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Evaluation of genetic diversity and drought tolerance among thirty-three dichondra (Dichondra repens) genotypes

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  • Dichondra (Dichondra repens) is an important ground cover plant and is also used as a herbal medicine in China. Objectives of this study were to evaluate phenotypic and genetic diversities among 33 genotypes by using 18 simple sequence repeat (SSR) markers and to further identify the drought tolerance of these germplasms based on five physiological parameters. Results showed that natural variations in phenotypes including plant height, leaf area, leaf thickness, and petiole length were observed among 33 genotypes under well-watered conditions. All 18 SSR primer pairs were found to be polymorphic and significant genetic variation was found in these genotypes. In addition, there were obvious differences in leaf relative water content (RWC), electrolyte leakage (EL), chlorophyll (Chl) content, photochemical efficiency (Fv/Fm), and performance index on absorption basis (PIABS) among 33 genotypes in response to a prolonged period of drought stress (46 d). Drought tolerance of 33 genotypes was then ranked by using subordinate function value analysis (SFVA) and the most drought-tolerant or -sensitive genotypes were identified as Dr5 or Dr29, respectively. Principal component analysis (PCA) further classified 33 genotypes into group I (drought-tolerant), group II (drought-sensitive), and group III (medium types). Current findings showed that 18 selected SSR primers could be potentially used to analyze genetic diversity and varietal identification in dichondra species. Drought-tolerant wild dichondra resources provide a rich genetic base for breeding of new cultivars.
  • One of the traditional techniques for increasing value and reducing agricultural produce spoilage is drying. Where more expensive alternative storage methods are used, this is especially crucial[1]. Through the addition of one or more energy sources, moisture from a product is removed throughout the drying process[2,3]. The physicochemical characteristics of the fruit are changed by drying, which can improve the flavor and texture of specific foods like raisins and dates[2]. It lowers the product's water activity (aw), and when the aw value drops to less than 0.6, it inhibits the growth and spread of spoiling bacteria[4]. Drying also reduces product weight, which reduces packing, storage, and shipping costs and ensures off-seasonal production[5,6]. The demand for dried fruit is rising globally as people become more health conscious[7].

    Worldwide pomegranate production is steadily rising, although large post-harvest losses are also common, according to reports[8]. When fruit is unsuitable for standard processing methods due to fruit cracking or sunburn, drying is a great way to reduce post-harvest losses because it extends shelf life and can be utilized to reduce food waste[9]. Numerous products, such as pharmaceuticals, snacks, cereals, quick drinks, and other confectionary items, employ dried pomegranate arils[10]. Dried pomegranate arils, also known as anardana, are utilized both medicinally and culinarily in several regions of the world, including India[11]. Dried arils can therefore be quite useful as value-added items that generate revenue. According to research conducted in the Indian Ramban area, anardana trade accounts for at least 41% of all annual household income[11].

    There are many different drying techniques. The most popular are freeze-drying, hot air drying, vacuum drying, and solar drying. Each technique has pros and cons in terms of the final product's quality and how efficiently it uses energy. Pre-drying procedures are frequently used in conjunction with drying. Pretreatment enhances the drying rate, product quality, and energy efficiency of the drying process. Enzymes that cause enzymatic browning, which lowers the product quality, are rendered inactive by pretreatments[1214]. Sensory qualities like color, texture, taste, scent, microbiological activity, and general acceptability are among the criteria that determine the quality of dried pomegranate arils[15]. These elements are crucial because they can have a big impact on customer preferences and, if not taken into full account, can lead to financial losses[16,17].

    Several studies[12,18,19] have looked at the impact of drying and pretreatment techniques on the general quality of the completed product. However, the combined impact of pretreatment and drying on quality was not sufficiently investigated. Reviews on pomegranate fruit often discuss the fruit's chemistry, nutritive value, and pharmacology. Therefore, by evaluating, highlighting, and reflecting on recent studies on pomegranate aril drying, this review seeks to close these gaps. This review paper compared and contrasted several pretreatment and drying setups with an emphasis on product quality.

    Heat, mass, and momentum exchanges all occur simultaneously during the drying of fruit materials in a sophisticated cellular architecture of biological tissue[20,21]. The characteristics of the material that affect the drying process are intricately dependent on size, shape, porosity, moisture content, and time[22]. For instance, the initial moisture level and the bioactive chemicals in pomegranate arils can vary depending on the cultivar and fruit ripeness. The understanding, engineering, and management of the drying process are further complicated by the intrinsic diversity of biological materials[23,24]. The mass, heat, and momentum transfer events that happen during a typical drying process are shown in Fig. 1[25]. Conduction and convection are the most common heat transfer methods, but radiation is typically only employed for high-end items due to its expensive cost[1,26]. Diffusion, capillary action, and bulk flow are only a few of the processes that might transfer mass. These mass transfer mechanisms must adapt to the ongoing physical changes in the material that take place as it dries out[22,27].

    Figure 1.  A visual representation of the drying processes of solid materials.

    Although it is native to Iran, the pomegranate (Punica granatum L.) is widely distributed worldwide[28]. It belongs to the family Lythraceae and is a deciduous shrub. It is a versatile plant that can be found growing in both semi-arid and subtropical climates. Pomegranates, however, need hot summer temperatures to ripen[29]. Pomegranate fruit has a non-uniform round shape and a range of hues depending on the cultivar and fruit development stage, including yellow, green, pink, deep red, deep purple, and black[30,31]. An outsized calyx crowns the fruit. The leading producers worldwide are Peru, Australia, South Africa, and Chile in the southern hemisphere, and India, China, and Iran in the northern hemisphere[32].

    Pomegranate is a one-of-a-kind fruit with distinct edible seeds (arils) that must be extracted by hand (Fig. 2)[33]. An aril is made up of a seed and fleshy, moist tissue surrounding the seed. Color, sweetness, juice content, and hardness of arils vary depending on cultivar and fruit maturity[30,31]. While the arils can be eaten fresh, they can also be made into jams, jellies, coloring agents, juices, vitamins, and anardana (dried arils). They can also be mixed into yoghurts, biscuits, and cereals[34]. Fresh pomegranate arils can be kept at 7 °C for up to 14 d without losing much quality. Dried pomegranate aril has an extremely low perishability, with a potential shelf life of more than 14 weeks in ambient air[35].

    Figure 2.  A typical breakdown of the material balance throughout the drying process for pomegranate aril.

    As demonstrated in Table 1, pomegranate is high in ellagitannins, gallic acids, ferulic acids, anthocyanins, flavonoids, fiber, and minerals like vitamin C, calcium, and phosphorus. Pomegranates' phenolic components and high vitamin C content (Table 1) have attracted the interest of both researchers and consumers due to their health advantages[3638]. As a result of its strong antioxidant activity and nutritional benefits, pomegranate is considered a superfruit. Pomegranate can also be employed in cosmetics and pharmacology due to its phytochemical and antioxidant qualities[39]. Pomegranate fruit extract (PFE) has bioactive elements that have been found to inhibit or prevent various types and levels of cancer[40]. Punicalagic acid, ellagic acid, urolithin, and luteolin are the most important pomegranate components known to have anticarcinogenic characteristics[40,41]. Pomegranate fruit has also been linked to the prevention of diseases such as Alzheimer's, hypertension, and diabetes[42,43]. Pomegranate supplements may also help during or after exercise because they have the potential to speed up hard exercise recovery[44].

    Table 1.  The nutritional composition of 100 g of pomegranate arils.
    NutrientValueUnit
    Water77.9g
    Energy346kJ
    Protein1.67g
    Total lipid fat1.17g
    Ash0.53g
    Total dietary fiber4g
    Total sugar13.7g
    Calcium10mg
    Phosphorus36mg
    Magnesium12mg
    Iron0.3mg
    Potassium236mg
    Sodium3mg
    Zinc0.35mg
    Vitamin C10.2mg
    Vitamin K16.4µg
    Vitamin E0.6mg
    Vitamin B-60.075mg
    Total choline7.6mg
    Folate38µg
    Adapted from United States Department of Agriculture (Agricultural Research Service), FoodData Central[52].
     | Show Table
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    Pretreatment procedures are utilized to improve the drying process's effect on product quality characteristics such as color, flavor, appearance, and some physicochemical aspects[45,46]. Figure 3 depicts the most often used pretreatment procedures[46]. A product is immersed in a chemical solution prior to drying in chemical pretreatment. Physical pretreatments, on the other hand, necessitate a physical alteration of the product. When drying with heat, Maillard reactions might occur, resulting in an unpleasant color change[47]. As a result, pretreatment techniques are critical in many drying applications, including the drying of pomegranate arils[45,48,49]. There is evidence that pretreatment reduces the product's exposure to heat by reducing drying time[50,51].

    Figure 3.  A classification of the numerous pretreatment techniques utilized in the drying process for pomegranate aril.

    Soaking in acidic solutions involves immersing the product to be dried in a hot acidic solution for many minutes before drying. Pretreatment with an acidic solution keeps the product's color and speeds up the drying process. The acidic solution suppresses polyphenol oxidase enzyme activity, slowing the rate of enzymatic browning (Fig. 3). Furthermore, numerous investigations[5355] have documented the retention of nutrients such as vitamin C in acidic solution pretreatment samples. Some acid-sensitive components, on the other hand, can be destroyed or leached away. As a result, while employing this strategy, this effect must be considered. In a pomegranate aril drying research, arils prepared with 3% citric acid had the highest sensory acceptance[56]. Vardin & Yilmaz[57] conducted research on the combined effect of acid blanching and subsequent drying temperature. The authors blanched the arils in 0.1% citric solution for 2 min at 80 ± 2 °C followed by drying at 55, 65, or 75 °C and discovered that drying at 55 °C had the maximum antioxidant capacity[46]. Understanding the connection between soaking in acid (balancing in acid) and the subsequent drying temperature is required to carry out the operation correctly.

