Introduction

Current demand for forest seedlings to establish commercial plantations and reforestation is constantly increasing. According to the ^{Comisión Nacional Forestal (2014)}, in the recent five years, its programs of forest plantations and reforestation used an annual production of more than 200 million seedlings. This growing demand requires development and innovation of new production systems to meet the demand, reduce time of seedlings in the nursery, optimize production inputs and favor quality standards.

The concept of seedling quality has different approaches, and some researchers focus on seedling production in the nursery (^{Ortega et al., 2006}; ^{Pinto et al., 2011}). From this perspective, seedling quality is a set of morphological and physiological properties that the seedling must have to achieve establishment, survival and successful growth in the field, and initial increase in height and diameter. The quality is a concept that, besides considering appropriate characteristics of the seedling in the nursery, it considers the attributes that have direct repercussions on establishment of forest plantations, in function of site conditions of the plantation (^{Rodríguez and Duryea, 2003}; ^{Palacios et al., 2009}; ^{Grossnickle, 2012}).

In nursery production schemes, it is common to produce seedlings with root balls, which leads to offer diverse containers in the market. Container design and the material with which it is manufactured define a series of culture variables: seedling dimensions, formation of the radical system (root architecture), number of seedlings to cultivate per unit of area, amount of substrate to use and species to produce. The volume and design of the container are the variables that have greatest impact on the size of the seedling in the nursery, particularly on the structural conformation of the root system and, consequently, on survival in the field (^{Jacobs et al., 2005}; ^{Prieto et al., 2006}; ^{Grossnickle, 2012}).

In Mexico, different types of containers for forest seedling production have been tested, but there is no single design because of the differences in growth habit of each species, production schemes, and soil and climate characteristics of the plantation site. Therefore, the most appropriate container depends on the objectives of the nursery, the species, and the production system, as well as site characteristics (^{Prieto et al., 2006}). Moreover, the color of the container has an important function in regions where temperatures are high and the use of colors that reflect solar radiation will minimize its effects.

In this context, the production of Pinus greggii Engelm. and P. oaxacana Mirov forest seedlings was evaluated in individual white, black and transparent containers, which had typical drainage design or lateral drainage. The objective was to evaluate the impact of container color, and the type of drainage on seedling quality. The hypothesis was that the seedling produced in containers with lateral drainage design would have a fibrous root system with more live roots, compared with those with typical drainage. Moreover, there would be no differences in terms of root development in the three colors of containers evaluated.

Materials and Methods

Seedlings of both species were produced in individual containers (220 mL) and with the same substrate: peat moss (50 %), composted pine bark (20 %), perlite (20 %) and vermiculite (10 %). Eight-to-nine-month slow release fertilizer (Osmocote Plus™ formula 15-9-12 N-P-K) was also added at a dosage of 7 g L^-1. Seedling permanence in the container was different for the two species, based on the time required for the seedlings to reach a suitable size, depending on their growth habits. For P. greggii, this time was six months, and for P. oaxacana it was eight months.

Seedlings were produced in the Forest Nursery of the Graduate Program in Forest Science of the Colegio de Postgraduados, Montecillo, Estado de Mexico, located at 19° 29' N and 98° 54) W, at an altitude of 2240 m. Climate type is C (Wo) (w) b (1) g', corresponding to temperate subhumid climate with summer rains, a mean annual rainfall of 750 mm and mean annual temperature of 15.5 °C, whose thermal oscillation is 5 to 7 °C (^{García, 1973}).

Seedling management in the nursery was light daily watering during germination and the first stage of development (six weeks), irrigation to saturation every other day during the stage of rapid growth and light daily watering every three days during the period of seedling hardening. Complementary fertilization with soluble products (Peters Professional™) was applied in the irrigation water once a week. During the rapid growth stage, the formula 20-20-20 (N-P-K) was used, and during the hardening stage 4-25-35 in dosages of 70 and 40 ppm, respectively.

