Plant material

The commercial saladette tomato hybrid 'Condor' (Ahern Seeds) with indeterminate growth was used. The experiment was carried out from April to August 2016 in a greenhouse located in the Plant Science Department’s Experimental Field at Autonomous Chapingo University, State of Mexico, Mexico (19° 29’ 25” LN and 98° 52’ 23” LW, at 2,240 masl), with an average annual temperature of 15.9 °C.

Growth bioregulators applied

Four commercial chemicals with growth bioregulatory activity were used: ethephon (Ethrel 240, Bayer^®, Mexico), calcium prohexadione (Apogee^®, BASF, USA), iodine (Q-2000 Plus^®, Quimcasa, Mexico) and sodium selenite (45 % sodium selenite^®, Retorte, Germany). First, 2.5 L of each bioregulator were prepared under laboratory conditions, transported to the experimental site (greenhouse) in amber bottles with a screw cap, and coated with aluminum foil to prevent their degradation by light. Each bioregulator was individually applied to the experimental units on a foliar basis with a 20-L portable manual backpack (Swissmex^®, Mexico).

Crop management

Sowing was carried out in 200-cavity polystyrene trays, with a mixture of peat moss and vermiculite (90:10) as substrate. After 35 days the transplant was carried out in gutters (with dimensions of 25 x 1 x 0.6 m) at a density of 8 plants·m^-2 of useful greenhouse area (without considering aisles), for which the plants were pruned at the fifth cluster and led to a single stem. The gutters were filled with red tezontle (igneous volcanic rock with high iron dioxide content), with a particle size of 3 to 5 mm in diameter.

The supply of essential elements for crop growth and development was carried out according to the parameters established by Steiner’s solution (^{Steiner, 1984}) (Table 1), supplemented with micronutrients (mg·L^-1) (iron [2], manganese [1], copper [0.05] and zinc [0.05]), with electrical conductivity values fluctuating between 2.5 and 3.0 dS·m^-1. Nutrients were supplied via irrigation (3 to 5 irrigations per day) with 0.30 to 3.0 L·plant^-1, depending on the weather conditions (environmental temperature and relative humidity) and phenological stages of the crop. Temperature was controlled manually by opening and closing the side windows protected with anti-aphid mesh. The fruits used for laboratory analysis were from the second and third bunches, with maturity stage six, that is, when the fruit had 90 % red coloration (^{Choi, Lee, Han, & Bunn, 1995}), which coincides with ripeness for consumption.

Experimental design

The experiment was conducted under a completely randomized experimental design with ten replicates, where the experimental unit consisted of eight plants. In total, two foliar applications of bioregulators (Table 2) were made per treatment, at 45 and 60 days after transplanting (dat). The control was maintained without application.

Evaluated variables

Fruit color. It was determined on the epidermis, in the equatorial part of the fruit, by means of a portable sphere spectrophotometer (SP-62, X-Rite^®, USA). The CIE 1976 color coordinates (L^* a^* b^*) (^{Voss, 1992}) were obtained, and from there the values of chromaticity (C* = [a²+b²]^1/2) and hue angle or °h (arctan^-1 [b/a]).

Fruit weight (g). It was obtained by means of a digital electronic scale (Scout Pro SP 602, Ohaus^®, USA), with a capacity of 0.6 kg and sensitivity of 0.01 g.

Polar and equatorial diameter (mm). They were measured with a Vernier caliper (CAL-6MP, Truper^®, Mexico) on the polar and equatorial plane of the fruit.

Roundness index (dimensionless). With polar and equatorial diameter data, this index was calculated using the expression RI = pd/ed, where pd and ed are the polar and equatorial diameter, respectively.

Firmness (N). It was determined on the epidermis and in the equatorial zone of the fruit with a digital texturometer (Compact Gauge, Mecmesin CE™, USA).

Total soluble solids (TSS, °Brix). They were quantified with a portable digital refractometer (PAL-1, Atago^®, USA), for which two drops of fruit juice were placed on the device’s optical reader.

Titratable acidity (TA, % citric acid). It was determined in accordance with the methodology proposed by the ^{Association of Official Analytical Chemists (AOAC, 1990)}. First, 20 g of fruit were homogenized with 50 mL of distilled water, then 10 mL of the mixture were taken and neutralized with a NaOH solution (0.1 N), in which 1 % phenolphthalein was used as an indicator.

