Foliar parameters

Temperature (°C) and chlorophyll (SPAD Units) in leaves were measured simultaneously with a Thermometer IR digital thermometer brand Radioshack and a Chlorophyll Meter SPAD 502 brand Konica Minolta respectively. The youngest and best developed leaf in each plant was taken as reference. Three measurements were made after each application every 5 days. Leaf transpiration (mmol H₂O m^-2 s^-1) and photosynthesis (µmol CO₂ m^-2 s^-1) were obtained with a LI-COR Model LI-6400 TX Portable Meter, in sheets described above.

These measurements were made twice, 5 days after the first application and 37 days after the second spray. Finally, the intrinsic efficiency of the water use ((µmolCO₂) (mmolH₂O)^-1) was obtained with the ratio of photosynthesis and leaf transpiration.

Yield

The total yield per plant resulted from the sum of the weight of fruits harvested in 10 cuts, using a Scout^® Pro scale with a capacity of 1 000 g.

Antioxidants

The vitamin C content in fruits was determined using the method reported by ^{Padayatt et al. (2001)}. The 10 g of pericarp of the fruit were macerated with 10 ml of 2% hydrochloric acid (v/v), then the mixture was homogenized in 40 ml of distilled water, filtered through gauze and collected in an Erlenmeyer flask. The 10 ml of the solution were taken and titrated with 2,6-dichlorophenolindophenol (1x10^-3 N), until the solution turned pink. The vitamin C content was determined using the following formula:

The lycopene content was obtained from 3 g of fresh pericarp weight of the fruit. The samples were placed in a frozen mortar containing 3 ml of phosphate buffer (pH 7) and ground, from the obtained mixture 2 ml were taken and placed in centrifuge tubes, 4 ml of the hexane-acetone mixture was added (3: 2), the mixture was stirred to separate and dissolve the pigments from the membranes (^{Davis et al., 2003}), centrifuged at 3 000 rpm for 10 min for phase separation, the colored phase was extracted and quantified at a wavelength of 450 nm on a Varian brand HPLC kit, model 500-MS. To quantify the lycopene content in the samples, a standard lycopene calibration curve (Sigma, Co) was constructed with a range of 0-40 mg mL^-1 previously dissolved in the aforementioned solution. The samples were compared to the calibration curve and the lycopene content was determined using a linear regression equation.

Mineral content

For the analysis of minerals (N, P, K, Mg and Ca) in fruits and leaves, three plants were randomly selected for each treatment. The mineral content in leaves was determined by carrying out three samplings in an interval of 20 days starting from the beginning of flowering. The determination of minerals in fruits was carried out in the first cut when 3 samples were taken at random from the 10 repetitions per treatment. The process of mineral analysis corresponding to Ca, Mg, K and P was carried out in two stages, extraction and quantification. For the extraction, the samples were dried in a Felisa Model FE-291 stove at 70 °C for 72 h, they were ground and one gram of the sample was subjected to the calcination process at 600 °C for two hours, the ashes obtained they were recovered with hydrochloric acid 1:1 and gauging at 100 m with distilled water. In the second phase the concentration of the minerals Ca, Mg, and K (mg kg^-1) were read in an atomic absorption spectrophotometer Varian Spectrc AA5, while the concentration of phosphorus (mg kg^-1). It was carried out by obtaining the absorbance by photocolorimetry with a wavelength of 560 nm. Nitrogen (%) was determined using the modified procedure of micro-Kjeldahl digestion (^{Jones, 1991}).

Total biomass

To determine the biomass (g), a destructive sampling of three randomly selected plants was carried out in each treatment. Samples were weighed on a Scout^® Pro scale with a scale of 0 - 1 000 g to obtain fresh weight, then the plants were placed in brown paper boses and placed inside a Felisa FE 291 drying ovens at 70 °C four days and then the dry weight was obtained.

