Introduction

Globally, Mexican lime (Citrus aurantifolia Swingle) is produced in several countries, including Mexico, Brazil, India, Peru and Egypt (^{Plattner, 2014}). The juice is used in the preparation of drinks and the peel for the extraction of pectin, whereas the essential oils are used in the perfume industry (^{Liu, Heying, & Tanumihardjo, 2012}). Nutritionally, Mexican lime is important for its contribution of ascorbic acid (vitamin C), minerals, citric acid and bioactive compounds such as limonin glucoside, a highly-oxygenated triterpenoid associated with anticancer activity (^{Jacob, Hasegawa, & Manners, 2000}).

In Mexico, production of Mexican lime, with spines or criollo (a native, regional variety), is estimated at 1.4 million tons (^{Servicio de Información Agroalimentaria y Pesquera [SIAP], 2014}), which is mainly sold in the domestic market, since only 2.3 % of production meets the color, size, juice content and health characteristics required for export (^{Plattner, 2014}). The fruits of Mexican lime with spines are small, with a high number of seeds, a thin epicarp and accelerated senescence; the latter results in a short shelf life, so a high percentage of the harvested fruit is destined for either industrial purposes or the fresh market.

Based on studies of natural genetic variation, conducted by the National Institute for Forestry, Agriculture and Livestock Research (INIFAP) through its Mexican Lime Breeding Program, three registered varieties have been generated: Colimex, Lise and Colimón. The first are trees with spines, which have high yield and larger fruit size; the second is similar to the first but in trees without spines, and the third produces seedless fruits (^{Robles-González, Carrillo-Medrano, Manzanilla- Ramírez, Velázquez-Monreal, & Medina-Urrutia, 2010}). In addition, the fruits of these varieties have better quality characteristics in terms of juice content, flavor components and refrigerated storage potential (^{Muñoz- Lazcano, Saucedo-Veloz, García-Osorio, & Robles- González, 2011}); however, their response to the use of bioregulators with phytohormonal effect, aimed at reducing the advance of deteriorative changes related to senescence, which significantly affect the quality of the fruits and limit their shelf life, is still unknown.

Physiologically, the fruit has non-climacteric behavior (^{Kader, 2000}), so in postharvest it shows changes associated with the phenomenon of senescence which, among other processes, involves a decrease in photosynthetic capacity and chlorophyll content, vitamin C losses and changes in membrane permeability, which favor the loss of water in cells and tissues (^{Goldschmidt, 2000}). These processes represent the main factors that significantly affect the quality and reduce the shelf life of Mexican lime fruits (^{Álvarez-Armenta et al., 2010}). In this regard, significant losses in the internal and external quality of untreated fruits of the Colimex, Lise and Colimon varieties, stored at 22 ± 2 °C for six days, have been reported (^{Muñoz- Lazcano et al., 2011}).

The use of plant hormones such as auxins (^{Agustí et al., 2002}), gibberellins (^{Jomori-Lye, Kluge, & Jacomino, 2003}) and cytokinins (^{Baéz-Sañudo, Tadeo, Primo-Millo, & Zacarías, 1993}) has been studied to assess their effect on the slowing of senescence-related changes in citrus fruits. In these fruits, pre-harvest applications with gibberellic acid (GA₃) have been evaluated in order to delay senescence, increase epicarp firmness, delay the harvest and control physiological disorders, all with contrasting results due to the effect of species, variety, dose, and application method, among other factors (^{Ritenour, Burton, & McCollum, 2005}). The aim of this research was to study the effect of pre-harvest treatment with GA₃ on controlling the progress of senescence in the fruit of three Mexican lime varieties, and its effect on fruit quality and shelf life.

Materials and methods

This study used Mexican lime fruits of the Colimex, Lise and Colimón varieties, obtained from the Tecomán Experimental Station, Colima, Mexico, belonging to the Institute for Forestry, Agriculture and Livestock Research (INIFAP), located at 32 masl, with a BS₁(h’) climate, considered as warm semi-dry, with an average temperature of 26 °C and mean annual rainfall of 750 mm.