    This entails immersing products in an alkaline solution. Alkaline solutions act by dissolving the wax covering on the fruit's surface, removing resistance to moisture transfer and increasing drying rate[58]. As a result, this pretreatment accelerates the drying process. However, the usage of alkaline solutions raises food safety concerns because the residue might be harmful to one's health[46,59]. In addition, although acidic solutions retain vitamin C, alkaline solutions leach it out and destroy it[46]. Samples dipped in ethyl oleate for roughly one minute revealed a considerably reduced drying rate: a 26.9, 28.5, and 27.2% decrease in drying time at drying air temperatures of 55, 65, and 75 °C, respectively, than the control[60].

    The fruit is dipped into a hypertonic solution, such as salt or sugar solutions, in this approach (Fig. 3). Because of the osmotic pressure differential, the hypertonic solution causes water to diffuse out of the fruit tissue[61]. When compared to other drying processes, osmotically pretreated dried products have great rehydration capability and little losses in quality parameters such as color, appearance, and nutrients[62]. Madhushree et al.[63] discovered that osmotic pretreatment (in 50oBrix sugar syrup concentrations) dried arils had high color retention. This could be owing to the samples' reduced exposure to oxygen when immersed in the sucrose solution. A separate investigation on the osmotic pretreatment of pomegranate arils with a 65°Brix sucrose solution revealed a decrease in drying rate in hot air drying at 70 °C compared to untreated control samples[64]. The scientists attributed the longer drying time (lower drying rate) to the creation of a dense sucrose layer beneath the fruit's surface, which created an additional barrier to moisture transfer. To that aim, the osmotic solution concentration must be assessed because it can result in prolonged drying times.

    Gaseous or liquid sulphur solutions have been used as a food preservation method and as a pretreatment in food drying procedures. Typically, sulphur solutions are utilized for their browning properties, both enzymatic and non-enzymatic[65]. In addition, sulfur pretreatment is associated with high vitamin C and A retention after drying, as well as inhibition of spoilage-related microbial proliferation[66].

    More et al.[23] compared physical pretreatments to chemical pretreatments with 1% potassium metabisulphide on arils. It was discovered that arils prepared with potassium metabisulphide had superior nutritional quality as well as improved color, flavor, taste, and overall acceptability (Table 2)[23]. As a result, processing of pomegranate arils with sulfur solutions can result in high-quality dried goods. Despite their anti-browning, antibacterial, antifungal, and nutrient retention qualities, sulphites might be harmful to one's health if the recommended dosage or daily intake is exceeded[67,68]. Furthermore, while the sulfur solutions maintain vitamins A and C, they deplete vitamin B1[65].

    Table 2.  Key findings in pomegranate aril pretreatment and drying studies.
    Pretreatment methodPretreatmentDrying techniqueKey findingsReference
    BlanchingWater blanching at 90 and 100 °CHot air oven dryingBlanched samples had a shorter drying time.[24]
    Water blanching at 80 °CHot air oven dryerBlanched samples had higher phytonutrient retention than unblanched samples.[69]
    Blanching using 0.1% citric solution
    at 80 ± 2 °C
    Cabinet tray dryerDrying process was shorter for blanched samples and there was a higher rate of bioactive compounds.[57]
    Sulphuring1% potassium metabisulphideSolar drying
    Cabinet tray dryer
    Freeze dryer
    Fruit of cv. Ganesh 1% potassium metabisulphide was of the highest quality and the highest acceptance.[23]
    Blanching and SulphuringHot water blanching 85 °C and 0.2% potassium metabisulphateMechanical dryer
    Solar dryer
    Keeping quality of mechanically dried arils was higher than the solar-dried arils.[70]
    Steam blanching, potassium metabisulphide and 0.3% Sulphur fumigationCabinet tray dryerThe highest dried aril quality was obtained from the combination of steam blanching and 0.3% Sulphur fumigation.[71]
    Steam blanching, sulphuring at 0.3%Vacuum dryer
    Hot oven dryer
    Sun drying
    Poly-tent house drying
    Room drying
    Sun drying had the highest moisture content reduction and the highest overall acceptance.[72]
    Hot water blanching 85 °C,
    potassium metabisulphite varying
    from 0.25% to 1%
    Hot air oven dryerThe best treatment was blanching in hot water at 85 °C for 1 min and then dipping the arils in 0.25% potassium metabisulphite.[70]
    Steam blanching, sulphuringSun drying
    Cabinet dryer
    Blanching reduced drying time. Cabinet drying of blanched samples without sulphuring was considered optimum for anthocyanins.[73]
    Acidic solution2%, 3% and 4% citric acidCabinet tray dryer

    3% acidic treatment was found to be the most acceptable.[56]
    Microwave100 and 200 W.Hot air oven dryer200 W pretreatment resulted in minimum energy utilization and drying time.[74]
    100 and 200 WHot air oven dryer200 W had the highest drying rate.[75]
    Osmotic treatmentSugar syrup, freezing at minus 18 °COpen sun drying,
    Solar tunnel dryer,
    Cabinet tray dryer
    Osmotic treatment and cabinet tray dryer produced dried arils with better physicochemical and sensory qualities.[63]
    • 100% pomegranate juice
    • 50% pomegranate and 50% chokeberry juice
    • 50% pomegranate and 50% apple
    • 50% apple and 50% chokeberry
    • 75% apple and 25% chokeberry
    Freeze drying
    Convective pre-drying vacuum microwave finish drying
    Vacuum-drying and freeze drying
    Pomegranate and chokeberry concentrated juice improved the quality of the dried arils.[12]
    Sucrose solutionHot air oven dryingPretreatment increased the drying time of the samples.[64]
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    The dipping or soaking of a product in an alcoholic solution, usually ethanol, is known as alcoholic pretreatment. Ethanol dissolves the cell wall components, which increases moisture loss and thus the drying rate[76]. Several fruits, including melon (Cucumis melo L.) and apples (Malus domestica), have been pretreated in alcoholic solutions before drying[77,78]. However, no investigations on the pretreatment of pomegranate arils with alcohol were reported. This could be owing to the aril's waxy layer, which could impede permeability and hence the efficacy of the alcohol pretreatment[79].

    In this method, the fruit is dipped, submerged, or sprayed with a liquid solution that forms a thin coating layer on the product's surface and then dried (Fig. 3). According to studies, the use of edible coatings can help to retain the color, texture, and nutrient retention of dried items[80]. It is critical to note that the drying pace and dried product quality are affected by the coating thickness, drying method, and coating solution. Most of the research on edible coverings for pomegranate arils has focused on cold preservation and packing.

    Acoustic cavitation is utilized to rupture cell walls using ultrasound pretreatment[81]. According to one investigation on the effect of sonification on osmotic dehydration and subsequent air drying of pomegranate arils, ultrasonography caused a 2-fold and 2.7-fold increase in water loss[82]. The authors hypothesized that ultrasonic promoted cell wall disintegration and enhanced permeability. While using ultrasonic improved color quality, it also reduced anthocyanin content when compared to osmotically dehydrated samples[82].

    Blanching is a pretreatment technique that involves rapidly heating and then cooling a product that will be dried[45]. Blanching can be used to inactivate enzymes that potentially degrade product quality, such as polyphenol oxidase, peroxidase, and polygalacturonase[4]. Unwanted sensory traits in color, flavor, texture, and nutritional aspects are examples[4,83]. Blanching also improves cell membrane permeability, resulting in a faster drying rate[45]. Furthermore, blanching kills bacteria that might cause product spoiling[45,83]. As indicated in Fig. 3, there are several blanching processes, including hot water blanching and modern technologies such as microwave blanching and infrared blanching.

    Adetoro et al.[18] discovered that blanching pomegranate arils in hot water accelerated drying rates compared to unblanched samples. In a separate investigation, the authors found that blanching arils at 90 °C for 30 s and drying at 60 °C had higher total anthocyanin content and radical scavenging activity than blanching at 100 °C for 60 s and drying at 60 °C. In another work, Karaaslan et al.,[69] blanched arils in water at 80 °C for 2 min to investigate the effects of temperature and pretreatment on the arils. The authors discovered that while 75 °C had the fastest drying time, 55 °C had the maximum anthocyanin concentration, phenolic content, and antioxidant capacity. In other studies, pretreatment procedures are combined to produce high-quality dried fruit products.

    Singh et al.[73] conducted a study to evaluate the drying of pomegranate seeds under various drying conditions. They discovered that blanched samples dried faster and had higher acidity than sulphured samples. Furthermore, the anthocyanin concentration of blanched samples was higher than that of blanched and sulphured samples using the mechanical dryer. The authors hypothesized that the reduced anthocyanin concentration seen after sun drying was caused by the long drying hours in the sun. They also suggested that pomegranate arils be dried using blanching rather than sulphuring to achieve the maximum nutritional quality. Sharma et al.[70] investigated ideal methods for drying pomegranate arils by blanching them in hot water at 85 °C for one minute and then immersing them in a solution of potassium metabisulphite with concentrations ranging from 0.25% to 1% for two minutes (Table 2). The highest potassium metabisulphite content resulted in the lowest acidity.

    Thakur et al.[71] used steam blanching for 30 s and 0.3% sulphur fumigation for one hour to standardize pretreatments for dried arils from wild pomegranate. The authors discovered that cabinet drying outperforms solar drying and open sun drying. The solar dryer ranked second in terms of sensory characteristics like texture, taste, and general acceptability. Sun-dried pomegranate arils, on the other hand, exhibited the highest reduction in moisture content and overall acceptance when compared to vacuum drying, hot air oven drying, polytent drying, and room temperature drying, according to Bakshi et al.[72].

