Effect of GA₃ and 2,4-D foliar application on the anatomy of date palm (Phoenix dactylifera L.) seedling leaf

Abstract

Two concentrations (10-5M and 10-3M) of both GA3 and 2,4-D were used as foliar spray to evaluate the response of date palm (Phoenix dactylifera L.) cv. Khedri seedlings. They affected some of the anatomical characteristics of the first leaf emerging after the beginning of the spray. The high concentration of GA3 increased the size of the midrib and its vascular bundle numbers. Both low and high concentrations of 2,4-D inhibited the formation of the midrib. 2,4-D in both low and high concentrations decreased the number of vessels in both protoxylem and metaxylem and also decreased their diameters, where as GA3 in low and high concentrations have less effect on the number of vessels and its diameters. GA3 in high concentration increased the number of vascular bundles in 1mm long of the leaf blade, while 2,4-D in low and high concentrations decreased their numbers. 10-3M of 2,4-D increased the size and layers of special hypodermal cells.

Introduction

Date palm (Phoenix dactylifera L., family Arecaceae) is one of the oldest cultivated trees in arid and semi-arid regions such as Saudi Arabia. It is valued mainly for its fruits (dates) as well as an ornamental tree. The dates are very rich in nutritive components, i.e. carbohydrate and contain different percentages of fat, minerals, protein, vitamins and a high percentage of dietary fiber (Fayadh and Al-Showiman, 1990; Al-Shahib and Marshall, 2003). Saudi Arabia is one of the major dates producing countries in the world and the estimated production of dates in 2006 was 986000 MT (Arab Agricultural Statistics Yearbook, 2009).

According to Fishel (2006) the plant growth regulators, such as auxins promote shoot elongation, thin tree fruit, and increase rooting and flower formation, gibberellins stimulate cell division and elongation; increase stalk length, and increase flower and fruit size, cytokinins stimulate cell division, bud initiation and root growth and prolong the storage life of flowers and vegetables, ethylene generators ripen and induce uniform ripening in fruit and vegetables.

The biological effects of applying plant growth regulators on plants have received much attention due to their important use in agriculture; in particular, the economical application of plant growth regulators on flowers and fruits (as parthenocarpic, thinning, and elongating agents) and on shoot (as a controlling agent of plant height and lateral branching (Salisbury and Ross, 1985, 1992; Whiting, 2007). On the other hand, investigators used plant growth regulators in plant tissue culture, with auxin and cytokinin being the most widely used plant growth regulators (Slater et al., 2003).

Gibberellins were found to enhance the stem length in higher plants due to cell elongation in the internodes and to stimulate the cell division, and increase volume of individual cells (Stowe and Yamaki, 1959; Cleland, 1969; Stefanini et al., 2002). These effects lead to stem elongation, internode length, leaf area and growth of dry mass, although plant species might react to certain GAs (Kerbauy, 2004). In general, GA₃ increases xylem differentiation in vascular cambium, stem and leaf length, and xylem fibers, but it decreases the amount of leaves produced by the apical meristem, xylem vessels and xylem parenchyma (Ammar and Hadad, 1982). However, to the knowledge of the authors, no study has been encountered concerning the effects of 2,4-D and GA₃ foliar application on the changes in the anatomical structure of date palm leaf (Phoenix dactylifera L.) seedling. Therefore, aim of the present experiment was to study the effect of foliar spray of gibberellic acid (GA₃) and 2,4-Dichlorophenoxyacetic acid (2,4-D) on the leaf anatomy of date palm seedling.

Materials and methods

Seeds of date palm (Phoenix dactylifera L.) c.v. Khedri were washed and soaked in double distilled water (DDW) with aeration for four days. Three selected seeds were sown in a pot containing field soil-peat moss mixture (1:1). After sowing, pots were kept in the greenhouse (temperature 30 °C and relative humidity 60%). After three months of sowing of plant, each seedling was transferred into a larger pot. Seedlings were irrigated with DDW two times a week, except every other two weeks with one nutrient solution (for composition of IX solution, see Al-Whaibi and Al-Ackhal, 1985). Seedlings were divided into five groups. Each group contained six seedlings. Control was only sprayed with IX nutrient solution and treatments were sprayed with IX solution containing 10⁻⁵ M or 10⁻³ M of either 2,4-D or GA₃. All spraying solutions contained four drops of Tween 20 to reduce surface tension. Seedlings were sprayed weekly at 10:00 a.m., until at least the fourth leaf emerged. Seedlings were then harvested and divided into shoots and roots. The first leaf emerging after the beginning of spray for each treatment was chosen. Segments (2 cm) of leaf blade were taken at the leaf base (3 cm from the blade base) middle and apex (3 cm from the blade apex). Each leaf segment was divided into 5 mm pieces and immediately fixed in FAA (Formalin: Acetic acid: Alcohol), dehydrated in ethanol, embedded in paraffin, sectioned at 10–20 μm thick using a rotary microtome, stained with safranin and light green and then mounted in Canada balsam. Sections were examined and photographed using Novel 002002 microscope with USB pc Camera 301+. Cell wall thickness was determined according to Doaigey et al., 1989. This procedure was repeated for the same length in both the middle and near the base of the leaf. Vessel diameter was also recorded at both middle and near the base of the leaf. Statistical analysis: The data were analyzed statistically with SPSS-12 statistical software (SPSS Inc., Chicago, IL, USA). Means were statistically compared by Duncan’s multiple – range test at p < 0.05% level.

