Propagación asexual de Pinus leiophylla Schiede ex Schltdl. et Cham.

Juan Carlos Cuevas-Cruz⁴; Marcos Jiménez-Casas¹*; Jesús Jasso-Mata¹; Paulino Pérez-Rodríguez²; Javier López-Uptón¹; Ángel Villegas-Monter³

¹ Postgrado en Ciencias Forestales, Colegio de Postgraduados. Carretera México-Texcoco km 36.5. C. P. 56230. Montecillo, Texcoco, Edo. de México. Correo-e: marcosjc@colpos.mx, Tel.: 595 95 20246 ext. 1454 (*Autor para correspondencia).

² Postgrado en Estadística, Colegio de Postgraduados. Carretera México-Texcoco km 36.5. C. P. 56230. Montecillo, Texcoco, Edo. de México.

³ Postgrado en Fruticultura, Colegio de Postgraduados. Carretera México-Texcoco km 36.5. C. P. 56230. Montecillo, Texcoco, Edo. de México.

Received: August 19, 2014.
Accepted: February 17, 2015.

ABSTRACT

The effect of substrate, type of cutting and indole 3-butyric acid (IBA) concentration on the rooting of Pinus leiophylla cuttings was evaluated with the goal of multiplying progenies of half-sib families of this species, which have showed superior growth and positive response against Toumeyella pinícola attack. Two types of substrates (100 % perlite vs. a mix of peat, perlite, vermiculite at a ratio of 1:1:1), two types of cuttings (apical vs. basal) and two concentrations of IBA (0 vs. 10,000 ppm) were used. Probability and percentage of rooting, growth of cuttings and characteristics of the roots were evaluated. Results indicate that using basal cuttings is 3.5 times more likely to induce rooting of P. leiophylla than using apical cuttings. The treatment consisting of the mixture of peat-perlite-vermiculite, a basal cutting and 10,000 ppm of IBA produced 45.3 % rooting (highest percentage), while the control only yielded 8.6 % rooting (perlite, basal cutting and without IBA). Interactions with the type of substrate showed significant differences (P ≤ 0.05) in the growth of the cuttings. The use of perlite and application of IBA promoted a greater number of roots, particularly in the basal cuttings of P. leiophylla.

Keywords: Indole 3-butyric acid, clone, root morphology, rooting of cuttings.

RESUMEN

Palabras clave: Ácido 3-indolbutírico, clon, morfología de raíces, enraizamiento de estacas.

INTRODUCTION

Propagation of the genus Pinus by rooting cuttings obtained from young buds is carried out to multiply progeny identified as superior, in order to obtain elite clones for reforestation and commercial plantations (Greenwood & Weir, 1994; Mori, Miyahara, Tsutsumi, & Kondo, 2011; Zobel & Talbert, 1984). A sexual seed orchard located in Montecillo, State of Mexico, has progeny of some families of Pinus leiophylla Schiede ex Schltdl. et Cham. with proven superiority in growth, seed production and resistance to Toumeyella pinicola attack. Vegetative propagation of this progeny is important to speed up production of plants with desirable traits, intended to reforest and restore degraded forest sites; however, there is no information reported on the vegetative propagation of P. leiophylla and little is known about the methods of rooting other Mexican pines.

The rooting of cuttings has been aided by the use of stems with juvenile characteristics and variable size, conditions of high relative humidity, controlled temperature and types of substrate (Lebude, Goldfarb, Blazich, Wise, & Frampton, 2004; Mori et al., 2011; Rosier, Frampton, Goldfarb, Blazich, & Wise, 2004). The factors that have been the most evaluated in studies on rooting conifers are: substrates with good aeration, drainage, water-holding capacity and no waterlogging; the juvenility of the cutting; and indole 3-butyric acid (IBA) at optimal concentrations (King, Arnold, Welsh, & Watson, 2011; Ragonezi et al., 2010; Rosier, Frampton, Goldfarb, Blazich, & Wise, 2004). However, the results indicate that the requirements of these factors vary depending on the species and genotype (Majada et al., 2011; Sharma & Verma, 2011).

