There were also differences among cultivars for photosynthetic assimilation, stomatal conductance, and transpiration at Riverside, but not at Somis, with ‘Haku Botan’ having significantly lower rates of assimilation, stomatal conductance and transpiration than ‘Wonderful’ at Riverside. No other differences for these physiological traits were detected. Because the main differences observed were in the Riverside, and included gs and E which describe water loss, examining water loss characteristics offers a promising direction for improving water-use efficiency during cultivar selection. The most interesting findings of this field study were that there were differences among cultivars and between sites for establishment rates, important physiological traits, such as A, E, gs, water potential, and precocity. These findings provide a glimpse into phenotypes of USDA-ARS NCGR cultivars and suggests there may be other cultivars in the national germplasm, or in other germplasm collections, that have even faster establishment rates, greater production efficiencies, and higher precocity than those represented in this study. This finding is important for not only to growers looking for cultivars that use less water, grow faster and have higher yields with less water for higher profits and more sustainable agricultural practices, but also for breeders who can use this information to make informed decisions regarding genotype selection for crosses. Hepaksoy et al. reported evidence that E and instantaneous water-use efficiency of pomegranate may be correlated with fruit split in some cultivars,container raspberries which means that those cultivars demonstrating differences for these physiological traits should be followed in the field to determine their effects on fruit split. The next step in this discovery of differences in physiological traits is to investigate why one cultivar is more efficient with water than another on a molecular level.
Literature regarding physiology of pomegranate trees is limited. The results of the present investigation are similar to investigations into physiological differences among pomegranate cultivars in other germplasm collections . Because information regarding precocity in pomegranate germplasm is limited, this study provides some insight to pomegranate reproductive biology in an experimental field trial setting. Water potential values agree with other investigations which included midday stem water potential , but data for predawn water potential are scant in the literature. These are the first known values for water potential in predawn hours for California-grown pomegranate. At Riverside, there were significant differences among cultivars for midday water potential, but not for predawn water potential. The low midday stem water potentials provide evidence that these trees were under water stress in the hot summer conditions of Riverside. These findings indicate that we are seeing differences in water uptake during the middle of the day, and that trees are rehydrated at night. We studied eleven unique pomegranate cultivars displaying very different phenotypes and found some interesting differences among them in terms of establishment rate. physiological traits, and precocity. Despite these differences, many of the cultivars investigated were determined to perform virtually the same as Wonderful in semi-arid and coastal climates, while others did not. Because we found differences among cultivars for various physiological traits relevant to water-use efficiency, the next steps would be to conduct more studies with more than four cultivars, and to also carry out these physiological and water relations measurements during different times of years and different times of day. It would also be important to investigate these cultivars on molecular, morphological or anatomical scales to determine the underlying causes of these differences among cultivars for breeding purposes. The pomegranate germplasm in the United States is highly diverse and is comprised of hundreds of cultivars sourced from domestic and international origins . Most pomegranate cultivars are in the public domain with the potential to be developed into commercial cultivars by breeders and made available to growers. Stover and Mercure indicated that cultivars with softer seeds or lower acidity could increase consumer demand for fresh fruit. The Wonderful cultivar, the industry standard in several countries, is a relatively tart, bitter, acidic, moderately hard-seeded fruit that has astringent juice compared to other cultivars previously analyzed from the collection at the United States Department of Agriculture – Agricultural Research Service National Clonal Germplasm Repository , Davis, CA .
Pomegranate cultivation in the United States is predominantly a monoculture of ‘Wonderful.’ Many of the 14 cultivars included in this study have been previously described to have one or more of the following traits: unique color, soft seeds, low acidity, or unique flavor . Many of these cultivars have had limited attention in the literature and have yet to be phenotyped for potential use by commercial growers, the food and beverage industries, or breeders. Since 2011, for unknown reasons, beverage companies began to only purchase ‘Wonderful’ juice from growers . This preference for Wonderful is likely due to industry demand for the red, sweet-tart juice of this cultivar. Additionally, since Wonderful juice was the primary cultivar selected for clinical trials testing the putative health effects of pomegranate juice on humans the reliance on this single cultivar has increased. To understand the factors influencing juice quality differences among pomegranate cultivars, analytical chemistry methods are used to evaluate the chemical composition and properties of their juices. The use of multiple methods to evaluate fruit juice allows for more robust quality analyses by overcoming the limitation of one method by employing another method with different strengths . Several methods exist to analyze the components and quality of fruit juices, including gas chromatography , liquid chromatography coupled with mass spectrometry , acid titration, refractometry , and spectrophotometry . While all of these methods are useful, they can be quite different in terms of sensitivity, selectivity and specificity. Additionally, several methods have disadvantages that include destruction of the sample, the inability to determine specific quantities or classes of metabolites, or the requirement of a separation of individual sample components prior to analysis. The ability of NMR to universally and rapidly detect and quantify organic compounds makes it an efficacious method for evaluating differences in chemical profiles among fruit juices from different cultivars without requiring a separation. In addition, NMR is advantageous because it accommodates relatively simple sample preparation, and allows compound identification and quantification without authentic standards . It has been demonstrated that NMR methodology is useful to the food industry for juice quality control including applications such as the detection of adulterants, quality control during mixture analysis of fruit juice products, and determination of juice authenticity .
