Abstract:
Anthocyanins are a group of important plant pigments, as they have reported health benefits, and are also responsible for generating marketable interest due to their novel colours, including orange (pelargonidin) red (cyanidin), blue (delphinidin) and purple (delphinidin and cyanidin derivatives). Out of these colours the most rarely seen is blue, which are mainly produced by delphinidin derivatives in the anthocyanin pathway. However, because delphinidin branches off the pathway, generally we see the two colours (cyanidin, red; delphinidin, blue) produced together to create a purple colour. The plants of interest to this thesis are kiwiberry (Actinidia sp.), which produce fruit with purple/blue pigmentation. They are relatively low production-wise in comparison with kiwifruit, so kiwiberries would benefit from a novel boost in their marketability to gain traction in the NZ economy. In this thesis I explore whether the downregulation of cyanidin coincides with an increase in delphinidin. This is done through the use of CRISPR-Cas9 guides which target the key cyanidin determining flavonoid 3’ hydroxylase (F3’H) genes of kiwiberry A. chinensis, A. arguta and A. eriantha. I also use N. tabacum as a model plant in which to test this hypothesis. The main findings of these experiments were a significant downregulation of anthocyanins in tobacco in both transient assays and stable transformation experiments. The most significant result was the change in cyanidin to delphinidin ratio, where in cells co-expressing the R2R3 MYB transcription factor, AcMYB110 the ratio was approximately 3:2, whereas in tobacco stably transformed with CRISPR-Cas9-F3’H guides this ratio changed to 1:3. Analogous transient fruit-flesh based assays were used, but further improvements of this technique are required. The results of these experiments will have implications for developing fruit with enhanced delphinidin levels, by downregulation of key biosynthetic genes in conjunction with upregulation of key transcription factors.