    The effect of pulsed electric field treatment on the behavior of microwave-assisted hot air drying of pomegranate arils was examined by Amiali et al.[84]. When compared to drying at 70 °C, they found that pulsed electric field treatment was only advantageous when the subsequent drying process was done at the lowest temperature (50 °C). The lower the temperature, the higher the overall phenolic concentration. Arils treated with a pulsed electric field had a 21.02% higher total phenolic content than untreated arils.

    Various pretreatment procedures on pomegranate arils have been utilized with the goal of conserving physicochemical, physical, and chemical properties or enhancing drying rates. However, pretreatment standardization is currently restricted and underexplored. Table 2 lists some of the methods for preparing dried pomegranate arils.

    Despite the fact that pomegranate is a fruit with numerous health and nutritional benefits, it is now a modest crop with limited marketability. The difficulty in collecting the interior edible seeds (arils) is the greatest impediment to realizing the full potential of this unusual fruit[29,31]. Only manual extraction of pomegranate arils for laboratory scale testing is described in the literature.

    Pomegranates are first cleaned and sorted for uniformity in color, size, shape, and weight before aril extraction[18,85]. All pomegranates should be washed. To avoid introducing bacteria into the arils when the fruit is sliced open, excess water from the fruit surface is dried before cutting[86]. Following that, the fruit is cut along the ridges and the segments are gently pulled apart to form a flower-like structure. The deconstructed pomegranate is then flipped over a bowl of water and gently tapped with a wooden spoon on the skin side. As a result, the arils will begin to come out without being broken. Once all arils have dropped out, the white membranes are skimmed off the surface of the water as it floats, the water is drained, the arils are separated, and the surplus water is gently patted off with a towel. This method involves cutting the pomegranate with a knife, which results in a loss of more than 30% of the arils owing to mechanical damage[86]. As a result, a better approach was required, such as the machinery developed by Schmilovitch et al.[87], which allows opening the fruit without cutting, extracting the arils with minimal damage, separating the arils from extraneous materials, and delivering clean arils to a packaging machine. This technology could be used to produce dried pomegranate on a greater scale.

    The drying time is determined by the pretreatment process, kind, and technique of dehydration used. The dehydration methods that will be investigated in this study are low-temperature drying and high-temperature drying.

    Low-temperature drying methods employ temperatures ranging from subzero to 50 °C[49,88]. These drying processes are time-consuming and are usually utilized for temperature-sensitive goods like herbs. Furthermore, low-temperature drying procedures reduce the risk of scorching the fruit, protecting heat-sensitive components such as vitamin C[89]. Freeze drying, vacuum drying, and sun drying are all low-temperature drying procedures[90]. Pomegranate arils dried using low-temperature procedures such as freeze drying and vacuum drying have a more acceptable look and nutrient composition than most high-temperature drying methods[12,91].

    Freeze-drying (FD) is a low-temperature technology frequently used for drying food samples for high-quality or heat-sensitive products[92]. It is also recognized as one of the most expensive, time-consuming, and energy-intensive procedures in the food industry[93]. It entails removing moisture from food ingredients under low temperature and high vacuum via ice sublimation[48]. Because the product is frequently frozen, it is also known as sublimation drying.

    Adetoro et al.[94] freeze-dried fresh pomegranate arils at a freezer temperature of −80 °C for 96 h to examine the effect of drying procedures on pomegranate arils. The researchers discovered that color, total phenolic compounds (TPC), total anthocyanin content (TAC), and radical-scavenging activity stability differed significantly from the hot air-drying procedure (Table 3). More et al.[23] investigated the effect of drying procedures on the quality of dried pomegranate arils from three varieties. The authors discovered that FD produced the greatest results in terms of color, flavor, taste, and nutritional factors across all cultivars. However, FD stood out due to its prolonged drying duration, 24−48 h, when compared to solar drying (17 h) and hot air drying (10 h). A study on the influence of freeze-drying on the color attributes of 'Assiuty' pomegranate arils revealed that FD had the best color attributes (L* value of 46.50 ± 4.4 and a* value of 13.97 ± 1.23)[95]. Gölükcü[91] discovered that the FD had the maximum phenolic matter content (5580 mg/kg), followed by vacuum, convective, and sun-dried pomegranate arils (Table 3). Caln-Sánchez et al.[96] investigated the chemical composition, antioxidant capability, and sensory quality of pomegranate arils and rind after exposure to FD. The investigators found that FD pomegranate arils retained the most sensory characteristics and punicalagin content. Similarly, Cano-Lamadrid et al.[12] discovered the best sensory profile and sweetness in FD pomegranate arils at 65 Pa for 24 h at −60 °C. The drying kinetics, total bioactive content, in-vitro bio accessibility of bioactive compounds, and color and microstructural features of pomegranate arils were also studied[97]. When compared to alternative drying methods, the FD was shown to be the best approach in terms of final product quality and has been highly recommended by multiple reviewers[48]. FD arils have been demonstrated to have a higher bioactive chemical content, less shrinking, and excellent color quality. The FD for pomegranate arils has a disadvantage in terms of bioactive chemical recovery when compared to other methods, as well as extensive drying times[97]. Furthermore, FD is costly due to high energy consumption and initial investment expenses[98].

    Table 3.  A summary of the many techniques for drying pomegranate arils.
    CultivarDrying methodDrying
    time (h)
    Drying conditionInitial moisture
    content (%)
    Final moisture
    content (%)
    Key findingsReference
    WonderfulFreeze dryer96−80 °C
    5,999.1 Pa
    74.7 (w.b.)FD showed a higher color shift (19.6% ± 2,77%) at week 4 compared to hot air drying at week 0.[94]
    Ganesh, Bhagwa and AraktaFreeze dryer24−48−45 °C79.9 (w.b.)
    80.5 (w.b.)
    78.9 (w.b.)
    9.65−9.9 (w.b.)
    9.8−10.2 (w.b.)
    9.8−9.9 (w.b.)
    Arakta pre-treated with 1% potassium metabisulphide had the highest ascorbic acid concentration (6.81 ± 0.07 mg 100 g−1).[23]
    AssiutyFreeze dryer36−70 °CTA (18.80, 2.80 mg 100 g−1), TP (608.09a, 41.26 mg 100 g−1), DPPH (68.91, 0.72%), and ABTS (2,956.59c, 120 mol trolox
    100 g−1) were all higher in frozen pomegranate arils than in freeze-dried arils.
    [95]
    Mollar de Elche24−60 °C, 65 Pa,81.5 (w.b.)FD showed higher anthocyanin content (646 mg kg−1) compared to osmotic drying and conventional drying.[12]
    Hicaznar36−20 °C, 100 Pa for
    12 h
    −70 °C, 0.26 Pa
    76.96 (w.b.)10 (w.b.)Arils dried using FD had the highest magnesium content (96.17 ± 6.95 mg kg−1), manganese content (0.96 ± 0.05 mg kg−1) and zinc content (3.93 ± 0.07 mg kg−1) compared to sun drying, hot air drying and vacuum drying.[91]
    KebenFreeze dryer57−55 °C77.6 ± 1 (d.b.)20 ± 1 (d.b.)FD had lower bioactive recovery during in-vitro gastrointestinal digestion (TPC of 2.92%, ABTS of 6.12% and CUPRAC of 38.85%) compared to vacuum drying, hot air drying and ultrasound-assisted vacuum drying.[97]
    KebenVacuum dryer10.855 °C77.6 ± 1 (d.b.)20 ± 1 (d.b.)Vacuum drying had the highest bio accessibility recovery of bioactive compounds at 10.32% compared to HAD, FD and ultrasound-assisted vacuum drying.[97]
    HicaznarVacuum dryer3.7
    4.6
    7.8
    75 °C
    65 °C
    55 °C
    85,000 Pa
    78.1 ± 0.2 (w.b.)16 (w.b.)Drying temperatures of 75°C resulted in higher degradation of anthocyanins, phenolic compounds and antioxidant capacity (20.0%, 51.0%, 29.7% ± 0.28% respectively).[99]
    HicaznarVacuum dryer2455 °C
    3 500 Pa
    76.96 (w.b.)10 (w.b.)FD had the highest quality attributes such as TAC-1288.73 mg/kg) compared to VD and HAD however, due to physical changes that were undesirable physical changes, VD and HAD are recommended for pomegranate aril drying.[91]
    Wild pomegranates (cultivar- unspecified)Vacuum dryer1342 ± 2 °CVD had a higher sensorial overall acceptance (17.1) compared to sun drying (16.2) and room temperature (12.6).[72]
    BasseinSun drying1715.73
    (unspecified)
    Drying rate of pomegranate arils is affected by tray load and the recommended tray load is 1.25 kg m−2[73]
    Wild pomegranates (cv. Unspecified)Solar poly-tunnel14014.9−28.4 °C
    48.5%−74% -RH
    0.639−0.944 -wind speed
    Arils dried in the solar poly-tunnel had higher ascorbic acid, anthocyanins, and phenol content, 12.7 mg 100 g−2, 28.12 mg 100 g−2, and 108.60 mg 100 g−2 respectively than open sun drying.[100]
    SefriIndirect solar dryer75
    10
    6
    4
    40 °C
    50 °C
    60 °C
    75 °C
    78 ± 0.1(w.b.)The optimal water activity for drying and storing arils is 0.3684 ± 0.03.[101]
    Wild pomegranates
    (cv. Unspecified)
    Solar tunnel30−45 °CArils from the Karsog location had the highest TSS, sugars, anthocyanin, total phenols and antioxidant activity[102]
    Microwave2.3
    1.3
    0.7
    270 W
    450 W
    630 W
    70.25 ± 0.5(w.b.)10(d.b.)Color changes increased from 6.77−13.11 with an increase in microwave power from 270−630 W and were lower compared to arils dried using HAD.[85]
    HicazMicrowave1.2
    0.6
    0.4
    210 W
    350 W
    490 W
    23.93 ± 1.4 (unspecified)22.2
    (unspecified)
    Based on quality parameters, a microwave drying power of 350 W was recommended for drying pomegranate arils.
    Sweet acid
    (cv. Unspecified)
    Infrared4.3
    2.2
    1.6
    50 °C
    60 °C
    70 °C
    78 ± 0.2 (w.b.)9 ± 0.2 (d.b.)
    22.2
    (unspecified)
    Drying time for infrared drying was less than for HAD.[103]
    HicazHAD2450, 60, 70 °C at
    1.0 m/s air velocity
    To obtain better dried aril quality, 60 °C was recommended for drying pomegranate arils.[104]
    Wild pomegranates
    (cv. Unspecified)
    HAD16.542 ± 2 °CSun drying resulted in a maximum loss of moisture compared to VD, HAD, poly-tent house drying and room drying.[72]
    Wild pomegranates
    (cv. Unspecified)
    HAD1062 ± 2 °CHAD achieved the highest total soluble solids (39.6°Brix) and drying rate compared to solar drying and open sun drying.[105]
    KebenHAD555 °C; with 1.3 m s−1 constant air velocity77.6 ± 1 (d.b.)20 ± 1 (d.b.)In comparison to hot air oven drying, ultrasound-assisted vacuum drying and freeze-drying have higher quality characteristics.[97]
    Bassein seedlessHAD5−660 ± 5 °C. Airflow in
    the dryer was 1.2−1.8 m s−1.
    8.98 ± 0.091For the finest preparation of anardana, blanched samples (with sulphur) should be dried in a cabinet.[73]
    Mollar de ElcheVacuum-
    microwave
    240, 360, 480 W and pressure ranging
    from 4,000−6,000 Pa
    80.4 (w.b.)Arils dried using vacuum microwave drying at 240 W had the highest sensorial scores for odour and aroma at 3.1 and 5.6 respectively.[106]
     | Show Table
    DownLoad: CSV

