Location of the experiment

The experiment was established in the greenhouse at the Jefatura de Ingeniería Forestal del Instituto Tecnológico Superior de Venustiano Carranza (ITSVC), located between the geographical coordinates 20°28'19.6" N and 97°41'56.4" W, at an altitude of 333 m, in Lázaro Cárdenas, Puebla.

Plant material and nutritional analysis

In July 2019, 25 pest-free trees, with best growth and stem straightness were selected from a four-year-old plantation of the hybrid P. elliottii var. elliottii × P. caribaea var. hondurensis located within the facilities of the ITSVC. A sample of foliage (approximately 200 g of green material) was obtained from each selected tree to analyze nutritional status. The samples were placed in paper bags and transported to the Laboratorio del Posgrado en Ciencias Forestales of Colegio de Postgraduados, where they were washed with running and distilled water and dried in a Shel-Lab oven (model 1675-S) for 48 hours at 70 °C. Subsequently, three samples of 60 g each of dry matter were ground and processed for chemical analysis at the Salvador Alcalde Blanco Laboratorio del Programa de Posgrado en Edafología at Colegio de Postgraduados. For each foliar sample, the concentrations of phosphorus, potassium, calcium, magnesium, iron, copper, zinc, manganese and boron were determined by wet digestion extraction using Merck^® sulfuric acid, Merck^® perchloric acid, and Merck ^® hydrogen peroxide (1.3:0.7:1, v:v:v:v). The extracts were analyzed with a plasma optical emission spectrometer (Agilent 725 Series ICP-OES). Nitrogen concentration was determined in a 10 mL aliquot of the extract described above, by distillation of the sample and assessed by titration with sulfuric acid (^{Alcántar and Sandoval, 1999}).

The foliar analyses were interpreted using the critical concentrations cited by ^{Dickens et al. (2016)} for Pinus elliottii Engelm. ex Parl. and those suggested by ^{Xu et al. (2000)} for Pinus elliottii × Pinus caribaea.

Application of fertilization treatments

In accordance with the foliar nutrient concentrations (Table 1), five fertilization treatments were prescribed, based on solutions prepared with tap water: T1 control (water only), T2 Nitrogen (80 g urea per tree), T3 Phosphorus (20 g phosphoric acid per tree), T4 Magnesium (10 g magnesium sulfate per tree), and T5 Nitrogen/Phosphorus (80 and 20 g urea and phosphoric acid per tree, respectively).

In November 2019, the experiment was conducted under a completely randomized design; each treatment was administered arbitrarily to five trees. A total of 25 trees were considered in the experiment. The solutions were applied within a trench of approximately 1 m in diameter around each tree.

After one month from fertilization, all individuals were pruned. Pruning was carried out at the tip of eight branches located in the second third of the crown of each tree, using scissors with a telescopic handle. The purpose of this action was to induce the production of adventitious shoots. During the experiment, continuous weeding was carried out in order to prevent competition for nutrients.

Collection and preparation of cuttings

By February 2020, the shoots (cuttings) had reached a length greater than 5 cm, and were harvested. At the base of each cutting, a bevel cut was made with a scalpel disinfected with alcohol (70 %). Then, the stakes were submerged in a (Adama México) captan solution (2 mL L^-1) for 5 minutes.

Planting and application of IBA

The basal section (2 cm) of each cutting was immersed in the IBA solution (Radix^® 3 000) for 20 seconds; for the control, only running water was used for the same period of time. Cuttings were then planted in a substrate mixture of peat moss, agrolite, and vermiculite (3:1:1). Prior to planting, the substrate was brought to field capacity with running water. The trial was established in rooting chambers 120 cm long by 60 cm wide and 15 cm high, which were built with wooden boards and polycarbonate sheets. During the experiment, a relative humidity of 80 % and an average temperature of 20 °C were recorded in the chambers.

Experimental design and evaluated variables

The experiment was set up under a completely randomized block design with factorial arrangement; each rooting chamber represented one block. IBA was considered as factor A with two levels (0 and 3 000 ppm), while factor F represented the fertilization (of mother or donor trees) with five levels (control, nitrogen, phosphorus, magnesium, and the combination of nitrogen and phosphorus), for a total of ten treatments in each block. The experiment was replicated four times with each experimental unit consisting of eight cuttings, so that the total number of cuttings was 320.

In April 2020, 11 weeks after planting, the number of live cuttings, number of rooted cutting, number of primary and secondary roots, length of primary roots and number of cuttings with callus formation were recorded. These were used to determine: the percentage of cutting survival, percentage of rooted cuttings, number of primary roots, length of the main root, number of secondary roots, and percentage of cuttings with callus formation. Cuttings were considered dead when more than half of their tissue, starting from the base, was necrotic at the time of evaluation. Cuttings were considered rooted when they presented at least one 0.5 cm long root.

