1 Introduction

Solanum lycopersicum is an annual crop and one of the popular fresh vegetables grown in tropical, subtropical, and temperate climates of Nepal. Alongside cauliflower, cabbage, broccoli and radish, tomato is also among the fresh vegetables grown the most in terms of area and productivity in Nepal (MoALD, 2023). It is a plant with angular and hairy stem, pinnately compound leaves that are lobed and alternate and flowers are borne in clusters on the main axis and on lateral branches (Mallick, 2021). Its fruit is globular/ovoid and may be either bilocular or multilocular consisting of about 50 to 200 seeds enclosed in a gelatinous membrane within the locular cavities (OECD, 2017). Nutritionally, tomato is rich in various vitamins such as Vitamins A, C, and K and carotenoids such as lycopene and carotene which act as antioxidants (Esguerra and Rolle, 2018). It is used for several purposes such as multiple culinary uses (salad, pickle, cooked vegetable, sauce, ketchup) while its high acidic content makes it popular for canning (Bhandari et al., 2016). It is a highly perishable climacteric vegetable that over ripens and softens quickly leading to decreased quality and limited shelf life (Batu, 2004).

During the recent fiscal year, the area of production of tomato has increased up to 22 911 hectares and resulted in a total production of 422 703 metric tonnes i.e., yield of 18.45 Mt/Ha (MoALD, 2023). The tomato industry has experienced a significant surge in consumption and demand, correlating with increased production. Particularly in the hilly areas where plastic house and off-season production during summer rainy seasons are trending with comparative advantages (Ghimire et al., 2017). Despite the promising demand, abundant opportunities, and heightened productivity potential there are considerable post-harvest losses. The adoption of post-harvest techniques lags among the farmers, primarily due to a lack of technical knowledge and insufficient extension visits (Dhakal and Maharjan, 2023). The alternate solutions such as use of “Cold storage” is not practical as well as feasible as green tomatoes develop chilling injury at temperatures lower than 12 °C (Rugkong, 2009), later inducing physiological disorders such as surface pitting (Soleimani Aghdam et al., 2012), total failure of fruit color development (Rugkong, 2009), increased susceptibility to Alternaria rot (Ding et al., 2002), and prove to be detrimental to tomato flavor quality (Maul et al., 2000). Other solutions such as modified atmospheric packaging, controlled atmospheric packaging, etc. require high investment cost, energy and technology which developing countries like Nepal cannot facilitate. For that reason, research on alternative postharvest management methods that are economically friendly, readily available, and feasible for use by marginal farmers in developing countries is imperative such as the use of CaCl₂ as mentioned by Arah et al. (2016). 

Exogenous treatment with GA₃ in tomatoes reduced TSS and pH, but maintained high levels of titratable acidity and ascorbic acid content. The optimum concentration for such effects was reported to be 0.1% and 40 mg/L, respectively (Pila et al., 2010; Singh and Patel, 2014). These findings indicate that GA₃ can delay respiration and metabolic activity, hence improving postharvest quality and extending shelf life under different storage conditions. This is in agreement with the findings of Demes et al. (2021) and Dhami et al. (2023).The treatments of calcium chloride of variable concentrations, such as 0.5%, 1%, 1.5%, and 2%, were found to exert a great impact on the post-harvest quality and shelf life of tomatoes by reducing TSS, pH, and sugar levels while maintaining higher titratable acidity and ascorbic acid content in tomatoes (Pila et al., 2010; Mazumder et al., 2021). These treatments effectively retard metabolic activity, slowing down the ripening process and hence increasing the shelf life. Very often, optimum effects were obtained at 1.5% or 2% CaCl₂, under both ambient and cold storage conditions (Abbasi et al, 2013; Demes et al., 2021; Dhami et al., 2023). Pre- and post-harvest treatments of SA have been found to influence tomatoes' quality and shelf-life characteristics, which are reflected in higher retention of ascorbic acid and titratable acidity, delay in accumulation of TSS, and extension in storage life. From these, the best results recorded varied between 0.4-1.2 mM on account of conditions prevalent for storage and the different varieties of tomato, reported earlier on by Baninaiem et al. (2016); Mandal et al. (2016); Kumar et al. (2018) and Chavan and Sakhale (2020). The present investigation aims to study the impact of exogenous application of plant growth regulators gibberellic acid, salicylic acid, and calcium chloride as a post-harvest treatment method on tomato chemical quality and shelf life under ambient conditions. 

