The concentrations of 7.5% and 11% sucrose were used to achieve osmotic balance between the strawberries and the impregnation solutions.Isochoric chambers , constructed from aluminum- 7075 with a type-II anodized coating, with a total volume capacity of 1.5 L and pressure rated up to 210 MPa, was used for the isochoric cold storage . Initially, a steel nut, serving as an ice nucleating agent, was placed at the base of the isochoric chamber to ensure that ice formation occurred away from the sample bags. Subsequently, a plastic spacer apparatus was inserted into the chamber, and the samples were transferred onto the plastic apparatus, with three bags placed in each chamber. The chambers were then sealed after being filled with water. The isochoric chambers were subsequently stored in chest freezers at −2 ◦C for 7 days. The freezing temperature of −2 ◦C was chosen based on preliminary work to avoid high pressures that would result in cell damage. In an isochoric environment, the temperature and the volume of unfrozen liquid in the chamber are correlated. At the triple point , about 45% of the initial volume remains unfrozen. As the temperature rises, this percentage increases, reaching 90% of the initial volume at −2 ◦C, 20 MPa. By carefully controlling temperature and pressure, strawberries can be kept within the unfrozen region of the system, ensuring they remain preserved without ice formation. Following the ICS, the chambers were relocated to a fridge at 5 ◦C and left overnight to allow the ice within the chamber to melt.The addition of sucrose and calcium chloride can influence microbial populations on strawberries. Sucrose, as a fermentable sugar, grow bags garden may promote the growth of certain microorganisms, including yeasts and molds, which utilize sugars as a primary energy source .

The reduction of microorganisms in strawberries treated with CaCl2 may be attributed to the ability of calcium salts to lower intracellular pH or reduce water activity.Visible signs of decay mark the point when strawberries are no longer acceptable for consumption. While fungicide applications during the growing cycle are the traditional method for controlling postharvest decay, their use is increasingly questioned due to sustainability and safety concerns, with bans in many countries. Alternatives like pulsed light, hypobaric treatment , or ultrasound help to slow decay but do not fully prevent spoilage. In contrast, the findings of this study demonstrate that the pressure applied during ICS successfully inactivated fungal growth on strawberries after a week. Furthermore, fungal growth remained undetectable during the subsequent 3 weeks of refrigerated storage at 4 ◦C. The classification of fruit decay as simply “rotten” and “non-rotten” is a limitation in our study. A more detailed decay assessment method will provide a more comprehensive understanding of the decay process and enable a better evaluation of treatment effectiveness.The changes in strawberry weight over the storage period are depicted in Figure 3. After 1 week of storage at 4 ◦C, RF and RF + C strawberries lost weight, while RF + S strawberries showed weight gain. As shown in Figure 3, the ICS strawberries in both impregnation solutions experienced weight gain after 1 week due to the solution penetrating the porous tissue and intercellular spaces through a hydrodynamic mechanism consisting of capillary action and pressure gradients. Throughout storage, the refrigerated strawberries experienced significant weight loss, reaching up to 80% by week 4. This was primarily due to moisture loss from evaporation between the fruit tissue and the surrounding environment as well as the respiration processes. Given their extremely thin skin, strawberry fruits are highly prone to rapid water loss. However, strawberries subjected to isochoric impregnation had limited weight loss compared to the refrigerated samples. This can be attributed to the mechanisms of isochoric impregnation, which involve pressure-induced mass transfer, allowing the liquid solution to penetrate the fruit’s porous structure.

This process not only enhances the retention of moisture within the fruit but also creates a barrier to water loss, effectively mitigating dehydration.The moisture content, pH, titratable acidity , and total soluble solid parameters of the strawberries are presented in Table 2. The moisture content of all the strawberries in solution either slightly increased in value or remained stable after 1 week, which could be due to an infusion of the solutions. However, the RF + S and RF + C samples had significantly lower moisture contents by weeks 3 and 4, albeit not as low as those of the RF sample. ICS impregnated strawberries exhibited less moisture loss than refrigerated strawberries , regardless of the solution used. The pH values of the RF + C and ICS + C samples were significantly lower than the others due to acidic nature of ascorbic acid. All samples showed a decrease in pH value throughout storage. The titratable acidity of fresh strawberries was 1.8%. All the strawberry samples showed an increase in TA during storage, in agreement with Vicente, Martínez, Civello, and Chaves. In addition, the TSS of all the strawberry samples significantly increased in value during storage. This was attributed to moisture loss, the breakdown of complex sugars into simpler sugars, the degradation of cell walls, and the overall decay of the fruit. After 4 weeks, the RF + S and RF + C strawberries became too small and shriveled, making it impossible to perform pH, TA, and TSS analyses.The hardness values of the strawberries are presented in Table 4. The RF strawberries showed a reduction in hardness during the refrigerated storage, especially between week 1 and week 2. This decrease in firmness is mainly related to biochemical alterations at the cell wall, middle lamella, and membrane levels due to the activity of pectin methylesterase, an enzyme that hydrolyzes pectin, leading to structural breakdown. However, the firmness of the RF sample during storage increased to a higher level than that of the fresh sample, which had been attributed to an increase in pectin viscosity.

