IntroductionTop

Pinus patula Schiede ex Schltdl. et Cham is one of the most economically important tree species among the subtropical pines natives to Mexico and Central America (Perry, 1991). Despite its restricted natural distribution range in the Sierra Madre Oriental, in eastern Mexico, the species has a fast growth rate and adaptive capacity to different soil and climate conditions, so it has been highly successful in forest plantations established in subtropical regions (Wormald, 1975; Velázquez et al., 2004), including southern and eastern Africa, South America and Australia (Perry, 1991; Dvorak et al., 2000); in South Africa, P. patula occupies about 50% of all the planted land (Malabadi & van Staden, 2005).

Due to the productive potential and wood quality of P. patula, there is a growing interest in Mexico in the genetic improvement of the species, with several progeny trials and seed orchards with phenotypically selected trees being established in the last decade in central Mexico (Salaya-Domínguez et al., 2012; Morales-Gonzalez et al., 2013). However, in most forest trees, particularly conifers, a long time is required before reaching sexual maturity (von Aderkas et al. 2004). Even when sexually mature scions from selected trees are grafted into seedlings, abundant production of genetically improved seed might take up to 10-15 years after grafting (Sirikul & Luukkanen, 1987; Cherry et al., 2007). In addition, variation in maturity and fertility among genotypes in the orchard, particularly during the early development of grafts, commonly leads to an irregular seed production and uneven clonal representation in the seed crop, negatively affecting genetic diversity and genetic gain in the seedling population (Harrison & Slee, 1991; Codesido & Merlo, 2007). An option to increase seed production and homogenize parental contribution in the seed crop of young grafts is to promote differentiation of reproductive structures in them. Several cultural practices and treatments have been successfully used to promote flowering in forest tree species, such as root pruning, water and high temperature stress, mineral nutrition, stem girdling and application of plant hormones (Philipson, 1990; Meilan, 1997; Owens et al., 2001).

Most treatments for flower induction are generally effective, but the highest production of reproductive structures is commonly obtained when plant hormones are applied, alone or combined with stem girdling (Pharis et al., 1987; Kong & von Aderkas, 2004; Wakushima, 2004). Gibberellic acid (GA) is the phytohormone most used to promote early flowering in forest trees, particularly the combination of GA₄ and GA₇, which has been very effective in several conifers (Philipson, 1990; Kong & von Aderkas, 2004), including various pine species (Wheeler & Bramlett, 1991; von Aderkas et al., 2004; Peng et al., 2011).

The success of GA_4/7 application for flower induction depends on age of trees, timing and method of application, dosage, and combined effect of other cultural practices (Ross, 1991, 1992; Philipson, 1992, 1996). Since gibberellins affect cell division and differentiation of reproductive structures (Ho & Schnekenburger, 1992; Kende & Zeevaart, 1997), as well as growth and development of female strobili (Owens, 1985), time of application is critical to get both male and female flower induction in these species (Ho & Eng, 1995; Siregar & Sweet, 1996; Wakushima et al., 1997). Thus, GA_4/7 must be applied during the period of shoot elongation, weeks before differentiation of reproductive structures would occur, in order to be effective (Ho & Eng, 1995; Siregar & Sweet, 1996; Muñoz-Gutiérrez et al., 2010).

In temperate pine species having only one shoot growing cycle, differentiation of pollen and seed cones occurs in late spring or early summer, before the rainy season, so GA_4/7 is usually applied in early spring (Ho & Eng, 1995). However, tropical and subtropical pine species have a more complex seasonal pattern of shoot growth (Sirikul & Luukkanen, 1987; Dick, 1995; Owens, 1995), with several overlapped shoot growing cycles per year (Gómez-Cárdenas et al., 1998; Isaza, 2008), so knowing the exact time for differentiation of reproductive buds is difficult, and the effective application of GA becomes more complicated (Sirikul & Luukkanen, 1987; Harrison & Slee, 1991; Codesido & Merlo, 2007). For these species, we need first to determine the appropriate timing of GA_4/7 application in order to develop effective treatments for early flowering.

In 2003, a clonal seed orchard of P. patula was established in central Mexico (municipality of Aquixtla, Puebla) as a part of a regional tree improvement program. The purpose of the orchard is to produce seed from phenotypically selected trees for commercial plantations within the natural range of the species in the region. This study was done to contribute in developing a cultural practice to accelerate and increase the production of genetically improved seed in orchards of the species; the specific objectives of the study were (1) to evaluate the effect of GA_4/7 injection and stem girdling at different times during the growing season on induction of male and female strobili on young P. patula grafts, and (2) to compare the effectiveness of strobili induction among clones with different cone production history.