2 Materials and Methods 

2.1 Procurement of tomato (sample)

The “Shrijana” variety of tomatoes, the most grown variety throughout the seasons in Nepal, was procured from farmers in Shankharapur municipality, Sankhu, Kathmandu. Fresh healthy tomatoes harvested in the turning and pink /breaker stage, evenly proportioned, unbruised, and with no injury and signs of disease were selected and collected.

2.2 Experimental design and treatment detail

This study employed the Completely Randomized Design (CRD), which consisted of 10 treatments, each replicated three times (Table 1). 

The tomatoes were cleaned, washed, sterilized using sodium hypochlorite (500 ppm for 10 min), and air dried before dipping in respective chemical solutions. A sample size of 10 tomatoes was allocated for each treatment. After treatment, the fruits were kept in a makeshift aluminum bowl. Data were taken in alternate day intervals until signs of decay or spoilage were observed and then, the chemical parameters were analyzed after 15 days of storage and on the 25^th day of storage until commercial condition. The ambient temperature of the storage room was noted. 

2.3 Parameters observed

2.3.1 Total soluble solids (TSS)

Determination of TSS which is the total soluble solid present in the unit volume of solution was carried out using a handheld Refractometer. A drop of the blended tomato juice sample was placed on the prism and the percentage of dry substances in it was read directly. The TSS value thus obtained is expressed in º Brix.

2.3.2 Titratable acidity and sugar acid ratio

The juice from the sample was extracted and filtered. After, the dilution of the extract i.e., 10 mL juice mixed with 100 mL of distilled water aliquot was formed. Three drops of phenolphthalein indicator were added to 25 mL of the aliquot. Titration was conducted using 0.1 N NaOH alkali solution until the pink endpoint was reached and persisted for 30 seconds. The quantity of alkali consumed was recorded for 3-4 readings. 

The volume of alkali consumed was measured by subtracting the initial reading from the final reading. Furthermore, the following formulae were used for the calculation of percentage acidity and the sugar-acid ratio:

2.3.3 pH

pH was determined using a digital pH meter. The juice was extracted from the experimental sample and filtered to remove solid particles. Then, pH meter probe was immersed into the extracted juice and kept stable for two minutes to ensure an accurate reading. To enhance the reliability of the measurement, the instrument was calibrated using standard buffer solutions before the measurement and cleaned it after each use to prevent cross-contamination. The final reading was recorded as the pH value of the sample, which was used for further analysis of its changes over storage time and under different treatments.

2.3.4 Ascorbic acid

Ascorbic acid or vitamin C can be determined by the redox titration method. In this method, dichlorophenolindophenol (DCPIP) was used as a dye for titration of the centrifuged sample until the sample changed its color to pink. The ascorbic acid measurement was calculated using the formula below (Ranganna, 2015).

100 mL of conical glass was filled with five milliliters of working standard using a pipette. After that, 10 mL of 4% oxalic acid was added, and the dye was titrated. The amount of ascorbic acid was equivalent to the amount of dye that was consumed until the moment at which pink coloring appeared. 

In 4% oxalic acid, two milliliters of the sample were extracted, and 12 milliliters of a known volume were prepared and centrifuged. A titration against the dye was performed using five milliliters of this supernatant, to which 10 milliliters of 4% oxalic acid was added. The quantity of titer in the formula corresponded to this dye consumption.

2.3.5 Shelf life

Tomato’s shelf life was determined by calculating the number of days required to attain the last stage of ripening and then at the point where the fruit could still be sold i.e., it remained acceptable for marketing. 