In this context, the aim of this study was to determine the effect of substrate type, cutting type and IBA concentration on the rooting of P. leiophylla cuttings, in order to generate information and protocols on the rooting ofprogeny cuttings ofthis species. Furthermore, this paper proposes an alternative methodology that can be adapted without major investments for cloning and vegetative propagation of species of Pinus.

MATERIALS AND METHODS

Location of the experiment

Plant material

In 2011, 800 seeds of eight half-sib families (100 seeds per family), collected from the P. leiophylla sexual seed orchard in the spring of 2009, were germinated. At nine months of germination, the plants were transplanted into 310 cm³ individual containers with peat-perlite-vermiculite substrate at a ratio of 2:1:1. The plants grew under greenhouse conditions until 11 months of age, continuing their development under 50 % shade mesh. At 14 months, the plants were pruned to a height of 15 cm and buds produced at the base of the stem were removed. At four months after pruning, new apical and basal buds of 8-12 cm in length were generated. The plants were fertilized with Peters Profesional^® (0.7 g·liter^-1) once a week during the first 2.5 months with the formula 7-4017 (N-P-K); subsequently and up to 4.5 months, 20-719 was applied, and finally 4-25-35 was applied until buds with juvenile characteristics were harvested.

Experimental conditions

The study was conducted in a rustic greenhouse with partial control of the temperature by opening and closing curtains, and without an irrigation system. Inside the greenhouse a plastic micro-tunnel (1.80 m wide x 2.5 m long x 1.5 m high) was installed; during the experiment, the average temperature in the microtunnel was 24 °C, with a minimum of 19 °C minimum and a maximum of 27.5 °C. The relative humidity was 77 % on average, with a 65 % minimum and a 90 % maximum.

Experimental design

The rooting of P. leiophylla was evaluated considering the following factors: two types of substrate (perlite and a mixture of peat-perlite-vermiculite [1:1:1]), two types of cuttings (basal and apical) and two concentrations of IBA (0 to 10,000 ppm), which were combined in a completely randomized design with a 2 x 2 x 2 factorial arrangement; i.e., eight treatments with three replications and 18 cuttings in each replication. Table 1 presents the eight treatments evaluated.

Rooting was evaluated in two cutting types (apical and basal). The basal-type cutting represented the sprouts emerging at the base of the plant's stem and the apical cutting refers to the sprouts that emerged at the apex. The sprouts were separated from the mother plants on the same day the experiment was established, obtaining cuttings of 8 cm in length and 1.5-2 mm in diameter; 2 cm of needles were removed from the base of each cutting. The basal section (2 cm) of the cutting was immersed in rooting compound (4-[1H-Indol-3-yl] butyric acid), removing the excess. The cuttings treated with rooting compound and those of the control treatment (no IBA) were placed in plastic trays (96 cm long, 46 cm wide and 18 cm high) containing perlite (pH 6.5-7.5) or the mixture of peat-perlite-vermiculite (pH 5.6-6.1), according to the treatment. Previously, the cuttings were wetted to field capacity using distilled water. The cuttings were sprayed with 1 g-liter¹ of fungicide (N- [trichloromethylthio] cyclohex-4-en-1,2-dicarboximide) at the time of their establishment. Finally, the trays were fully covered with gauge-200 transparent plastic, in order to facilitate a microclimate within them and thus retain the moisture, a product of the transpiration of the cuttings. As a result, watering was unnecessary during the 84-day experiment since the relative humidity stayed within the required percentages (average relative humidity of 77 %, minimum 65 % and maximum 90 %), according to the monitoring carried out every eight days.

Variables assessed and data analysis

A cutting from each experimental unit was randomly selected and extracted at 60 and 80 days after the experiment was established to review progress in root formation. Based on both samples, it was decided to perform the final evaluation at 84 days into the experiment. The probability of rooting, the percentage of rooting and growth of the cuttings were determined based on the factors studied; a rooted cutting was considered to be one that had at least one first-order root ≥ 1 cm in length. The number of first- and second-order roots in the rooted cuttings was counted, and the length of the longest root for both root types was measured.

