An analytical pre-breeding method for flavonoid screening in grapefruit
Van der Loo, Almari M. J.
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The citrus industry forms a major part of the agronomic and economic sectors of South Africa, and supports the livelihood of an estimated 1 million South Africans. The citrus industry has a long history in South Africa with the first exports of fruit dating back to 1907. Presently the South African industry consists of 120 commercial citrus varieties, of which grapefruit accounts for 11% of the total citrus orchards planted in the 2015/16 production season. Grapefruit are naturally rich in flavonoids, with naringin as the major flavonoid, best known for its distinct bitterness. Global breeding and selection programmes for new cultivars focus on increased yield, pest and disease resistance, and improved nutritional content. Citrus fruit quality is influenced by several genetic and environmental factors. The aim of this study was to provide the pre-breeding programme of the Agricultural Research Council (ARC) with a flavonoid screening technique for grapefruit germplasm, which takes into account factors such as variety differences, as well as the fruit location within the tree canopy. The physical and chemical traits as well as the naringin and naringenin content of three grapefruit varieties (Star Ruby, Sweetheart and Marsh) grown and harvested over two seasons (2015 - 2016 and 2016 - 2017), were evaluated. For determination of the naringin and naringenin content of the fruit juice, a high performance liquid chromatography (HPLC)-UV/Vis method was optimised, using the Accela 600 HPLC system with an Accela UV/Vis Detector. Successful resolution and retention times were achieved using an Accucore C18 (2.3 μm particle size, 50×3 mm i.d) column at 0.818 ml min-1 flow rate, with a gradient of acetonitrile:water at a constant temperature of 25°C. The method gave acceptable linearity for both naringin and naringenin with a R2 value of 0.999 in both cases. A limit of detection (LOD) and limit of quantification (LOQ) of 1.77 mg 100 ml-1, 5.35 mg 100 ml-1 for naringin and 0.23 mg 100 ml-1, 0.72 mg 100 ml-1 for naringenin was obtained. The inter and intra-day repeatability was determined by injecting the standard calibration solutions six times per day over three consecutive days obtaining a relative standard deviation (RSD) (%) consistently lower than 5%. There were significant varietal differences for fruit size, °brix, pH and naringin content due to season and canopy position. Fruit in different canopy positions varied in physical traits such as fruit mass, circumference, peel mass and segment mass. As for the chemical composition of the fruit, the northern quadrant fruit were highest in °brix content, and also had a high °brix:acid ratio. The effect of quadrant sampling on flavonoid content indicated no significant differences. The interactions between the physical and chemical traits were constant over both seasons. This study indicated that when screening fruit in a citrus breeding programme for new or improved flavonoid traits, such as the naringin content, the sampling quadrant does not seem to have an effect, but genetic differences and climatic differences between seasons would affect the naringin content. This study demonstrated that the sampling of fruit for determining naringin/naringenin content in grapefruit can be simplified, which is beneficial for screening of a large number of possible parents with regard to naringin/naringenin content in a breeding programme.