red-staining microspores was used to analyse the inheritance of the introduced GBSS genes. A comparison of the results from microspore staining and Southern hybridisation indicated that, in three tetraploid transgenics, the gene was probably inserted before (duplex), and in all others after, chromosome doubling (simplex). The partial complementation was not due to methylation of the HPAII/MSPI site in the promoter region. Partially complemented plants had low levels of mRNA as was found when the GBSS expression levels were inhibited by anti-sense technology.

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

“…A decreased amylose content in potato starch can be obtained by mutation in the GBSS gene such as in the amf mutant [9] or by introduction of antisense genes coding for GBSS [34]. Indications for a third possibility were found during complementation studies of the amf mutant with a wild-type GBSS gene of potato [18,2]. When complementing the potato amfmutant, transformants were found with full and partial complementation.…”

“…In the present study, a partially complemented amf mutant was crossed with a wild-type potato, in order to investigate the effect of co-suppression on complementation in the presence of an endogenous wild-type GBSS allele. In parallel with the complementation experiments [2], the cloned wild-type GBSS gene was introduced in a wild-type potato genotype. The characteristics of these transgenic plants and those resulting from transformation with other GBSS sequences will be outlined.…”

Starch meets biotechnology : in planta modification of starch composition and functionalities

“…We suggest that a further increase in phosphate content is only possible when more places are available to deposit phosphate groups in the starch granules resulting from a change in the starch molecular structure. A similar phenomenon has been observed in earlier studies, demonstrating that introduction of additional copies of the granule-bound starch synthase (GBSS) gene into potato plants did not increase amylose content above the wild-type level (Flipse et al, 1994;Flipse et al, 1996a). The authors reasoned that the restricted amount of amylose content was due to the limited space existed to deposit amylose molecules in starch granules.…”

“…This is supported by the earlier studies, showing elongated and branched chains are preferably phosphorylated (Blennow et al, 2002;Schwall et al, 2000;Takeda and Hizukuri, 1982). A similar phenomenon has been observed in transgenic potato plants overexpressing the granule-bound starch synthase (GBSS) gene (Flipse et al, 1994;Flipse et al, 1996a). The authors found that the amylose content in transformants did not increase above the wild-type level, therefore, they suggested that there is limited space existing in starch granules to deposit amylose molecules.…”

Antisense Waxy Genes with Highly Active Promoters Effectively Suppress Waxy Gene Expression in Transgenic Rice

“…Extensive studies aimed at controlling the quality of starch in potato tubers have been performed . Transformation of the amylose-deficient amf mutant of potato with the GBSS gene led to amylose synthesis (van der Leij et al 1991, Flipse et al 1994, whereas various levels of reduction in GBSS protein and amylose were observed in transgenic potato tubers with the antisense GBSS gene connected to the CaMV35S promoter or GBSS promoter , Salehuzzaman et al 1993, Kuipers et al 1994, Kuipers et al 1995. The role of AGPase in starch accumulation (Muller-Rober et al 1992, Stark et al 1992) and the functions of soluble starch synthesis and starch branching enzyme (Flipse et al 1996, Kortstee et al 1996, Jobing et al 1999 have been investigated by the use of sense and antisense genes in transgenic potatos.…”