Localization and vegetal material

The seeds of the 99 family used for plant production were gathered on May 2012, at the seed orchard of Campo Experimental El Palmar, in Veracruz, which belongs to the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. The seeds were sowed on April 2013, in a greenhouse of Colegio de Postgraduados, in Texcoco, Estado de México. A 2:1:1 peat, perlite, and vermiculite mix was used as substrate; it was contained in 1 L. polyethylene forestry tray cells, with 10 g of controlled-release fertilizer per cell. Germination began on the 15^th day, and was finished on the 35^th day, with a 95 % growth. Subsequently, each month, frequent irrigation and fungicide (captan^® 1 g L^-1) were applied to the plant.

Mini-cutting production

Pruning at 6 and 12 months were carried out to stimulate the growth of juvenile material, 6 cm over plant collar. In order to evaluate the impact of the cutting’s position in the rooting of each bud, the apical, intermediate, and basal sections were separated. The selected buds were 25 cm in length, and 1 cm in diameter. The leaves of each twig were cut, and three 7 cm-long cuttings were obtained.

Substrate preparation

Substrate consisted of: perlite and 2:1:1 peat, perlite, and vermiculite mix. Both were sterilized for 40 minutes with water vapor; then, they were cooled and placed in a rooting chamber.

Establishing the experiment

Irrigation and temperature were controlled in a semiautomatic 2.7x1.2x0.7 meter rooting chamber. Irrigation was scheduled to distribute 2250 mL of distilled water in 10 daily irrigations. Relative humidity was kept over 80 % and temperature fluctuated between 18 and 25 °C during the day; when temperature reached 25 °C, an automatic fan cooled the chamber until temperature reached 18 °C.

As asepsis control, cuttings were placed in a 5 % Captán^® solution during 15 min; then, to establish the best dose for rooting, cuttings were immersed in 1500, 5000 y 10 000 ppm of Radix^® solutions, during 5 seconds; industrial talcum powder was used as control.

Substrates were humidified with distilled water. Cuttings were placed in 4 cm deep holes and with a 10x10 cm spacing, pressing the substratum around them, without damaging the tissues.

Experimental design and variables evaluation

This experiment had a completely random design, with a 2x3x4 factorial arrangement, as follows: two types of substrate, three cutting cut position, and four different IBA concentrations. Ten repetitions were established (10 cuttings per treatment, 240 in total). The experiment was carried out during the fall-winter season (August 26^th 2014 January 13^th 2015); the presence of pathogens was monitored by means of regular evaluation, and 20 weeks later the percentage of live cuttings, cuttings rooted by treatment, the number and length of primary and secondary roots per cutting were evaluated.

Prior to analyzing variance, the variable expressed as a percentage (Y) was transformed with the arccosine function of the square root of the original value expressed as a decimal fraction T= arcosenoY. The number of roots was transformed using the [T = log₁₀ (Y )], logarithm function, the average values were transformed back to the original units, using the [Y =100 seno² (T )] function for the percentage variable and the [Y =10 ^T ] function for the count variable (^{Itoh et al., 2002}; ^{Syros et al., 2004}; ^{Muñoz et al., 2009}). Treatments means were compared using Tukey test (p≤0.05). Analysis were carried out with SAS for personal computers (^{SAS Institute, Inc., 2004}). An statistical analysis -using the following linear modelwas carried out to obtain variance and error estimators for each variable:

where: Y _ijkl is the response variable of i ^th type of cutting, with the j ^th IBA dose, in the k ^th type of substrate; μ is general average; E _i is the fixed effect of i ^th type of cutting (apical, intermediate, and basal); D _j is the fixed effect of the j ^th IBA dose (0, 1500, 5 000 y 10 000 ppm); S _k is the fixed effect of the k ^th type of substrate (2:1:1 peat, perlite, and vermiculite mix vs. perlite); EDi _j is the interaction between the i ^th type of cutting and the j ^th IBA dose; ES _ik is the effect of the interaction between the i ^th type of cutting and the k ^th type of substrate; DS _jk is the effect of the interaction between the j ^th IBA dose and the k ^th type of substratum; EDS _ijk is the interaction between the i ^th type of cutting, the j ^th IBA dose, and the k ^th type of substrate; and, E _ijkl is the random error related to Y _ijkl observation.

Effect of the substrate

The highest survival and rooted cuttings percentage occurred with perlite (Table 2). Perlite increased rooting and survival, in comparison with the 2:1:1 peat, perlite, and vermiculite mixture, in a two to one ratio. Primary root length was greater with mixture than with perlite, in a two to one ratio.

The best rooting with perlite is the result of its physical features, such as porosity, aeration, drainage, and pH (^{Saranga and Cameron, 2007}). ^{Bonfil et al. (2007)}, in order to keep moderate temperatures and to allow appropriate aeration, a substratum must provide constant humidity. Perlite has these features, which favour the rooting of: Pinus leiophylla Schiede ex Schltdl. et Cham. (^{Cuevas et al., 2015}); Pinus pinaster Ait. (^{Majada et al., 2010}); Pinus elliottii Engelm. var. elliottii Little & Dorman, Pinus caribea var. hondurensis Barr. & Golf. (^{Hunt et al., 2011}), Juniperus scopulorum Sarg., and Thuja occidentalis L. (^{Bielenin, 2003}).

