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A mature, sweet orange cultivar derived from 'Valencia' with high Agrobacterium transformation efficiency

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  • Received: 11 June 2024
    Revised: 07 August 2024
    Accepted: 19 September 2024
    Published online: 04 November 2024
    Fruit Research  4 Article number: e036 (2024)  |  Cite this article
  • Transformation efficiencies of sweet orange cultivars 'Florida EV1' and 'Valencia', recalcitrant to Agrobacterium transformation, were investigated using liquid culture in We-V™ vessels. The two mature cultivars were transformed using Agrobacterium with a vector containing selectable markers, nptII and TIPS-EPSPS, and the GFP reporter. Transgenics were identified with GFP in liquid culture at 0, 100, and 200 mg·L−1 kanamycin or in the semi-solid control with 100 mg·L−1 kanamycin. For 'Florida EV1', there were significant differences in the mean transformation efficiency based on the number of shoots screened (TES) at all kanamycin concentrations. Selection at 200 mg·L−1 was better than at lower concentrations in liquid or semi-solid control medium with 100 mg·L−1 kanamycin. The variable TEE, transformation efficiency based on the number of explants, did not discern differences. The means ± standard errors for TES at 200 mg·L−1 were 7.9% ± 2.7% for 'Florida EV1' and 2.4% ± 1.7% for 'Valencia'. In total, 74 transgenics were produced in 'Florida EV1', whereas seven were generated in 'Valencia'. Obtaining transgenics in 'Florida EV1' was easy; fewer shoots were screened at 200 mg·L−1. 'Florida EV1' exhibited better regeneration ability, and all transgenics survived on glyphosate medium, suggesting the TIPS-EPSPS selectable marker could be useful in transformation. Molecular analyses confirmed their transgenic nature. 'Florida EV1' trees produced fruit earlier than 'Valencia' in less than two years. 'Florida EV1' could accelerate the production of HLB disease-resistant trees.
  • 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
    DownLoad: CSV

    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.

  • Supplementary Table S1 The number of nodal budsticks and shoots at each glyphosate concentration in the two cultivars.
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  • Cite this article

    Canton M, Peraza-Guerra O, Wu H, Grosser J, Mou Z, et al. 2024. A mature, sweet orange cultivar derived from 'Valencia' with high Agrobacterium transformation efficiency. Fruit Research 4: e036 doi: 10.48130/frures-0024-0030
    Canton M, Peraza-Guerra O, Wu H, Grosser J, Mou Z, et al. 2024. A mature, sweet orange cultivar derived from 'Valencia' with high Agrobacterium transformation efficiency. Fruit Research 4: e036 doi: 10.48130/frures-0024-0030

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ARTICLE   Open Access    

A mature, sweet orange cultivar derived from 'Valencia' with high Agrobacterium transformation efficiency

Fruit Research  4 Article number: e036  (2024)  |  Cite this article

Abstract: Transformation efficiencies of sweet orange cultivars 'Florida EV1' and 'Valencia', recalcitrant to Agrobacterium transformation, were investigated using liquid culture in We-V™ vessels. The two mature cultivars were transformed using Agrobacterium with a vector containing selectable markers, nptII and TIPS-EPSPS, and the GFP reporter. Transgenics were identified with GFP in liquid culture at 0, 100, and 200 mg·L−1 kanamycin or in the semi-solid control with 100 mg·L−1 kanamycin. For 'Florida EV1', there were significant differences in the mean transformation efficiency based on the number of shoots screened (TES) at all kanamycin concentrations. Selection at 200 mg·L−1 was better than at lower concentrations in liquid or semi-solid control medium with 100 mg·L−1 kanamycin. The variable TEE, transformation efficiency based on the number of explants, did not discern differences. The means ± standard errors for TES at 200 mg·L−1 were 7.9% ± 2.7% for 'Florida EV1' and 2.4% ± 1.7% for 'Valencia'. In total, 74 transgenics were produced in 'Florida EV1', whereas seven were generated in 'Valencia'. Obtaining transgenics in 'Florida EV1' was easy; fewer shoots were screened at 200 mg·L−1. 'Florida EV1' exhibited better regeneration ability, and all transgenics survived on glyphosate medium, suggesting the TIPS-EPSPS selectable marker could be useful in transformation. Molecular analyses confirmed their transgenic nature. 'Florida EV1' trees produced fruit earlier than 'Valencia' in less than two years. 'Florida EV1' could accelerate the production of HLB disease-resistant trees.

