Acclimation of croton and hibiscus seedlings in response to the application of indobultiric acid and humic acid for rooting

Submitted on 01/21/2013 and aproved on 04/24/ 2015. Study financed by F APEMIG (APQ-02395-10). 2 Universidade Federal de Viçosa, Florestal, Minas Gerais, Brazil. lilian.estrela@ufv .br; marihus@ufv .br Universidade Federal de Viçosa, Florestal, Minas Gerais, Brazil. matheus.simoes@ufv .br; reges.oliveira@ufv .br 4 Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. herminia@ufv .br; vhav@ufv.br *Corresponding author: marihus@ufv .br Acclimation of croton and hibiscus seedlings in response to the application of indobultiric acid and humic acid for rooting


INTRODUCTION
According to Ibraflor (2012), the area used for growing plants and ornamental flowers all over Brazil is 12 thousand hectares, with around nine thousand farmers and a farm average size of 1.5 hectares.This creates 194,000 direct jobs, of which, 96,000 (49.5%) are related to production, 6,000 (3.1%) are related with distribution, 77,000 (39.7%) are distributed in retail and 15,000 (7.7%) in other functions, especially support, and with a per capita consumption of R$ 20.00 per inhabitant.
With the objective of increasing yield and optimization of acclimation phase of ornamental seedlings, the following has been studied: the use of fitted substrates (Yamamoto et al., 2007;Lima et al., 2008) and containers (Cunha et al., 2005), the association between plants with micorhizal fungi and diazotrophic bacteria (Weber et al., 2003;Baldotto, 2010), the use of growth regulators (Lima et al., 2008), among others.The objective of such efforts is to accelerate plant growth and to reduce the impact of transference from nursery to the field, increasing yield and seedling quality in less time and at lower costs.
Amongst ornamental plants, it stood out species used for its blooming foliage, such as croton, and flower-producer species, hibiscus, for example.Croton (Codianeum variegatum L. Rumph) of the Euphorbiaceae family is a set of semi-hardwood shrubs with 2.0 to 3.0 m of height, latescent, leathery and very attractive leaves, due to their size and shape (Lorenzi & Souza, 2008).Hibiscus (Hibiscus rosasinensis L.), belongs to Malvaceae Family, is a group of hardwood shrubs, with solitary and red, pink or white flowers, formed all over the year (Lorenzi & Souza, 2008).Because such plants are very appreciated by the population, they need fast propagation methods, with low cost and that assures the formation of vigorous, high-quality seedlings.
Therefore, plant propagation by cuttings is a proper method since it allows obtaining a great amount of even, early seedlings with genetic characteristics of the matrix plant (Hartmann et al., 2002).Adventitious rooting of cuttings involves the action of auxins, plant hormones transported to the base of the cutting, which act on the formation of meristematic centers, inducing root formation (Hartmann et al., 2002).Synthetic auxins, plant growth regulators, such as indobultiric acid (IBA) are used to promote rooting of ornamental plant cuttings at commercial scale (Lima et al., 2008).The application of humic acid, the bioactive fraction of humificated organic matter in ornamental plant cuttings may promote the adventitious rooting, which is another technological option (Baldotto et al., 2012).
Humic acid (HA) is formed by heterogeneous molecular aggregates and stabilized by hydrogen bonds and hydrophobic interactions (Piccolo, 2001).It acts in the growth and development of many plants for agronomic purposes.Those effects are reflected in the acceleration of the development of roots and aerial part (Canellas et al., 2006;Baldotto et al., 2009;Silva et al., 2011;Baldotto et al., 2012).
The objective of this work was to study the growth characteristics and nutritional contents of croton and hibiscus plants over seedling acclimation in response to the concentrations of indobultiric acid (IBA) and humic acid (HA) applied over adventitious rooting of the cuttings.
The apical stem semi-hardwood cuttings were collected in September, sectioned at 15 cm of length and four apical leaves were maintained.
Humic acid was isolated from vermicompounds and previously characterized by Baldotto et al. (2007) and Busato (2008).The bases of the cuttings were soaked in solutions of HA for 24 hours (Baldotto et al., 2009), and in solutions of IBA for ten seconds (Lima et al., 2008).For this procedure, the cuttings were placed in plastic glasses with 50 mL of the solutions corresponding to the different treatments (Baldotto et al., 2012).
Seedlings were in the nursery covered by polyethylene mesh in 50% for acclimation.The experimental unit was one plant per plastic bag.The experiment was carried out in a random block design, with five replicates, totalling 100 experimental units.

