Project Abstract

<p>                <b>ABSTRACT&nbsp;</b></p><p>This work evaluated the rooting of Pinus caribaea var. hondurensis Morelet cuttings under the action of different levels of plant growth regulators. The cuttings consisted of 4-6 cm long shoots of P. caribaea var. hondurensis Morelet with their basal needles removed. The basal part of the cuttings were treated for 2 seconds with the following treatments 1- NAA 2000mg L-1; 2- NAA 4000mg L-1; 3- NAA 6000mg L-1; 4- NAA 2000mg L-1 + PBZ 100mg L-1; 5- NAA 4000mg L-1 + PBZ; 6- NAA 6000mg L-1 + PBZ; 7- IBA 2000mg L-1; 8- IBA 4000mg L-1; 9- IBA 6000mg L-1; 10-IBA 2000mg L-1 + PBZ; 11- IBA 4000mg L-1 + PBZ; 12- IBA 6000mg L-1 + PBZ; and a control. After receiving the treatment, the cuttings were planted in tubes containing 50% carbonized rice hulls and 50% vermiculite. The evaluations, performed 60 days after planting, showed that P. caribaea var. hondurensis cuttings treated with IBA produced a higher percentage of rooted cuttings than those treated with NAA; the most effective treatment was IBA 4000mg L-1 plus 100mg L-1 paclobutrazol.&nbsp;</p><p><b>Key words Auxins, paclobutrazol, Pinus caribaea </b> <br></p>

Project Overview

<p> <b>INTRODUCTION&nbsp;</b></p><p>Cloning or vegetative propagation of trees has been a useful tool in traditional tree improvement, and the great promise of clones is for production forestry (Libby, 1986). The majority of gymnosperms, especially in the genus Pinus, are sexually propagated by seeds. With regard to large scale propagation and preservation of selected genetic characters, sexual reproduction poses a risk of loss of these characteristics. On the other hand, vegetative propagation is the form by which desired individual characteristics are maintained, resulting in a plant that is genetically identical to the original donor plant, thus becoming the preferred method of propagation (Oliveira, 1989). Conifers may be vegetatively multiplied in several different ways, for example via grafting, cuttings, adventitious shoots or somatic embryos. Comercial production of Pinus radiata by tissue culture methods is in development in New Zealand (Gleed, 1985). Propagating plants with particularly desirable characteristics is a very common practice in horticulture, used extensively with some forestry species, particularly in high productivity, fiber farming systems (e.g., plantations of hybrid poplars, eucalyptus, and Caribbean pines). Rooting in Pinus cuttings depends on a series of factors that can be controlled by cutting collection time, cutting size, whether the needles are kept or not, condition and age of the source plant, plant nutritional condition, prunning, type and health of the cutting, season when the cuttings are collected, rooting-promoting treatments, condition and type of substrates, irrigation, moisture, temperature, and other factors (Silva, 1985). The process of root formation at the base of a cutting may be divided into three stages: initiation, elongation of root initials, and root growth and development (Hartmann et al., 2002). Three groups of chemicals may be applied to regulate and/or influence the rooting process in some way: a) hormones, which induce the initial meristematic activity (Wiesman et al., 1989; Davis and Haissig, 1990) and stimulate the elongation and development of roots formed (Hartmann et al., 2002; Wiesman et al., 1989); b) nutritional elements which promote growth of the new roots (Wiesman and Lavee, 1995); c) protecting agents, such as biocides, which enable the cuttings to resist attack by pathogens during the entire rooting period. Studies on the physiology of auxin action showed that auxin was involved in such varied plant activities as stem growth, adventitious root formation (Went, 1934; Haissig and Davis, 1994), lateral bud inhibition, abscission of leaves and fruits, and activation of cambial cells. Indole-3- acetic acid (IAA) was identified as a naturally occurring compound having considerable auxin activity (Haissig and Davis, 1994). IAA was subsequently tested for its activity in promoting roots on stem segments, and in 1935, investigators demonstrated the practical use of this material in stimulating root formation on cuttings (Thimann and Koepfli, 1935). About the same time it was shown (Zimmerman and Wilcoxon, 1935) that two synthetic materials, indole-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA), were even more effective than the naturally occurring or synthetic IAA for rooting. Today, IBA and NAA are still the most widely used auxins for rooting stem cuttings and for rooting tissue-cultureproduced microcuttings. It has been repeatedly confirmed that auxin is required for initiation of adventitious roots on stems, and indeed, it has been shown that divisions of the first root initial cells are dependent upon either applied or endogenous auxins (Strömquist and Hansen, 1980; Gaspar and Hofinger, 1988). IBA has been found to occur naturally. The formation of root