The amylose - free potato mutant as a model plant to study gene expression and gene silencing

Abstract

In this thesis, gene-expression and gene silencing were examined for Granule Bound Starch Synthase (GBSS) which catalyses the formation of amylose and Branching Enzyme (BE) which catalyses the formation of amylopectin. The (GBSS) deficient, with iodine, red staining amylose-free (amf) potato mutant was used in order to facilitate the experiments; GBSS-gene expression and BE-gene silencing resulted in blue staining starch.A dosage effect of the wild-type GBSS-allele on GBSS-activity and amylose content was found in a tetraploid dosage population. The presence of amylose had a distinct influence on the physico-chemical properties of the starch.Insertion of the wild-type GBSS-gene in the amf -mutant resulted in fully and partially complemented plants. The minimum number of independently segregating active GBSS-inserts was estimated by genetic analysis after microspore staining. The complemented phenotype was normally transmitted to the F1 for the fully complemented, but not for the partially complemented plants. For one plant the partial complementation was correlated with the presence of a block of five inserts. This block was also capable of inhibiting the endogenous GBSS-gene in a wild- type. This indicates that partial complementation can be caused by co-suppression, besides low transgene expression. Co-suppression was also obtained when the full size (GBSS) sequence or (GBSS) cDNA were introduced in a wild-type potato.Introduction of the distal 1.5 kb cDNA coding for BE in both sense and antisense orientation resulted in several transgenic plants with a small blue core in these starch granules. This could indicate the presence of loosely branched amylopectin. in the core of the starch granules. The expression of the endogenous BE-gene was largely or fully inhibited as judged by the absence of BE mRNA and protein. This did not result in a measurable effect on the branching degree, but resulted in altered physico-chemical properties of the starch compared to amf -starch.It is concluded that this study shows that the amf -mutant is successfully used as a model plant to examine different aspects of gene expression