Inhibitory Effect of Jasmonic Acid and Ethylene on Epicotyl Growth and Bud Induction in the Maritime Pine, Pinus Pinaster Soland. in Ait Introduction

Two independent parameters, epicotyl height (cm) and number of induced buds were studied on Pinus pinaster explants to analyse the effects of three phytohormones (6-benzylaminopurine, jasmonic acid, ethylene) which were combined or not in 11 different treatments. Epicotyle length diminished significantly in relation to the control medium (medium without exogen phytohormones) in presence of jasmonic acid, 6-benzylaminopurine or Ethephon (which is converted to ethylene in plants) in any of treatments. Concentrations of 100 μM of jasmonic acid and Ethephon had a greater inhibitory effect than the treatments with 10 μM. In addition to that, jasmonic acid was a stronger inhibitor than Ethephon in any of the tried combinations. There were no significant differences between the control treatment and the treatments with only 10 μM of jasmonic acid or Ethephon. However, 10 μM 6-benzylaminopurine induced bud formation. The different combinations of 6-benzylaminopurine with jasmonic acid and Ethephon showed that concentrations of 10 to 100 μM did not affect the number of induced buds. Jasmonic acid had an inhibitory effect which Ethephon only showed when combined with 100 μM of jasmonic acid and 10 μM of 6-benzylaminopurine. Three response groups were defined by cluster analysis: group 1 produced the greatest mean number of buds (4 to 5) and a mean epicotyl growth of 1 to 1.5 cm; group 2 produced 2 to 4 buds and a mean growth of 0.5 to 1.2 cm; group 3 produced only one bud and a mean epicotyl length of 1.2 to 2 cm.


Introduction
In most species, once the programmes for genetic improvement are advanced and outstanding individuals are available, one may resort to cloning of the selected genotypes to obtain the greatest possible descent, particularly if the improved trait does not present high heritability.The maritime pine, Pinus pinaster does not spontaneously spread through vegetative propagation.Propagation by cuttings or plantlets has been tried with reduced success.In vitro culture is introduced as an alternative that could generate an important number of copies of the same genotype (Tang et al., 2006).However, like other types of vegetative propagation, in vitro culture of conifers is not easy, and becomes more difficult as the individual to be cloned gets older (Abdullah et al., 1987).Another aspect that conditions the experiment is the influence of the genotype; there are genotypes which are more successful than others (Tang et al., 2001).In vitro culture has the additional advantage of allowing the conservation of a great number of genotypes during a theoretically unlimited period of time in a reduced space through the establishment of a bank of in vitro germplasm.As the first step for clone propagation of outstanding individuals of Pinus pinaster, the in vitro technique has been refined by using young material, i.e., newly germinated plantlets.
To our knowledge this is the first studies that have been carried out with Pinus pinaster, on the effect of phytohormones of maturity on in vitro bud induction and growth.Jasmonic acid and Ethephon (2-chloroethyl phosphonic acid, a plant growth regulator which is converted to ethylene by plant metabolism) have been tested alone or in a different combinations with the 6benzylaminopurine cytokinin.The latter is generally used as a phytohormone in the micropropagation of plants to activate dormant lateral buds, induce cell division and shoot formation and delay senescence.Ethylene participates in the regulation of a large variety of developmental processes, from seed germination to cell elongation, fruit ripening, organ senescence and abscission (Stepanova and Alonso, 2005).Jasmonic acid is usually present in the picomolar range per gram of fresh leaf tissue, and can quickly increase under external stimuli.Some organs and tissues exhibit more than 10 times the level found in leaves, which suggests that these high levels have different functions in the regulation of particular processes of development (Wasternack and Hause, 2002).
Jasmonates are signal compounds that appear not only as a defence against infections, and to promote tolerance to salinity and drought, but also in a variety of physiological mechanisms related to biotic and abiotic stress (Creelmann andMullet, 1995, 1997;Davis et al., 2002;Pedranzani et al., 2003).Thus, we studied the individual and combined effects of the three phytohormones: 6-benzylaminopurine, jasmonic acid and ethylene.
Jasmonic acid and ethylene are phytohormones associated with stress, and they are involved in the regulation of the expression of genes intervening in maturity and senescence (Bohnert et al., 1995;Park et al., 1994).It has been recently shown that jasmonic acid is involved in the response of Pinus pinaster to stress due to cold and drought, and that its phenotypic expression is probably related to Spanish genotypes (Pedranzani et al., 2007).A number of positive and negative interactions between ethylene and jasmonate have been described both at physiological and molecular levels (Ellis and Turner, 2001).Both hormones participate in the defence response to a variety of fungal pathogens.Similarly, both hormones positively interact on the transcription of several wounding-regulated genes and on the formation of the apical hook (Rojo et al., 2003).Analysis of the regulation of gene expression in response to both ethylene and jasmonic acid in different genetic backgrounds has uncovered one of the best-characterized examples of interactions between hormones at the molecular level (Lorenzo et al., 2003).
In the current paper, two independent parameters, epicotyl growth (cm) and number of induced buds were studied on Pinus pinaster explants to analyse the effects of three phytohormones combined in 11 different treatments.