Growth analysis
At 90 days of acclimation, plants were collect for measurement of the following variables: plant height (H), measured by distance from the plant collar to the leaf apex, using a meter tape; crown diameter (CRD); stem diameter (STD), measured using a digital model Starret 727 pachimeter; number of leaves (NL); root fresh matter mass (RFM) and aerial part fresh matter (APFM); root dry matter mass (RDM) and aerial part dry matter (APDM), obtained by drying in air ventilation oven at 60 o C for seven days and then weighed.

Nutritional analysis
After drying, leaves were ground in a Wiley-type mill, coupled to a 60-mesh wire cm -2.Then, the resulting powder was submitted to sulphuric digestion combined with hydrogen peroxide for determination of total contents of nitrogen (N), phosphorus, (P), potassium (K), calcium (Ca) and magnesium (Mg), sulphur (S), zinc (Zn), iron (Fe), manganese (Mn), copper (Cu) and boron (B).The Nessler method was used for N; content of P was obtained by molecular absorption spectrophotometry (colorimetry), after reaction with C vitamin and ammonium molybdate at the wavelength of 725 nm; K was determined by flame photometry.Contents of Ca, Mg, Cu, Fe, Mn and Zn were all obtained by molecular absorption spectrophotometry and content of S was achieved by turbidimetry and B was determined by colorimetry (Embrapa, 2009).

Statistical analysis
The evaluation results were submitted to analysis of variance and the treatment effects, for qualitative analysis, were unfolded in average contrasts, according to Alvarez & Alvarez (2006).For quantitative factors, the regression equations were adjusted among the assessed variables and concentrations of IBA and HA.The F test and factor unfolding were applied between 1, 5 and 10% of probability.Regarding regression analysis, the angular coefficients of the equations were tested when presented determination coefficient higher than 0.60.Regression equations were used to determine concentration of maximum physical efficiency of aerial part dry matter, in function of the concentrations of IBA and HA.Values of maximum efficiency concentration were replaced in the regression equation of each variable to estimate them for this condition.