Lycopene (mg·100 g^-1 fresh weight). The lycopene concentration was quantified according to the method modified by ^{Sadler, Davis, and Dezman (1990)}. First, 20 g of tissue were taken and homogenized with 50 mL of distilled water. The mixture obtained was placed in a flask covered with aluminium foil and dried at 38 °C. Subsequently, 0.1 g of the pulp was taken and placed in a test tube covered with aluminium foil, to which 30 mL of a mixture of hexane, ethanol and acetone (2:1:1) were added and stirred for 10 min. After this time, 18 mL of distilled water were added and stirred again for 5 min. The mixture was separated into two phases (aqueous and organic). With separation flasks, the volume of the organic phase was taken and indicated, and its absorbance was determined at 470 nm. The lycopene content was calculated using the formula indicated by ^{Inbaraj and Chen (2008)}.

With one fruit per experimental unit and ten replicates, color, weight, polar and equatorial diameter, and roundness index were evaluated. For firmness, TSS, TA and lycopene content, two fruits per experimental unit and three replicates were used.

Statistical analysis

The data obtained were verified for normality and homogeneity of variances with the Kolmogorov-Smirnov and Bartlett tests, respectively (^{Sokal & Rohlf, 1995}). Subsequently, one-way analysis of variance and Tukey’s test (P ≤ 0.05) were performed using Statistical Analysis Software (^{SAS Institute, 2002}).

Color

No statistical differences were detected in the brightness and hue (°h) of the fruits among the evaluated treatments (Table 3), which was confirmed visually with the presence of red fruits, but with orange hue tendencies (°h between 51.88 and 59.51) and with low brightness values (L* between 29.33 and 31.07). In this regard, the onset of tomato fruit ripening is characterized by the production of phytoene, a colorless compound related to color development, as it induces the synthesis and accumulation of lycopene (red) (^{Carrillo-López & Yahia, 2014}; ^{Casierra-Posada & Aguilar-Avendaño, 2008}), which coincides with the decrease in brightness of the red color (^{Becvort-Azcurra et al., 2012}).

Fruits from plants treated with 200 mg·L^-1 of calcium prohexadione and 3 mL·L^-1 of iodine showed greater color purity with respect to the control (33.10); however, they did not surpass what was shown by the other treatments, with values fluctuating between 34.00 and 40.53. In contrast, ^{Islam et al. (2018)}, when evaluating various growth bioregulators (sodium selenate and potassium iodide at a concentration of 1 mg·L^-1) in ‘Unicorn’ cherry tomato at harvest, reported no significant variation in relation to color (a*/b*), with values between 0.65 and 0.66. On the other hand, ^{Pezzarossa et al. (2014)} indicate that selenium decreases the ripening rate by temporarily inhibiting the ethylene biosynthesis pathway, in addition to contributing to the antioxidant defense system against reactive oxygen species, caused by abiotic factors (presence of salts or heavy metals) (^{Caffagni et al., 2012}).

Fruit weight

Among the fruits harvested, the heaviest (133.71 g) were those from plants sprayed with 1.6 mL·L^-1 of ethephon (Table 3), representing a 62.65 % increase with respect to the control plants (80.36 g). In this sense, ^{Atta-Aly, Riad, Lacheene, and Beltagy (1999)}, when making foliar applications with 100 mg·L^-1 of ethephon on 'Castle Rock' tomato flower bunches, obtained a significant increase in fruit fresh weight (123 g) compared to the control (113 g). These same authors point out that the dose used is not the most suitable for inducing flower abscission, but it is sufficient to change the growth pattern of the fruit; that is, it increases the cell division and elongation processes (^{Marzouk & Kassem, 2011}). This may suggest that applying a lower concentration, such as the one used in this study, may be positively involved in cell division and elongation of newly-set fruits, favoring a larger size, as indicated by ^{Uchanski and Blalock (2013)}.