Material
Commercial seeds of Pinus pinaster Soland. in Ait. from Sierra de Guadarrama, were provided by El Serranillo, gathered in 1998/99 and were from different progenies.The three phytohormones, Ethephon (2chloroethylphosphonic acid), jasmonic acid and 6benzylaminopurine were acquired from Merck and Sigma.The phytohormones were added to the culture media after autoclaving.

Sterilization of seeds
Pine seeds were immersed in water for 12 hours.Those that did not float were surface sterilized with 70% ethanol for 2 min., then washed in sterile water for 15-20 min., then in calcium hypochlorite at 3%, for 15-20 min.and finally three times in sterile water for 15-20 min.

Germination
Once sterilized, 20 seeds were placed on moist sterile vermiculite in jars of 10 cm in diameter by 10 cm in height.The seeds were incubated at 22ºC, with a relative humidity of 75-85%, and a photoperiod of 16 light: 8 hours darkness.After the pericarp was loose, the cotyledons sprouted and the radicle emerged, the seedlings were surface sterilised with calcium hypochlorite at 1%, before sowing them in the culture medium.EFFECT OF JASMONIC ACID AND ETHYLENE OF Pinus pinaster

In vitro setting
Seedlings used as explants had the radicle cut, and only a 0.5 cm end left.This portion was introduced in the culture medium.

Bud induction and growth
The medium used to cultivate buds was Campbell and Durzan (1975) macronutrients with Murashige and Skoog (1962) micronutrients, with vitamins (George, 1993).Iron (80 mg/L, sucrose (20 g/L), and agar (8 g/L) were added.Three phytohormones (6-benzylaminopurine, jasmonic acid and Ethephon) were added to the basic medium in 11 different combinations (Figs. 2 and 3).Both the number of induced buds and growth (epicotyl height, apical needles included, in cm) were registered for each treatment after 23 days in culture.

Statistical analysis
Differences in the mean number of buds and growth in the 11 treatments were analyzed by one way ANOVA.The Tukey test was used for post hoc comparisons between pairs.The effects of the different hormones (6benzylaminopurine, jasmonic acid and Ethephon) and the quadratic effects of jasmonic acid and Ethephon were analyzed by regression analysis.A general GLM model was performed for height and number of buds.In addition, a generalised GLZ model for number of buds was performed, due to the lack of normality in this variable.This model was consistent with the GLM model, thus only the GLM model is presented here.All these analy-ses were performed with STATISTICA.The relationship between the active variables (epicotyl height and bud number) was tested by main components analysis (SPAD programme).

Results
Main components analysis for the active variables, epicotyl growth (cm) and number of buds, yielded an angle of about 90º (Fig. 1), thus showing that they were unrelated variables.

Epicotyl growth
In figure 2, the mean values for epicotyl height in each of the 11 treatments are presented; for each treatment error bars indicate the standard deviation of the replicates.ANOVA test indicated statistically significant differences (P < 0.0001).Significant differences between pairs are indicated in Table 1.The GLM model for regression analysis was significant (P < 0.0001) and explained 31% of the variability.The equation obtained was Height = 1.7592 -0.0332*BA -0.0374*E + 0.0003*JA 2 + 0.0002*E 2 , where BA is the effect attributed to 6-benzylaminopurine, JA is that of jasmonic acid and E is that of Ethephon all the single and quadratic effects were significant.Therefore, the phytohormones had an inhibitory effect on epicotyl growth.