Growth analysis
The results of the growth analyses of croton plants at 90 days of acclimatation, in response to the application of IBA and HA, revealed some modifications in the initial performance, when differences among means (Table 1) were found in the average contrasts (Table 1) and in the regression equations (Table 2) adjusted for the plant growth data in response to the application of plant regulators.
Overall, for growth traits of croton seedlings at 90 days of acclimation, in response to the application of IBA and HA, no differences for most of the analysed variables were found, when compared to the control (Table 1).In this same table, when comparing the use of plant regulators, it was possible to verify that IBA application incremented CRD by 11.33% and NL by 20.82%, in croton plants, in comparison to HA application.It is assumed that, endogenous levels of auxins in croton plants are sufficient to stimulate the Rev. Ceres, Viçosa, v. 62, n.3, p. 284-293, mai-jun, 2015 formation of adventitious roots, allowing this species to be classified as one with easy vegetative propagation by cutting (Baldotto et al., 2012), which can be seen in the acclimation period, when there is a lack of differences during growth among plants treated or not with IBA or HA.According to Trewavas & Cleland (1983), the low effect with the application of growth regulator may point to the low tissue sensitivity to the presence of the promotor, in addition to the high endogenous concentration of auxin.
Most of the regression equations adjusted among dependent variables (growth traits) and increasing concentrations of plant regulators (IBA and HA), were curvilinear (quadratic, quadratic roots, cubic and cubic roots) for growth variables in the aerial part and in the roots of croton (Table 2).The variable chosen for identification of the maximum concentration of physical efficiency (MFE) was the dry matter of the aerial part.Regarding IBA, the concentration that provided MFE of aerial part dry matter was 1089.37 mg L -1 and for HA, due to the lack of adjustment, the MFE concentration was estimated by the mean of values of the aerial part dry matter (Table 2).Concentrations of MFE were replaced in the regression equation for each variable in Table 2, in order to calculate it for this condition.It was found that the treatment with IBA incremented height (9.40%), crown diameter (0.88%), stem diameter (0.71%), number of leaves (26%), aerial part dry matter mass (27.60%), root fresh matter mass (13.24%) and root dry matter mass (5.63%) in croton plants.
In hibiscus, the results of growth analysis in response to the application of increasing concentrations of IBA and HA showed the effects of those plant regulators (Table 3).The average contrasts (Table 3) showed that the treated hibiscus plants were superior than the control ones for crown diameter, root fresh matter mass and root dry matter mass with IBA; and crown diameter and root fresh matter mass with HA.It can also be seen in this table that the comparison among the plant regulators showed that the use of IBA incremented all variables in comparison to HA, except crown diameter.Indobultiric acid is a synthetic product that, at proper concentrations, acts in the formation of meristematic centers and adventitious roots (Hartmann et al., 2002;Pizzatto et al., 2011;Baldotto et al., 2012), which favours dry mater accumulation in the root system during seedling acclimatation.Similarly, HA Table 1.Means, average contrasts, relative increments (RI), residual mean square (RMS) and coefficient of variation (CV) for growth traits of croton plants at 90 days of acclimation in response to the application of indobultiric acid (IBA) and humic acid (HA) applied in five concentrations (0, 250, 500, 1000, 2000 mg L -1 of IBA and 0, 10, 20, 30, 40 mmol L -1 of C from HA), carried out in the random block design with 5 replicates.
(3) Average contrasts: control versus IBA; control versus HA; IBA versus HA; Relative increments: 100 (x-y)/y, where x is the mean of the treatment with the highest value and y is the mean of the treatment with the lowest value.*,** and º = significant between 1, 5 and 10% of probability by the F test. (1) Sources of variation: show the plant regulators and the concentrations; (-) = control; IBA = indobultiric acid; HA = humic acid. (2)Growth traits: H = height; CRD = crown diameter; STD = stem diameter; NL = number of leaves; APFM = aerial part fresh matter mass; RFM = root fresh matter mass; APDM = aerial part dry matter mass; RDM = root dry matter mass.
(3) Average contrasts: control versus IBA; control versus HA; IBA versus HA; Relative increments: 100 (x-y)/y, where x is the mean in the treatment with the highest value and y is the mean of the treatment with the lowest value.**, * and º = significant among 1, 5 and 10% of probability by the F test.
The regression equations adjusted for growth of hibiscus plants in response to concentrations of IBA and HA are in Table 4.They were, mostly, curvilinear (quadratic, quadratic roots, cubic, cubic root) for growth of roots and aerial section in hibiscus.It was not possible to adjust regression equation only for crown diameter for IBA treated plants and the mean among the dots in the curve was estimated.
The concentrations that resulted in the greatest accumulation of aerial part dry matter mass in hibiscus plants, that is, the most efficient ones for a seedling with more reserves, were 977.77mg L -1 of IBA and 26.83 mmol L -1 of C in the form of HA.The rapid accumulation of dry matter in the shoots is desirable to guarantee energy for the post-planting (Lorenzi & Souza, 2008).