    Vacuum drying (VD) is the process of subjecting items to low pressure in a vacuum. Because of the low pressure, water has a lower boiling point, allowing samples to be dried at low temperatures. As a result, VD is appropriate for heat and/or oxygen-sensitive items. During VD, heat transmission can occur by conduction, radiation, or microwave energy. VD is distinguished by faster drying times when compared to FD, and the products are not initially frozen as necessary for FD[107]. This low-temperature operation, combined with the elimination of oxygen during vacuum drying, allows nutrients and bioactive components such as phenolic compounds and vitamins to be retained[108,109].

    Ozay-Arancioglu et al.[97] investigated the influence of drying methods on dried pomegranate arils by comparing four distinct drying techniques: FD, VD, ultra-assisted vacuum drying, and hot air drying. They discovered that VD had better antioxidant capacity values than the samples tested for ABTS following FD. According to Gölükcü[91], VD is second only to FD as the finest choice for producing dried pomegranate (Hicazar) arils. Another study found that arils dried at 55 °C had higher phytonutrient levels than those dried at 65 and 75 °C under vacuum conditions[69]. When compared to other drying procedures, vacuum drying produces products with higher levels of phytochemical components. However, drying times range from 7.8 to 24 h at 55 °C, contributing to high costs, and products can only be dried in batches[91,99,110].

    Sun drying is one of the oldest and most used methods of drying. Sun drying is a low-cost, renewable energy-based drying process. In a nutshell, products are laid out on a flat area where they can be fully exposed to the sun for as long as possible. Because the drying process is dependent on solar radiation, the temperature is low and the drying process can be lengthy, taking approximately 15 d for pomegranate arils[60]. Furthermore, exposure to light and oxygen can lead to decreased preservation of substances like vitamin C. Furthermore, solar drying is an uncontrolled process with substantial risks of pest contamination, dust exposure, and product remoistening at night. To increase safety, solar dryers and solar tunnels are proposed to reduce pest and dust contamination[111]. Solar dryers use a contained environment comprised of a transparent or opaque cover, resulting in either direct or indirect drying[112]. The indirect drier system captures solar heat and transfers it to the product drying chamber via a second solar collector. A solar tunnel dryer is typically large in size and has a clear cover (Fig. 4). To regulate drying conditions such as temperature and relative humidity within the tunnel, solar tunnels often require a forced convection facility. A solar tunnel dryer may also include a solar air heater[111]. Solar dryers have the potential to boost drying temperatures, resulting in a quicker drying time[113].

    Figure 4.  This image depicts a sample of wild pomegranate arils being dried in a solar tunnel drier. Reprinted from Thakur et al.[102].

    In a comparison of hot air drying (60 °C), solar tunnel drying, and sun drying by Madushree et al.[63], hot air-dried arils were shown to have the highest quality. However, of all the drying techniques, solar drying had the greatest L* values (lightness), a desired quality. This was due to the comparatively low temperature of sun drying. Additionally, Bakshi et al.[72] discovered that when compared to vacuum drying, oven drying (42 ± 2 °C), and room drying (23 ± 2 °C), sun dried arils had the highest sensory overall acceptance and the lowest moisture content. In their comparative analysis of drying techniques, Singh et al.[73] discovered that hot air-dried pomegranate arils had the greatest anthocyanin and acidity contents. But in hot-air oven-dried samples, undesirable non-enzymatic browning was most pronounced. Sharma & Thakur[100] demonstrated that the quality of arils dried in solar polytunnels was superior to that of arils dried in the open sun (Fig. 4). The ascorbic acid, anthocyanins, and phenols were found to be significantly greater in the sun polytunnel dried arils, according to the authors. They also received superior sensory ratings for color, texture, taste, and acceptability.

    Temperatures above 50 °C are used in high-temperature drying processes[88]. These drying processes are energy intensive, have large operating expenses, and so are costly. These technologies rely on fossil fuels, which pollute the environment where they are generated and utilized, and their continued usage is seriously harming our environment[114]. The drying mechanism is designed such that there is a controlled direct or indirect heat transmission to the product, leading in moisture elimination. These drying procedures may not be suited for some foods because they may induce nutritional breakdown[115]. Furthermore, high temperatures might cause product shrinkage and distortion. Hot-air drying ovens, steam drying, heat pump drying, and spray drying are all examples of high-temperature drying processes.

    Using forced convection, hot air oven drying (HAD) eliminates moisture from materials. Objects dry out through evaporation when hot air is forced through and around the substance. As a result, the dried product's flavor, color, nutrients, and ability to rehydrate may alter unintentionally[97,104,106]. The HAD techniques were shown to have a comparatively high total color change by Ozay-Arancioglu et al.[97]. As shown in Fig. 5, hot air-dried arils were darker than freeze-dried and vacuum-dried arils.

    Figure 5.  Illustration of dried pomegranate arils that have been dried using different methods. (a) Freeze drying, (b) vacuum drying, and (c) hot air drying. Adapted from Ozay-Arancioglu et al.[97].

    Başlar et al.[99] prepared dried aril samples using the hot air-drying process and subjected them to various quality assessments. Fresh aril samples were dried at three different temperatures (55, 65, and 75 °C). According to the authors' findings, high temperatures and short drying times are optimal for retaining valuable food biocomponents. However, whereas bioactive chemical losses increased over time, they degraded faster at higher temperatures. The antioxidant activity, on the other hand, decreased with drying time and was unaffected by drying temperatures. Horuz & Maskan[104] investigated the effect of hot air drying on pomegranate aril cv. Hicaz at three different drying temperatures and compared quality metrics such color, shrinkage, rehydration capacity, and drying time (Table 3). The authors suggested 60 °C for pomegranate aril HAD. The authors also discovered that shrinkage was greater in HAD than in microwave drying. In a second study, researchers discovered that the optimal drying temperature for retaining bioactive chemicals when drying pomegranate arils (cv. Hicaznar) in a hot air dryer was 65 °C[116]. When compared to the sun drying method for anardana made from wild pomegranate, Bhat et al.[105] discovered HAD dried arils with maximum acidity of 13.72%, phenols of 110.7 mg per 100 g, total sugars (24.2%), and reducing sugars (21.2%).

    However, Bakshi et al.[72] carried out a study with lower temperatures in which they studied the influence of different drying processes on the moisture content of dried pomegranate aril (cv. Wild). Lower temperatures were employed to gain insight into the quality of the dried product when compared to the low temperature drying methods used in the study, such as sun drying, poly tent house drying, room drying, and VD. When compared to alternative drying methods, the authors discovered that HAD (42 ± 2 °C ) for 16.5 h and drying in room at normal air (23 ± 2 °C ) for 10−12 d produced in the greatest loss of moisture from fresh arils of wild pomegranate (75.12%).

    Singh et al.[73] evaluated the influence of different drying conditions on the quality of dried pomegranate arils (Bassien Seedless) samples (Table 3). The scientists discovered that sun-drying preserved more MC while drying was faster with a HAD dryer and generally recommended it as a better strategy for preparing dried pomegranate arils.

    HAD drying of pomegranate arils is a standard drying procedure that can be utilized in commercial settings. Although HAD does not generate high-quality goods like FD, it does provide better TSS, TA, and antioxidant capacity stability. Furthermore, although having a higher rate of bioactive component degradation, higher temperatures may result in higher retention compared to approaches such as solar drying due to the short drying times.

    Electrical current is passed through the pomegranate aril during electric drying techniques including ohmic heating. The intrinsic resistance of the aril induces internal heating as the electrical current flows through it[117,118]. Ohmic heating is typically employed for liquid, viscous, and particle-containing foods[119]. Regardless of the meal's densities, food products prepared using this approach are heated quickly and uniformly[120].

    Dielectric techniques, on the other hand, use electromagnetic waves to directly produce heat inside the product, such as microwave, radio frequency drying, and infrared radiation[121]. Dielectric heating results from the conversion of electromagnetic energy to kinetic energy by dipolar molecules oscillating in accordance with the rapidly oscillating electric field[122,123]. Compared to traditional methods like hot air drying, dielectric technologies dry materials more quickly[124]. Additionally, the items are of a higher caliber than those produced by traditional drying techniques.