Materials and methodsTop

ResultsTop

Percentage of trees with reproductive structures

Overall, 48% and 82% of grafts produced male and female strobili, respectively, in FT₁; the respective values in FT₂ were 79% and 66% (Table 1). No significant differences among girdling levels, GA_4/7 dose or application dates were detected with the chi-square test in both trials, but the percentage of trees with male and female strobili differed among clones (Table 1). All clones produced male and female strobili in the FT₂ with percentages varying from 67 to 100% for male and from 38 to 95% for female strobili (Table 1).

Table 1. Percentage of P. patula grafts (%) with male and female strobili in each trial (FT₁ and FT₂), by clone, GA_4/7 dose, application date, and girdling level

In FT₁, 92% of grafts receiving 1.27 g cm^–2 GA_4/7 in September produced female strobili versus 75% in the control treatment; in FT₂ respective values when applied in July were 92 and 25% (Table 1). On the other hand, 96% of grafts produced female strobili in FT₁ when treatments were applied in September, and 71% when applied in October, whereas in FT₂, 96% of grafts produced female structures when treated in July, and 54% when treated in September (Table 1).

Production of male and female strobili

Significant differences (p < 0.05) among clones, but not among application dates, GA dose, girdling levels or their interactions, were found in the production of male and female strobili in FT₁ (Table 2); stem girdling and GA_4/7 application did not have a positive effect on differentiation of reproductive structures in this trial. In FT₂, however, a significant main effect (p < 0.05) of clones and application dates was found for all traits measured on male and female strobili (Table 2). Treatment effects and clone X treatment interaction were significant only for male strobili in this second trial. No other interaction effects were significant in the analysis across application dates. When treatments applied in early July were analyzed separately (to include the control), treatment effects were significant (p < 0.05) for both male and female strobili production, but clone differences were significant only for total and upper crown female strobili, and the interaction clone X treatment was not significant. Thus, except for the interaction effect detected in production of male strobili, the clones tested responded similarly to the treatments and application dates.

Table 2. Statistical significance values (p) obtained from analysis of variance for factors included in the flower induction trials of P. patula.

The timing of treatment application was critical for cone stimulation, as shown by the contrasting results in both trials. In FT_1, when treatments were applied in late summer and early fall, none of the traits was affected. However, in FT₂, treatments applied in early summer (on July 3) increased substantially the production of both male and female strobili (5-fold increase in male strobili and 3-fold increase in female strobili per tree) as compared to the treatments applied in early September (Table 3).

Table 3. Effect of application date, GA_4/7 dose and partial stem girdling on the average number of male and female strobili formed in the two flower induction trials (FT₁ and FT₂) for P. patula

In FT₂, combined application of GA_4/7 and partial stem girdling had a positive effect on induction of both male and female strobili, as compared to the application of GA_4/7 alone, but the effect was significant only for male ones. GA_4/7-treated grafts produced on average 71.9 male strobili per branch in 34.2 % of the branches in the lower whorls and 14.7 female strobili per tree, whereas grafts receiving the combined treatment produced 101.1 male strobili per branch in 48.7% of branches and 22.8 female strobili per tree (Table 3). This represents a 100 and 55% increase in the number of male and female strobili, respectively.

When applied in July, use of GA_4/7 and partial stem girdling increased production of male strobili by 84% and female strobili by 89 % with respect to the use of GA_4/7 alone. GA_4/7 applied in July also caused a 3-fold increase in the number of male strobili (as a result of almost doubling the percentage of branches with strobili and the number of strobili per branch), and a 13-fold increase in the number of female strobili per tree, as compared to the control (19.7 vs. 1.4, Table 3). Thus, the combined application of GA_4/7 and partial stem girdling in July caused a 5-fold increase in male strobili and over a 25-fold increase in female strobili per tree, compared to the control (Table 3). However, when GA_4/7 alone or combined with partial stem girdling were used in September, the production of male strobili was similar to the control, and the number of female strobili per tree was only 8.4 and 9.9, respectively (data not shown).

Production of male and female strobili also varied among clones in each trial. Overall, in FT₁ less male strobili per branch, but more female strobili per tree, were formed than in FT₂ (Table 4), primarily because of the different clones included in each trial and the variation in cone production in previous years. However, for clone 33 included in both trails, male and female strobili production increased in the second trial.