    • Agrobacterium transformation efficiency is influenced by the plant genotype or cultivar, the effectiveness of the selection system, the reporter gene used to identify the transgenic shoots and the meristematic nature of the plant cells. Highly embryogenic cell cultures and meristematic cells in organogenic explants yield the best results in plant transformation. Generally, cultivars and genotypes with high transformation efficiency will regenerate more transgenics and edited plants[1].

      Agrobacterium-plant interactions are complex and only partially understood[2]. Recalcitrance among different species might be due to any number of reasons stemming from the physiology of the donor plant, in vitro explant manipulations, plant stress physiology[3], or other problems[411]. In mature citrus explants, cells in the cambial ring become competent for transformation and regeneration[12], but there is immense cultivar variation in Agrobacterium transformation efficiency (Zale unpublished).

      Mature 'Valencia' (Citrus sinensis L. Osbeck) citrus scions form shoots in tissue culture, but few are transgenic, so it is considered recalcitrant to Agrobacterium. Transformation efficiency based on the number of shoots (TES) and based on the number of explants (TEE) is consistently less than 5% on semi-solid medium with 100 mg·L−1 kanamycin[1315]. In practice, its transformation efficiency is considerably less than 5%, and higher transformation efficiencies depend upon other factors, such as the re-invigoration of the mature scion donor plant by budding[16].

      'Florida Early Valencia 1' ('Florida EV1') is a somaclone derived from 'Valencia' in tissue culture[17,18]. 'Florida EV1' matures from December to January in central Florida (USA), while 'Valencia' matures from early to late March. The term somaclonal variant was introduced by Larkin & Scowcroft in 1981 to describe the genetic variation in plants regenerated from cell cultures[19]. These alterations might be genetic or epigenetic changes induced by the stress of tissue culture or due to auxins and cytokinins generating disorganized cell growth[11,20,21]. In wheat, three near-isogenic lines identified in tissue culture differed in regeneration ability and transformation ability, but the most amenable wheat line had two translocations[9]. Somaclonal variation can create unwanted variations but is also a potential source of useful genetic diversity[19].

      Citrus biotechnology has traditionally used the neomycin phosphotransferase II (nptII) gene for selection, which, unfortunately, is riddled by escaped shoots[22]. Canton et al. determined the best concentrations of kanamycin in liquid vs. semi-solid selection medium to screen two mature citrus rootstocks by GUS staining[23]. They found that selection at stringent kanamycin concentrations (150 to 200 mg·L−1) in liquid culture was optimal. The present research aims to apply this same technology using the GFP reporter to mature sweet orange scions.

    • The TIPS-EPSPS vector has been previously described[24]. The Citrus sinensis EPSPS gene was mutated at two sites with site-directed mutagenesis and shown to confer tolerance to glyphosate in immature citrus[24]. In the present study, it was transformed into Agrobacterium tumefaciens, strain AGL1, with the appropriate antibiotics and kanamycin bacterial selection at the recommended concentrations[25]. The vector also contains the neomycin phosphotransferase II (nptII) gene as a secondary selectable marker which encodes an aminoglycoside phosphotransferase that confers resistance to kanamycin in plants, and the enhanced green fluorescent gene (egfp) as the reporter in plants (Fig. 1).

      Figure 1. 

      Schematic map of the TIPS-EPSPS vector in pBI101 vector backbone for Agrobacterium-mediated genetic transformation. The nptII, eGFP, and TIPS-EPSPS are driven by the NOS promoter, prolD promoter, and double 35S promoter, respectively. LB, left border; RB, right border; NOSp, nopaline synthase promoter; prolD, A. rhizogenes rolD promoter; 2×35Sp, Enhanced Cauliflower Mosaic Virus promoter; nptII, neomycin phosphotransferase; p(A), CaMV polyadenylation signal; Tnos, nopaline synthase terminator; T35, Cauliflower Mosaic Virus terminator; eGFP, enhanced green fluorescence protein; TIPS, citrus shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) modified to obtain glyphosate resistance[24].

    • Mature internodal explants of 'Valencia SPB-1-14-19' and 'Florida EV1' were used in transformation experiments[18]. Mature scions were budded onto immature five-month-old Citrus volkameriana (L.) rootstocks[23]. Conditions in the growth room were maintained with a 12 h light cycle photoperiod provided by cool white, fluorescent lamps, with 50% humidity and a temperature of 26 ± 4 °C. Mature scion budsticks were collected at three months, and prepared accordingly[23].