Foliar parameters

The effect obtained when applying bioregulators on the temperature of the leaf is shown in Figure 1. The individual treatments with gibberellin A_4/7 when compared with the control, significantly increased leaf temperature (DMS p≤ 0.05), whereas Applications with P-Ca and 6-BAP at 50 mg L^-1 showed the lowest temperature in the tissue. ^{Ferreyra et al. (2002)} have related the temperature of the plant with the evolution of the hydric state of the plant; effect that they relate to the variations of water supply to tissues. ^{Goldhamer et al. (1999)} and ^{Matthews et al. (1987)} indicate that the temperature of the plant is related to the water stress of the atmosphere adjacent to it.

Figure 1 Effect of bioregulators on leaf temperature of tomato plants hybrid “Raptor-F1” saladette. Each point represents the average of ten repetitions. *= statistical differences. DMS (p≤ 0.05). 

The gibberellins are hormones that increase cellular elongation, causing a greater flow of water to the tissue, a condition that would cause a greater fluctuation of the temperature of the leaf and manifest a faster water deficit (^{Ramírez et al., 2016a}) as it can appreciate in Table 1 where the water efficiency ((µmolCO₂) (mmolH₂O)^-1) shown by the plants under treatment were lower than the control in the first sampling. The P-Ca is a plant retardant that causes in plants a partial closure of stomata and therefore a reduction in water loss through the leaf (^{Rademacher, 2004}) which causes this hormone to slightly increase the temperature of the plant. leaf having an intrinsic efficiency of water use ((µmolCO₂) (mmolH₂O)^-1) (Table 1) lower than the control.

The chlorophyll content in the leaves of all treatments with bioregulators was lower than the control (Figure 2). The levels of chlorophyll in the leaves treated with P-Ca were close to the control. ^{Bekheta et al. (2009)} showed in bean plants that P-Ca at 10, 20 and 30 mg L^-1 increased the pigment content. ^{Fletcher and Hofestra (1985)} proposed that the optimal application of bioregulators cause improvements in various plant plants such as increased levels of chlorophyll and a wide range of chloroplasts. The authors carried out their studies with plants in the open field. The present study was carried out in a greenhouse and with different concentrations of bioregulators. The lower amount of chlorophyll observed among them could reflect the concept of supra-optimal concentration reported in other crops (^{Rademacher, 2000}).

Figure 2 Effect of bioregulators on the chlorophyll content of hybrid saladette tomato plants “Raptor-F1”. Each point represents the average of ten repetitions. *= statistical differences (DMS p≤ 0.05). 

It is important to point out that chlorophyll is an exponential growth factor in the plant that often adopts a curve that grows at the beginning of the crop but that usually decreases at the end of the production cycle (^{Zermeño et al., 2015}). This natural behavior was modified by applying AG_4/7 at 50 and 100 mg L^-1 and with the combination of AG_4/7 and 6-BAP at 50 and 100 mg L^-1. It can be observed that days after the applications, the greenness of the leaves (represented in SPAD units) is reduced immediately and managed to recover 15 days after the application (Figure 2). P-Ca follows a natural pattern of chlorophyll production, which could be explained by its ability to inhibit the formation of biologically active gibberellins (^{Ramírez et al., 2003}; ^{Ramírez et al., 2016a}). Plants sprinkled with 6-BAP at concentrations of 50 and 100 mg L^-1 showed values lower than P-Ca; however, the behavior of the chlorophyll curve was not affected, this agrees with that reported by ^{Ros et al. (2004)} who observed these same trends when making 6-BAP applications in a cotton crop.

In the Table 1 shows the effects of bioregulators on photosynthesis (µmol CO₂ m^-2 s^-1), leaf transpiration (mmol H₂O m^-2 s^-1) and the intrinsic efficiency of water use ((µmolCO₂) (mmolH₂O)^-1) in sheets. It was observed, in the majority of the treatments with bioregulators that they did not modify these physiological processes adversely and therefore they can be considered very positive for future studies that allow to broaden the knowledge about photosynthesis and stomatal conductance in tomato under greenhouse conditions.