    Microwave drying (MD) is one of the emerging drying technologies. Unlike other techniques, MD utilizes volumetric heating to rapidly dehydrate the sample material[104]. Some studies[85,104] have indicated that arils desiccated at 150 W microwave power and 58 bar (abs) pressure produced the highest quality arils. In another study, microwave power of 80 W and vacuum pressure of 60 mm Hg provided the highest drying efficiency and qualitative attributes, including color and texture[125]. Horuz & Maskan[104] observed that microwave-dried pomegranate samples had lower levels of shrinkage and bulk density than hot air-dried samples. The authors also noted that microwave drying caused a greater loss of color in terms of total color difference (E) compared to air drying. It was observed that microwave-dried samples had a brownish hue.

    Drying with MD reduces drying time, but essential quality parameters, such as color, are sacrificed. Since the product is directly heated, the lack of heating uniformity during MD, which is difficult to control and could contribute to product burning has been cited as a disadvantage[126].

    Infrared drying is an effective technique of drying in which the product is heated directly without the use of air as the drying medium. In a comparative study of drying methods (hot air drying and infrared drying) to dry pomegranate arils, the authors discovered that infrared drying effectively dried pomegranate arils and that the polyphenol content in arils dried using infrared drying was higher at 50 and 60 °C than in arils dried using hot air drying at similar temperatures[103]. Therefore, pomegranate arils can be infrared-dried at 50 °C for optimal nutrient retention[103]. In contrast, a distinct study dried pomegranate arils under near-infrared vacuum conditions and found that drying at 60 °C and 20 kPa air pressure resulted in optimal colour retention and shrinkage[127].

    Although infrared drying is a rapid drying method, it is challenging to control due to parameters such as infrared intensity and radiation distance, and its energy consumption is unpredictable[26].

    Drying kinetics is the study of how factors that influence the removal of moisture from products during a drying process interact[110]. The drying kinetics of a substance is dependent on its thermal and mass transport properties. Understanding drying kinetics relates to process variables, and therefore aids in identifying suitable drying methods and controlling drying processes[57,96,116]. For optimal operating conditions, drying kinetics can be used to estimate drying time, energy requirements, and drying efficiency[2]. Due to the complexity of the drying phenomenon, however, mathematical models describing the drying kinetic of biological tissues are devised based on the time history of the moisture ratio from a controlled drying experiment[128]. There are numerous mathematical models, but Table 4 provides a summary of the most used models.

    Table 4.  A summary of the mathematical equations that are most commonly used to model the drying kinetics of pomegranate arils is shown here.
    Model nameModel expressionReference
    PageMR = exp(-ktn)[129]
    NewtonMR = exp(-kt)[130]
    Henderson and PabisMR = aexp(-kt)[131]
    Midili et alMR = aexp(-ktn) + bt[132]
    Wang and SinghMR = 1 + at + btn[133]
    Two TermMR = (aexp(-k0 t) + bexp(-k1 t))[134]
    LogarithmicMR = aexp(-kt) + c[135]
     | Show Table
    DownLoad: CSV

    The most common models that best describe the hot air drying of pomegranate arils include the Logarithmic, Midili and Page models[24,99]. Baslar et al.[99] found the Logarithmic model as the best in describing hot air drying of arils at 55, 65 and 75 °C. In another study, it was found the Sigmoid model describing the kinetics of hot air drying of pomegranate arils at similar drying temperatures[116].

    In their study on the infrared drying of pomegranate arils, Briki et al.[103] discovered that the Midili model was the most accurate representation of the drying kinetics. In a similar manner, it was discovered that the Midili model provided the best fit to the experimental data for drying using a combination of infrared and hot air[136]. Another investigation indicated that the Aghbashlo model provided the greatest fit for the data obtained from near-infrared vacuum drying at a temperature of 60 °C[127].

    Based on measurement data from three pomegranate cultivars (cvs. 'Acco', 'Herkaswitz', and 'Wonderful') at 60 °C, Adetoro et al. demonstrated cultivar as another influencing factor in selecting an optimal model. While the drying data of the blanched samples of all the cultivars in this investigation were best fit by the Logarithmic model, the unblanched samples of 'Acco' and 'Herkaswitz' and 'Wonderful' were best fitted by the Page and Midili models, respectively[24].

    The bioactive chemicals retained and drying periods may be affected by the pretreatment process, although frequently the drying kinetics of both untreated and pretreated samples may be described by the same model. The Page and Modified Page were determined to be the best models to fit the drying data of both blanched and unblanched pomegranate arils under vacuum air drying by Karaslaan et al.[69]. In a different investigation, the Page and Modified Page were shown to be the model that best suited the drying data of pomegranate arils that had been bathed in citric acid and dried by hot air[57]. Although the drying rate of pre-treated samples is higher than that of untreated samples, the scientists noted that the same models were found to best reflect the drying kinetics. The Page, Logarithmic, and Midili drying models are the most popular and effective drying models for pomegranate arils employing HAD, MD, and VD. The mathematical models that were utilized to explain the drying kinetics of pomegranate arils are summarized in Table 5.

    Table 5.  A synopsis of the results of mathematical modeling of the kinetics of drying pomegranate arils.
    CultivarDrying methodDrying parametersPretreatmentSuitable drying modelReference
    cv. HicazHAD55, 65, 75 °C0.1% citric acidPage and Modified Page[57]
    cv. HicaznarHAD55, 65, 75 °CSigmoid[116]
    HAD55, 55, 60 °CPage[137]
    HAD50, 60, 70 °CPage[85]
    MD270, 450, and 630 WPage[85]
    cv. HicaznarVD55, 65, 75 °CHot water blanchingPage and Modified page[69]
    HAD45, 50, 55, 60, 65, and 70 °CMicrowaveMidili[75]
    cvs. Acco, Herskawitz and WonderfulHAD60 °CHot water blanching (at 90 and 100 °C, each for 30 s
    and 60 s)
    Logarithmic, Page,
    Midili for unblanched arils
    Midili and Page for blanched
    [24]
     | Show Table
    DownLoad: CSV

    The drying procedure aids in reducing bacterial growth, which can result in reducing spoilage. However, while food is being dried, other changes may occur that degrade its quality. To determine the product's expiration life, chemical, physical, physicochemical, and microbiological changes are monitored[138,139]. These modifications are affected by stowage, environment, and packaging methods. The most significant factor affecting the integrity of stored food products is temperature[140]. Consequently, most shelf-life experiments are designed to evaluate the temperature-time history in relation to changes in product quality[138,141]. To accurately evaluate quality changes and safety, shelf-life evaluations should ideally simulate actual storage conditions[142]. In the case of desiccated goods, the actual storage period is lengthy, and the evaluation of shelf-life can become time-consuming and expensive. When the actual storage time is lengthy for practical reasons, an accelerated shelf-life test or analysis of the worst-case scenario is employed[142]. Ordinarily, the end of shelf life is determined by relevant food legislation, guidelines issued by enforcement authorities or agencies, guidelines issued by independent professional organizations, current industrial best practices, self-imposed end-point assessment, and market data[142].

    Appropriate packaging material can help to reduce quality losses. The packaging and shelf-life tests on dried pomegranate arils are summarized in Table 6. Sharma et al.[70] examined the packaging of dried pomegranate arils with high-density polyethylene (HDPE), low-density polyethylene (LDPE), and polypropylene (PP) films. For 12 months, samples were held at 7 °C and ambient (14−39 °C). They found that HDPE retained the most color, total tannins, and acidity while gaining the least moisture. The authors proposed a safe storage time of 6 and 9 months for HDPE packed dried pomegranate arils under ambient and refrigerated conditions, respectively.

    Table 6.  A list of the several types of containers used to store dried pomegranate arils.
    Drying methodDrying temperaturePackaging materialStorage period (months)Shelf-life performanceReference
    MC drier
    Solar cabinet drier
    Open sun
    62−64 °C
    50−55 °C
    18−24 °C
    Aluminum laminated polyethylene pouch, polyethylene pouches and thermoform trays.6Aluminum laminated polyethylene pouches were best for packaging.[105]
    Microwave-vacuum drying38 °CHigh-density polypropylene (HDPP) and aluminum laminated polyethylene (ALP).3−6Pomegranate arils stored showed that ALP is more protective than HDPP.[35]
    Solar tunnel30−45 °CGunny bags, aluminum laminated polyethylene pouches (ALP) and vacuum-sealed aluminum laminated polyethylene pouches (ALPV).12Both refrigerated and ambient storage can securely preserve dried pomegranate samples for 12 months. Best performance was ALP with vacuum and cold storage.[144]
    Hot air dryer60 °CAluminum laminated polyethylene pouch (ALP), polyethylene pouch (PEP), and thermofoam tray (TT) covered in shrinkable polypropylene transparent sheet.6Moisture absorbers aid in the preservation of samples.[100]
     | Show Table
    DownLoad: CSV

    Bhat et al.[105] compared aluminum-laminated polyethylene (ALP, 99.8 g m−2) and polyethylene pouches (93.9 g m−2) each storing 100 g dried pomegranate arils and stored at ambient (15−25 °C). The authors discovered that aluminum laminated polyethylene pouches performed best after a 6-month storage period. Dak et al. [35] compared HDPP and ALP under accelerated shelf-life conditions (38 ± 1 °C and 90% ± 1% relative humidity) and evaluated the correlation between packaging material, storage period, and anthocyanin, phenolics, TSS, TA, and microbial count. The authors estimated that HDPP and ALP have shelf lives of 96 and 187 d, respectively. The above two research confirmed that ALP had the best performance features. This could be owing to the pouches' thickness and the opaque barrier of the aluminum lamination. The use of opaque packaging material may extend the shelf life of pomegranate arils by minimizing photodegradation of components such as carotenoids, flavonoids, and lipids, which can alter qualitative qualities such as aroma, texture, and color[143]. Based on safe consumption criteria, Mokapane et al.[19] proposed a shelf life of 5 months for citric acid pretreatment and dried arils wrapped in kraft paper pouches.