    • Agrobacterium strain AGL1 was grown as described and used in transformations[15]. Plant transformations and tissue cultures were performed, and cultures were placed in a dark incubation with 100 mg·L−1 kanamycin selection for three weeks[23]. After three weeks, the explants were placed in liquid culture vessels with 3M paper or paper towels[23]. The explants were placed on top of this paper in the liquid culture vessels at 0, 100, and 200 mg·L−1 kanamycin and grown for four weeks[23]. A semi-solid medium with 100 mg·L−1 kanamycin was used as a control for three weeks in the dark incubation and four weeks in the light incubation[23]. Micrografting and secondary grafting of some but not all GFP positive shoots were performed[15,23].

    • A glyphosate assay was developed to test whether the transgenic, nodal budsticks positive for the EPSPS transgene could sprout shoots on glyphosate medium. Roundup Super Concentrate (50.2% active ingredient, containing 3.7 lb glyphosate acid equivalent per US gallon or 2.6 M glyphosate) was used as a selection system in transgenic vs. wild-type (WT) nodal budsticks in semi-solid MT medium (Phytotechnology Labs, Lenexa, KS, USA) with 30 g·L−1 sucrose at pH 5.7 and different glyphosate concentrations (0, 2.6, 6.5, and 13.1 mM)[24,26]. Transgenic and WT budsticks were prepared[23], cut into nodal budsticks with one node per budstick, and plated onto the glyphosate medium. Three to six nodal budsticks per petri dish were plated onto the semi-solid medium and incubated in a chamber with a 12 h photoperiod, 45 μmol·m−2·s−1 light intensity, at 26 ± 2 °C. Sprouted shoots per nodal budstick were recorded after 28 days and analyzed using ANOVA.

    • Shoots were examined for GFP fluorescence with a Nikon SMZ 745T stereoscope (Nikon, Melville, NY, USA) equipped with a NIGHTSEA fluorescence adapter (NIGHTSEA, Lexington, MA, USA) and a blue filter. These shoots were micrografted onto decapitated seedlings[15,23]. Transgenic lines were confirmed by PCR using primers for EPSPS, nptII, and gfp to amplify fragments of 455, 239 and 713 bp, respectively. The oligonucleotides used to amplify these genes were as follows: nptII (nptII-F: GTGGAGAGGCTATTCGGCTATGA and nptII-R: CTTCGCCCAATAGCAGCCAGT), gfp (gfp-F: CTGACCGGATCGGCACATTA, and gfp-R: CTTGTAGTTGCCGTCGTCCT) and EPSPS (EPSPS-F: AGAGGACACGCTGAAATCAC and EPSPS-R: AAGCATATGGTGAATATCTTCGC). Genomic DNA extractions were performed with the PowerPlant® DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA). The PCR reactions were set to a 15 μL volume total, containing 0.15 mM of each primer, 7.5 μL of Dream Taq PCR Master Mix (2X) (ThermoFisher Scientific®, Atlanta, GA, USA), and 20 ng of DNA template. The cycling parameters were programmed into an MJ Mini™ thermal cycler (Bio-Rad®, Hercules, CA, USA) with a preliminary denaturation at 95 °C for 3 min; followed by 35 cycles of 95 °C for 30 s; an annealing step at 62 °C for 30 s, and an extension step at 72 °C for 1 min, followed by a final 5 min extension at 72 °C. Polymerase chain reaction products were analyzed on 1.5% agarose gel electrophoresis with a 100 bp EZ Load molecular ruler (Bio-Rad®, Hercules, CA, USA).

    • A duplex TaqMan real-time quantitative PCR (qPCR) assay determined the transgene copy number in some transgenic citrus lines vs a known, single copy, 'Hamlin' control[27,28]. Amplification of the nptII gene and the internal control gene CsLTP (Citrus sinensis lipid transfer protein) was analyzed in each transgenic line against the 'Hamlin' control known to contain only one copy of the nptII gene (supplied by the Mou lab, University of Florida, Microbiology and Cell Science, Gainesville, FL, USA). Primer probe combinations were standardized with the 'Hamlin' single copy nptII control reference line. A serial dilution was performed starting with 100 ng of total DNA, and Ct values were plotted vs the log nanograms of total DNA. The R-squared value (coefficient of determination) for the standard curves for both nptII and CsLTP genes was 0.99. The slope of the plot was −3.5 for nptII and −3.49 for CsLTP, giving an efficiency of 1.93 (93%) (Table 1). This is in the range (90%−97%) of good efficiency for a duplex qPCR.