Nutritional status
Nutritional contents of croton plants at 90 days of acclimation ranged in response to the application of IBA and AH (Table 5).There was a better performance of IBA treated plants than the ones treated with HA and control.
The use of IBA in croton plants increased contents of N, P, S, Fe and B, when compared to control (Table 5).In the same table, it is found that the use of HA resulted in the increase in N, P, K, Ca, Mg, S and Cu, as the contents of B decreased when compared to the control.Overall, when the two plant regulators were compared, it was found that the contents of macronutrients N, K, and S achieved their peak when HA was applied, compared to IBA, which was superior only for the content of B.
Regression equations were calculated for nutritional contents of croton plants (Table 6) in response to concentrations of plant regulators.The ways of response found in the experiment were, mostly, curvilinear (quadratic, quadratic roots, cubic, cubic roots) with good adjustments (R > 0.70) and most of them showed positive increment rates.
For hibiscus, increases in almost all nutritional variables were found (Table 7) in response to the HA application.The bio-stimulant action of humic Rev. Ceres, Viçosa, v. 62, n.3, p. 284-293, mai-jun, 2015 substances comprehends the activity of ATPases, enzymes related to both absorption of nutrients such as "acid growth", mechanisms that have been used to explain its bioactivity (Canellas et al., 2006).Humic acid also stimulates the formation of root hair (Silva et al., 2011), increasing the absorption area of nutrients by the roots.
The regression equations for contents of plant nutrients in function of the increasing concentrations of IBA and HA are presented in Table 8.The variable chosen to estimate the concentration of maximum physical efficiency was aerial part dry matter mass, whose values were 977.77mg L -1 for IBA and 26.83 mmol L -1 of C, for HA.Such concentration of HA resulted in an accumulation of dry matter 61% higher than the control.Significant effect for IBA was not found.Humic acid acts in the formation of meristematic centers, especially in the formation of adventitious root, which, in turn, increment the nutrient absorption and plant growth.Thus, the results obtained show that the use of HA in hibiscus cuttings accelerates plant growth in seedling acclimation.The efficiency of propagation benefits production and commercialization of ornamental plants.
Concentrations of IBA and HA of MFE were replaced in the regression equations of each variable of Table 8, to estimate them for that condition.It was found that the treatment with HA incremented contents of P (89%), K (67%), Ca (21%), Mg (76%), S (132%) and Zn (9%).The use of IBA also resulted in a greater accumulation of some nutrients than in the control, but this better nutritional composition was not converted into greater content of dry matter.
Finally, the results show that seedling production of croton and hibiscus by using synthetic hormones and bioactive fractions of organic matter (IBA and HA) is an efficient alternative since propagation of croton by cuttings for seedling production was benefited with the application of IBA, which incremented the nutritional status when compared to the control.In addition, for hibiscus, both stimulants presented positive effects in growth and seedling development.
The results support data of Baldotto et al. (2012), in the rooting phase of seedlings of croton and hibiscus.Therefore, the effect in rooting leads to a greater efficiency in the following phase, acclimation of seedlings of those ornamental plants.

Sources of variation (1)
Table 7. Nutritional contents of hibiscus plants at 90 days of acclimation in response to the application of indolbutiruc acid (IBA) and humic acid (HA) applied in five concentrations (0, 250, 500, 1000, 2000 mg L -1 of IBA and 0, 10, 20, 30, 40 mmol L -1 of C from HA), carried out in a random block design with 5 replicates (1) Sources of variation: show the plant regulators and the concentrations. (2)Nutritional contents: correspond to the contents of N = nitrogen; P = phosphorus; K = potassium; Ca = calcium; Mg = magnesium; S = sulphur; Zn = zinc; Fe = iron; MN = manganese; Cu = copper and B = boron. (2) Average contrasts: control versus IBA; control versus HA; IBA versus HA; Relative increments: 100 (x-y) /y, where x is the mean of the treatment with the highest value and y is the mean of the treatment with the lowest value.**, * and º = significant among 1, 5 and 10% of probability by the F test, respectively.

CONCLUSIONS
The results achieved in this experiment show that the response to application of plant regulators depends on plant genotype and concentration.
The use of IBA and HA at the indicated concentrations favours acclimation of hibiscus plants propagated by cutting, reducing production time of seedlings and benefiting production and commercialization of ornamental plants.
Regarded to croton, the use of IBA at the indicated concentration is also recommended.

Table 3 .
Means , average contrasts, relative increments (RI), residual mean square (RMS) and coefficient of variation (CV) for growth traits of hibiscus plants at 90 days of acclimation in response to the application of indobulitiric acid (IBA) and humic acid (HA) applied in five concentrations (0, 250, 500, 1000, 2000 mg L -1 of IBA and 0, 10, 20, 30, 40 mmol L -1 of C from HA), carried out in random block design with 5 replicates