Table 4. Clone and trial differences in the number of male and female strobili formed in the two flower induction trials (FT₁ and FT₂) for P. patula

In FT₁, clones in the “high production” group produced on average over 10 times as much male and female strobili as those in the “low production” group (Table 4). Clone 19 was the most prolific, with over 110 male strobili per branch in 82.5 % of branches at each whorl and over 100 female strobili per tree (Table 4). In the “low production” group, clones 14 and 46 behaved mostly as females, with around 10 female strobili per tree, and clone 12 mostly as male. In FT₂, clones 27 and 32 were quite fertile with 100 or more male strobili per branch and almost 30 female cones, but clones 33 and 34 had much lower production of reproductive structures for both sexes (Table 4). The significant clone X treatment interaction detected for male strobili in FT₂ revealed a larger effect of the combined use of GA_4/7 and partial stem girdling, compared to the use of GA_4/7 alone, on the proportion of branches and number of male strobili per branch on clones 27 and 33 than on clones 32 and 34 (Figure 1).

Figure 1. Effect of clone and treatment in the proportion of branches with male strobili (a, b), number of male strobili per branch (c, d), and number of female strobili per tree (e, f) in two separate flower induction trials for P. patula: FT1 (a, c, e) and FT2 (b, d, f). In both trials, the bars represent the mean value ± s. e. across application dates, except for the control, which was applied only in the first date. In FT1, clones are grouped as “low” or “high” based on cone production in previous years, and treatments represent the GA4/7 dose used in mg cm–2 cross-sectional area of stem. In FT2, “GA” represents 1.27 mg cm–2 GA4/7 and “Gi” is partial stem girdling (see text).

DiscussionTop

Injection of a commercial mixture of GA_4/7 combined with partial stem girdling were successful in stimulating male and female cone production in young P. patula clones. Even though similar enhancing effects of GA_4/7 applied alone or combined with other cultural treatments have been reported for several temperate, subtropical and tropical pine species (Greenwood, 1982; Sirikul & Luukkanen, 1987; Harrison & Slee, 1991; von Aderkas et al., 2004; Kong & von Aderkas, 2004; Codesido & Merlo, 2007; Peng et al., 2011) no previous information was found for P. patula, so it seems to be the first report of effective flowering induction with GA_4/7 and stem girdling for this species. The effects of treatments were primarily on the amount of strobili formed per tree. A trend to increase the proportion of trees with a positive response was also observed, particularly in FT₂, but the differences were not enough to be declared significant with the contingency chi-square test. The overall percentage of trees with female strobili was relatively stable in both trials; however, the percentage of trees with male strobili increased in the second trial. In addition, although all clones included in the FT₂ produced male and female strobili, the proportion of ramets forming cones varied widely among them, and the response in strobili induction in this study seems to be dependent on several factors.

The contrasting results obtained in FT₂ for treatments applied in July, compared to when they were applied in September, confirm that treatments in FT₁ were applied too late in the growing season. In that particular year most probably the buds were already differentiated by the time the physiological stimuli produced by injecting GA_4/7 or girdling the stem reached the tree crown. Previous studies of flowering induction in most conifers, particularly in Pinus species, have emphasized the importance of optimum application time of treatments to stimulate differentiation of reproductive structures (Greenwood, 1982; Wakushima et al., 1997; Isaza, 2009). In those studies, the optimum period for GA application to promote flowering in Pinus species has ranged from March to September (Luukanen & Johanson, 1980; Greenwood, 1982; Harrison & Slee, 1991; Siregar & Sweet, 1996; Wakushima et al., 1997; Isaza, 2008), depending upon the species and the particular environmental conditions where the studies were done.

We could not find any published information about the timing of differentiation for reproductive structures in P. patula, but the striking differences in the effectiveness of GA_4/7 and stem girdling found among both trials indicates that it must be occurring sometime in mid-summer, since treatments applied after August did not have a positive effect on sexual differentiation of primordia, and it commonly takes 1-3 weeks after injecting GA or doing the girdling in the stem to detect any changes in phytohormones or related metabolites in the tree crown (Kong et al., 2008, 2012). In the region where the flowering induction was done, young trees of Pinus patula have a shoot growing period that extends from February up to December at least, with 4-5 successive and overlapping shoot elongation cycles (Gómez-Cárdenas et al., 1998), so it is difficult to pinpoint the exact timing when the morphological changes associated with bud formation and differentiation are occurring in the terminal leader or first-order lateral branches. On the other hand, the optimum timing for GA_4/7 and stem girdling application might differ with any change in the environmental growing conditions, size of trees, application method, or other factors influencing either the time of strobili differentiation or the distance from the application point in the stem to the crown, as has been shown in other Pinaceae species (Chalupka, 1980; Daoust et al., 1995; Siregar & Sweet, 1996; Almqvist, 2003; Kong & von Aderkas, 2004; Isaza, 2008).