      Table 1.  The linear range and standard curves for endogenous CsLTP and nptII genes.

      Gene Linear range (CT) Regression equation Correlation coefficient
      nptII 21.07−26.16 f(x) = −3.505x + 21.987 0.999
      CsLTP 21.92−25.32 f(x) = −3.491x + 21.126 0.999
    • Oligonucleotides and probes were designed and synthesized (Eurofins Genomics, Louisville, KY, USA) for the nptII transgene[29,30]. The oligonucleotide and probe sequences for the nptII gene were as follows: nptII-F: ATCATGGTGGAAAATGGCCG, nptII-R: GCCAACGCTATGTCCTGATAG and nptII-probe: [FAM]-TTCTGGATTCATCGACTGTGGC-[BHQ1], yielding an 86 bp product. The Citrus sinensis lipid transfer protein (CsLTP) gene was used as the endogenous single copy control[27,28]. The oligonucleotide and probe sequences for the CsLTP gene were as follows: CsLTP-F: CGGATCAATCCCTAACCTCAAC, CsLTP-R: GTCAGTGGAGATGCTGATCTTG and CsLTP-probe: [TxRed]-CGAGCTTGTGGAGTCAGCATTCCT-[BHQ2] and they yield a 94 bp PCR product.

    • Duplexed TaqMan PCR reactions were performed in 12.5 μL volume in a 96-well format using a QuantStudio™ 3 Real-Time PCR system (Applied Biosystems, Foster, CA, USA). The reaction mixture contained 6.25 μL of 2x TaqPath™ master mix (Applied Biosystems, Waltham, MA, USA), 1 μL DNA (~50 ng), and 900 nM nptII-F/R primers, 900 nM CsLTP-F/R primers and 100 nM each gene-specific probe. The PCR program consisted of initial denaturation at 95 °C for 3 min, 40 cycles of 10 s at 95 °C, 20 s at 55 °C, and the fluorescence was collected at 55 °C. A no-template control and non-transgenic plant DNA were included. The instrument software determined the Ct values. The log10 of the DNA dilution series was plotted vs the Ct values obtained for each dilution. The PCR efficiency (E) was calculated using the following equation (E=10(−1/slope))[31]. This formula was used to calculate the copy number for each transgenic line and control as formulated by Pfaff[32].

      Ratio=(Etarget)(DeltaCt(target)(controlsample))(Ereference)(deltaCT(reference)(controlsample))
    • Transformation response variables included the total number of shoots greater than 2 mm (SL > 2) and the number of positive shoots (PS). The mean transformation efficiencies were based on the number of explants (TEE, the number of positive shoots/number of explants × 100), and transformation efficiencies based on the number of shoots screened (TES, the number of positive shoots/the number of shoots screened × 100). Data collection occurred after seven weeks. ANOVA was performed separately for each cultivar and comparisons were made within each cultivar. For 'Florida EV1' the statistics were calculated for 60 explants per vessel with ten replicates at each kanamycin concentration (0, 100, and 200 mg·L−1) in liquid medium and with ten replicates in semi-solid medium at the standard kanamycin concentration (100 mg·L−1) . For 'Valencia', the statistics were calculated based on 60 explants per vessel with seven replicates at each kanamycin concentration (0, 100, and 200 mg·L−1) in liquid medium and seven replicates in the semi-solid medium at the standard kanamycin concentration (100 mg·L−1). The square root transformation was used to transform the PS variable before ANOVA. Percentage data were divided by 100 and transformed into the arcsine transformation before ANOVAs. The Mann-Whitney non-parametric statistics test was used to compare the regeneration ability of each cultivar (SL > 2/number of explants) across all treatment levels and at 0 mg·L−1 kanamycin. Similarly, a non-parametric Mann-Whitney test compared the median number of positive shoots (PS) between 'Florida EV1' and 'Valencia'. Glyphosate tolerance was assessed after 28 d as the number of shoots that sprouted per nodal budstick at four different glyphosate concentrations (0, 2.6, 6.5, and 13.5 mM glyphosate) and used in ANOVAs. Descriptive statistics, ANOVAs, and multiple comparisons with Fisher's LSD were used to analyze the variables in the cultivars studied. All tests were calculated with Minitab Version 21.