On the other hand, applying calcium prohexadione caused an alteration in the synthesis of active gibberellins in the apexes of the stems (^{Altintas, 2011}) and an increase in the translocation of photoassimilates via phloem by the fruit (^{Çetinbaş et al., 2015}), as also reported by ^{Uchanski and Blalock (2013)} in cayenne pepper (Capsicum annuum L.) ‘Mesilla’. In contrast, ^{Meland and Kaiser (2011)} found a linear reduction between fruit weight and an increase in ethephon concentration applied to apple (Malus sylvestris [L.] Mill. var. domestica [Borkh] ‘Summerred’) two weeks after flowering. On the other hand, the treatment with calcium prohexadione showed a reduction in the number of fruits and a better distribution of available photoassimilates (^{Jiang et al., 2011}). These results contrast with those reported in date palm (Phoenix dactylifera L.) (^{Mohammed-Al-Saif, Issa-Alebidi, Sultan-Al-Obeed, & Saad-Soliman, 2017}), fig (Ficus carica) (^{Crisosto et al., 2010}) and macadamia (Macadamia integrifolia) (^{Trueman, McConchie, & Turnbull, 2002}). The variability is attributed to the planting season, the applied concentration and the phenological state of the crop (^{Crisosto et al., 2010}; ^{Shinozaki et al., 2015}).

Firmness

Among the fruit quality parameters most appreciated by the consumer is the firmness of the pulp (^{Figueroa-Cares et al., 2018}), which is related to its morphological characteristics and the agronomic management provided in pre- (adequate nutritional and water input, as well as the control of fungal and bacterial diseases) and postharvest (harvest index and appropriate conservation methods) (^{Casierra-Posada & Aguilar-Avendaño, 2008}). In this research, applying 5 mL·L^-1 of iodine resulted in the highest firmness value (3.26 N) with respect to the control, while the values obtained with 200 mg·L^-1 of calcium prohexadione and 3 mL·L^-1 of iodine were the lowest for this variable (Table 3).

^{Saure (2014)} indicates that obtaining adequate fruit growth requires increasing the synthesis and concentration of gibberellins (GB), of which the most important in the pericarp of the tomato fruit are those of the GB1 and GB20 types (^{Bohner, Hedden, Bora-Haber, & Bangerth, 1988}). The GB1 content during fruit growth and development is high, and the Ca²⁺ concentration is reduced; this is generated by the plant to allow cell expansion and increase membrane permeability, which is restored when the fruit has reached physiological maturity (^{Marschner, 1995}). In this sense, applying a GB synthesis inhibitor, such as calcium prohexadione, is usually an alternative to reduce the risk of calcium deficiency in the early stages of fruit growth and development (^{Saure, 2014}).

For its part, iodine is a non-essential microelement for plants (except for some aquatic species); however, it does have some beneficial effects (^{Caffagni et al., 2012}) as it has been reported that it can induce a decrease in the respiration process and ethylene synthesis in cherry tomato fruits (^{Islam et al., 2018}), which maintains the integrity of the cell wall (^{Dhall & Singh, 2013}; ^{Saure, 2014}). This is desirable if the fruit’s shelf life is to be increased.

^{Islam et al. (2018)} mention that 'Unicorn' cherry tomato fruits sprayed with 1 mg·L^-1 of potassium iodide had firmness values of approximately 15.86 N, which was similar to the application of 1 mg·L^-1 of sodium selenate (16.82 N). In this study, sodium selenite treatments showed no significant variation (Table 3). The disparity between what has been studied and what has been reported in the literature, according to ^{Caffagni et al. (2012)}, may be associated with the iodine uptake and accumulation capacity of the tissues, which in most cases is a function of the applied concentration, time of application, age, and type and morphology of the organs (leaves, roots and fruits) (^{Kiferle et al., 2013}; ^{Landini, Gonzali, & Perata, 2011}).

Equatorial and polar diameter

Fruit growth directly influences its characteristic shape, and a quantitative way to determine it is by equatorial and polar diameter (^{Figueroa-Cares et al., 2018}; ^{Montoya-Holguin, Cortés-Osorio, & Chaves-Osorio, 2014}). In this study, none of these variables showed significant statistical differences among treatments, whose values were 43.33 to 55.6 mm and 59 to 77.14 mm, for equatorial and polar diameter, respectively (Table 4). In contrast, ^{Atta-Aly et al. (1999)}, with the application of 100 mg·L^-1 of ethephon in ‘Castle Rock’ tomato (S. lycopersicum L.) plants at 10, 12, 18, 24, 30, 35, 40, 45, 50, 55 and 60 days after floral opening, found a greater response from day 30 in fruit diameter (from 5.71 to 6.98 cm), behavior associated with the increase in the number and size of cells (^{Shinozaki et al., 2015}). These same authors mention that concentrations of 300 mg·L^-1 of ethephon, applied in days after floral opening, cause the formation of abscission points and flower fall, a situation that did not occur in this study because the applied ethephon dose was much lower.