Additionally, water loss from the outer layer can increase fruit density, lower gas permeability, reduce oxygen levels, and elevate internal carbon dioxide concentrations, which may contribute to the firmer texture. The refrigerated strawberries also showed an increase in hardness throughout the storage period, likely attributed to a significant loss of internal water content in the cells. However, the samples had high standard deviations since certain strawberries remained quite firm while others were very soft. The breakdown in texture may have been caused by the increase in aerobic microbial counts, which could have led to the production of pectinolytic enzymes responsible for tissue softening. The ICS strawberries were harder than the RF strawberries for the same solution. Also, the strawberries impregnated with the solution containing sucrose, CaCl2, and AA were harder than the strawberries impregnated only with the sucrose solution. The increase in hardness was attributed to the addition of exogenous calcium ions to the strawberry fruit via impregnation with pressure. Calcium ions are essential for maintaining fruit quality by inhibiting the activity of polygalacturonase , an enzyme responsible for breaking down cell wall components such as pectins. Furthermore, Ca2+ binds with demethylesterified pectin backbones to form a pectin–Ca2+ network, which strengthens the mechanical properties of the cell wall and helps preserve fruit texture. Koushesh Saba and Sogvar reported that Ca2+ helped maintain and enhance the integrity and mechanical properties of the cell wall, effectively preventing the softening of fruits.Figure 5 shows the total anthocyanin content of the strawberries during storage. The total anthocyanin content of the fresh strawberries was 20.2 ± 4.4 mg/g dry matter . The strawberries stored in sucrose solution for one week did not show a significant change in the total anthocyanin content , whereas the strawberries in the sucrose + CaCl2 + AA solution showed a significant decrease in the total anthocyanin content . The high anthocyanin content in the RF + S samples can be attributed to the sucrose treatment, grow bag for tomato which stimulates anthocyanin accumulation by upregulating the expression of genes involved in anthocyanin biosynthesis. The CaCl2 treatment positively influenced the retention of monomeric anthocyanins during storage by facilitating pectin–anthocyanin binding. The presence of ascorbic acid accelerated anthocyanin degradation and led to a loss of color, indicating a direct interaction between the two molecules. The lower pH in the strawberries impregnated with the AA solution could also contribute to the reduced anthocyanin content, since the stability of anthocyanins is influenced by pH.The total anthocyanin content of the refrigerated samples significantly decreased during storage . The anthocyanin degradation might be associated with water loss during storage, leading to physiological stress and accelerating fruit senescence.

Water loss led to membrane disintegration and leakage of cellular contents, both of which contributed to the decrease in anthocyanin concentration. In addition, the increase in enzyme activity, such as polyphenol oxidase, may also have contributed to the reduction in anthocyanin content in the strawberries during storage. The ICS samples showed significantly lower anthocyanin contents after one week compared to the refrigerated samples. This initial decrease can likely be attributed to the effects of pressure and impregnation during isochoric treatment, which may introduce physical stress and promote anthocyanin degradation. At week 4, the ICS samples exhibited significantly higher total anthocyanin contents compared to the RF, RF + S, and RF + C samples , becoming redder and darker over time due to the synthesis of anthocyanins, the pigments responsible for the red color in strawberries.This study examined the effects of isochoric cold storage at −2 ◦C/48 MPa in combination with isochoric impregnation with sucrose solution or sucrose solution containing calcium chloride and ascorbic acid on the quality of strawberries. The refrigerated strawberries at 4 ◦C experienced growth of mesophilic aerobic bacteria, yeasts, and molds over the 4-week storage period, whereas isochoric impregnation effectively inhibited the growth of these microorganisms over the same period. After 4 weeks, refrigeration at 4 ◦C resulted in significant weight loss in the strawberries, with reductions of 79% in the CaCl2 solution and 82% in the sucrose solution. In contrast, ICS helped minimize weight loss, with reductions of 68% in the CaCl2 solution and 60% in the sucrose solution during refrigerated storage. Also, ICS strawberries in the presence of CaCl2 and ascorbic acid showed better mechanical properties, color stability, and higher nutrient content than those in the sucrose solution or under refrigeration. Overall, ICS with sucrose, CaCl2, and ascorbic acid impregnation proved to be a highly promising postharvest technology for extending the shelf life of strawberries for up to 4 weeks. This study highlights the potential of ICS not only for improving the storage stability of strawberries but also as a sustainable alternative to conventional methods. Future research should focus on scaling up this technology and evaluating its feasibility for commercial applications, offering a pathway to reduce postharvest losses and meet the growing demand for longer-lasting, high-quality fresh produce.Whole genome duplications , also known as polyploidy, are an important recurrent process over evolutionary time that have contributed to the origin of novel phenotypes and driven species diversification across eukaryotes . Polyploids are species that contain three or more complete sets of chromosomes in each nucleus, ranging from triploid to dodecaploid. For example, two rounds of whole genome duplication, termed 1 R and 2 R events, are unique to vertebrates. 1 R preceded the origin of crown vertebrates, while 2 R occurred in the lineage leading to bony vertebrates after the divergence of the cyclostome lineage. Many retained duplicated genes from these two ancient polyploidy events have functionally diverged and are associated with the evolution of several novel structures including the neural crest, cartilage, bones and/or adipose tissue. Similar patterns have also been reported following ancient polyploidy events in yeast and plants. Polyploids often evolve novel phenotypes and show greater phenotypic plasticity, which may explain certain polyploid lineages surviving mass-extinction events and exhibiting subsequent shifts in net diversification rates. There are two main categories of polyploids; autopolyploids and allopolyploids. Autopolyploids are formed from genome doublinginvolving a single diploid progenitor species, while the formation of allopolyploids involves genome doubling after hybridization of two or more diploid progenitor species. Newly formed allopolyploid genomes may experience instability, as the previously separate genomes of each diploid progenitor species, known as subgenomes, have evolved independently and now coexist in a single nucleus.