Total biomass

Figure 3 shows the total fresh and dry biomass of the treatments. P-Ca and 6-BAP at 50 mg L^-1 were similar (p≤ 0.05) to the control in the variable Total Fresh Weight (PFT). In previous P-Ca investigations in doses higher than 100 mg L^-1 increased the number of leaves, stem diameter and number of internodes as well as the fresh and dry biomass of the plant (^{Ramírez et al., 2016a}). In Golden Delicious apple tree this relationship was also observed (^{Ramírez et al., 2003}). This demonstrates the fact that P-Ca is a biologically active gibberellin synthesis blocker (^{Rademacher, 2004}) that acts as an apical growth retardant and stimulates the synthesis of cytokinins causing the vegetative increase of other organs of the plant (^{Costa, et. al., 2004a}). This experience would lead to consider in the present study the non-effect due to low dose of those bioregulators used in tomato.

Figure 3 Influence of bioregulators on fresh and total dry biomass in tomato plants hybrid “Raptor-F1” saladette. Each bar represents the average of ten repetitions. Bars with the same letter are statistically equal (DMS p≤ 0.05). 

In total dry weight (PST) (Figure 3) we observed a statistically significant difference (p≤ 0.05) among the treatments where AG_4/7 + 6-BAP showed to be effective when increasing the dry matter of the plants. These effects have been observed in cocoa by ^{Cardenas-Hernandez et al. (2010)} who evaluated the effect of gibberellic acid and 6-benzylaminopurine on the development of buds in grafts of that species. At a general level, these results support the postulate that gibberellins and 6-BAP promote cell elongation, increasing the growth of the plant (^{El Fouly et al., 1988}; ^{Wilson-García et al., 2007}).

The application of any bioregulator did not alter the content of N, Mg and Ca in leaves (Table 2). According to ^{Gutiérrez (1997)} the flow of minerals in plants is varied since it depends on the availability and demand between organs, as well as the phenological stage of the same; however, these elements are required for the metabolism of the plant since they fulfill structural functions in organic molecules, in the energy and ion reserve and redox reactions (^{Aven et al., 1992}). Based on these experiences, the effects on the physiology of the tomato observed in this study (Table 2) are interesting since they do not observe detriment in nutrient levels.

In most treatments, the phosphorus content was similar to the control; whereas, the potassium content was higher in the AG_4/7 + 6-BAP treatments at any dose (Table 2). The efficiency of bioregulators in plant nutrition has been related to the translocation of nutrients to certain tissues (^{Medjdoub et al., 2002}; ^{Rademacher, 2004}) where a stimulus or inhibition of their appearance or development can be caused (^{Ramírez et al., 2010}).

The Table 3 shows the nitrogen levels accumulated in the fruit. The treatments AG_4/7 at 50mg L^-1 and AG_4/7+ 6-BAP at 100 mg L^-1 presented a greater amount of this element, surpassing the control in 2.1 and 8% respectively. It has been shown that nitrogen is directly related to the protein content and is also a promoter of the synthesis and production of sugar in fruits (^{Kjellbom and Larsson, 1984}). In a study conducted by ^{Ramírez et al. (2016a)} it was shown that AG_4/7 at 100 mg L^-1 increased the content of brix degrees and firmness and prolonged the shelf life in the fruits. The above is relevant since the higher sugar content in the fruit there is the possibility of a shorter shelf life (^{Siller-Cepeda et al., 2004}).

The content of phosphorus in fruits varied between treatments (Table 3). The bioregulators AG_4/7 and 6-BAP at 100 mg L^-1 showed increases of 9.4 and 9.7% respectively in relation to the control. ^{Gutiérrez (1997)} showed that phosphorus is translocated in greater quantity to the fruits when they begin their development. The potassium content was not affected by the bioregulators and they maintained levels similar to the control.

^{Ramírez et al. (2010)} showed that P-Ca does not affect K levels in peel tomato fruits. ^{Barrera et al. (2008)} reported that the potassium content in the plant ranges between 2.7% and 4.5%, influences photosynthesis, transport of carbohydrates and plays an important role as an antagonistic element of nitrogen in fruits, besides regulating the entry and metabolism of nutrients. In human medicine, potassium occupies the third place among the minerals that act most in our body and plays an important role in most vital functions (^{Hopkinson et al., 1998}); however, ^{Hobson and Davies (1980)} indicate that a high concentration of potassium in fruit is associated with a low quality of it.