Each species was evaluated indepently with a completely randomized experimental design and factorial array for drainage design and container color. The design included two levels grouped by containers with typical drainage (holes at the bottom of the container) and containers with lateral drainage, which included the same holes on the bottom as well as 5 mm diameter circular holes in the lateral walls (Figure 1). The colors of the containers were white, black and transparent. The combination of the two factors resulted in six container types (treatments) used in the greenhouse experiment (Figure 1). The experimental unit comprised a rack containing 25 seedlings; each type of container with each species was replicated five times.

Figure 1 Nomenclature of treatments used in the factors (container color and design). 

Seedlings were evaluated six (P. greggii) and eight (P. oaxacana) months after sowing. For each type of container, a sample of 12 seedlings located in the central part of each experimental unit was used, disregarding the seedlings on the edges to avoid edge effects. In the evaluation, height and stem diameter at the root neck were measured. To avoid damage to the shoot and root, each seedling was extracted from the container carefully, the substrate was eliminated with running water and loss of roots was avoided during the process.

The root was separated from the foliage by cutting at the root neck. The volume of the radical system was measured using a method based on the Archimedes Principle (^{Landis et al., 2010}). Excess water was then eliminated with absorbent paper and packed separately in paper bags labeled. Dry weight of the samples was determined after drying in an oven for 72 h at 70 °C. With these data, the woodiness index (ratio of root volume over root dry weight), sturdiness quotient (ratio between seedling height and diameter), ratio between shoot dry weight and root dry weight (shoot/root ratio), and the Dickson quality index (DQI) were calculated with equation 1:

(1)

Root growth potential (RGP) was measured in 15 seedlings in each type of container. The test consisted of identifying the seedling's live roots at the end of the nursery phase. For this process, the substrate was washed from the root ball and live white roots longer than 1 cm were counted and cut from the radical system manually. Five seedlings per container type (subsamples of five seedlings with three replications) were then transplanted to 10 L pots with a mixture of composted pine bark (79 %) and perlite (30 %). RGP was assessed 28 d after establishment. This consisted of measuring newly emitted roots, which were identified by their white color and length of more than 1 cm. Accumulated length and dry weight of new roots per seedling were obtained. These variables permitted evaluation of the tree's capacity to emit new roots.

Data of each species were analyzed with a two-factor ANOVA (drainage design and container color). Means were compared with the Tukey test (p< 0.05) to contrast the differences in the values of morphological variables and growth potential.

Results and Discussion

Seedling quality morphological standards

In the analysis of variance, the species were examined separately because of the differences in morphological characteristics that are attributed to the seedlings' growth habit in the nursery.

In P. greggii significant differences in diameter were found for the color, and for the drainage factor, differences were found in height, diameter and root biomass (Table 1). There were no significant differences in any variable by effect of the interaction color ( design (data not shown).

Table 1 Comparison of means (p< 0.05) for factors and variables in Pinus greggii seedlings in the final nursery stage. 

^† Diám.: Root collar diameter; Vol.: Volume of root system; Fib.: Root fibrosity; I. de esbeltez: Sturdiness quotient; Rel. PA/R: Shoot/root ratio; ICD: Dickson quality index; Transp: Transparent; S. A.: Without lateral drainage holes; C. A.: With lateral drainage.

The analysis of variance of P. oaxacana container color revealed differences only in DQI. For the factor drainage design, there were significant differences in all the morphological variables and in DQI (Table 2). The interaction of factors (color × design) had no significant effect (data not shown).

Table 2 Comparison of means (p< 0.05) for factors and variables in Pinus oaxacana nursery seedlings. 

^† Diám.: Root collar diameter; Vol.: Volume of root system; Fib.: Root fibrosity; I. de esbeltez: Sturdiness quotient; Rel. PA/R: Shoot/root ratio; ICD: Dickson quality index; Transp: Transparent; S. A.: Without lateral drainage holes; C. A.: With lateral drainage.

