Geographic location

The study was carried out in the Experimental Field of the School of Higher Studies of Xalostoc (EESuX) of the Autonomous University of the State of Morelos (UAEM), Parque Industrial Cuautla, Cd. De Ayala, Morelos, during the year 2016. 6 000 m², planted on a sandy-clay crumb soil, with a pH of 7.03, located at the geographical coordinates of 18° 49’ north latitude and 99° 01’ west longitude, at an altitude of 1 330 meters above sea level, with a warm climate prevailing subhumid (^{INEGI, 2009}).

Vegetal material

The rootstocks and varieties were obtained from the certified nursery ‘Cazones’, located in Cazones de Herrera, Veracruz. 149 one-year-old plants were purchased after grafting. The rootstocks used were: Amblycarpa (Citrus amblycarpa Ochse), Naranjo Agrio (Citrus aurantium L.), Limon Volkamerian (Citrus volkameriana Pasq) and Citrange C-35 (Poncirustrifoliata [L.] Raf. X Citrus sinensis L.), with the four varieties of orange ‘Campbell’, ‘Hamlin’, ‘Mars’ and ‘Jaffa’. Sixteen rootstock-variety combinations were made (Table 1).

The plants were certified by the Xalapa High Technology Laboratory, SC (LATEX) registered with SENASICA in Xalapa, Veracruz, they were free of the citrus sadness virus (VTC). They were transferred to the experiment area and kept in a greenhouse to be acclimatized until the transplant.

Plantation density and topological arrangement

Trees were planted in a rectangular spatial arrangement with a completely random design, where the experimental unit was a tree, with seven to ten replications (Table 1). The planting distance was 7 m between rows and 4 m between plants, giving a planting density of 357 plants per ha. The transplant was carried out in April 2015, in strains 35 to 50 cm deep, 3 kg of vermicompost was added to the bottom of each strain and the plant was subsequently placed. Irrigation was applied to avoid stress and eliminate porous space.

Agronomic management of the plantation

The agronomic management of the plantation was according to that recommended by ^{Lugo et al. (2009}); ^{Ariza et al. (2009}). Drip irrigation was performed every other day. It was fertilized with the 150-60-60 dose (N-P-K) according to the soil analysis and nutritional requirements. Triple fertilizer was applied 17 to 20 cm from the plant in two lateral furrows, 100 g were deposited on each side. Foliar fertilization was carried out with Bayfolan Forte^® using an Echo^® brand motorized backpack sprayer at 4 ml L^-1 of water each month. Formation pruning was performed every two months, eliminating suckers developed on the main stem of the rootstock, the cuts were covered with vinyl paint.

Agronomic variables evaluated

Stem diameter (mm) and plant height (cm). They were evaluated monthly with a Caliper^® brand digital vernier and a tape measure in all the plants. The stem diameter was taken at 20 cm above ground level.

Flowering-fruiting. The number of flowers in the selected plants was evaluated when the first flower buds appeared every 15 days. The number of fruits in each plant was also evaluated.

Chlorophyll content. It was determined quickly, directly and non-destructively. The readings were performed with a portable chlorophyllometer (Minolta Chlorophyll Meter SPAD-502^®; ^{Markwell et al., 1995}). Two leaf readings were taken from the top and two leaf readings from the bottom, making a total of 3 evaluations every 4 months in each of the combinations.

Cup diameter and height. They were evaluated one year after transplant, when the crown was fully formed. The diameter was measured using a tape measure. The height of the tree canopy was determined with the support of two completely straight thin iron rods, one rod was placed horizontally at the base of the canopy and another at the top of the tree, taking measurements from the bottom up.

Statistical analysis

The data were processed with the statistical program SAS^® version 9 ^{SAS (1996)}, applying a simple analysis of variance (Anova) and comparison test of means according to Tukey, after checking the parametric assumptions of Normality.

Stem diameter and plant height

The stem diameter (mm) and the plant height (cm) evaluated in the different combinations showed significant statistical differences (p< 0.05; Table 2). The combinations: Volkamerian-Mars, Volkamerian-Jaffa, Volkamerian-Campbell and Volkamerian-Hamlin were the ones that threw the greatest thickness with 30.83, 27.37, 26.39 and 26.33 mm respectively, without statistical differences between them (p> 0.05), while cultivars that were grafted on Naranjo Agrio showed the lowest values (p< 0.05), with the Naranjo Agrio-Jaffa combination showing the smallest diameter of the tree trunk (10.45 mm).

The height of orange trees was greater when the Volkamerian rootstock was used (p< 0.05, Table 2), the Volkamerian-Jaffa combination was the one that presented the greatest growth with 82.6 cm, without significant difference with Volkamerian-Hamlin, Volkamerian-Mars and the Citrange C-35-Campbell combination (p> 0.05), coinciding with that reported by ^{Anderson (2012}).