primordium cells depends on the endogenous auxins in the cutting and on a synergic compound such as a diphenol. These substances lead to the synthesis of ribonucleic acid (RNA), which act upon root primordium initiation (Hartmann et al., 2002). The application of some plant growth retardants, together with auxin, has been used to improve the rooting capacity of cuttings in some species (Pan and Zhao, 1994; Wiesman and Lavee, 1995). Triazoles have been reported to affect some physiological processes during rooting (Davis and Haissig, 1990; Wiesman and Lavee, 1995). Paclobutrazol (triazol), a gibberellin synthesis inhibitor (Rademacher et al., 1984), keeps the gibberellin levels in the cuttings at low values; gibberellins are considered substances that inhibit rooting (Hartmann et al., 2002). Moreover, paclobutrazol increases the sink capacity at the base of the cutting for carbohydrates and/or hormones (Wiesman and Lavee, 1995). Thus, the objective of this work was to analyze the rooting potential of Pinus caribaea var. hondurensis Morelet cuttings under the action of different concentrations of plant growth regulators.&nbsp;</p><p><b>MATERIALS AND METHODS</b>&nbsp;</p><p>Pinus caribaea var. hondurensis Morelet shoots were collected from a clonal garden. The cuttings were 4 to 6cm in length with the basal needles eliminated. Later, the cuttings were treated with plant growth regulators, planted in tubes and taken to a intermitent mist system in a enclosed polyethylene propagation house for 60 days. The cuttings were treated with two auxins, indole-3- butyric acid (IBA) and α-naphthalene acetic acid (NAA), and with paclobutrazol (PBZ), by immersing the base of the cuttings in a gel solution for 2 seconds. The following treatments were used: 1- NAA 2000mg L-1; 2- NAA 4000mg L-1; 3- NAA 6000mg L-1; 4- NAA 2000mg L-1 + PBZ 100mg L-1; 5- NAA 4000mg L-1 + PBZ; 6- NAA 6000mg L-1 + PBZ; 7- IBA 2000mg L-1; 8- IBA 4000mg L-1; 9- IBA 6000mg L-1; 10- IBA 2000mg L -1 + PBZ; 11- IBA 4000mg L-1 + PBZ; 12- IBA 6000mg L-1 + PBZ; and a control. <br></p><p> After treatment, the cuttings were planted in tubes containing 50% carbonized rice hulls and 50% vermiculite. Evaluations were performed 60 days after planting, including: rooting percentage, total sugars contents, reducing sugars contents and phenol contents. The biochemical analyses were conducted with cutting samples on the day the experiment was started and at the end of the experiment. The total phenol content analysis in P. caribaea var. hondurensis cuttings was performed according to the official method prescribed by the Association of Official Analytical Chemists - A.O.A.C. (1984), adapted by Skorupa et al. (1998). The total sugars and reducing sugars analyses were performed by the Somogy-Nelson method (Nelson, 1944). The experiment was carried out as a completely randomized design with 13 treatments containing 4 replicates with 8 cuttings each. The data were submitted to analysis of variance (F test) and the means compared by Tukey test at the 5% probability level. The data were transformed into arc sin sqrt((x + 0.5)/100) for statistical analysis.<b>&nbsp;</b></p><p><b>RESULTS AND DISCUSSION</b>&nbsp;</p><p>Percentage of rooted cuttings A significant difference in NAA concentrations (F = 19.89) was observed for P. caribaea cuttings. The rooting percentage in the NAA 2000 mg L-1 treatment (53.13%) was higher than in other treatments (Table 1), therefore, indicating that this plant growth regulator was effective in promoting rooting in cuttings of this species. NAA at 4000 and 6000 mg L-1 showed lower rooting percentages. The increase of NAA concentration was accompanied by the rooting percentage decrease, suggesting that high NAA concentrations were phytotoxic for the root formation process. Thus, it could be suggested that the concentration that promoted the highest rooting percentage was between 0 and 2000mg L-1 . Through statistical analysis, it was observed that a significant difference occurred between treatments containing IBA and the control (F= 24.20), but no difference was observed between the different IBA concentrations used. A value of 95.31% rooted cuttings were observed at the concentration of 4000 mg L-1 IBA (Table 2). With regard to treatments with and without PBZ, no significant differences were observed, i.e., they did not contribute towards the root regeneration process. According to Wiesman and Lavee (1995), paclobutrazol applied in combination with IBA increased the rooting capacity of cuttings, by increasing root formation. This plant growth retardant has effects on the partition of photoassimilates, on the water status of the cuttings, and has been described as a promoter of adventitious roots in many species (Steffens and Wang, 1986). Paclobutrazol, in addition to stimulating root growth, as also mentioned by Davis et al. (1985) and Steffens and Wang (1986), increased rooting potential in this work.