2.3.6 Marketable fruit (%)

The percentage of marketable fruit was calculated to evaluate the postharvest quality and shelf-life maintenance of the treated samples. The calculation was performed using the following formula: 

2.4 Statistical analysis

Data was systematically arranged using observed parameters, and analysis of variance was performed using EXCEL and GENSTAT. The available literature was used to assist in the analysis of the data and the discussion of the findings.

3 Results and Analysis 

3.1 Total soluble solid (º Brix)

ANOVA showed statistically similar values between treatments (GA₃, CaCl₂, SA, and the control) and TSS in the tomatoes (Table 2). The TSS values ranged from 4.35º Brix to 5.26º Brix during the 15-day storage period, where control had the maximum TSS value of 5.26º Brix. Among the treatments, T4 (0.5% CaCl₂) exhibited the lowest TSS value, followed by T1 (0.1% GA₃) than other treatments. 

3.2 Titratable acidity

Titratable acidity is the total acid concentration in fruits. TA decreased showing a significant difference (p<0.001) with a grand mean of 0.53% on the 15^th day of storage (Table 3). The highest TA was identified in T1 (0.1% GA₃) with 0.58% and T6 (1.5% CaCl₂) with 0.57%, while the lowest TA was found in the control (0.49%). Except for these, SA also proved effective in maintaining higher TA levels valued at 0.56% with treatment T9 (0.3 mM SA). However, after 25 days of storage, CaCl₂-treated fruits showed maximum retention of titratable acidity (0.47%) than gibberellic acid-treated tomatoes valued at 0.43%. 

3.3 Total sugar-acid ratio

The statistical result revealed that there was a significant difference in total sugar acid ratio (p<0.05) with the lower sugar acid ratio found in T1 (0.1% GA₃) and T6 (1.5% CaCl₂) and the highest sugar acid ratio found in T10 or the control (Figure 1). After 25 days of storage, an increased sugar-acid ratio was observed in the remaining treatments (T1, T2, T3, T4, T5, and T6) signifying the increased concentration of sugar and decreasing acidity levels. 

3.4 pH

A highly significant difference (p<0.01) was observed in tomatoes treated with GA₃, CaCl₂, and SA during the storage period of 15 days. The highest pH was recorded in T10 (control) while the lowest pH was in T3 (0.3% GA₃) (Table 4). As the acidity decreases with ripening, pH is bound to increase. Therefore, on 25^th day, pH levels were comparatively increased than on the 15^th day in all the remaining treatments where tomatoes treated with CaCl₂ had the lowest pH values. This relationship between acidity and pH is explained by Demes et al. (2021) by stating that the fluctuations in pH can be attributed to the decreasing levels of acidity caused by the increased activity of citric acid glyoxylase during ripening. 

3.5 Ascorbic acid

The effects of GA₃, CaCl₂, and SA on the ascorbic acid content of tomatoes during the storage period of fruits are shown in Table 5. There was a significant difference in ascorbic acid content among the treated fruits and untreated fruits (p<0.001). Treatments T1 (0.1% GA₃), T3 (0.3% GA₃), and T6 (1.5% CaCl₂) retained higher amounts of ascorbic acid valued at 14.23, 11.73, and 11.43 mg/100g of tomato fruit, where the maximum retention was observed in T1. A similar pattern was observed on the 25^th day, even with increased ripening and increased degradation of ascorbic acid, among the remaining treatments (T1, T2, T3, T4, T5, and T6) T1 managed to retain higher ascorbic acid (12.5 mg/100 g) than the control set had in the 15^th day. While the other treatments had ascorbic content levels ranging from 6.89 to 7.75 mg/100 g.