The seedling DQI variables (root fibrosity, sturdiness quotient and shoot/root ratio) were not significantly different by effect of the factors color and container design (Tables 1 and 2). In contrast, P. oaxacana exhibited significant differences in DQI by effect of the two factors. However, even though there are differences in morphological variables, the results are within the quality standards of the Mexican norms (^{CONAFOR, 2014}). The morphological analysis showed no difference for container color. The decision to use one or the other is influenced by the factor design, rather than by container color.

One aspect of the effect of container color to analyze has to do directly with alteration of substrate temperature, which affects root development during the growing period of the seedling in nursery (^{Landis et al., 1990}). The consequences of this phenomenon are critical in nurseries located in high temperature regions (> 35 °C), where the properties of absorption and heat conduction in the container are important (^{Landis et al., 1990}). High temperatures in the substrate inhibit root development and can cause seedling death (^{Landis et al., 2010}). The effect, however, varies among species and varieties. Monitoring temperatures during seedling production revealed a differential gradient of 7 °C when comparing the containers, with extreme temperatures (black without lateral holes vs. white with lateral holes) where 36 °C was recorded in the environmental condition of the production site.

There were significant differences for the type of drainage in most of the variables in both species (Tables 1 and 2). In P. greggii, the container with typical drainage had the highest values for height (28.4 cm), diameter (4.1 mm), shoot biomass (3.3 g), root biomass (0.8 g) and root volume (5.8 cm³). The container with lateral holes had lower values of height (26.7 cm), diameter (3.9 mm), shoot biomass (3.0 g), root biomass (0.7 g), and root volume (5.5 cm³). DQI did not differ significantly.

With P. oaxacana, the tendency was similar to P. greggii regarding the factor drainage design. The seedlings produced in the containers with typical drainage had higher values than seedlings produced in containers with lateral holes: height (25.7 vs. 23.7 cm), diameter (6.5 vs. 5.9 mm), shoot biomass (8.3 vs. 6.2 g), root biomass (2.2 vs. 1.6 g) and total biomass (10.4 vs. 7.7 g), and root volume (11.0 vs. 7.4 cm³). There were statistical differences only in DQI (1.34 vs. 0.97), the container without holes having the higher value (Tables 1 and 2).

Of the nursery seedling morphological quality variables, seedling height contributed little to quality, since there is no correlation with seedling survival in the plantation site (^{Grossnickle, 2012}) or it is negative (^{Domínguez-Lerena et al., 2006}; ^{Pinto et al., 2011}). However, diameter gives an approximation of the cross section of water transport, mechanical resistance and relative ability to tolerate high temperatures at ground level (^{Barajas- Rodríguez et al., 2004}; ^{Grossnickle, 2005}; ^{Pinto et al., 2011}). The most used morphological seedling quality variables are diameter and height (^{Luis et al., 2004}), but it is important to identify other variables that provide more information on characteristics of the ideal seedling.

In this sense, the inclusion of the different components of biomass and root volume, as well as the quality indexes derived from these variables permitted a broader approach in comparing the effect of the factors color and drainage design on seedling quality of the two species. Seedling quality, from the perspective of morphological standards, gives an idea of the seedling's ability to overcome the adverse conditions of the plantation site, but these variables are not determinant when its physiological functioning is considered (^{Davis and Jacobs, 2005}; ^{Landis et al., 2010}; ^{Grossnickle, 2012}). Better development of the morphological variables in the containers designed without holes may motivate the decision to use them. However, it is important to analyze the root architecture developed in one or the other design. In this respect, the holes in the walls of the container improve the root ball architecture by inducing lateral root pruning, with which spiral root development is prevented (^{Torrente and Peman, 2004}; ^{Ortega et al., 2006}; ^{Landis et al., 2010}).