</p><p> Table 1 - Average rooting percentage of Pinus caribaea var. hondurensis Morelet, submitted to NAA treatments with and without the addition of paclobutrazol (PBZ). Treatments with PBZa without PBZa Meana, b Control 31.25 ns NAA 2000mg L-1 59.38 46.88 53.13 Ba NAA 4000mg L-1 18.78 9.38 14.08 Bb NAA 6000mg L-1 0.00 15.63 7.81 Bb Mean 26.05 A 23.96 A C.V.= 41.75% aMeans followed by different capital letters in the rows and small letters in the columns are significantly different (Tukey test at p≤0.05); ns= non significant. bMeans followed by different capital letters in the mean column are significantly different of the table 2 mean column (Tukey test at p ≤ 0.05).&nbsp;</p><p> The percentage results of rooted cuttings treated with NAA or IBA showed that cuttings treated with IBA presented, significantly, a higher root formation response in comparison with NAA (F= 6.90), suggesting that this auxin was more effective in initiating roots in this species at these concentrations. Nelson et al. (1992), using four different auxins (IAA, IBA, NAA, and indole-3- propil) in P. taeda, P. elliotti var. elliotti, and P. palustris showed that IAA and IBA presented good results, while NAA and indole-3-propil showed poorer results in the rooting of cuttings of these species. When IBA is applied to stem cuttings or microcuttings to stimulate rooting, it is, in part, converted to IAA (Vander Krieken et al., 1992). IBA may also enhance rooting via increased internal-free IBA, or may synergistically modify the action of IAA or the endogenous synthesis of IAA; IBA can enhance tissue sensitivity for IAA and increase rooting (Vander Krieken et al., 1993). According to Hartmann et al. (2002), IBA is the best auxin for general use because it is nontoxic to plants over a wide concentration range than NAA, and is effective in promoting rooting of a large number of plant species. Total Sugars Content There was a significant difference between the control and the other treatments (F= 18.82 and F= 75.51, respectively) for P. caribaeae cuttings treated with either NAA or IBA. A significant interaction was also observed between NAA or IBA concentrations and the addition or not of PBZ (F= 8.32 and F= 5.44, respectively) on total sugars content. Cuttings treated with 4000 mg L-1 NAA + PBZ and 6000 mg L-1 IBA showed the lowest total sugars content, while those treated with 2000 mg L -1 NAA and 4000 mg L-1 IBA + PBZ showed the highest total sugars content (Tables 3 and 4). The same treatments showed the highest rooting percentages (Tables 1 and 2). The total sugars content analysis in Pinus cuttings, immediately after they were prepared, before being placed in the rooting medium was 14.63%. Thus, a reduction higher than 50% in total sugars content from the beginning of the rooting process until the evaluation period was observed, indicating the use of these sugars in the process. Reducing Sugars Contents The statistical analysis showed a significant difference between treatments with NAA or IBA and the control for reducing sugars content. In treatments with NAA there was a significant interaction between NAA concentrations and the presence or not of PBZ. Among the NAA treatments, 6000 mg L-1 NAA and 2000 mg L-1 NAA + PBZ showed the highest reducing sugars contents, while the lowest content was shown by the 2000 mg L-1 NAA treatment. The treatments containing IBA at 6000 mg L-1 and IBA at 2000 mg L-1 showed the lowest and highest reducing sugars contents, respectively (Tables 5 and 6).&nbsp;</p><p>Table 2 - Mean rooting percentage of Pinus caribaea var. hondurensis Morellet cuttings, submitted to IBA treatments with and without the addition of paclobutrazol (PBZ). Treatments with PBZa without PBZa Meana, b Control 31.25* IBA 2000mg L-1 93.75 84.38 89.06 Aa IBA 4000mg L-1 100.00 90.63 95.31 Aa IBA 6000mg L-1 78.13 84.38 81.25 Aa Mean 90.63 A 86.46 A&nbsp;</p><p>C.V.= 23.11% a Means followed by different capital letters in the rows and small letters in the columns are significantly different (Tukey test at p≤0.05). *significant at p≤ 0.05. bMeans followed by different capital letters in the mean column are significantly different of the table 1 mean column (Tukey test at p ≤ 0.05).&nbsp;</p><p>Table 3 - Mean total sugars contents in Pinus caribaea var. hondurensis Morelet cuttings, submitted to NAA treatments with and without the addition of paclobutrazol (PBZ). Treatments with PBZa without PBZa Meana Control 6.56* NAA 2000mg L-1 4.86 Ba 7.61 Aa 6.23 NAA 4000mg L-1 3.91 Bb 6.81 Ab 5.36 NAA 6000mg L-1 4.71 Ba 6.22 Ab 5.47 Mean 4.49 6.88&nbsp;</p><p>C.V.= 6.42% aMeans followed by different capital letters in the rows and small letters in the columns are significantly different (Tukey test at p≤0.05). *significant at p≤ 0.05. &nbsp; &nbsp; <br></p><p> The reducing sugars content analysis before initiation of the rooting process was 3.2%; thus, a small increase in this content was observed, probably because of the conversion of total sugars, since reduction occurred, into reducing sugars. <br></p>