Data analysis

With the average values of the response variables, the statistical analysis was carried out using the Mixed procedure in the statistical analysis package SAS (Statistical Analysis System^©) version 9.4 (^{SAS, 2013}). In order to verify the assumptions of the parametric variance analysis, the Shapiro Wilk test for normality and the Levene and Barlett test for homogeneity were used. Then, the variables expressed in percentage (rooting, callus and survival) were transformed with the arc-sine function of the square root of the original value expressed in decimal fraction, since the data in the original scale did not meet the criteria of normality and homogeneity of variance. A similar procedure was carried out for the variables number of rooted cuttings, number of primary and secondary roots, and length of primary roots, which showed a Poisson-type distribution; in this case, the square root of the original value was used. After analysis, the average values per treatment were retransformed to the original units. The variance analysis model used was as follows:

Where:

Y _ijk = ijk ^th observation in i ^th block containing the j ^th level of factor A and the k ^th level of factor F

μ = Overall average

B _i = Effect of the i ^th block

A _j = Effect of the i ^th level of factor A (auxin)

F _k = Effect of the i ^th level of factor F (fertilization)

AF _jk = Interaction between the i ^th level of factor A and factor F

ε _ijk = Experimental error

For variables that showed significant differences, Tukey's mean comparison test (α= 0.05) was applied to determine statistical differences between the levels of the factors under study.

Effects of IBA and fertilization of the mother plant

IBA and fertilization treatments had significant effects on survival of the cuttings, number of rooted cuttings, main root length, number of primary roots (except for IBA), and number of secondary roots; whereas, only the rooted cuttings showed interaction between treatments (Table 1). On the other hand, neither treatment influenced the observed callus development in live un-rooted cuttings (Table 2).

In general, the cuttings maintained a survival rate above 50 %, which is an acceptable percentage for propagation by this technique (Table 3). IBA increased cutting survival by 25 % and also increased the rooting rate by just over 35 %, compared to the controls (Table 3). According to these results, it can be deduced that auxin has a direct influence on root promotion, particularly in those cuttings that probably required stimulation from the external hormonal regulator for their development. The presence of roots in the cuttings allows them to reactivate their physiological functions and form a seedling. Otherwise, unrooted cuttings survive only until the reserves in their tissues are exhausted, and then die. This coincides with what has been pointed out by ^{Fachinello et al. (2005)}: the survival of the cutting depends not only on its quality (nutritional status, vigor and juvenility) but also on the rapid formation of adventitious roots, which are responsible for providing water and nutrients.

The use of IBA in the promotion of adventitious roots in cuttings is well documented; this hormone accelerates the developmental and root formation process in the shortest possible time, thus ensuring the survival of the cutting (^{Endres et al., 2007}; ^{Gratieri-Sossella et al., 2008}; ^{Latsague-Vidal et al., 2010}).

A small percentage of cuttings showed the proliferation of callus, an undesirable effect, since these failed to develop adventitious roots; however, its formation was not linked to the treatments (Table 3). Callus cells lack connection to conductive tissues, are fragile and remain undifferentiated at the level of the root structure (^{Heaman and Owens, 2011}). For some species, such as sequoia, the development of callus is considered an indispensable initial stage that precedes root formation (^{Navroski et al., 2014}). In this work, no factor promoting callus production in the cuttings was determined, but the environment may be relevant in this process.

IBA had no significant effect on the number of primary roots, but it did influence secondary root production and primary root length (Figure 1). When compared to the untreated controls, the cuttings treated with IBA increased the production of secondary roots by more than 30 %, while the length of the main root increased by 35 % (Figure 1).

Longitud de raíces primarias = Length of primary roots; Number of roots = Número de raíces; Primarias = Primary; Secundarias = Secondary.

A) Length of primary roots, B) Number of primary and secondary roots. Within a root type, different letters in the error bars indicate significant differences between treatments (α=0.05).

Figure 1 Effect of Indolebutyric Acid (IBA) dosage on root production in Pinus elliottii var. elliottii × Pinus caribaea var. hondurensis hybrid cuttings.  

In conifers that are difficult to root, IBA is an excellent promoter of adventitious roots. In cuttings of Pinus leiophylla Schiede ex Schltdl. et Cham., IBA not only induces rooting of the cuttings but also elongates the primary roots and increases the number of primary and secondary roots (^{Cuevas et al., 2015}); a similar effect was found in the present study. However, little or no effect of IBA on root morphology has been documented; therefore, the variation in the responses to IBA is considered to depend on the applied dose, as well as on the predisposition of the cutting to rooting (^{De Oliveira et al., 2020}).

Auxins facilitate rooting by stimulating cambium activity and mobilization of reserves to the site of root initiation, where cells divide and differentiate in order to generate primordia (^{Dhillon et al., 2011}; ^{Brondani et al., 2012}). Therefore, the application of IBA on cuttings is desirable to improve root morphology and quality, which in turn is important for plant growth and development, since it increases the area of nutrient and water absorption (^{Davis and Jacobs, 2005}).