3.6 Shelf life days and marketable fruit% 

The data shows shelf life and marketable fruit % were significantly (p < 0.05) affected by GA₃, CaCl_2, and SA during the storage period (Figure 2). The fruits treated with T1, T6, and T9 extended the average shelf life to almost twice that of the control (15 days). Fruits treated with T1 (0.1% GA₃) had the longest possible average shelf life of 29 days followed by those treated with T6 (CaCl₂ 1.5%) at 27 days and T9 (0.3 mM SA) at 24 days. In the same fashion, the results for marketable fruit% follow almost the exact pattern where T1, T6, and T9 have twice the amount of marketable fruit than in control (35.57%) i.e., 65.2%, 64.94%, and 64.74% respectively. 

4 Discussion

During ripening, fruits undergo significant changes in their carbohydrate composition and organic acids. The TSS content increases with the breakdown of starch and sugar accumulation with storage time, as observed in the study throughout the storage period. 0.1% GA₃-treated tomatoes recorded the lowest TSS values, signifying the effectiveness of GA₃ in delaying the ripening process. However, despite these variations in TSS values, ANOVA results indicate these differences are not statistically significant. This was similar to the study conducted by Bhattarai and Gautam (2009) and Senevirathna and Daundasekera (2010). Furthermore, Genanew (2013) also observed the corresponding insignificancy of CaCl₂ to TSS of tomato fruits during storage and suggested it could be due to less reduction by volume in solids, unlike liquids and gases. Shafiee et al. (2010) results on strawberries reported no effects of salicylic acid on the TSS values. 

On the contrary, as the storage period increased, the TA values were observed to decrease. This could be due to the fruit's utilization of the acids for metabolic activities of living tissues, resulting in the depletion of organic acids during storage (Bhattarai and Gautam, 2009). When compared to the control, the values were much higher in the treated tomatoes. GA₃ and CaCl₂ were the most effective treatments for the maintenance of TA levels. In this regard, the view of Pila et al. (2010) is noteworthy that calcium-treated fruits had significantly higher retention of TA which might be due to the reduction of metabolic changes of organic acid into water and carbon dioxide. Furthermore, Singh and Patel (2014) also reported the supremacy of GA₃ over other treatments including borax and KHCO₃ for having the highest TA (1.05%) on the 6^th day of storage. The ability of GA₃ to maintain higher titratable acidity may be attributed to its role in suppressing ethylene biosynthesis and delaying ripening-related metabolic processes, also observed by Devkota et al. (2019) and Senjaliya et al. (2015) in tomato fruits. Ünal et al. (2021) also reported higher values of TA at 10 days in tomatoes treated with salicylic acid. 

These sugar levels and acidity are crucial for determining the quality and taste of tomato fruit, both in its raw state and when processed (Lambeth et al., 1964). The relationship between the acidity and soluble solids i.e., ‘Sugar acid ratio’ is taken as the measurement of fruit maturity rather than assessing the acidity or soluble solids individually (Gustavo et al., 2003). Gautam and Bhattarai (2006) stated that sugar/acid balance and the astringent compound result in “Flavor”. The higher sugar-acid ratio in T10 indicates a higher concentration of sugar than acid which correlates to higher level ripening and maturity. On the contrary, the lower TSS: acid ratio in treated tomatoes signifies that these treatments have potential anti-ripening effects (Asghari and Aghdam, 2010). 

pH in tomatoes progressively increases with maturation and is found to have increased with ripening (Yamaguchi, 1960). All treatments exhibited comparatively lower pH than the control however, except those treated with CaCl₂ as it seemed to have little effect on the pH of tomatoes which was also in the case of a study conducted by Senevirathna and Daundasekera (2010) in tomatoes. All the concentrations of SA were able to maintain significantly lower levels of pH. Similar findings were reported by Pila et al. (2010) in tomatoes stored in ambient conditions. 