Prior to harvest, two groups of three trees each were formed; gibberellic acid (GA₃) was applied to the first group when the fruits reached 30 ± 2 mm in diameter; applications were made to the second group on two occasions, when the fruits were 20 ± 2 mm and 30 ± 2 mm in diameter. A third group of six trees remained untreated. The GA₃ solution with a concentration of 30 mg∙L^-1 was prepared with granulated ACTIVOL^® 40 % GS; Penetrator^® Plus at a ratio of 10 mL∙L^-1 was used as surfactant. At 9:00 am the fruits were sprayed with 10 L of this solution per tree.

The harvest of the fruits, with and without GA₃, was carried out when they reached 39 ± 2 mm in diameter (in accordance with paragraphs 4 and 5 of the Mexican Standard: NMX-FF-087-SCFI-2001, Secretaría de Agrícultura, Ganadería, Desarrollo Rural, Pesca y Alimentación ^{[SAGARPA], 2014}). After 12 h they were transferred to the laboratory where they were conditioned for 12 h at room temperature, and subsequently selected on the basis of overall health.

Based on the above, the factors studied in this research were:

Number of pre-harvest GA₃ applications (zero, one or two). When two applications occurred, the fruits reached 20 ± 2 mm in diameter in the first, and 30 ± 2 mm in the second. In the case of one application, the fruits had 30 ± 2 mm in diameter.

Application of water-based wax in postharvest (with and without). This treatment consisted of immersing the fruits into the water-based wax (14 % Carnauba wax solids); then they were dried with airflow at room temperature.

From these levels, four treatments were generated (with a sample size of 60 fruits), which were: a pre-harvest GA₃ application and postharvest waxing (1-GA₃), two GA₃ applications and waxing (2-GA₃), postharvest waxing and without any treatment (control).

Subsequently, the fruits were stored for 10 days (22 ± 2 °C and 60 ± 5 % relative humidity), after which the following variables were determined:

Fruit weight loss (%). It was obtained with respect to initial fruit weight.

Color index (CI). This parameter was determined in whole fruit using the expression CI = 1000a / bL (^{Jiménez, Cuquerella, & Martínez, 1981}); parameters L, a, b were obtained with a HunterLab Model D25A optical sensor system, Reston, Virginia, USA.

Chlorophyll concentration (mg∙100 g^-1). It was obtained from the epicarp by spectrometry, performing the extraction with acetone (^{AOAC, 1990}).

Titratable acidity (% with respect to citric acid).

Total soluble solids (TSS, in %). It was determine by the methods described by ^{AOAC (1990)}.

Ascorbic acid concentration (mg ascorbic acid∙100 mL^-1). It was determined by the 2,6-dichlorophenol-indophenol method (^{AOAC, 1980}).

Determination of non-destructive variables, CI and weight loss, as well as destructive ones: chlorophyll, citric acid, TSS and ascorbic acid. It was performed on a sample composed of five replications of four fruits each. An analysis of variance based on a factorial treatment design in a completely randomized arrangement was performed, comparing the means of the treatments by the Tukey test (P ≤ 0.05) using the Statistical Analysis System statistical package (^{SAS, 2002}).

Results and discussion

Significant differences in weight loss due to the treatment effect were observed (Table 1), being lower in fruits treated with one or two GA₃ applications compared to those treated with wax and those in the control. Several researchers (^{Báez-Sañudo et al., 1993}; ^{Tafolla-Arellano, González-León, Tiznado-Hernández, Zacarías-García, & Báez-Sañudo, 2013}) have reported the cuticle’s role in regulating water loss in fruits, as well as the effect, in Clementine mandarin (Citrus reticulata [Hort] Ex Tanaka, cv. Nules), of GA₃ treatments in maintaining the permeability of the flavedo cuticles by preventing their breaking due to changes in the lipid fraction related to senescence, which results in reducing water losses by transpiration; this allows GA₃ to take similar action in Mexican lime fruits.

Regarding the variety effect, Colimón fruits had the greatest weight loss (Table 2), a response suggested in the hypothesis concerning differences in changes related to the lipid fraction of cuticular waxes among the lime varieties studied. On the other hand, it has been reported that Mexican lime fruits with a weight loss of less than 6 - 7 % are suitable for marketing purposes (^{Muñoz-Lazcano et al., 2011}), which allows establishing that only GA₃-treated fruits had this condition after the established storage period and temperature. The variety × treatment interaction (Table 3) indicated that the lower weight loss corresponded to the treatments with GA₃, establishing that a single application was favorable for reducing this problem.