    • In separate experiments, mature 'Florida EV1' and 'Valencia' sweet orange scions were transformed with the TIPS-EPSPS vector (Fig. 1), which possesses two selectable markers (P-Nos::nptII::T-Nos and 2x35S::TIPS-EPSPS::p(A)) in Agrobacterium strain AGL1. TIPS-EPSPS confers resistance to glyphosate[24]. A schematic representation of the process is shown in Fig. 2. A total of 74 'Florida EV1' transgenics and seven 'Valencia' transgenics were identified by GFP fluorescence in We-V™ liquid culture vessels with gravity wells using three levels of kanamycin selection (0, 100, and 200 mg·L−1 kanamycin) vs semi-solid medium at 100 mg·L−1 kanamycin (Table 2 & Fig. 2). The results of these experiments were analyzed separately using ANOVAs.

      Figure 2. 

      Schematic representation of the transformation process using We-V™ liquid culture vessels with gravity wells with different kanamycin concentrations of (a) 0, (b) 100, and (c) 200 mg·L−1.

      Table 2.  'Florida EV1' and 'Valencia' and the variables that were measured (shoots longer than 2 mm (SL > 2) and positive shoots (PS)) in liquid and semi-solid medium.

      Cultivar1 Kanamycin
      (mg·L−1)
      Medium Explants SL > 22 PS3
      'Florida EV1' 0 Liquid 600 875 17
      'Florida EV1' 100 Liquid 600 424 22
      'Florida EV1' 200 Liquid 600 285 18
      'Florida EV1' 100 Semi-solid 600 535 17
      'Valencia' 0 Liquid 420 361 0
      'Valencia' 100 Liquid 420 281 2
      'Valencia' 200 Liquid 420 177 4
      'Valencia' 100 Semi-solid 420 247 1
      1The two cultivars were tested and analyzed separately. 2SL > 2, shoots longer than 2 mm. 3PS, GFP positive shoots.

      For 'Florida EV1', the mean shoot length greater than 2 mm (SL > 2) variable in the 100 and 200 mg·L−1 kanamycin treatments in liquid media and the 100 mg·L−1 control in the semi-solid medium was significantly less (p < 0.05) than the 0 mg·L−1 kanamycin liquid control treatment (Table 3 & Fig. 3a). Fewer shoots (n = 285) were screened at 200 mg·L−1 compared to the 0 mg·L−1 control (n = 875) (Table 2). The mean TES (transformation efficiency based on the number of positive shoots/total number of shoots × 100) variable at 200 mg·L−1 kanamycin in liquid medium was 7.9% ± 2.7; at 100 mg·L−1 kanamycin in liquid medium, it was 5.4% ± 1.4 (Table 3). However, the latter treatment generated more transgenics, although it was not statistically different from the mean TES value at 200 mg·L−1 (Fig. 3a). The mean TES value of 7.9% was significantly greater than the 0 mg·L−1 kanamycin liquid medium control and the 100 mg·L−1 kanamycin semi-solid control (p < 0.05), showing that liquid medium selection is superior (Table 3 & Fig. 3a). The mean TEE variable was not significant (Table 3 & Fig. 3b), and the number of positive shoots (PS) was not significant because transgenics were produced at every kanamycin level (Table 2). Transgenics readily regenerated in 'Florida EV1' at all kanamycin concentrations.

      Table 3.  The means for the number of positive shoots (PS), shoots with lengths greater than 2 mm (SL > 2), and transformation efficiencies (TES and TEE) at three concentrations of kanamycin in liquid and semi-solid medium for 'Florida EV1' and 'Valencia'.

      Cultivar1 Kanamycin (mg·L−1) Medium Mean PS2 ± SE3 Mean SL > 24 ± SE Mean TES5 ± SE Mean TEE6 ± SE
      'Florida EV1' 0 Liquid 1.7 ± 0.4 87.5a ± 14.3 2.4b ± 0.7 2.8 ± 0.7
      'Florida EV1' 100 Liquid 2.2 ± 0.6 42.4b ± 3.7 5.4ab ± 1.4 3.7 ± 1.0
      'Florida EV1' 200 Liquid 1.8 ± 0.5 28.5b ± 3.2 7.9a ± 2.7 3.0 ± 0.8
      'Florida EV1' 100 Semi-solid 1.7 ± 0.4 53.5b ± 8.7 2.9b ± 0.7 3.2 ± 1.0
      'Valencia' 0 Liquid 0 ± 0 51.6a ± 4.4 0 ± 0 0 ± 0
      'Valencia' 100 Liquid 0.3 ± 0.2 40.1b ± 2.0 0.7 ± 0.4 0.5 ± 0.3
      'Valencia' 200 Liquid 0.6 ± 0.4 25.3c ± 2.7 2.4 ± 1.7 0.9 ± 0.7
      'Valencia' 100 Semi-solid 0.1 ± 0.1 35.3bc ± 5.5 0.3 ± 0.3 0.2 ± 0.2
      1The two cultivars were tested and analyzed separately. 2PS, GFP positive shoots. 3SE, Standard error. 4SL > 2, the number of shoots longer than 2 mm. 5TES, transformation efficiency based on the number of shoots. 6TEE, transformation efficiency based on the number of explants.