The concentration of ascorbic acid acts as an indicator of the fruit’s development stage and overall health. A decrease in ascorbic acid typically indicates senescence in fruit while an increase shows that the fruit is still in the ripening stage (Esteves et al., 1984).The findings of this study align with that of Pila et al. (2010) who observed the highest ascorbic acid content retention in tomato fruits treated with 0.1% GA₃ and concluded all three treatments of gibberellic acid, calcium chloride, and salicylic acid were beneficial in retarding degradation of ascorbic acid content. Likewise, in the case of calcium chloride higher concentrations were more effective in the retention of ascorbic acid i.e., 1.5% CaCl₂ (11.43 mg/100 g)> 1% CaCl₂ (10.01 mg/100 g) > 0.5% CaCl₂ (9.87 mg/100 g). Similar findings were observed by Mazumder et al. (2021) that the ascorbic acid content decreased with ripening, however, higher retention in ascorbic acid content was found in tomatoes treated with 1% to 2% CaCl₂ harvested at the breaker stage after 10 days of storage duration. Such trends in tomatoes treated with CaCl₂ were also observed by Chepngeno et al. (2016). The SA-treated tomatoes exhibited higher ascorbic acid content than the control, among which the highest observed in T9 (0.3 mM SA) valued at 10.36 mg/100 g. In justification of our result, Baninaiem et al. (2016) reported that SA-treated tomato fruits showed comparatively higher levels of ascorbic acid than the control set and suggested SA effectively protects the cell wall by decreasing the expression of degrading enzymes, slows down the ripening and hence reduces the degradation of ascorbic acid (concurrently with degradation of fruit tissues). The studies done by Changwal et al. (2021) also mentioned SA was found helpful in maintaining higher levels of ascorbic acid in tomatoes. 

Our findings on the shelf-life extension of tomatoes with the results of Pila et al. (2010) who reported that the fruits treated with 0.1% GA₃, 1.5% CaCl_2, and 0.4mM SA had the most significant extension of the shelf-life by 18, 17 and 15 days respectively. This can be attributed to the negative roles of GA₃ in the ripening of tomatoes (Li et al., 2019; Dhami et al., 2023). Similarly, higher concentrations of CaCl₂ (1% and 1.5%) resulted in tomatoes' longer shelf life as Bhattarai and Gautam (2009) reported that the higher the concentration of CaCl₂, the higher the shelf-life. Moreover, fruits treated with SA did exhibit longer shelf-life days than the control, however, it wasn't as significant as other treatments. This discrepancy might be due to the concentrations of SA used to treat fruits and storage temperature, contrary to Mandal et al. (2016) who compared lower concentrations of SA (0.2, 0.4, 0.6, and 0.8 mM) with higher concentrations of SA (1 and 1.2 mM) at refrigerated conditions and found that higher concentrations of SA were significantly effective in extending the shelf life of tomatoes up to 32.75 days. Likewise, the treatment of tomatoes with 0.75 mM salicylic acid prolonged the shelf life by 7 days along with a lower weight loss percentage, and was proved to be more effective than oxalic acid (Kant et al., 2013). 

The use of 0.1% GA₃ and 1.5% CaCl₂ as a postharvest treatment could provide a cost-effective solution which are rather readily available, and simple to prepare and use for smallholder farmers in Nepal. These plant growth regulators will not only extend the tomatoes' shelf life but also provide the farmers an opportunity to negotiate better prices for their hard work. This could potentially reduce postharvest losses and prove to be functional for commercial tomato farmers, retailers/wholesalers with improved marketability, and ultimately consumers. Future studies should investigate the efficacy of these treatments on the same variety as well as different varieties under varied environmental conditions to ensure broader applicability.

Authors’ contributions

PD handled the sample collection, data collection, data analysis, and article writing. During the research period, KR served as the primary supervisor and helped with the article preparation in addition to coming up with the research topic. CB aided on the manuscript draft. All authors read and approved the final manuscript. 

Acknowledgments

The authors sincerely acknowledge Himalayan College of Agricultural Sciences and Technology (HICAST), Purbanchal University, Kirtipur, Kathmandu and Prime Minister Agriculture Modernization Project (PMAMP), PIU Bhaktapur, Nepal for providing the opportunity to conduct this research. 

Conflict of Interest Disclosure

The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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