The CI results revealed significant differences due to the effect of the treatments, with the fruits treated with one or two GA₃ applications being those that after 10 days of storage at 22 ± 2 °C had values corresponding to a greener tone, which was confirmed when the treatments showed a higher chlorophyll concentration in the epicarp compared to those that were only waxed and the control (Table 1). This allows assuming that the pre-harvest application of GA₃, at the dose and fruit growth stages established, is effective in slowing the degradation of chlorophyll due to the effect of the progress of senescence in postharvest, thus prolonging shelf life.

Increases in chlorophyll content and delay of senescence due to GA₃ applications in citrus as have been reported by several researchers studying ways to delay the harvest (^{García-Luís, Herrero-Villén, & Guardiola, 1992}; ^{Mcdonald, Greany, Shaw, & Mccollum, 1997}; ^{Porat et al., 2001}). On the other hand, the fruits of the Colimex and Lise varieties presented, significantly, CI values corresponding to a greener tone, compared to the Colimón variety. This suggests that the latter has greater metabolic activity in postharvest, resulting in a faster degradation of chlorophyll; the lowest concentration of this pigment quantified in this variety confirms this response (Table 2). The results of the variety . treatment interaction (Table 3) confirm the positive effect of GA₃ applications in slowing the loss of the epicarp’s green color, with only one application being effective.

The statistical analysis showed that, in treatments with GA₃, TSS increased significantly compared to the waxed fruit and the control (Tables 1 and 3). For its part, the percentage of TSS was higher in the Colimex and Lise varieties than in Colimón (Tables 2 and 3). As in other citruses, sugar accumulation in Mexican lime fruits occurs during growth and maturation, by the source-demand mechanism (^{Iglesias et al., 2007}). Postharvest, the sugar content tends to decrease with advancing senescence, due to the interconversion of sugars to other compounds (^{Yun et al, 2013}.), which explains the higher TSS concentration in GA₃-treated fruits due to presenting less progress of this physiological event.

In relation to citric acid, treatments with GA₃ and waxing had, after 10 days at 22 ± 2 °C, a significantly higher concentration compared to the control (Table 1). It has been reported (^{El-Otmani & Coggins, 1991}) that, in citruses, treatment with GA₃ applied pre-harvest decreases the postharvest loss of citric acid; this same behavior has been observed in Mexican lime fruits with GA₃ applications and stored after harvest, without waxing, at 9 ± 1 °C for 35 days (^{Álvarez et al., 2010}). It should be noted that no significant differences among varieties were observed (Table 2); the same occurred with the variety x treatment interaction (Table 3), so it is assumed that the treatment did not affect the concentration of this compound.

As for the ascorbic acid concentration, although no significant differences between the treatment and variety factors were observed (Tables 1 and 2), the variety . treatment interaction (Table 3) showed that the fruits with GA₃ applications significantly increased their concentration of this vitamin, particularly with two applications; moreover, this response was more evident in the Colimex and Lise varieties. It has been noted that, in postharvest, the decrease in ascorbic acid occurs due to, among other factors, conditions that favor water loss (^{Lee & Kader, 2000}), a response that was evidenced by the fruits of the Colimex and Lise varieties, which had less weight loss.

Conclusions

Pre-harvest treatments with GA₃, at a concentration of 30 mg∙L^-1, in fruits of Mexican lime varieties Colimex, Colimón and Lise, with one (20 ± 2 mm in diameter) or two applications (20 ± 2 and 30 ± 2 mm in diameter), waxed postharvest (14 % Carnauba) and stored at 20 ± 2 °C for 10 days, slow the progress of senescence by presenting less physiological weight loss, an epicarp with a higher chlorophyll concentration and greener tone, and a higher concentration of citric acid and TSS in juice. For cost reasons, a single application is recommended. The treatment with two applications has a higher concentration of ascorbic acid, which increases the nutritional quality. Delay of senescence, as a function of the above-mentioned parameters, is more effective in the Colimex and Lise varieties, keeping, therefore, their quality longer, resulting in a longer shelf life compared to the Colimón variety.