Fertilization treatments favored the survival of cuttings in a differentiated manner, but always above 50 % (Table 3). Donor plants treated with N+P generated cuttings with a survival rate of more than 85 %, while the cuttings of control plants did not reach 40 %. These results demonstrate the importance of having a donor plant in good nutritional condition to produce quality cuttings, and are in agreement with other authors, in the sense that adequate fertilization of the donor plants influences the level of carbohydrates, auxins and other compounds in the tissues of the cuttings, which keep them alive while initiating the process of rhizogenesis (^{Kanmegne et al., 2017}).

Fertilization treatments promoted root formation. The highest number of rooted cuttings was recorded with the N+P and P-only treatments, which increased rooting by more than 80 %, with respect to the control (Table 3).

In Pinus radiata D. Don, N-based fertilization of donor hedges had a positive effect on the induction of adventitious roots on cuttings (^{Hernández and Rubilar, 2012}). Nitrogen is a constituent of proteins and nucleic acids; moreover, its participation in plant growth is essential; when N is absorbed by the plant in the form of nitrate (NO₃ ^-) or ammonium (NH₄ ⁺), it is incorporated into the meristems for plant growth and development. In the cambium of the cuttings, N is required for the formation of adventitious roots (^{Emer et al., 2019}).

Phosphorus is an element that enhances plant metabolism and favors absorption; combined with N and an optimal dose of auxin, it favors a better absorption of nutrients and root development in the cuttings (^{Henry and Blazich, 1992}). A significant relationship was observed between P concentration and rooting potential, which reinforces the hypothesis that P is important in post-transplant survival via the promotion of root extension in the field (^{Planelles, 2004}; ^{Oliet et al., 2005}). In general, the nutritional status of the plant affects the processes of production of new roots (^{Villar et al., 2004}; ^{Oliet et al., 2006}).

The length of primary roots and the number of primary and secondary roots were also significantly affected by fertilization treatments (Figure 2). In particular, the N+P treatment increases by more than 80 % the number of primary and secondary roots in the cuttings, with respect to the control without fertilization; whereas the other fertilization treatments outperformed the control by more than 35 % (Figure 2A). Similar increases were obtained in the length of the primary root of the cuttings (Figure 2B).

Number of roots = Número de raíces; Longitud de raíces primarias = Length of primary roots; Primarias = Primary; Secundarias = Secondary; Nutrimento aplicado = Applied nutrient; Nitrógeno+Fósforo = Nitrogen+ Phosphorus; Magnesio = Magnesium; Testigo = Control.

A) Number of primary and secondary roots present in cuttings, B) Length of primary roots of cuttings.

Figure 2 Effect of fertilizer on root production in Pinus elliottii var. elliottii × Pinus caribaea var. hondurensis hybrid cuttings. 

The length of the main root and the number of primary and secondary roots conform the architecture of the plant root system. Therefore, they are important parameters for the plant produced from a cutting. Fertilization of the mother plant contributes to the carbohydrate content of the cuttings, which are in high demand during the formation of adventitious roots (^{Otiende et al., 2017}). In this work, fertilization improved these parameters considerably. It is likely that, in the untreated cuttings, the available carbohydrates did not cover the requirement for the generation of new roots; in cuttings photosynthesis does not provide sufficient carbohydrates for the processes involved in rhizogenesis (^{Kanmegne et al., 2017}).

As for the interactions of the IBA dosage by fertilizer type, significant differences (P˂0.05) were obtained only for the percentage of rooted cuttings (Table 1). The percentage of rooted cuttings from fertilizer-treated trees increased with the IBA treatment, while those from unfertilized (control) trees decreased (Figure 3).

Estacas enraizadas = Rooted cuttings.

Figure 3 Interaction between mother plant fertilization treatments and IBA dosage in rooted cuttings of the hybrid Pinus elliottii var. elliottii × Pinus caribaea var. hondurensis. 

This interaction implies that rooting capacity depends not only on the exogenous application of IBA, but also on the quality and vigor of the cutting; therefore, the nutritional status of the cutting is of paramount importance in the process of rhizogenesis for the hybrid in question. Nutrient mobilization within the cutting is known to be limited during the root initiation phase, but not during growth and development.

In this case, unfertilized cuttings were not potentiated with auxin, as were the fertilized cuttings. Probably, in the former, the low level of carbohydrates or other metabolic compounds in the tissue limited the rhizogenesis process and reduced the capacity to form roots. Thus, from a nutritional point of view, root initiation depends, to a large extent, on the initial nutrient levels found in the portion of the cutting where they will form. Hence the fundamental role of mother plant nutrition on the subsequent rooting of cuttings (^{Mesén et al., 2001}; ^{Leakey, 2014}).

The formation of adventitious roots is a process directed by growth regulators, but it is affected by multiple factors that include, among others, the nutritional state of the cutting; thus, mineral nutrition is important in the conformation of organic structures, activation of enzymatic reactions, and osmoregulation, among others (^{Geiss et al., 2018}).