Effect of the IBA dose

In comparison with control, a 1500 ppm IBA increased rooting by 10 %; higher doses decreased it, probably as a result of toxicity. According to ^{Mesén et al. (1997)} some forestry species -such as Eucalyptus, Cupressus lusitánica Mill., Alnus acuminata Kunth y Tectona grandis L. (^{Husen y Pal, 2003})do not tolerate concentrations higher than 2000 ppm. With a IBA of 1500 ppm, the number of primary and secondary roots was three times and twice than in the control group. The length of primary and secondary roots did not have a significant effect (p≤0.05) with IBA, in comparison with the control group. However, there was more than 30 % difference (p≤0.05) in cutting survival between the best and worst treatments.

With a 5000 ppm dose, the number of primary and secondary roots was five and three times higher (p≤0.05) than the control group. Once cuttings have established their roots, enhanced IBA doses increase the transportation of substances towards the cutting’s base, as a result of which more roots are developed (^{Mesén et al., 1997}).

The lowest percentage of rooted cuttings was obtained with 10 000 ppm (Table 2), attributable to auxin intoxication. Cuttings without IBA showed two times more rooting than with 10 000 ppm; this indicates that cuttings have enough endogenous auxins to foster rooting.

Auxins foster adventitious root development in cutting rooting (^{Bielenin, 2003}; ^{Castrillón et al., 2008}; ^{Kumar et al., 2011}). ^{Castillo et al. (2013)} suggest using IBA in species with difficult rooting. Rooted cuttings percentage showed significant differences (p≤0.05) between IBA doses. Auxins stimulated cambium activity and reserve mobilization towards the place where roots begin (^{Dhillon et al., 2011}). IBA improves rooting in Pinus jaliscana Pérez de la Rosa (^{Aparicio et al., 2006}); Viccinium meridionale Swartz (^{Castrillón et al., 2008}); Taxus globosa Schltdl. (^{Muñoz et al., 2009}); Olea europaea L. (^{Denaxa et al., 2012}), and Gmelina arborea Roxb. (^{Ruíz et al., 2005}).

Effect of the position of mini-cuttings

Survival and rooting percentages of apical cuttings were 20 and 50 % higher, respectively, than those of intermediate and basal cuttings (Table 2). Apical cuttings favored the amount of primary and secondary roots -twice and four times, respectively-, with regard to intermediate and basal cuttings. Additionally, the primary roots of apical cuttings were twice longer than intermediates (which was the second best). There were no differences with regard to the length of secondary roots.

Apical cutting showed the best rooting, perhaps because of some physiological or anatomical condition related to the presence of leaf buds. According to ^{Hartmann et al. (2013)}, apical cuttings root better than lateral cuttings, because apical leaf buds produce plentiful auxins, and stimulate radical development (^{Moore, 1984}). ^{Ruíz et al. (2005)} noted that the degree of juvenility, tissue lignification, reserves content, and, in particular, the level of endogenous phytohormones are the factors involved in enchancing the rooting capacity of Gmelina arborea apical cuttings.

In some cases, basal cuttings root better, like in Populus, where it doubles intermediate and apical cuttings (^{Zalesny et al., 2003}). ^{Cuevas et al. (2015)} noted that the rooting of P. leiophylla basal cuttings is 3.5 more likely to succeed. The reason for this variation between species is unknown, and it is yet to be researched. In this study, although apical cuttings had the best rooting response, using basal cuttings to propagate C. odorata select clones has not been rejected. However, increasing the rooting of basal cuttings percentage requires constant experimentation; therefore, most of the material would be used to maximize plant production.

Effect of the interaction between factors

Only the substrate-type of cutting interaction showed a significant effect in the length of secondary roots (Table 1). The combination of apical cutting with substrate (2:1:1 peat, perlite, and vermiculite mix) favored longer secondary roots, 96±11 mm. Root length decreased gradually according to the type of cutting, from the longest (apical) to the shortest (basal); however, root length decrease was greater with mixture (intermediate position cuttings; Figure 1).

Figure 1 Interaction of cutting position and substrate in the length of secondary roots. Each point represents the average with the standard deviation. 

The root length induced in cuttings can show the speed of rooting; the longest cuttings had less time to put down roots (^{Ruiz et al., 2005}). In our study, the secondary roots of apical cuttings probably appeared first in the mixture’s substrate than in the perlite. This could prove advantageous, as it would decrease the time that the cuttings stay in the propagation unit, the contamination risks, and the maintenance costs. However, this substrate is still not recommended, because it is still not known if the length of cutting roots established in perlite can reach the size than those in the mixture, in one or two weeks.

These results show that there is a big potential for C. odorata vegetal propagation through cutting rooting. More research is necessary to determine if the greatest rooting percentage can be achieved in less time. Other IBA concentrations (around 1500 ppm), high quality clones, and apical sections could also be evaluated.