Compared across application dates, partial stem girdling was more effective on improving the effect of gibberellin in the production of male strobili than female ones, but when applied in July the combined treatment is effective for both sexes, or even more so for female strobili (compared to the control treatment). Thus, either the physiological stimuli produced by stem girdling and GA_4/7 injection differ (Pharis et al., 1987; Ross, 1992; Dick, 1995; Kong et al., 2012), or the differentiation of both type of reproductive structures does not occur at the same time in P. patula, as has been found in other conifers (Chalupka, 1980; Pharis et al., 1987; Peng et al., 2011; Kong et al., 2012). Male strobili are mainly produced in the lower crown branches whereas female strobili are concentrated in the middle and upper crown branches (Chalupka, 1980), so differences among the effect of GA_4/7 alone or combined with partial stem girdling might also be related to the differing distance of the respective branches to the point on the stem where the treatment was applied. Though not statistically significant, data in Table 3 for FT₂ shows that the relative effect of the combined application of GA_4/7 and partial stem girdling on female strobili was higher in the lower section of the crown. An anatomical and phenological study aiming to determine the timing of differentiation for both male and female primordia in P. patula would allow to improve the effectiveness of the flowering induction treatments and even promote sex conversion of male to female primordia in the lower branches to have a more balanced sex ratio, as has been shown to occur in other pine species (Wakushima, 2004; Kong et al., 2015).

The variation among clones in the amount of reproductive structures produced (either male or female strobili) in P. patula is common to most tree species studied so far (Longman, 1982; Harrison & Slee, 1991; Siregar & Sweet, 1996; Codesido & Merlo, 2007; Isaza, 2009; Peng et al., 2011) and it is generally due to genetic variation among them in sexual precocity, fecundity or sex behavior (i.e., maleness or femaleness). Contrasting with studies in other pine species where the clonal response to the induction treatments varied between “good” and “poor” flower producer clones (Harrison & Slee, 1991; Siregar & Sweet, 1996; Codesido & Merlo, 2007; Isaza, 2008), in our study the effect of GA_4/7 and stem girdling on female strobili was similar for all the clones tested (as indicated by the lack of clone group (or clone) by treatment interaction in both trials), regardless of their precocity and reproductive output in previous years or during the year of measurement. We recognize that the inability to detect a significant clone by treatment interaction for female strobili, particularly in FT₂, might be partially due to the structure of treatments used, the limited number of replications available and the intrinsic variability in the response among ramets, since the relative change in production of female strobili due to the use of GA_4/7 combined with stem girdling, compared to the use of GA_4/7 alone, or to the control treatment included in July, varies among clones (Figure 1). Despite this, the significant clone X treatment interaction detected for male strobili and the variation among clones in relative amounts of female strobili produced in FT₂ due to the combined effect of GA_4/7 and stem girdling were not related to the average clonal fecundity in the measurement year.

The increase in the amount of reproductive structures observed in clone 33 (a “poor” producer clone) from FT₁ to FT₂ might be due to age or changes in environmental conditions from year to year but, most probably, it was due to the timing of induction treatments. In FT₂, clone 33 produced only 21.3 male strobili per branch and 0.7 female strobili per tree in the control treatment, but it produced 87.3 male per branch and 16.0 female strobili per tree when GA_4/7 and stem girdling were applied in July (data not included). Thus, these cultural treatments could be implemented as standard management practices in the seed orchard to enhance cone and seed production and to increase the genetic diversity in the seed crop by incorporating a larger number of clones, particularly those that would normally produce a low number of male or female strobili without the stimulus of the induction treatment.

ConclusionsTop

Results from this study show that the combined use of GA_4/7 injected to the trunk and stem girdling effectively promote production of male and female strobili in young P. patula grafts, when they are applied on time to induce differentiation of the reproductive structures. Under the study conditions the most appropriate application date was in early July. Even though a broad clone variation in reproductive capacity was found in the study, the response to the induction treatments was not related to their fecundity in previous or during the evaluation years. Thus, the operational use of these cultural treatments would allow to accelerate and increase both seed production and genetic diversity in the seed crop of the seed orchard.