    In a second investigation, Thakur et al.[144] examined the effectiveness of gunny bags, ALP, and ALP combined with vacuum for storing dried pomegranate arils for a period of one year. According to the authors' findings, ALP performed best when performed under vacuum. The various types of containers that are used to store dried pomegranate arils are broken down into categories and shown in Fig. 6.

    Figure 6.  Different types of storage bags for dried pomegranate arils. (a) Aluminum-laminated polyethylene pouches, (b) vacuum-sealed aluminum-laminated polyethylene pouches, (c) gunny bags and (d) transparent polyethylene pouch. From Thakur et al.[144], modified.

    Sharma & Thakur[100] investigated the effect of active packaging on the quality features of dried wild pomegranate arils over a 6-month storage period. Salt or sugar sachets were inserted in ALP pouches or Thermofoam trays that had been wrapped in shrinkable polypropylene transparent film or polyethylene pouches. The researchers reported that for arils dried in a mechanical drier, ALP pouches had the best quality retention of criteria such as ascorbic acid, anthocyanins, total phenols, color, and texture. Furthermore, the inclusion of salt or sugar in active packing aids in the production of high-quality dried arils. However, salt-based active packaging had somewhat higher TA, ascorbic acid content, total sugars, anthocyanin content, and total phenols than sugar-based active packaging.

    The shelf life of dried pomegranate aril can range anywhere from three months to a year depending on the pretreatment, drying procedures and packaging that were used. There is, however, no definitive guideline about the influence that pretreatment and drying procedures have on the quality characteristics of the product when it is being stored. Many studies on the shelf life of dried pomegranate arils focus on examining the influence that different types of packaging material have on the storage life. These studies pay less attention to the physical and microbiological changes that accompany quality alterations. Additionally, a specialized shelf-life testing process and quality standard for dried pomegranate arils can be difficult to locate in the literature.

    While high temperatures have a positive influence on the drying rate, it has a negative effect on the product's texture, color, and nutritional content. Because of this, lower temperatures are often ideal for retaining the pomegranate arils' nutritional value and maintaining their consistency. Freeze dryers offer the best result in this regard. Freeze-drying, on the other hand, is a time- and money-consuming process. When paired with other types of pretreatment, inexpensive procedures such as sun dryers can be adjusted to produce high retention on chemicals that are virtually identical to those obtained in freeze dryers. However, before implementation, it is necessary to examine the intended outcome of the pretreated arils. This is because pretreatments affect both the drying rate and the retention of nutrients. Therefore, in order to completely optimize the process, it is necessary to have an awareness of the interaction that occurs between the pretreatments and the subsequent drying procedure. It is proposed that recommendations be formulated to assist in the manufacture and marketing of dried pomegranate arils that are consistent, nutritious, and hygienically safe.

    The authors confirm contribution to the paper as follows: study conception and design: Ambaw A, Opara UL; project administration and supervision: Ambaw A, Opara UL; draft manuscript preparation: Maphosa B; manuscript review and editing: Ambaw A. all authors reviewed the results and approved the final version of the manuscript.

    Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

    This work is based on the research supported wholly/in part by the National Research Foundation of South Africa (Grant No. 64813). The opinions, findings and conclusions or recommendations expressed are those of the author(s) alone, and the NRF accepts no liability whatsoever in this regard. Research reported in this publication was supported in part by the Foundation for Food and Agriculture Research under award number 434—grant ID: DFs-18- 0000000008.

  • The authors declare that they have no conflict of interest.

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  • Cite this article

    Tan M, Ling Y, Peng Y, Li Z. 2022. Evaluation of genetic diversity and drought tolerance among thirty-three dichondra (Dichondra repens) genotypes. Grass Research 2:8 doi: 10.48130/GR-2022-0008
    Tan M, Ling Y, Peng Y, Li Z. 2022. Evaluation of genetic diversity and drought tolerance among thirty-three dichondra (Dichondra repens) genotypes. Grass Research 2:8 doi: 10.48130/GR-2022-0008

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Evaluation of genetic diversity and drought tolerance among thirty-three dichondra (Dichondra repens) genotypes

Grass Research  2 Article number: 8  (2022)  |  Cite this article

Abstract: Dichondra (Dichondra repens) is an important ground cover plant and is also used as a herbal medicine in China. Objectives of this study were to evaluate phenotypic and genetic diversities among 33 genotypes by using 18 simple sequence repeat (SSR) markers and to further identify the drought tolerance of these germplasms based on five physiological parameters. Results showed that natural variations in phenotypes including plant height, leaf area, leaf thickness, and petiole length were observed among 33 genotypes under well-watered conditions. All 18 SSR primer pairs were found to be polymorphic and significant genetic variation was found in these genotypes. In addition, there were obvious differences in leaf relative water content (RWC), electrolyte leakage (EL), chlorophyll (Chl) content, photochemical efficiency (Fv/Fm), and performance index on absorption basis (PIABS) among 33 genotypes in response to a prolonged period of drought stress (46 d). Drought tolerance of 33 genotypes was then ranked by using subordinate function value analysis (SFVA) and the most drought-tolerant or -sensitive genotypes were identified as Dr5 or Dr29, respectively. Principal component analysis (PCA) further classified 33 genotypes into group I (drought-tolerant), group II (drought-sensitive), and group III (medium types). Current findings showed that 18 selected SSR primers could be potentially used to analyze genetic diversity and varietal identification in dichondra species. Drought-tolerant wild dichondra resources provide a rich genetic base for breeding of new cultivars.

    • Dichondra (Dichondra repens) is a perennial convolvulaceous plant that is wildly used as a ground cover for landscaping, ecological restoration, and weed control due to its ability to form a dense and low-growing sward[1, 2]. Previous studies have demonstrated that dichondra was able to establish a denser greensward for weed suppression than other ground cover plants such as creeping red fescue (Festuca rubra) and white clover (Trifolium repens) in an apple orchard[3, 4], but did not cause reduction in fruit yield[5]. In addition, dichondra is also a main constituent in many traditional herbal beverages in China and its extracts including n-butanol, vanillin, umbelliferone, and scopoletin exhibit antinociceptive effect, antibacterial activity, and anti-inflammation for treatment of icterohepatitis, dysentery, hydrops, or other diseases[68]. There are more than five species of the genus Dichondra in the world and most of them are distributed in the Americas. Up to now, only one wild species is found in China[8]. Requirement for new dichondra cultivars to be used for park and home landscaping is increasing in virtue due to their creeping growth habit and no need for mowing. However, the breeding of dichondra species is far behind other ground cover plants.

      Global warming aggravates the frequency of extreme weather events such as high temperature and drought worldwide. Drought stress causes a lack of water availability in plants resulting in growth retardation and a decline in utility value[9]. Screening and evaluation of relative drought-tolerant genotypes play pivotal roles in breeding for stress-tolerant new cultivars. Multiple molecular markers including microsatellite or simple sequence repeat (SSR), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), and random-amplified polymorphic DNA (RAPD) markers have been applied for selection and evaluation of diverse plant resources[10]. Among them, SSR markers exhibit outstanding characteristics of chromosome-specific location, co-dominant inheritance, and better interspecific transferability, and has become an important tool for molecular breeding[11]. Earlier studies by Varshney et al. and Powell et al. proved that SSRs were found to be more polymorphic than other molecular markers[12, 13]. Kumar et al. reviewed the importance of SSR markers for molecular breeding of salt-tolerant Brassica genotypes[14]. Maqbool et al. evaluated drought tolerance of 40 chickpea (Cicer arietinum) genotypes based on the change in seed yield and genetic diversity via SSR markers, which provided basic information for breeding of drought-tolerant chickpea genotypes[15].

      Understanding of genetic diversity and drought tolerance of different dichondra genotypes could help geneticists or breeders to interpret germplasm architecture or breed new cultivars. In addition, selection and utilization of drought-tolerant dichondra genotypes could be propitious to decrease in maintenance and management costs in the field. Objectives of this study were to evaluate morphological variation, genetic diversity via SSRs, and drought tolerance based on changes in five physiological parameters including leaf relative water content (RWC), electrolyte leakage (EL), chlorophyll (Chl), photosystem II photochemical efficiency (Fv/Fm), and performance index on absorption basis (PIABS) of 33 dichondra genotypes (three cultivars and 30 wild genotypes collected from southwest China). These physiological parameters have been widely used for evaluating stress tolerance in various plant species, since they indicate water status, cell membrane stability, and photosynthetic capacity[1619]. Current findings will provide potential materials for breeding program and further exploration of drought-resistant mechanism by using drought-tolerant and -sensitive dichondra genotypes.

    • Figure 1 showed leaf sizes among 33 genotypes under normal conditions. There were significant variations in plant height, leaf area, leaf thickness, and petiole length among 33 genotypes (Fig. 2ad). Dr9 exhibited the highest plant height and the greatest leaf area, whereas Dr12 showed the shortest plant height and Dr28 had smallest leaf area compared to other genotypes (Fig. 2a & b). The value of leaf thickness of all genotypes was more than 0.1 mm except Dr20 (Fig. 2c). The biggest value of leaf thickness was also observed in Dr9 (Fig. 2c). Dr26 and Dr28 had smaller petiole lengths than other genotypes under normal condition (Fig. 2d). Table 1 showed amplification results using 18 SSR primers. A total of 256 bands were amplified by these SSR primers and the total number of polymorphic bands reached 228 (Table 1). Primer C24 or IBM13 exhibited the highest or lowest polymorphism information content (PIC) than other primers, respectively (Table 1). Cluster analysis found that the average variation range of genetic similarity coefficient was from 0.56 to 0.89 among 33 genotypes (Fig. 3). New cultivars 'Xiaoshao' (Dr32) and 'Duliujiang' (Dr33) had closer genetic relationship, and commercial cultivar 'Silver Falls' (Dr30) showed closer genetic relationship with Dr31 (Fig. 3).