The pomegranate juice industry in the United States has experienced high profile lawsuits and federal investigations involving both the false advertising of commercial juice composition and health benefits to humans through consumption . Therefore, for the pomegranate industry, it is important to be able to detect beverage adulterants, such as sucrose and high fructose corn syrup, and distinguish between the juices of different species and cultivars for economic, quality evaluation and public health reasons. Knowing differences in juice quality among cultivars is important because the cultivar is more influential in determining fruit juice composition than site of cultivation, year of harvest, or length of storage . According to Hasnaoui et al. ,draining pots citric acid is the chief determinant of sour flavor in pomegranate juice regardless of the sugar concentration. This means that if a juice has a high citric acid concentration, it will have a sour flavor whether or not its sugar concentration is relatively high. Sweet or “low acid” pomegranates typically have a citric acid concentration below 0.50% . Standards have been recommended for titratable acidity and total soluble solids of ‘Wonderful’ pomegranate. Because citric acid is the predominant organic acid in pomegranate juice, TA is expressed in citric acid equivalents. Minimum maturity of ‘Wonderful’ is reached when TA declines to 1.85 TA and growers in California, USA typically harvest fruit after reaching at least 15 °Brix . Maturity index is also a measure of maturity in fruit crops and is calculated as the ratio between °Brix and TA , and is used as a tool by growers to determine fruit maturity. The optimum MI for ‘Wonderful’ is greater than 8.1, at which point the fruit is considered ready to pick. The objectives of this research were 1) to compare fruit juice quality parameters and metabolites of promising preselected pomegranate cultivars from the USDA NCGR collection with the industry standard, Wonderful; 2) to identify cultivars in the USDA national collection that could serve as candidates to diversify the largely single cultivar pomegranate industry; 3) to gain a refined understanding of the nutritional composition and profiles of pomegranate juices utilizing advanced analytical chemistry tools among pomegranate cultivars conserved at the NCGR; and 4) to compare juice expressed from a diverse set of pomegranates to the commercial standard 100% ‘Wonderful’ pomegranate juice.Solvents, chemicals, and reagents used to measure total phenolics , antioxidant activity and TA of the pomegranate juice were of analytical grade. Solvents used were ethanol and methanol, manufactured by Acros Organics and Fisher Scientific , respectively. Chemicals used were gallic acid, manufactured by Acros Organics and 2,2-diphenyl-1-picrylhydrazyl, manufactured by EMD Chemicals . Reagents used were Folin-Ciocalteu reagent, manufactured by MP Biomedicals LLC , and sodium carbonate manufactured by Mallinckrodt . Potassium solution, from Sigma Aldrich was used for ion chromatography.
Deuterated solvents and 2,2-dimethyl-2-silapentane-5-sulfonate-d6 sodium salt from Cambridge Isotope laboratories were used as well. This research was conducted over two years with fruit sourced from pomegranate cultivars preselected for their fruit quality from the USDA-ARS NCGR for Tree Fruit and Nut Crops and Grapes in Davis, CA. The pomegranate cultivars analyzed were preselected based on fruit quality and consumer taste panel results and include: Al Sirin Nar, Ambrosia, Blaze, Desertnyi, Eversweet, Golden Globe, Green Globe, Haku Botan, Loffani, Parfianka, Phoenicia, Purple Heart, Sakerdze, and Wonderful . The cultivars in this study that have been described as soft-seeded include Desertnyi, Eversweet, and Parfianka. Low acidity cultivars include Ambrosia, Desertnyi, Eversweet, Golden Globe, Green Globe, and Loffani. Wonderful fresh fruit and commercial juice were included as a control and as the standard to compare the other cultivars in this study. Up to twelve fruits of each cultivar were harvested on 14 October 2014, 15 September 2015, and 15 October 2015. After phytosanitary inspection, fruits were wrapped in paper, shipped in shipping tape-sealed cardboard containers by ground and received within two days, and then stored at 6 °C and 98% relative humidity until processing. Fresh market quality fruit, as defined by being well-filled, mature, unblemished and greater than 250 g, were chosen for juice analysis from each of the 14cultivars. Two cultivars, Loffani and Green Globe were only available for one harvest date . There were two methods of juicing in this study: aril-pressed juice and peel-pressed juice. Peel-pressed juice is more common in the USA industry, but aril pressed commercial juice exists. For aril-pressed juice, fruits were halved and 100 undamaged arils were manually removed and placed in a polyethylene bag. These arils were pressed manually to express the juice directly into a 15 mL test tube; this juice type is called aril-pressed. Peel-pressed juice samples consisted of halved pomegranates that were pressed with peel and septa intact with a juice press exerting approximately 69 MPa. Juice samples from 2014 consisted of aril-pressed juices from a single fruit. Juice samples analyzed in 2015 utilized aril-pressed and peel-pressed juices and both juicing methods consisted of composite samples from multiple fruit per cultivar. Samples derived from both juicing methods were centrifuged at 1000 g for 5 min using a Becton Dickinson DYNAC Centrifuge and aliquots of the supernatants were used for all analyses. For methods utilizing spectrophotometry, supernatants were hundredfold diluted in 6:4 methanol:deionizied water as described below. Juice TA was measured with a Hanna HI 84532 fruit juice TA mini-titrator for samples prepared by mixing a centrifuged juice aliquot with 45 mL of deionized water. Results are expressed in citric acid equivalents as a percent acid in the juice sample. Both high and low acidity titrants were used to titrate high acid and low acid pomegranate juices, respectively. Juice samples were autotitrated with a OH- -based titrant solution to an endpoint pH of 8.1 . One sample was run per juice sample.The 1H spectra were recorded with a Bruker Avance NMR spectrometer operating at 599.58 MHz, using a 5 mm BBI probe. Concentrations of organic acids, sugars, and ethanol were determined using 1H NMR. For this analysis, the juice sample was centrifuged again for 10 min at 12,000 g to remove any particles formed during storage.