Who pointed out that Volkamerian induces vigorous and large plants, as with the results reported by ^{Milla et al. (2009}) in Tahiti lime tree, where plants grafted on Volkamerian presented larger sizes than those grafted on mandarin Cleopatra and Naranjo Agrio. ^{Arrieta et al. (2010}) in studies carried out on different rootstocks, they reached greater height and diameter with the Volkamerian and Amblycarpa patterns.

This coincides with the results obtained by ^{Girardi et al. (2007}), reporting that lemon-type rootstocks were more vigorous and developed faster from nursery than tangerines. ^{Zamora et al. (2003}) obtained similar results when carrying out a study on the selection of rootstocks for ‘Valencia’ orange in calcimorphic soils, they found that the grafts made on Volkamerian, produced the largest and leafiest trees.

The rapid growth of the stem of Volkamerian Lemon has also been pointed out by ^{Girardi et al. (2007}), who stated that it is a vigorous rootstock in the nursery. This has a larger stem diameter, indicating that both variables are related. The stem diameter is very important in the nursery stage, because the grafting is done when the stem is greater than 0.55 cm. This reaches this thickness faster, which represents less time in the acclimatization phase, lower production costs and an excellent alternative for nurserymen.

The smallest trees were obtained with the Naranjo Agrio pattern (p< 0.05; less than 43 cm). ^{Del Valle et al. (1981}) showed that the orange trees grafted on Amblicarpa and Volkamerian showed greater height and thickness than those grafted on Naranjo Agrio (p<0.05), which agrees with what was obtained in this work and with what was reported by ^{Pérez et al. (2003}) and ^{Navejas et al. (2010}).

From which it follows that the choice of a pattern for conventional use constitutes an important decision, which must be based on the characteristics and qualities that it presents, both in the juvenile phase and in field conditions, given that the behavior of the patterns are variable in relation to their adaptability to climatic conditions, soils, tolerance to diseases and pests, also having a marked influence on the behavior of the graft and therefore on vigor, earliness, production level and the quality of the fruits of the different varieties of orange.

Flowering-fruiting

When comparing the different rootstock-cultivar combinations in relation to flowering, statistical differences were observed between them (p< 0.05; Table 2), detecting that the Jaffa variety grafted on the Volkamerian pattern had the highest emission of flower buds per tree (p< 0.05) with an average of 2.54 flowers every 15 days, during the evaluated period and differs significantly with the rest of the combinations.

The lowest number of flowers was observed in the Hamlin, Jaffa and Campbell varieties grafted on Amblycarpa and Hamlin on Volkamerian with 0.7 flowers/tree, without statistical differences between them (p> 0.05). The highest number of fruits achieved was in the Volkamerian-Jaffa combination (1.89; p> 0.05). The Amblycarpa-Hamlin, Amblycarpa-Campbell, Amblycarpa-Jaffa, Volkamerian-Hamlin, Citrange C-35-Jaffa and Naranjo Agrio-Hamlin combinations reported the lowest number of fruits, with no differences between them (p> 0.05).

In Mexico, the species of C. macrophylla and C. volkameriana are commonly used as rootstocks for the propagation and establishment of Mexican lemon trees (^{Bermúdez et al., 2017}). Three lemon species are cultivated: sour (Mexican) lemon Citrus aurantifolia, Persian lemon Citrus latifolia and Italian lemon Citrus limon (^{SIAP, 2018}).

Most of the citrus fruits in the state of Morelos are established on Lemon Volkamerian (C. volkameriana) and some on Naranjo Agrio (C. aurantium; ^{Lugo et al., 2009}). Confirming the acceptance of this lemon hybrid as a good producer of large, vigorous and productive plants.

Chlorophyll content

The chlorophyll content (SPAD units), indicator of the green color of the leaves, was a parameter that did not show great variation during the study. In the first evaluation, the Volkamerian-Mars and Volkamerian-Jaffa combination presented the highest values, with 86.35 and 84.04 (SPAD units) respectively, with no statistical differences between them or with the rest of the combinations (p> 0.05), only with the combination Naranjo Agrio-Jaffa that obtained the lowest value (p< 0.05; 66.09 SPAD units). During the second evaluation, the Hamlin, Jaffa and Mars varieties grafted onto Volkamerian with 83.51, 82.62, 85.15 (SPAD units) and the Amblycarpa-Campbell combination (83.87 SPAD units) were those that showed the highest chlorophyll content, without statistical differences between them nor with the rest of the combinations (p> 0.05). In this period the Naranjo Agrio-Campbell combination yielded the lowest value (75.65 SPAD units).