A slight and lower content of magnesium in fruits resulted in most of the treatments with bioregulators (Table 3). It is possible that this variation is caused by the concentration of hormones used and by the physiological state of the moment in the development-maturation of the fruit. ^{Gutiérrez (1997)} mentions that the flow of Mg in plants is varied and depends on the availability and demand between organs of the plant and the stage of development of the same. On the other hand, ^{Betancourt and Pierre (2013)} point out that the order of Mg extraction in tomato is of leaf > stem > fruit > root, making it possible to demonstrate that the amount of Mg that accumulates in fruits depends on the amount stored in stems and leaves.

This is reflected when comparing the Mg found in leaves (Table 2) where the plants sprinkled with the bioregulators were similar to the control, condition that could cause an unbalanced shipment of Mg towards the fruits. The previous thing, would reflect the greater amount of Mg observed in leaves when being compared with fruits. These data coincide with those published by ^{Fayad et al. (2002)} who reported a greater Mg accumulation in the vegetative part of the tomato in relation to the fruit.

It can be observed that the calcium content was higher in the treatment 6-BAP at 50 mg L^-1 (Table 3) while P-Ca at 50 mg L^-1 also showed a greater tendency to control. ^{Betancourt and Pierre (2013)} observed that the level of Ca accumulated in tomato fruits was gradual starting from leaves > stems and that generally the accumulation in fruits is usually very low, this presumably because the Ca has little mobility in the phloem, transported in the plant basically through the xylem (^{Malone et al., 2002}). It is important to consider then that the high level of Ca with 6-BAP and P-Ca at 50 mg L^-1 highlights the potential of these bioregulars as good alternatives to increase the quality of the tomato fruit.

Performance

Figure 4 shows that the yield in the 6-BAP and P-Ca treatments in 50 mg L^-1 was similar to the control. This behavior could be due to the concentration effect. Previous investigations (^{Ramírez et al., 2010}) report that P-Ca at 200 mg L^-1 increased yield 83% in rind tomato. This influence that P-Ca has had on increasing yield was also obtained in pear (^{Costa et al., 2004a}) and apple tree (^{Greene, 1996}; ^{Unrath, 1999}; ^{Basak and Rademacher, 2000}). The rest of the treatments had a tendency to have a lower yield than the control. This effect may reflect greater stimulus to vegetative development which competed with floral formation and therefore less yield (^{Rademacher, 2004}).

Figure 4 Influence of bioregulators on the performance of tomato plants hybrid “Raptor-F1” saladette. Each bar represents the average of ten repetitions. Bars with the same letter are statistically equal (DMS p≤ 0.05). 

Antioxidants

The effect of the treatments on the lycopene and vitamin C content in tomato fruits is shown in Table 4. The combination of AG_4/7 + 6-BAP at 100 mg L^-1 caused significant increases in both antioxidants; these, surpassed the control in 55.4% and 47.8% in lycopene and vitamin C respectively. The individual concentrations of AG_4/7 showed trends in higher content of the two antioxidants; while P-Ca also reflected that effect for vitamin C.

Previous research shows that P-Ca increases the content of vitamin C and lycopene in red tomatoes (^{Ramírez et al., 2003}) and habanero peppers (^{Ramírez et al., 2016b}). These findings allowed P-Ca to be classified as a retardant that increases the antioxidant content, which affects good health in humans since it strengthens the immune system that protects against diseases such as diabetes, cancer and blood pressure (^{Ramírez et al., 2010}). Little information exists on the stimulation of gibberellins and cytokinins in the synthesis of lycopene and vitamin C in tomato (^{Ramírez et al., 2010}). Therefore, more research is needed on this subject.

The higher content of lycopene and vitamin C observed in several treatments with bioregulators gives an added value to the fruit. This characteristic allows a higher price in the international market than that of a tomato with normal levels of these antioxidants (^{Ramírez et al., 2016a}), which compensates for a yield equal to or perhaps less than that of the control.