Bud induction
The mean number of induced buds under the different treatments is presented in figure 3. The ANOVA test showed highly significant differences between treatments (P < 0.0001).Post hoc analysis with the Tukey test indicated several significant differences between pairs (Table 2).The control treatment (1) was not statistically different from treatments 3 (jasmonic acid) and 6 (Ethephon), in which only the apical buds were developed; the other treatments produced a significantly greater number of buds.Treatment 2 (6-benzylaminopurine) increased significantly the number of buds to a mean 4.78, which represented the highest one obtained in this study.The GLM model for regression analysis yielded a significant P value (<0.0001) and explained a 49% of the variability.The equation obtained was: number of buds = 1.3680 + 0.3046*BA -0.0747*JA -0.0211*E + 0.0006JA 2 + 0.0002*E 2 , where BA is the effect attributed to 6benzylaminopurine, JA is that of jasmonic acid and E is that of Ethephon.The single and quadratic effect of Ethephon were not significant, while the effects of 6benzylaminopurine and jasmonic acid (both single and quadratic) were highly significant.The parameter esti- mates indicated that the greater effect was produced by 6-benzylaminopurine (estimate= 0.3046), that stimulated the production of lateral buds.This can be interpreted as the number of buds increased at a rate of 0.3 by unit of 6benzylaminopurine.
The effect of jasmonic acid alone on the number of buds was inhibitory (estimate= -0.0747): a decrease rate of 0.07 buds per unit of jasmonic acid was expected with the 10 μM concentration, while at the expected decrease in number of buds was lower (r 2 =0.0006) at the 100 μM concentration.The phytohormone combinations of treatments 4 (6-benzylaminopurine: jasmonic acid, 10:10) and 5 (6-benzylaminopurine: jasmonic acid, 10:100) produced mean values of 3.22 and 3.06 buds respectively.When increasing jasmonic acid concentration from 10 μM to 100 μM in presence of 6benzylaminopurine, there was no further significant inhibition increase (Table 2).Treatments 7 (6benzylaminopurine: Ethephon, 10:10) and 8 (6benzylaminopurine: Ethephon, 10:100) induced 4.06 and 4.50 buds respectively.As it can be observed in figure 3, treatments 7, 8, 9 and 10 did not present significant differences in the mean bud number (4.06; 4.50; 4.45; 4.00).Those values represented a significant in-crease in relation to the control treatment (1).Finally, medium 11 (6-benzylaminopurine: jasmonic acid: Ethephon, 10:100:10) with 2.19 buds per explant showed an intermediate level of response between treatments 1, 3 and 6, which produced only one bud; treatments 4 and 5, which produced 3 buds; and media 2, 7, 8, 9 and 10, which produced 4 or more buds.These data confirmed that jasmonic acid had a stronger inhibitory effect than of Ethephon on bud induction.On the other hand, there was a concentration effect, i.e., in presence of 6benzylaminopurine and Ethephon, inhibition was higher with 100 μM than with 10 μM of jasmonic acid.

Cluster analysis
Main components analysis (SPAD) was performed on the data reported above, with the objective of correlating the global effects of the treatments used, which were grouped according to the recorded height and bud number.A cluster analysis represented in a direct hierarchical tree grouped the 11 treatments in three groups (Fig. 4).In the first group, treatments 2, 7, 8 and 9 were included; in the second one, treatments 4, 5, 10 and 11; and, in the third one, treatments 1, 3 and 6.