      Figure 3. 

      TES and TEE interval graphs for 'Florida EV1'. (a) The means, standard errors (SE) for TES (transformation efficiency based on the number of positive shoots/total number of shoots × 100), and multiple comparisons are shown in liquid medium at 0, 100, and 200 mg·L−1 and in semi-solid medium at 100 mg·L−1. (b) The means and standard errors (SE) for TEE (transformation efficiency based on the number of positive shoots/explants × 100) are shown in liquid medium at 0, 100, and 200 mg·L−1 and in semi-solid medium at 100 mg·L−1.

      For 'Valencia', the mean SL > 2 variable at 0 mg·L−1 was significantly greater (p < 0.05) than the other treatments (Table 3), but there were fewer shoots to screen (n = 177) at 200 mg·L−1 kanamycin (Table 2). In contrast, 361 shoots grew in the 0 mg·L−1 control. No significant differences existed for the TES or TEE variables (Table 3, Fig. 4a & b). Two transgenics were identified by GFP fluorescence at 100 mg·L−1, while four were identified at 200 mg·L−1 kanamycin in liquid medium, so selection was increased at higher kanamycin concentrations. Only one transgenic developed in the semi-solid control medium with 100 mg·L−1 (Table 2). No transgenics were produced without kanamycin selection in the liquid medium, and the PS variable was not significant (Table 3). One 'Valencia' transgenic died after grafting.

      Figure 4. 

      TES and TEE interval graphs for 'Valencia'. (a) The means and standard errors for TES (transformation efficiency based on the number of positive shoots/number of shoots × 100) are shown in liquid medium at 0, 100, and 200 mg·L−1 and in semi-solid medium at 100 mg·L−1. (b) The means and standard errors (SE) for TEE (transformation efficiency based on the number of positive shoots/explants × 100) are shown in liquid medium at 0, 100, and 200 mg·L−1 and in semi-solid medium at 100 mg·L−1.

      A nonparametric Mann-Whitney test was used to compare the median values for regeneration ability (the number of shoots per explant) between the two cultivars. Across all treatments, the median value for 'Florida EV1' was 0.71 (n = 40), which was significantly greater (p < 0.05) than the median value for 'Valencia' at 0.63 (n = 28). Yet in the control treatment at 0 mg·L−1 kanamycin in liquid medium, the median regeneration ability for 'Florida EV1' was 1.19 (n = 10) was not significantly different from 'Valencia's' median regeneration ability at 0.98 (n = 7). A Mann-Whitney test was also used to compare both cultivars' median values for GFP positive shoots (PS). The median value for 'Florida EV1' was 1.5 (n = 40), while the median value for 'Valencia' was 0 (n = 28), indicating a highly significant difference (p = 0.00) in the medium number of positive shoots between the two cultivars.

    • To test whether glyphosate can be used as a selectable marker in mature tissue transformation, an experiment to show glyphosate tolerance conferred by the EPSPS selectable marker was conducted with four levels of glyphosate (0, 2.6, 6.5, and 13.1 mM) with the transgenic lines and wild-type (WT). Nodal budsticks were plated on glyphosate medium, and the sprouting of shoots was measured after 28 d. An ANOVA of the data comparing transgenic and WT nodal budsticks for their ability to sprout shoots at these concentrations of glyphosate medium revealed significant differences (p < 0.05) between the two cultivars. Across all treatments and the control, 'Florida EV1' sprouted more shoots per node than 'Valencia' (Figs 5 & 6). At 0 mM glyphosate, 19 shoots sprouted from 12 nodal budsticks in 'Florida EV1', whereas in 'Valencia', 12 shoots sprouted from 12 nodal budsticks. Transgenic 'Florida EV1' had a mean of 0.9 (SE ± 0.1) sprouted shoots, while 'Valencia' had a mean of 0.5 (SE ± 0.1) shoots. Citrus lines 3 and 22 died on the glyphosate medium (photo not shown) (Fig. 5), whereas line 14 died after secondary grafting before this experiment. There was no significant difference among the different glyphosate concentrations, indicating that the lowest concentration (2.6 mM) was enough to select for herbicide resistance in transgenic mature citrus scions. All nodal budsticks of WT 'Florida EV1' and 'Valencia' died at all concentrations of glyphosate (Fig. 5, Supplementary Table S1).