The variable p_ijk is the probability of rooting and it is assumed that it depends on the substrate type, cutting type and IBA concentration. In the model it is assumed that the response variable Y_ijk ~ Bernoulli(p_ijk), Y_ijk = 0 (cutting not rooted), Y_ijk = 1 (rooted cutting); D1 = 0 if the substrate type is perlite and 1 if it is the mixture, D2 = 0 if the cutting type is apical and 1 if it is basal; finally, D3 = 0 if the IBA concentration is zero (0 ppm) and 1 if the concentration is 10,000 ppm. The model was fitted using PROC LOGISTIC in SAS 9.1 software (Statistical Analysis System [SAS], 2004).

The rooting percentage and cutting growth were analyzed with the GLM procedure in SAS 9.1 software (SAS, 2004). Comparison of means was performed with the Tukey test (P = 0.05). Prior to analysis of variance, the variable rooting percentage was transformed with the arcsine function (y = arcsin p), because the data did not meet with the assumption of normality and homogeneity of variance. The count variables (number of roots) were also transformed using the natural logarithm function (y' = ln [counts]), given that when analyzing the data they were not fitted to the Poisson distribution (λ). Subsequently, the mean values of rooting percentage and number of roots were re-transformed to the original units.

RESULTS AND DISCUSSION

Effect of substrate type on rooting of P. leiophylla cuttings

Table 2 shows the result of the binary regression model for the analysis of the probability of rooting P. leiophylla cuttings. Based on this analysis, the substrate type had no effect on rooting probability (X² = 1.0, gl = 1, P = 0.314). On the other hand, Table 3 presents the analysis of variance for each factor evaluated. This table shows that substrate type had no effect on rooting percentage, growth of cuttings or root length, but it did have an effect (P ≤ 0.05) on the number of first- and second-order roots. According to the comparison of means shown in Table 4, the highest number of first- and second-order roots (2.4 and 4.5, respectively) was generated by using perlite as substrate. On average 26 % rooting was obtained in the trial.

Substrate has proved to be very important in root production, both in terms of number and length; for example, the use of sawdust fosters a greater number of roots than pine bark, due to the fact that the former has neutral pH (Santelices & Cabello, 2006). In our study, perlite with pH 6.5 to 7.5 (commercial form) could be an important factor for the development and growth of first- and second-order roots, in contrast to what has been observed with peat that has acid values (Rodríguez-Macías et al., 2010). Although this research could confirm what has been reported by other authors on the importance that neutral pH values in substrate have in favoring rhizogenesis (Hartmann & Kester, 2001), in our case, given the number and growth of roots, other physicochemical studies with perlite are needed to clarify its effects on rooting cuttings. The results suggest that the use of perlite as substrate is a viable option in the rooting of P. leiophylla cuttings, compared to peat-perlite-vermiculite (1:1:1) substrate, due to the economic aspects and benefits obtained in the root morphology produced.

Effect of cutting type on rooting of P. leiophylla cuttings

The basal cuttings of P. leiophylla are more juvenile in structure and chronology than apical ones, which could explain the results obtained in both rooting percentage and root morphology (length and number of second-order roots) of the cutting types tested. In conifers, several studies have shown that the juvenility of the explant is required to increase the rooting percentage and that positively influences the length, area and volume of the root (Foster, Stelzer, & McRae, 2000). In Gmelina arborea Roxb., the position of the cutting is the only factor that affects the rooting percentage (Ruíz-García, Vargas-Hernández, Cetina-Alcalá, & Villegas-Monter, 2005). The characteristics of the stem from where the cuttings are taken, such as juvenility, ontogeny and position, as well as the management of the mother plants, are the factors that most affect the percentage of rooted cuttings in Taxus globosa Schltdl. (Muñoz-Gutiérrez, Vargas-Hernández, López-Upton, & Soto-Hernández, 2009), Abies fraseri (Pursh) Poir. (Rosier, Frampton, Goldfarb, Wise, & Blazich, 2005) and Pinus virginiana Mill. (Rosier et al., 2006).

