Antisense RNA mediated inhibition of granule - bound starch synthase gene expression in potato

<p>Potato starch and its derivatives are widely used in several fields of application. The manufacturing of most products requires the modification of native starch with respect to, for example, viscosity and physical stability. In addition to the currently used physical, chemical and biochemical derivatization and gelatinization of extracted starch, modification of the starch biosynthetic pathway <em>in planta</em> is regarded as a valuable approach for altering the quality and quantity of potato starch.<p>This thesis describes the application of antisense RNA technology for the modification of the composition of potato tuber starch. Starch consists of amylose and arnylopectin, and is predominantly synthesized in amyloplasts of tubers and seeds, where it is deposited in starch granules. Several enzymes are involved in the biosynthesis of starch, of which granule-bound starch synthase (GBSS) catalyses the formation of amylose. Generally, starch contains 20% of amylose and 80% of arnylopectin. However, mutants are known in which the ratio between amylose and arnylopectin has changed. An example is the amylose-free ( <em>amf</em> ) potato mutant, of which the starch exclusively contains arnylopectin due to a recessive mutation in the GBSS-gene. The suppression of the expression of specific genes can also be achieved by means of antisense inhibition, which is supposed to be based on the formation of an RNA-duplex between the target mRNA and the antisense RNA, thus blocking the transport of the mRNA from the nucleus.<p>To inhibit the (GBSS) gene expression, several antisense genes were introduced into the potato genome via <em>Agrobacterium</em> -mediated transformation. Among transgenic clones in which inhibition of (GBSS) gene expression was observed. two types could be discerned:<br/>A. Transgenic clones with completely inhibited (GBSS) gene expression. These clones lacked the (GBSS) protein, (GBSS) activity and amylose. The complete inhibition was demonstrated in microtubers, greenhouse grown tubers and field grown tubers, and was maintained in several tuber generations. The composition of tuber starch from these clones and from the <em>amf</em> -mutant was similar.<br/>B. Transgenic clones with incompletely inhibited (GBSS) gene expression. In these clones the (GBSS) protein content, (GBSS) activity and amylose content were reduced as compared to non-transformed clones. With respect to the extent of inhibition, variation was observed between transgenic clones which was supposed to result from position effects. Variation was also observed between tubers of individual plants and within individual tubers. This type of variation was suggested to be caused by factors related to tuber development.<p>Microscopic analysis of iodine stained tuber starch granules from transgenic clones showed that the reduced amount of amylose was not equally distributed throughout the entire granule, but was confined to the central core of the granule. Iodine-staining of starch granules yielded a blue core, in which the amylose content equalled that of wildtype starch, and a red, amylose-free, outer part. The size of the blue core was shown to be positively related to the amylose content, as determined in starch isolated from the transgenic clones. This finding led to the hypothesis that at a certain point in starch granule development the available amount of (GBSS) protein will become insufficient for the formation of starch with a normal amylose content. From this point onwards, amylose-free starch will be produced throughout further starch granule development.<p>With respect to the effectiveness of antisense inhibition, the influence of the composition of the antisense gene on the percentage of transgenic clones with complete or incomplete inhibition was studied. To this end, eleven antisense genes were constructed, based on the full length (GBSS) cDNA, the genomic (GBSS) coding region, and some cDNA- and genomic fragments. Furthermore, a comparison was made between the 35S CaMV promoter and the potato (GBSS) promoter. The extent of inhibition of GBSS gene expression was studied in at least 30 transgenic clones per antisense gene construct. In addition to construct-related differences, the number of T- DNA insertions was found to be positively related to the extent of antisense inhibition. It was concluded that the chance for complete inhibition of (GBSS) gene expression was highest in transgenic clones with three or more copies of the antisense gene based on the full length (GBSS) cDNA driven by the (GBSS) promoter.<p>In order to evaluate the applicability of the antisense (GBSS) system, two antisense genes based on the full length (GBSS) cDNA and driven by the (GBSS) or the 35S CaMV promoter were introduced into several potato cultivars via transformation with <em>A</em> . <em>tumefaciens.</em> The trangenic clones of one of the cultivars were analysed for the extent of antisense inhibition and its stability in a field trial based on in vitro plantlets. Complete inhibiton of (GBSS) gene expression, resulting in the formation of amylose-free starch, was demonstrated in mature tubers of four of these clones. Other characteristics, such as the total starch- and sugar content, the total yield and the dry matter content, did not deviate significantly as compared to the non-transformed cultivar. This led to the conclusion that antisense inhibition has good perspectives for the formation of amylose-free starch in potato tubers. In 1993, three of the transgenic clones with amylose-free tuber starch that are described in this thesis, were submitted to the Board for Plant Breeder's Rights for registration