      Figure 5. 

      Transgenic lines and wildtype (WT) nodal budsticks at two glyphosate concentrations (0 and 2.6 mM). Nodal budsticks of 'Florida EV1' (EV1) (samples 5, 12, 16, 17, 22, and WT) and 'Valencia' (Val) (samples 28 and WT) were plated on glyphosate (Gly) medium to show tolerance conferred by the TIPS-EPSPS transgene. Two additional concentrations of glyphosate were used but are not shown (Supplementary Table S1). 'Florida EV1' line 22 was later shown not to possess the EPSPS transgene.

      Figure 6. 

      Sprouted shoots per nodal budstick at four concentrations of glyphosate in semi-solid medium for 'Florida EV1' and 'Valencia'. In some treatments, 'Florida EV1' sprouted more than one shoot per nodal budstick.

    • PCR tests were performed for the transgenes (nptII, egfp, and EPSPS) in 23 'Florida EV1' and six 'Valencia' transgenic lines. All transgenic lines analyzed carried the nptII and gfp genes at the correct sizes (nptII 239 bp; egfp 713 bp) (Fig. 7). The 455 bp EPSPS transgene was present in all samples except 'Florida EV1' lines 3, 10, 14, and 22. Line 23 of 'Florida EV1' could not be tested with the EPSPS primers because it died.

      Figure 7. 

      PCR confirmation of transgenic lines transformed with the Mu-EPSPS vector and selected in liquid medium with gravity wells. (1a) 'Florida EV1' 1-23 samples, PCR for nptII 239 bp; (1b) egfp 713 bp; (1c) EPSPS 455 bp. Samples 3, 10, 14, and 22 contained the nptII gene but not the EPSPS gene. (2a) 'Valencia' 24-29 samples, PCR for nptII 239 bp; (2b) egfp 713 bp; (2c) EPSPS 455 bp. M: DNA ladder; P: plasmid; WT: wild-type.

      The 'Florida EV1' plants selected for duplexed TaqMan real-time PCR for copy number analysis were the ones that had the greatest number of shoots on the glyphosate selection medium (Supplementary Table S1) as well as the 'Valencia' transgenics. Eight transgenic lines were analyzed: four from 'Florida EV1' and four from 'Valencia' (Table 3). The maximum copy number determined was two. For 'Florida EV1', there were three transgenic lines with a single nptII copy and one with two copies of this gene. In the case of 'Valencia', there were two lines with one copy and two lines with two copies.

    • There are no previous reports of transformation experiments with mature 'Florida EV1'. Earlier, we transformed 'Valencia' with the EHA101 Agrobacterium strain and the GFP reporter[15] and obtained similar transformation efficiency for TEE as reported here if you calculate the efficiency based on the same number of explants in semi-solid medium at 100 mg·L−1 kanamycin. From the present study, we can conclude that liquid medium doubles the transformation efficiency compared to semi-solid medium with same kanamycin concentration. One additional report cited a higher, but still low, mature 'Valencia' transformation efficiency[14] , but another report produced no transgenics in mature 'Valencia' whatsoever[13].

      The most important measures in screening mature citrus shoots for transformation or gene editing in mature citrus are the number of shoots that must be examined that are long enough to be micrografted (SL > 2 mm) and the number of positive shoots (PS). Thus, the variable of transformation efficiency based on the number of shoots (TES) is the most valuable for our purposes[15,23,24,33]. In contrast, transformation efficiency based on the number of explants (TEE) better reflects the starting material (explants) required to initiate an experiment to find positive shoots. 'Florida EV1' is much more amenable to transformation and shoot regeneration, and it has repeatedly proven so in our transformation facility (unpublished results).

      In both mature and immature citrus biotechnology, the standard for kanamycin selection is 100 mg·L−1 in a semi-solid medium, except nptII is a poor selectable marker because it permits many escaped non-transgenic shoots to grow[15,22]. However, it is useful in both immature and mature citrus transformation and gene editing because it does not kill the explants. At higher concentrations of kanamycin in liquid medium, fewer escaped shoots grew from mature rootstock explants as determined by GUS assays[23]. From that study and the present work, it can be concluded that screening shoots of mature citrus scion explants in liquid medium with high concentrations of kanamycin is superior to screening shoots on semi-solid medium with 100 mg·L−1 kanamycin. Transgenics were even produced at 0 mg·L−1 kanamycin, which is a normal occurrence for mature citrus[33].