In the third evaluation, the combinations had a similar behavior to the second, highlighting again the combinations with Volkamerian which reached the highest chlorophyll units per leaves evaluated without difference with the others (p< 0.05), only with the Naranjo Agrio-Campbell combination that yielded the lowest chlorophyll content per leaf (p< 0.05; 75.66 SPAD units).

^{Achor et al. (2010}) cited that the SPAD readings are only taken from a partial part of the leaf, so as the destruction of chloroplast is not uniform, the sensor can capture areas with different concentrations of chlorophyll in the same leaf. In this case, the rootstock cannot be directly related to the chlorophyll content in the varieties evaluated, since this may be influenced by other factors such as fertilization and the agronomic management offered to the plantation.

Some authors, such as ^{Lizana and Errazuriz (1980}) have inferred that low temperatures, especially at night, could induce a disappearance of chlorophyll and an increase in the synthesis of carotenes, before the fruit is ripe. Other trials justified that after 15 days; from the application of chelates to the soil, the SPAD readings, which indicate the foliar content of chlorophyll, increased significantly with the doses of 50 and 100 g of Fe EDDHA per tree to values of almost 47 (SPAD index), corresponding to the green color. Common of healthy leaves and that indicates a normal content of chlorophyll in them (^{Sánchez et al., 2015}).

Cup height (AC)

The Hamlin and Mars varieties on the Volkamerian pattern presented the highest crown height (p< 0.05) with 139.1 cm and 135 cm respectively, without statistical differences between them (p> 0.05) in contrast to the Jaffa and Mars varieties grafted on Naranjo Agrio exhibited the lowest crown height (p< 0.05) with 67.7 cm and 62.6 cm, with no differences between them (p> 0.05).

This is in agreement with what several researchers have pointed out about the ability of the Volkamerian rootstock to induce large size (^{Stenzel and Neves, 2004}). However, it differs with that reported by ^{Curti et al. (2012}) in Persian lemon grafts made on Volkamerian, as those with the lowest height and volume of the glass.

Other researchers such as ^{Ledo et al. (2008}) pointed out that the rootstocks should form small crowns, with high productive efficiency in relation to the volume of the same, in order to increase the density of plantation and the production of the orchard. The Jaffa and Mars varieties grafted onto Naranjo Agrario were those with the lowest crown height at 67.7 cm and 62.6 cm, with no differences between them.

The results show that there are rootstocks different from the Naranjo Agrio, which is the one that has traditionally been used in citrus farming throughout the years, which can be used as an alternative to achieve high productions and more leafy trees.

Cup diameter (DC)

The Jaffa and Mars cultivars grafted on Volkamerian presented the highest crown diameter (p< 0.05) with 172 cm and 175.5 cm respectively, with no differences between them (p> 0.05). On the other hand, the Jaffa cultivar grafted on Amblycarpa presented the smallest crown diameter (p< 0.05) with 80.6 cm. ^{Figuereido et al. (2002)} and ^{Quijano et al. (2002}) indicated that the Persian lime plants grafted on Volkamerian presented the highest height and diameter of the crown than those grafted on ‘Cleopatra’ and ‘Carrizo’.

The graft/pattern relationship is important since the pattern constitutes the base where the crown will develop (graft) and the future production of the tree depends on it. ^{Reyes and Ruiz (1983}) rated the most leafy and grafted grafts as those that were made on Volkamerian, Rugoso lemon, Cravo lemon, which coincides with the results obtained, however it differs from that reported by ^{Pérez et al. (2003}) who cataloged Amblicarpa as the pattern that offered the highest height, rootstock and graft diameters, crown and crown volume.

According to ^{Orozco (1995}) the grafts made on Volkamerian constitute an alternative since they provide leafy trees and tolerant to the conditions of calcareous soils, but they have the disadvantage that they are sensitive to the sadness of citrus fruits, an aspect that must be taken into account time to plant them. ^{Avilan (1993}) pointed out that the knowledge of a reference pattern is important because its genetic characteristics can induce plants different development capacities in the aerial part.

From which it follows that it is significant to know the behavior of each combination in the plant production phase since their interactions affect the development of the graft, accelerating or retarding it, when presenting differences in compatibility according to the grafted varieties. The differences between rootstocks may be related to their response to limiting water conditions, as in the case of deficit irrigation or drought stress (^{Pérez et al., 2008}).

Greater aerial part/root ratio implies more leaf area and therefore, greater transpiratory surface, which results in greater absorption and transport of water and nutrients. The incorporation of new patterns is invaluable both for the good development of the graft and for its behavior against different pathogens, giving quality to the fruits and accelerating their production.