On the other hand, the use of calcium prohexadione is related to lower vegetative growth; that is, it acts as a growth retardant by inhibiting the synthesis of GB located at the apex of the stems (^{Schmitzer, Veberic, & Stampar, 2012}), which induces the differentiation of floral buds and an increase in fruit set (^{Altintas, 2011}). ^{Çetinbaş et al. (2015)} indicate that calcium prohexadione blocks the synthesis route of active GB, since it shortens shoot length, which decreases the competition for photoassimilates among newly-set fruits and reduces vegetative growth (branches and leaves) (^{Ramírez et al., 2012}). This could be associated with the presence of larger fruits (^{Crisosto et al., 2010}).

Roundness index

The relationship between the equatorial and polar index expressed as the fruit roundness index was not significant (P ≤ 0.05) among treatments (Table 4), which indicates the presence of the characteristic "flattened" pattern (values < 1); that is, fruits with greater polar than equatorial diameter (^{Montoya-Holguin et al., 2014}). In this sense, ^{Figueroa-Cares et al. (2018)}, when evaluating commercial varieties and native genotypes of cherry tomato, also do not report significant statistical variation in this variable. ^{Becvort-Azcurra et al. (2012)} mention that this characteristic shows greater correlation with the genotype than with environmental aspects and agronomic management.

Total soluble solids (TSS) and titratable acidity (TA)

Application of ethephon, calcium prohexadione, iodine and sodium selenite did not show significant statistical difference with respect to the control in terms of TSS accumulation in harvested fruits (Table 5), whose values were between 4.15 and 5.15 °Brix. These results coincide with the findings reported by ^{Islam et al. (2018)}, who indicate that they did not find significant changes in TSS in 'Unicorn' cherry tomato when they evaluated individual applications of potassium iodide (6.67 °Brix) and sodium selenite (6.69 °Brix) at a concentration of 1 mg·L^-1 at five weeks after harvest.

Soluble sugars in the form of disaccharides (sucrose) and monosaccharides (fructose and glucose) are among the main compounds that provide or favor the presence of the characteristic flavor in fruit and vegetable products (^{Beckles, 2012}). However, another minor factor is the synthesis and accumulation of organic acids (citric, malic and tartaric), of which citric acid predominates in the case of tomato (^{Caicedo-Orjuela & Galvis-Venegas, 2012}). In this sense, preharvest application of 125 mg·L^-1 of sodium selenite allowed obtaining fruits with the highest TA (Table 5), while ^{Islam et al. (2018)}, when applying 1 mg·L^-1 of potassium iodide and sodium selenate in 'Unicorn' cherry tomato fruits, found no significant statistical variation in TA; however, their reported values are higher (1.03 and 1.04 % citric acid for each treatment, respectively). Additionally, ^{Lee et al. (2007)} report higher TA values in 'Super Momotaro' tomato fruits with sodium selenium applications at 30 dat, that is, under conditions very similar to the present study.

Lycopene

Table 5 shows that the lycopene content of the fruits harvested from plants treated with bioregulators had, in most cases, a similar behavior in relation to the control, except the treatments with 50 mg·L^-1 of calcium prohexadione and 3 mL·L^-1 of iodine, whose concentration of this carotenoid decreased significantly (8.92 and 9.15 mg·100 g^-1, respectively). In contrast, ^{Ramírez et al. (2012)}, when applying 125, 175 and 200 mg·L^-1 of calcium prohexadione in 'Floradade' tomato fruits with 10 true leaves (vegetative growth), report a four- to eight-fold increase compared to the control. These authors found their best result when they evaluated fruits with green shoulders (maturity stage four), that is, when the fruit had more than 30 % red coloration, but not exceeding 60 % (^{Choi et al., 1995}). ^{Altintas (2011)} points out that prohexadione may be involved in the modification of the synthesis routes of several secondary metabolites, including carotenoids such as lycopene, and impact on antioxidant capacity, thus providing health benefits (^{Ramírez et al., 2008}).

Based on the conditions of this research and the results obtained, the bioregulators that had a greater response were ethephon, iodine and sodium selenite, without observing significant differences in relation to calcium prohexadione. If the goal is to increase the cell division and elongation processes, and obtain fruits of greater weight, the use of ethephon is suggested. Conversely, if the aim is to decrease respiration and ethylene production, and increase fruit firmness and shelf life, iodine application may be an excellent alternative. These recommendations are related to the fact that they are relatively inexpensive products and are available from commercial agrochemical product stores.