The overexpression of the Or-gene in potato tubers

Vitamin A deficiency remains one of the major nutritional problems and affects an estimated 250 million people worldwide. Plant carotenoids are the primary dietary source of provitamin-A (β-carotene) and play a critical role in human nutrition and health. Potato is the third most important food crop in the world following wheat and rice. It represents a major proportion of daily calorie intake in many countries, with more than a billion people consuming potato tubers on a daily basis. Potato is therefore a key target crop where enhancing carotenoid concentration can have real impact on human health. Introducing a worthwhile trait such as the cauliflower Or-gene into potato is hypothesised to have a positive impact on vitamin-A status. The Or-gene was isolated from an orange cauliflower mutant and encodes a plastid-targeted protein containing a cysteine-rich zinc finger domain. The Or-gene controls carotenoid accumulation by inducing the formation of chromoplasts, which provide a metabolic sink to sequester and deposit carotenoids. This research focused on enhancing carotenoid accumulation in potato tubers by expressing the cauliflower Or-Wild transcript. Potato cultivars Iwa, Desiree, Summer Delight and Agria representing diverse genetic backgrounds and tuber colours ranging from white, cream and pale yellow through to yellow were used in this study. The Or-wild transcript under the control of the patatin promoter was constructed into the pMOA33 binary vector and transgenic potato plants have been produced by Agrobacterium-mediated transformation expressing the cauliflower Or-Wild transcript. The efficient transformation procedure using disarmed Agrobacterium strain EHA105 was applied to produce around 40 transgenic lines from each cultivar through independent transformation events. Stable integration and expression of the Or-Wild transgene in independently derived potato transgenic plants were confirmed through molecular analyses. Transformation efficiency analysis showed that the potato transformation protocol used is genotype-dependent. Of the four genotypes tested, Iwa and Summer Delight were highly responsive genotypes, whereas Agria and Desiree were less responsive. All the rooted transgenic plantlets were successfully acclimatized to a greenhouse. The majority were observed to have a phenotypically normal appearance. However, a range of off types was observed and attributed to somaclonal variation. These may have been due to the in vitro establishment techniques (medium constituents) or may have pre-existed from the sources of explants. The RT-PCR analysis of RNA from tubers of selected transgenic lines confirmed expression of Or-Wild gene in tubers. Total carotenoid concentrations were determined in tubers of the above selected transgenic lines and non-transgenic control. Among the controls, Agria, the yellow-fleshed cultivar, had the highest total carotenoid content (mean 0.134, SD 0.022 mg g⁻¹ DW), substantially higher than all other cultivars tested. The lowest total carotenoid concentration was found in Iwa, a white-fleshed cultivar (mean 0.009, SD 0.002 mg g⁻¹ DW). The total carotenoid content shows a positive relationship with the colour intensity of tuber flesh. The transgenic tubers carrying the Or-Wild transgene show markedly higher carotenoid levels compared with respective non-transgenic controls. The transgenic lines from cultivars Agria and Summer Delight show 2-3-fold higher total carotenoid content compared with controls. Whereas, the transgenic lines from cultivars Desiree and Iwa show 2-fold higher total carotenoid content compared with controls. Among the transgenic lines, Agria line -7 (mean 0.326, SD 0.055 mg g⁻¹ DW); Desiree line - 3 (mean 0.078, SD 0.018 mg g⁻¹ DW); Iwa line - 35 (mean 0.026, SD 0.002 mg g⁻¹ DW); Summer Delight line - 2 (mean 0.156, SD 0.017 mg g⁻¹ DW) produced substantially higher total carotenoid content than all other transgenic lines tested in each cultivar. The differences in total carotenoid content between lines may be due to different levels of expression of the same transgene in tubers. It is recognized that total carotenoid composition varies as a function of such as stage of maturity, cultivar, sample handling, and analytical method. Since the tubers were harvested well before its full maturity, the total carotenoid content reported in this study may be an approximation of the potential to be derived from the expression of the Or-Wild transgene function in those transgenic tubers. The Or-gene is hypothesised to control carotenoid accumulation by inducing the formation of chromoplasts which provide a metabolic sink to sequester and deposit carotenoids. The successful demonstration of increased carotenoid accumulation represents a promising strategy for maximally improving the nutritional quality of food crops. This research developed potato plant material, genetic and biochemical data that increased our understanding of carotenoid accumulation in potato tubers of selected cultivars and set essential infrastructure for future detailed studies in this field.