      Figure 1. 

      Phenotypic differences in leaves of 33 Dichondra repens genotypes under normal conditions.

      Figure 2. 

      Differences in (a) plant height, (b) leaf area, (c) leaf thickness, and (d) petiole length among 33 Dichondra repens genotypes under normal conditions. Vertical bars represent standard errors of the mean (n = 10).

      Table 1.  Amplification results among 33 Dichondra repens using different SSR primers.

      Primer
      name
      Total number of
      amplified bands
      Number of
      polymorphic bands
      PPB (%)PIC
      C241818100.000.338
      C271919100.000.295
      C3017952.940.163
      C331313100.000.314
      C51111090.910.354
      C601818100.000.193
      C661313100.000.342
      C6788100.000.230
      C71231982.610.230
      Z25211362.900.163
      Z3733100.000.266
      Z57191684.210.266
      Z699888.890.215
      Z1131616100.000.292
      Z1351111100.000.279
      SSR111212100.000.305
      IBM1344100.000.111
      IBM445211885.710.292
      Total256228
      Average14.2212.6791.590.258

      Figure 3. 

      Cluster analysis of 33 Dichondra repens genotypes based on SSR markers.

    • Dr29 completely died after 46 d of drought stress, so no physiological parameters were detected (Figs 4 & 5). Obvious variations in RWC and EL among 33 genotypes were observed, as demonstrated by drought stress index (DSI) of RWC and EL (Fig. 4a & b). Dr5, Dr18, and Dr33 showed higher DSI of RWC than other genotypes, and smallest DSI of RWC was detected in Dr8 and Dr27 (Fig. 4a). Dr9 or Dr33 had the biggest or smallest DSI of EL than other genotypes, respectively (Fig. 4b). Dr5, Dr9, Dr3, and Dr4 exhibited higher DSI of Chl as compared to other genotypes, whereas Dr28, Dr27, Dr20, and Dr30 had lower DSI of Chl than other genotypes (Fig. 5a). DSI of Fv/Fm of Dr1, Dr2, Dr3, Dr4, or Dr5 was greater than 1.0, but DSI of Fv/Fm of Dr8, Dr14, or Dr27 was less than 0.5 (Fig. 5b). DSI of Fv/Fm of other genotypes ranged from 0.5 to 1.0 (Fig. 5b). The highest DSI of PIABS was detected in Dr5, and DSI of PIABS of Dr7, Dr8, Dr14, or Dr27 were close to 0.0 (Fig. 5c).

      Figure 4. 

      Differences in drought stress index of (a) relative water content (RWC) and (b) electrolyte leakage (EL) among 33 Dichondra repens genotypes. Vertical bars represent standard errors of the mean (n = 4).

      Figure 5. 

      Differences in drought stress index of (a) chlorophyll (Chl), (b) photosystem II photochemical efficiency (Fv/Fm), and (c) performance index on absorption basis (PIABS) among 33 Dichondra repens genotypes. Vertical bars represent standard errors of the mean (n = 4).

    • Drought tolerance among 33 genotypes was evaluated synthetically based on subordinate function value (SFV) (Table 2). Dr5 had the largest SFV of RWC, and the second or third largest SFV of RWC was found in Dr18 and Dr33, respectively. Dr33 (top), Dr31 (second), and Dr32 (third) showed bigger SFV of EL than other genotypes (Table 2). Maximum SFV of Chl was detected in Dr5. The top three genotypes with bigger SFV of Fv/Fm than other genotypes in the sequences were Dr3, Dr5, and Dr4. Dr5 had the maximum SFV of PIABS as compared to other genotypes, whereas Dr27 exhibited the minimum SFV of PIABS than other genotypes except Dr29. Comprehensive ranking showed Dr29, Dr28, and Dr27 exhibited lower drought tolerance than other genotypes. Out of the 33 genotypes, drought tolerance of Dr5, Dr33, or Dr3 ranked first, second, or third, respectively (Table 2). Heat map showed variations in five physiological parameters among 33 genotypes in response to drought stress (Fig. 6a). 33 genotypes could be divided into three groups based on principal component analysis (PCA) analysis (Fig. 6b). The first group included eight genotypes (Dr5, Dr33, Dr9, Dr1, Dr2, Dr32, Dr3, and Dr4) with better tolerance than other genotypes, and the second group consisted of four genotypes (Dr29, Dr27, Dr8, and Dr14) which had lower drought tolerance than other genotypes. The remaining 21 genotypes were aggregated to form the third group and their drought tolerance was intermediate between the first group and second group (Fig. 6b).

      Table 2.  Membership function values of five physiological parameters and comprehensive evaluation of drought tolerance among 33 Dichondra repens genotypes.

      Material No.RWCELChlFv/FmPIABSAverageOrder
      Dr50.8360.6410.9320.8790.6420.7861
      Dr330.7580.8550.7190.8490.4740.7312
      Dr30.6520.6180.8980.9040.4550.7063
      Dr40.6810.6120.8470.8680.4620.6944
      Dr320.6420.7060.6370.8230.4610.6545
      Dr20.5750.6130.7220.8390.4330.6366
      Dr10.5360.5720.7980.8420.3910.6287
      Dr180.8240.5770.7520.6580.3120.6258
      Dr310.5950.7660.6960.7420.2950.6199
      Dr90.4580.3280.9170.8510.4440.60010
      Dr60.6860.4340.5700.7640.4120.57311
      Dr250.5920.5450.7450.7230.2530.57212
      Dr170.7350.3140.7080.7270.3610.56913
      Dr160.4980.4400.7360.7570.2960.54514
      Dr220.6560.5690.5100.6520.2520.52815
      Dr190.5390.3960.7030.6430.3060.51716
      Dr100.6670.4520.7030.5920.1730.51717
      Dr120.5120.4300.6720.6710.2480.50618
      Dr230.5230.4530.5450.6790.2740.49519
      Dr210.6080.6170.4280.5730.2080.48720
      Dr240.5580.4220.4550.6340.2720.46821
      Dr70.7170.3710.5790.5390.1060.46222
      Dr150.4850.4440.5410.6050.2070.45623
      Dr300.5370.4840.3630.6470.2430.45524
      Dr260.4250.4550.5230.6350.2300.45325
      Dr130.4310.3910.7180.5470.1630.45026
      Dr110.5120.4830.6520.4600.1420.45027
      Dr200.6760.2900.1880.7000.2550.42228
      Dr80.3620.4120.5670.3290.1440.36329
      Dr140.4060.2810.6200.2780.1450.34630
      Dr280.5050.2710.2720.5140.1560.34431
      Dr270.3230.2950.3210.2940.1130.26932
      Dr290.0000.0000.0000.0000.0000.00033

      Figure 6. 

      Changes in (a) heat map and (b) principal component analysis (PCA) based on five different physiological parameters. RWC, relative water content; EL, electrolyte leakage; Chl, chlorophyll; Fv/Fm, photosystem II photochemical efficiency; PIABS, performance index on absorption basis.

    • Wild dichondra is widely distributed in southwest China, but the problem is that lack of enough research has slowed down breeding and utilization of these wild resources. In the past 30 years, SSRs have been widely used to evaluate genetic diversity in various plant species[2022]. In our current study, significant genetic variation was detected among 33 dichondra genotypes through using 18 selected SSR markers that were developed from convolvulaceous sweet potato (Dioscorea esculenta). Excellent transferability of SSR markers cross related species has been demonstrated in many previous studies. For example, SSR markers from barley (Hordeum vulgare) exhibited good interspecific transferability in wheat (Triticum aestivum) and rye (Secale cereale)[23]. Mutual transferability of SSR between wheat and rye was also very high[24]. In addition, tall fescue (Festuca arundinacea) SSR markers could be applied for evaluation of genetic relationships in meadow fescue (Festuca pratensis), tetraploid fescue (Festuca arundinacea), and ryegrass (Lolium perenne)[25]. Our study found that all 18 primers were found to be polymorphic when they were applied to 33 dichondra genotypes, which indicated these primer pairs could be used for analysis of genetic diversity and cultivar identification in dichondra species. In addition, phenotypic variations in plant height, leaf area, leaf thickness, and petiole length were also observed among 33 dichondra genotypes under well-watered condition. Diverse morphological variability and genetic variation are beneficial to screen suitable accessions for stress adaptation, because variation in morphological characters often indicates genetic differences in one particular plant species, which provides abundant gene resources for screening new cultivars differing in drought tolerance.

      Although many previous studies have been conducted to identify drought-tolerant plant genotypes in the field or under controlled conditions[2629], selection and identification of dichondra genotypes with better drought tolerance have not been reported so far. Leaf RWC and EL are two important indicators of drought tolerance, as the RWC reflects leaf water status and the EL indicates cell membrane stability when plants suffer from drought stress[30]. Both of them have been applied to evaluate plant drought tolerance. Ahmed et al. found that drought tolerance of wheat was positively related to higher RWC and cell membrane stability which could be used to screen drought-tolerant genotypes at the seedling stage[31]. Drought-tolerant bermudagrass (Cynodon dactylon) also showed higher RWC and lower EL than drought-sensitive accessions in response to drought stress[18]. Dhanda et al. reported that cell membrane stability was the most important trait for evaluation of drought tolerance among thirty wheat cultivars[32]. Our current study demonstrated that Dr5, Dr18, and Dr33 could maintain higher leaf RWC and lower EL than other dichondra genotypes, whereas Dr8 exhibited the lowest RWC and the highest EL under drought stress. Those genotypes with higher leaf RWC and lower EL in response to drought stress could be recognized as potential breeding materials for developing drought-tolerant varieties.