In root collar diameter, P. oaxacana exhibited growth differentiated in the containers with typical drainage and with lateral holes. In the former, it was 10 % higher than the latter (Figure 2). Total biomass (root and shoot biomass) was not different between the designs for P. greggii, but in P. oaxacana, the typical container was 25 % higher than the container with lateral holes (Figure 3).

Figure 2 Root collar diameter of the two pine species in function of the factor drainage design. 

Figure 3 Total biomass of the two pine species in function of the factor drainage design. 

There was a 33 % difference in volume of P. oaxacana root system (Figure 4), but in that of P. greggii there was no effect of container design. The DQI of seedlings grown in typical containers was 28 % higher than those grown in containers with lateral drainage in P. oaxacana. However, in P. greggii, there were no differences between container drainage designs (Figure 5).

Figure 4 Root volume of the two pine species in function of the factor drainage design. 

Figure 5 Dickson quality index (ICD) of the two pine species in function of the factor drainage design. 

Although the seedlings produced in containers with lateral drainage had lower values than those produced in typical containers, the variables are within the seedling quality standards defined by the Comisión Nacional Forestal for different species, according to the Mexican Norm NMX-AA-170-SCFI-2014, under which forest nurseries are certified (^{CONAFOR, 2014}).

The plants grown in containers with lateral holes were at a disadvantage as the design causes greater drying than the typical container. When using lateral drainage containers, it is necessary to consider seedling moisture requirements so that the produced seedlings meet quality standards.

Root growth potential (RGP)

The container design caused significant differences in number of live roots between the species P. greggii and P. oaxacana at the beginning of the test. In the final phase of the test, number of roots emitted, accumulated length and dry weight were not different (p> 0.05) between the two species for the factors container design and color (Tables 3 and 4).

Table 3 Comparison of means (p<0.05) for root growth potential factors and variables of P. greggii. 

Table 4 Comparison of means (p<0.05) for root growth potential factors and variables of P. oaxacana. 

It is important to determine RGP because it is related to the seedling's ability to grow roots when it is established in an optimum environment (^{Oliet et al., 2003}; ^{Campo et al., 2008}). The test consists of evaluating root growth as a response to its physiological condition (^{Landis et al., 1990}; ^{Campo et al., 2008}). RGP is, from a broader perspective, an indicator of the seedling's performance attributes because it integrates a broad spectrum of morphological and physiological features (^{Gazal et al., 2004}; ^{Campo et al., 2008}; ^{Landis et al., 2010}). In most cases, when this test was used as a seedling quality criterion, it correlated 75 % with survival and performance in the field (^{Landis et al., 2010}).

Because no significant differences in RGP were observed in the evaluation of the test for the descriptive factors of the container (color and drainage design) (Tables 3 and 4), it is concluded that seedling quality, from a perspective of analysis of its physiological functioning, is good enough to survive in the plantation site. In the RGP evaluation, at the end of the test, according to ^{Campo et al. (2008)}, the values tend to become equal, and thus, it is recommendable to define protocols for these species.

At the beginning of RGP test, seedlings in containers with lateral drainage had a higher number of live roots than those in containers with typical drainage in P. greggii (220.3 vs. 167.2; Table 3) and in P. oaxacana (372.8 vs. 246.5; Table 4). The number of live roots in the radical system indicates the quantity of active radical growth meristems in the root ball, which give the seedling a higher probability of establishment in different site conditions (^{Barajas-Rodríguez et al., 2004}; ^{Landis et al., 2010}; ^{Grossnickle, 2012}).

Conclusions

The kind of drainage affected the morphological quality variables of the two species of seedlings evaluated. The containers with lateral drainage had seedlings with smaller dimensions than those with no lateral holes, although the average dimensions of the seedlings in the two container types meet nursery seedling quality standards established by CONAFOR.

The seedlings grown in containers with lateral drainage had a higher number of live roots than those in typical containers without lateral drainage holes. Besides, container color affected only root neck diameter in P. greggii, while in P. oaxacana the effect was observed only in the Dickson quality index.