      'Florida EV1' lines 3, 10, 14, and 22 were positive for the nptII transgene but negative for the EPSPS transgene. EPSPS, on the 3' end of the T-DNA, was most likely truncated during T-DNA transfer because it is on the left border, which is not protected by the VirD2 protein[9,34,35].

      The 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) genes in plants and microbes occur in nature and have evolved naturally in response to glyphosate selection pressure. Several glyphosate-resistant EPSPS variants have been successfully engineered to impart herbicide resistance in a variety of crop plants[36,37]. The highly efficient citrus TIPS-EPSPS glyphosate selection system can not only serve as an alternative to antibiotic-based selection methods in the genetic transformation of citrus, but it can also facilitate the production of intragenic citrus plants. This present work suggests that it can be used as a selectable marker in mature citrus.

      Liquid culture maximizes the availability of water and nutrients and eliminates the adverse effects of contaminants in agar, which might contain impurities that limit plant growth[3842]. The shoots in the medium avoid hyperhydricity and anoxia because the vessel is aerated[43]. A thin layer of 3M paper or paper towel is added to the vessel, allowing the explants to contact the liquid without being submerged and suffering hypoxia. The gravity wells gradually dispense liquid as the explants and shoots use it. Simple systems such as stationary thin films often yield high-quality plant growth without the complexities and cost of mechanized systems that require shakers and aeration tubes[44]. Liquid selection in mature citrus rootstock reduces labor and costs and increases transformation efficiency[23], and the results were confirmed here with mature citrus scions. The liquid medium allows for larger, more elongated shoots for micrografting, better multiplication, and easier subculturing.

    • 'Valencia' is one of the most important citrus cultivars grown in the US, yet it is recalcitrant to Agrobacterium transformation. Mature 'Florida EV1', a 'Valencia' somaclone, has better transformation efficiency and regeneration ability than 'Valencia'. 'Florida EV1' performed remarkably well at all kanamycin concentrations in liquid and semi-solid medium. The TES variable in liquid selection in We-V™ vessels with gravity wells at stringent kanamycin concentrations (200 mg·L−1) was significantly better for identifying transgenics than in semi-solid medium at 100 mg·L−1 and the controls. A glyphosate assay with mature nodal budsticks suggests that the TIPS-EPSPS gene, which confers resistance to glyphosate, can be used as a selectable marker in mature citrus. 'Florida EV1' is being used in gene editing to alleviate disease problems afflicting the citrus industry in Florida. Transgenic and WT 'Florida EV1' trees produced fruit earlier than 'Valencia', which agrees with field performance data[18]. Future research will investigate the reasons for the differences in transformation efficiency and regeneration ability at the DNA sequence and chromosomal level between 'Florida EV1' and 'Valencia'.

    • The authors confirm contribution to the paper as follows: study conceptualization and design: Grosser J, Mou Z, Zale J; data curation: Canton M, Zale J; formal analysis: Canton M, Zale J; investigation: Canton M, Peraza-Guerra O, Wu H; methodology: Canton M, Peraza-Guerra O, Wu H; validation: Canton M, Zale J; draft manuscript preparation: Canton M, Peraza-Guerra O, Zale J; visualization: Canton M, Zale J; resources: Grosser J, Mou Z, Zale J; funding acquisition: Zale J; supervision: Zale J. All authors reviewed the results and approved the final version of the manuscript.

    • All data generated or analyzed during this study are included in this published article and its supplementary information files.

      • We wish to acknowledge the University of Florida Institute of Food and Agricultural Sciences (IFAS) and the Citrus Research and Education Center (CREC) for Citrus Initiative Funding.

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

      • Supplementary Table S1 The number of nodal budsticks and shoots at each glyphosate concentration in the two cultivars.
      • Copyright: © 2024 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 (7)  Table (3) References (44)
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    Canton M, Peraza-Guerra O, Wu H, Grosser J, Mou Z, et al. 2024. A mature, sweet orange cultivar derived from 'Valencia' with high Agrobacterium transformation efficiency. Fruit Research 4: e036 doi: 10.48130/frures-0024-0030
    Canton M, Peraza-Guerra O, Wu H, Grosser J, Mou Z, et al. 2024. A mature, sweet orange cultivar derived from 'Valencia' with high Agrobacterium transformation efficiency. Fruit Research 4: e036 doi: 10.48130/frures-0024-0030

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