Discussion
Plant responses to exogenous or endogenous phytohormones are usually the result of the activation of more than one signalling and response pathway.In this study, Pinus pinaster seedlings with the radicle cut were submitted to 11 different phytohormones combinations.The responses to these conditions were measured in terms of epicotyl growth and the number of lateral buds induced.
Epcotyle growth diminished significantly in relation to the control medium in presence of jasmonic acid, 6benzylaminopurine or Ethephon, either alone or in combination.The treatments with concentrations of 100 μM jasmonic acid and Ethephon had a stronger inhibitory effect than the treatments with 10 μM.As it can be observed in figure 2, if different treatments that contain one, two or three phytohormones in concentrations of 10 μM (2, 3, 4, 6, 7 y 9) are compared in pairs, the P values over 0.05 show there are no significant differences except in case 2:4.In this comparison, the inhibitory effects of jasmonic acid were slightly added to those of 6benzylaminopurine.Strangely enough, not significant differences were observed when 6-benzylaminopurine was combined with either Ethephon alone or jasmonic acid plus Ethephon.In other words, each of the phytohormones had on its own, the same inhibitory effect on epicotyl growth as when combined in concentrations of 10 μM.Comparing with one another the media in which at least one of the phytohormones was present at a concentration of 100 μM (5, 8, 10 and 11), a P value over 0.05 was observed.This pointed that the growth inhibition produced was not significant and that all these treatments form a more homogenous group than the one with concentrations of 10 μM.The comparison of the media of 10 μM and 100 μM presented extreme cases like medium 8 (8 vs. 2, 8 vs 3, 8 vs. 4, 8 vs. 6, 8 vs. 7 and 8 vs. 9) without significant differences, and the opposite case of medium 11 (11 vs. 2, 11 vs. 3, 11 vs. 4, 11 vs. 6, 11 vs. 7 and 11 vs. 9).The rest of the comparisons gave intermediate results which are also more complex to interpret.In addition to that, and although values did not reach statistical significance, jasmonic acid had a stronger inhibitory effect than Ethephon in the tried combinations.When treatments 3 vs.6, 4 vs. 7, 5 vs. 8 and 11 vs. 10 were compared, P values over 0.05 were obtained which showed that differences were not significant.However, the comparison of 6-benzylaminopurine alone or in combination with jasmonic acid (2 vs. 4 or 2 vs. 5) gave null P values which showed that values obtained were different; this was not the case for the combined treatment of 6-benzylaminopurine and Ethephon (2 vs. 7 and 2 vs. 8).
Regarding the number of induced buds, there were no significant differences between the control (treatment 1) and the jasmonic acid and Ethephon treatments.However, jasmonic acid had a strong bud-inducing effect (Fig. 3).The concentrations of 10 μM or 100 μM of jasmonic acid combined with 6-benzylaminopurine (4 and 5) did not produce any significant differences in bud number.The same was observed for the concentrations of 10 μM or 100 μM of Ethephon with 6-benzylaminopurine (7 and 8).This indicated that the difference of concentration did not intervene in the number of induced buds; it also pointed that it was important to consider the type of phytohormone to combine with 6-benzylaminopurine.Jasmonic acid had an inhibitory effect that Ethephon did not produce.The comparison of the p-values of 4:7 and 4:8 showed that the differences were significant.The identical response of treatments 2, 7, 8, 9 and 10 showed that there was no difference between the combinations of one, two or three phytohormones, as long as 6-benzylaminopurine was combined with Ethephon at 10 μM or 100 μM with or without jasmonic acid at 10 μM.In all these media, four or more buds per explant were induced.Treatment 11 with its mean two buds per explant, showed the inhibitory effect of 100 μM of jasmonic acid on bud number and got statistically near to the values of treatments 1, 3, and 6 (one bud), and also of the values of media 4 and 5 with about three buds per explant.Jasmonic acid is usually in the picomolar range in leaf tissue, while other tissues exhibit more than 10 times such level, suggesting that jasmonic acid may act in a variety of physiological mechanisms in non-leaf tissue (Wasternack and Hause, 2002).Our results in Pinus pinaster showed that 6-benzylaminopurine induced bud formation, while jasmonic acid had an inhibitory effect; Ethephon, however, showed only an inhibitory effect in medium 11.Moreover, high levels of jasmonic acid in combination with Ethephon strongly inhibited the induction of lateral buds, a situation that was not observed in absence of Ethephon.
The global effect of the different treatments caused on height and bud number was grouped by cluster analysis.Group 1 was composed by treatments 2, 7, 8, and 9, which showed a large mean number of buds (4-5) and a mean growth of 1-1.5 cm.This confirmed the bud-inducing effect of 6-benzylaminopurine and the positive effect of the different phytohormone combinations (6benzylaminopurine and Ethephon at both concentrations, and the combination of 6-benzylaminopurine, jasmonic acid and Ethephon, but only at 10 μM).
However, group 2 was composed by treatments 4, 5, 10, and 11, which showed 2-4 buds and an epicotyl growth of 0.5-1.2cm, indicated the inhibitory effect of jasmonic acid on the effect of 6-benzylaminopurine.
Finally, the control treatment (treatment 1) was put together with treatments 3 and 6 in group 3, were the bud number was 1 and growth was only 1.2-2 cm.This showed that buds were only obtained if 6benzylaminopurine was added to the culture medium.
The complexities of the interactions between the different hormonal treatments will require further studies.However the results presented in the current one indicated that similar signalling pathways act in jasmonic acid and ethylene regulations in a Gymnosperm.

FIGURE 1 .
FIGURE 1. SPAD analysis of the main components for epicotyl height and number of induced buds showing no interrelation between these variables (the angle is near 90º).

FIGURE 2 .
FIGURE 2. Mean values for epicotyl growth (cm) in the different treatments.Data are the mean ± standard deviations.

FIGURE 4 .
FIGURE 4. SPAD analysis of the main components for height and bud number cluster (represented in direct hierarchical tree) showed the existence of three groups of treatments.

TABLE 2 . Effects of different phytohormone treatments on number of induced buds: probabilities after post hoc Tukey tests.
FIGURE 3. Mean values for the number of lateral buds induced in the different treatments.Data are the mean ± standard deviations.