Effect of IBA on rooting of P. leiophylla cuttings

IBA concentration had no significant effect (X² = 1.5, gl = 1, P = 0.2193) on rooting probability (Table 2); it was only significant (P = 0.0194) for the number of first-order roots (Table 3). This suggests that P. leiophylla cuttings are able to root without the application of IBA. Aparicio-Rentería, Juárez-Cerrillo, and Sánchez-Velázquez (2014) obtained a similar response in Pinus patula Schl. et Cham. cuttings, which rooted with percentages above 90 % without the application of growth regulators. However, in our study, IBA contributed to the formation of a larger number of first-order roots (2.4 roots with IBA and 1.6 without it) (Table 4). Similar results have also been obtained in studies with species of pine and eucalyptus, where the IBA stimulated the production of a higher number of roots and other attributes related to root morphology (Majada et al., 2011; Navarrete-Luna & Vargas-Hernández, 2005). On the other hand, Hinesley, Blazich, and Snelling (1994) evaluated various IBA concentrations (0.0, 5.0, 10.0 and 15.0 g·liter^-1) in Chamaecyparis thyoides (L.) B. S. P., and found that the number of first-order roots increased linearly (r = 0.70). Production of first-order roots provides increased water uptake and nutrient translocation, which improves plant growth and development (Davis & Jacobs, 2005). IBA application is therefore desirable in order to increase the production of first-order roots and improve the morphological attributes of P. leiophylla cuttings.

Effect of interactions on rooting of P. leiophylla cuttings

Only the interaction of the three factors (substrate type*cutting type*IBA concentration) had an effect (P = 0.0219) on rooting percentage (Table 3), which ranged from 45.3 % in the substrate composed of peat-perlite-vermiculite, with basal cuttings and IBA(10,000 ppm), to 8.6 % in perlite, with apical cuttings and IBA (0 ppm). Figure 1 shows the effect of the eight evaluated treatments on the rooting percentage.

The substrate*cutting and substrate*IBA interactions, and the triple interaction showed significant effects (P ≤ 0.05) on cutting growth during the rooting process (Table 3). The peat-perlite-vermiculite substrate favored greater cutting growth (3.1 cm), while some of the substrate*IBA interactions and the triple interaction (P ≤ 0.05) affected cutting growth (Figure 2). Rooting percentages of P. leiophylla are within the range of those reported by several authors for other species of Pinus (Lebude et al., 2004; Mori et al., 2011; Rosier et al., 2006). It should be noted that most of the published studies were carried out with controlled temperature and humidity, testing different misting systems that in many cases create disease problems (Preece, 2003). In the present study we used "moderate" technology constituted by microtunnels, where the hermetically-sealed plastic cover (Ramírez-Villalobos, Urdaneta-Fernández, & Vargas-Simon, 2004) kept the relative humidity high, thereby avoiding the need for watering during the experiment.

The number of first-order roots was not affected by any of the interactions, while the number of second-order roots showed an effect (P ≤ 0.05) as a result of the cutting type*IBA and substrate type*IBA interactions, as well as the triple interaction (Table 3). Figure 3 shows a graphical representation of these interactions. The basal cutting*IBA(0 ppm) interaction resulted in 5.3 secondary roots, while the apical cutting*IBA(0 ppm) interaction only fostered 2.1 roots (Figure 3a); the perlite*IBA(10,000 ppm) interaction also formed more than double the number of roots of the other interactions (Figure 3b); finally, the difference between the triple interaction with the largest and smallest number of roots was 8.7 secondary roots (Figure 3c). The results show that the exogenous IBA concentration favors the formation of P. leiophylla roots depending on the cutting type and substrate type; in G. arborea, it has been observed that root formation is favored by the interaction between the IBA concentration and cutting type (Ruíz et al., 2005).

Regarding root length, only the substrate type*IBA interaction had an effect (P = 0.0053) on the length of primary roots (Table 3), where perlite*IBA (10,000 ppm) had the greatest length (10 cm) as shown in Figure 4. The cutting type*IBA interaction and the triple interaction affected (P ≤ 0.05) the length of secondary roots (Table 3). Figure 5 shows that the cutting type*IBA interaction presented a 1.6-cm difference between the largest and smallest length, while the combination of the three factors had a 2.5-cm difference between the largest and smallest length. King et al. (2011) also observed increased root length as a result of the interaction of factors in Taxodium distichum (L.) Rich., obtaining root lengths of 3.8-11.9 cm depending on the substrate type and IBA concentration.

The use of basal cuttings with 10,000 ppm of IBA and perlite as the substrate is the most feasible treatment for rooting cuttings from 18-month-old P. leiophylla plants. The technique and the method used in this study could represent an affordable option for vegetative propagation of conifers without large investments in infrastructure.

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