      Drought-tolerant plants could delay Chl degradation to maintain higher photosynthesis under water-deficit condition[33]. It has been found that the maintenance of higher Chl content is a common characteristic in drought-tolerant plant genotypes[18, 31]. Apart from Chl content, Fv/Fm and PIABS also are critical parameters for evaluation of stress tolerance in plant species, as Fv/Fm represents photosystem II photochemical efficiency and PIABS indicates health status of photosynthetic organs[34, 35]. It has been found that higher Chl content, Fv/Fm, or PIABS were the superior indicators with regard to better tolerance to heat stress in creeping bentgrass (Agrostis stolonifera) accessions[17], salt stress in white clover germplasm[16], and drought stress in sour cherry (Prunus cerasus) genotypes[36]. Dichondra genotypes exhibited significant variations in Chl content, Fv/Fm, and PIABS in response to a prolonged period of drought stress. Higher Chl content, Fv/Fm, and PIABS were found in Dr3, Dr4, Dr5, Dr9, and Dr33 which could be potential drought-tolerant genotypes.

      Drought tolerance evaluated by one particular parameter is often one-sided. Subordinate function value analysis (SFVA) has been applied to comprehensively evaluate drought tolerance of diverse plant accessions based on different parameters[28, 37, 38]. The most promising drought-tolerant dichondra genotypes (Dr5, Dr33, Dr3, Dr4, and Dr32) were screened based on the SFVA in our current study. In addition, those 33 dichondra genotypes were classified into three distinct groups according to the analysis of PCA. Group I included 8 genotypes (Dr1, Dr2, Dr3, Dr4, Dr5, Dr9, Dr32, and Dr33) which were identified to be drought-tolerant candidates and group II contained four genotypes (Dr8, Dr14, Dr27, and Dr29) which were recognized as drought-sensitive accessions. The remaining 21 dichondra genotypes were classified into group III, which was intermediate between group I and III for drought tolerance. Similar results were found in the study of Badr et al. who reported that PCA analysis could clearly separate out drought-tolerant maize (Zea mays) genotypes from 40 accessions[39]. Analytic results from SFVA were consistent with the findings based on the analysis of PCA. These selected drought-tolerant genotypes offer available materials for breeders to develop new dichondra cultivars.

    • A total of 18 SSR primer pairs were applied to evaluate genetic diversity of 33 dichondra genotypes and all primer pairs were found to be polymorphic. Natural variations in phenotypes including plant height, leaf area, leaf thickness, and petiole length were also observed among 33 genotypes under the well-watered condition. Drought tolerance of 33 genotypes was ranked by using SFVA, and the most tolerant genotype was Dr5 and most drought-sensitive genotype was Dr29. In addition, PCA analysis could classify 33 genotypes into group I (drought-tolerant), group II (drought-sensitive), and group III (medium types). Current findings showed that 18 selected SSR primer pairs could be used to potentially analyze genetic diversity and varietal identification in dichondra species. Selected drought-tolerant wild resources provide a rich genetic base for the breeding of new cultivars.

    • Thirty wild dichondra genotypes and three commercial cultivars 'Silver falls', 'Xiaoshao', and 'Duliujiang' were collected from the Field Gene Bank at Sichuan Agricultural University (Table 3) and transplanted into polyvinyl chloride (PVC) tubes (33 cm in length, and 11 cm in diameter). All PVC tubes were filled with same mixtures of soil and sand (v:v, 1:1). Plants were cultivated in a greenhouse from July 14th to August 30th, 2020 (average temperature about 27/18 °C day/night and 800 μmol m−2∙s−1 photosynthetically active radiation) and fertilized weekly with full Hoagland's solution[40]. For drought treatment, plants were then divided into two groups: one group was irrigated three times a week to avoid soil drought as well-watered control, and another group was subjected to drought stress by stop irrigating for 46 d. Leaves were collected for detecting physiological parameters and SSR markers. Each genotype was replicated four times (four tubes) under normal condition or drought stress.

      Table 3.  Test 33 Dichondra repens materials and their sources.

      Material No.OriginAltitude (m)
      Dr1Zhongjiang, Sichuan600
      Dr2Pingtang, Guizhou848
      Dr3Dushan, Guizhou1010
      Dr4Tianzhu, Guizhou350
      Dr5Naxi, Sichuan404
      Dr6Dayi, Sichuan310
      Dr7Bishan, Chongqing350
      Dr8Jining, Yunnan1890
      Dr9Xifeng,Guizhou990
      Dr10Xishui, Guizhou1169
      Dr11Sinan, Guizhou730
      Dr12Jiangkou, Guizhou475
      Dr13Tongren, Guizhou415
      Dr14Zhenyuan, Guizhou382
      Dr15Danzai, Guizhou894
      Dr16Sandu, Guizhou500
      Dr17Sandu, Guizhou780
      Dr18Dujun, Guizhou842
      Dr19Shuicheng, Guizhou1193
      Dr20Liuzhi, Guizhou1035
      Dr21Anshun, Guizhou1278
      Dr22Qinglong, Guizhou1393
      Dr23Jin’an, Guizhou1336
      Dr24Panzhou, Guizhou1532
      Dr25Xingren, Guizhou1336
      Dr26Anlong, Guizhou1250
      Dr27Wangmo, Guizhou653
      Dr28Ziyun, Guizhou1160
      Dr29Huishui, Guizhou980
      Dr30 (‘Silver Falls’)USA-
      Dr31Xichou, Yunnan1108
      Dr32 (‘Xiaoshao’)Yiliang, Yunnan1970
      Dr33 (‘Duliujiang’)Sandu, Guizhou600
    • A vernier caliper was used to measure leaf thickness and leaf area (S) which was calculated based on the formula S = π × [(length + width) / 4]2. Plant height and petiole length were measured by using a ruler, and 10 independent plants were selected randomly from each tube for the measurement of these phenotypic parameters. For leaf RWC, fresh leaves were cut from plants and weighted instantly to record fresh weight (FW). These leaves were then soaked in deionized water for 10 h and turgid weight (TW) was weighted. All leaves were put in an oven at 80 °C for 72 h to detect dry weight (DW). The RWC was calculated as RWC (%) = [(FW − DW) / (TW −DW)] × 100)[41]. To detect leaf EL, fresh leaves (0.15 g) were soaked in 40 mL of deionized water for 24 h at 25 °C and initial conductivity of solution (Cinitial) was measured by using a conductivity meter (YSI Model 32, Yellow Spring, OH). Max conductivity of solution (Cmax) was detected after leaves were autoclaved at 120 °C for 20 min. The EL was calculated as the ratio of Cinitial to Cmax[42]. For Chl content, leaves were soaked in 15 mL of dimethyl sulfoxide for 48 h and absorbance was detected at 645 and 663 nm with a spectrophotometer (Spectronic Instruments, Rochester, NY, USA)[43]. For Fv/Fm and PIABS, leaves were kept in darkness for 15 min and a fluorescence meter (Pocket PEA Chl Fluorimeter, Hansatech Instruments Ltd, UK) was used to record Fv/Fm and PIABS[44].

    • Total DNA was extracted from approximately 0.1 g of fresh leaf tissues by using an assay kit purchased from Tiangen Biotech Co., LTD, Beijing, China. A Hoefer Dyna Quant 200 (Amersham Biosciences, Piscataway, NJ, USA) was used to detect DNA concentration which was adjusted to 10 ng ∙ μL−1 of final concentration using purified water. PCR reaction was conducted by using 7.5 μL of 2× Mix (P2015, Dongsheng Biotech), 3 μL of DNA, 1.5 μL of 0.6 μmol∙L−1 each primer, and 3 μL of purified water. A total of 18 primer sequences which were developed from sweet potato and their annealing temperature were recorded in Supplmental Table S1[45]. PCR products were electrophoresed in 6% polyacrylamide denaturing gels under 200 V for 30 min and then 400 V for 1.5 h. For SSR bands detection, gels were silver-stained and then captured using a camera. Gel images were analyzed by using the software Gel Analyzer 19.1 (www.gelanalyzer.com) to estimate base pair size of bands. Polymorphism was determined based on absence or presence of SSR locus.

    • Variations in phenotypic and physiological parameters were analyzed by Statistix 8.1 (Tallahassee, FL, USA). PCA biplot analysis was performed by using SPSS 20 (IBM, Armonk, NY, USA). Drought tolerance was evaluated by using SFVA based on five physiological parameters (RWC, EL, Chl, Fv/fm, and PIABS)[17]. DSI was calculated according to the formula DSI = (value of parameter under drought stress) / (value of parameter under normal condition) × 100. Cluster analysis of 33 Dichondra micrantha genotypes based on SSR markers was conducted by using NTSYSPC2.10e and MEGA 6 (Tokyo Metropolitan University, Hachioji, Tokyo, Japan)[46].

    • We appreciate Prof. Youmin Gan who collected wild dichondra resources from southwest China and established the Field Gene Bank at Sichuan Agricultural University.

      • The authors declare that they have no conflict of interest.

      • Copyright: © 2022 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (6)  Table (3) References (46)
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    Tan M, Ling Y, Peng Y, Li Z. 2022. Evaluation of genetic diversity and drought tolerance among thirty-three dichondra (Dichondra repens) genotypes. Grass Research 2:8 doi: 10.48130/GR-2022-0008
    Tan M, Ling Y, Peng Y, Li Z. 2022. Evaluation of genetic diversity and drought tolerance among thirty-three dichondra (Dichondra repens) genotypes. Grass Research 2:8 doi: 10.48130/GR-2022-0008

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