| Flavonoids are widely distributed diverse groups of plant secondary metabolites with important functions in protection against UV light damage, pigment production, pollen growth, male fertility, and plant-microbe signaling. Moreover, they contribute dietary health benefits to humans due to their antioxidant, anticancer, anti-inflammatory, and antithrombogenic properties. In citrus, flavanone glycosides are the main group of flavonoids that accumulate in entire plants; roots, leaves and fruits. However, their physiological roles) in citrus plants are not known. The presence of some of these compounds affects the taste of citrus fruits, rather than influencing their color. Flavanone rutinosides are tasteless, whereas flavanone neohesperidosides, for instance naringin, give a bitter taste to fruit and fruit juice products. Since consumers' food choice is driven by taste, the bitterness of naringin in citrus reduces the acceptability of fresh fruit and juice products in commercial markets and affects the consumers' having benefit of them. The main objective of this research is to manipulate the citrus flavanoid biosynthetic pathway in order to reduce bitter taste by altering the production of flavanone neohesperidosides. Molecular genetic and transformation techniques were applied to achieve this goal. In order to suppress the expression of the target gene, sense and antisense constructs containing cDNAs of chalcone synthase (CHS) and chalcone isomerase (CHI) genes, whose products catalyze the first two steps in the biosynthetic pathway, were exploited in grapefruit transformation experiments. The 1,2 rhamnosyl transferase (1,2 RT) gene, which catalyzes the last biochemical step in the formation of naringin, was also utilized for the genetic transformation of C. paradisi. A sense construct was used for overexpression and a hairpin-forming construct of this gene was employed to express dsRNA, which interferes with target gene expression. Transformation efficiency varied depending on gene transformation construct. All of the resulting transgenic plants were analyzed by HPLC for their flavonoid composition. A few transgenic plants showed lower naringin levels than the control. According to southern blot analysis most of the transgenic plants had one to three transgene copy numbers. Some of the plants that were significantly different based on statistical analyzes, were selected to analyze for their target gene transcript level. However, no direct correlation was observed between target gene transcript and naringin level. Our initial studies are with the grapefruit; however, the approach could be applied to other citrus types that have bitter tasting fruit. |