Results

The internal characteristics of the first leaf emerged after the beginning of spray for each treatment (near leaf base, major and minor veins) were summarized and compared with those of the control. The observed features of this leaf in transverse section were described sequentially and in brief starting with the upper epidermis proceeding through to the mesophyll, vascular bundles and lower epidermis.

Upper epidermis

Foliar application of GA₃ enhanced thickness of the cuticle as compared to the control and 2,4-D (Fig. 3 (13–16)). The effect of low and high concentrations of GA₃ and 2,4-D foliar spray was not found significant on epidermal cells, ovate to tabular (irregular shapes); except over the multilayer hypodermis where cells were palisade-like forming a zip-like shape (Bulliform cells) with thin cell walls. They were not affected either by low or high concentrations of GA₃ and 2,4-D treatments (Fig. 3 (13–16)).

Figure 3.

(13–18): Part of T.S. of seedling leaf midrib showing bulliform cells (Green arrow), Hypodermis (Red arrow): (13) Control, (14) 2,4-D × 10⁻⁵ treatment, (15) 2,4-D × 10⁻³ treatment, (16) GA₃ × 10⁻⁵ treatment, (17) GA₃ × 10⁻³ treatment, (18) Control, fiber strand (Blue arrow).

Hypodermis

One or 2(5) layers of parenchymatous cells with thin cellulosic walls, were devoid of chloroplasts in control, the multilayer hypodermal cells under the bulliform cells became slightly large in cell size and limited to 1–3 layers by the effect of low concentrations of GA₃ and 2,4D treatments (Fig. 3(14,16)). These cells were also affected by the high concentration of 2,4-D treatment as they became larger in size and increased in layers (up to six layers) (Fig. 3(15)). However, they were not affected by the high concentration of GA₃ treatments (Fig. 3(17)).

Fiber strands

Small, many cells with lignified cell walls, were present under upper and lower epidermises. They were not obviously affected by low and high concentrations of GA₃ and 2,4-D treatments, except the lumen of the cell became slightly wide at high concentration of GA₃ and 2,4-D foliar spray. Some of the outer cells of fiber strands contained silica bodies which were not affected by either low or high concentrations of GA₃ and 2,4-D treatments (Figs. 1 and 3(1–6,18)).

Figure 1.

(1-6): T.S. of seedling leaf midrib. (1) Control, (2) 2,4-D × 10⁻⁵ treatment, (3) 2,4-D × 10⁻³ treatment, (4) GA₃ × 10⁻⁵ treatment, (5) GA₃ × 10⁻³ treatment, (6) Control, part of T. S. of leaf midrib, showing epidermis (Violet arrow), hypodermis (Brown arrow), mesophyll (White arrow), fiber strand (Blue arrow), and vascular bundle (Black arrow).

Vascular bundles

Two types, (a) large vascular bundles (VBs) surrounded with one layer of bundle sheath having cells devoid of chloroplasts and some of them contain silica bodies (Fig. 2(7,12)). Protoxylem with narrow vessels, metaxylem with wide vessels, 8–20 vessels in each vascular bundle were found in control plants (Fig. 2(7). However, they became less in the number of vessels (5–13 vessels) in plants that received low and high concentrations of 2,4-D treatment. An increase in number (10–24 vessels) was found at the high concentration of GA₃ × 10⁻³ (Table 1, Fig. 2(8–11)). Foliar application of GA₃ and 2,4-D had a significant change in the number of VBs (Table 1). A number of large vascular bundles were present in all the areas (top, middle, and base) of the leaf. Plants treated with higher dose of GA₃ exhibited greater number of VBs at all areas of leaf.

Figure 2.

(7-12): T.S. of seedling leaf midrib. Fig. 2(7–11), show Large vascular bundle (Black arrow): (7) Control, (8) 2,4-D × 10⁻⁵ treatment, (9) 2,4-D × 10⁻³ treatment, (10) GA₃ × 10⁻⁵ treatment, (11) GA₃ × 10⁻³ treatment. Fig. 2(12) Control, part of T S. of leaf showing epidermis (Violet arrow), hypodermis (Brown arrow), silica bodies (Yellow arrow), and mesophyll (White arrow).

Table 1.

Effect of GA₃ and 2,4-D treatment of date palm seeding on vascular bundle numbers and its vessel diameters. Data in the same column followed by the same letter do not differ statistically at P<0.05 (Duncan’s multiple-range test). Average of four determinations.

Treatment	Number of vascular bundles in 1 mm length
	Near top of the leaf	Middle of the leaf	Near base of the leaf
Control	6.40 ± 0.16ab	6.40 ± 0.16ab	6.50 ± 0.16b
GA3 × 10−3 M	6.90 ± 0.27a	6.70 ± 0.15a	7.20 ± 0.13a
GA3 × 10−5 M	6.10 ± 0.17b	6.10 ± 0.10b	6.20 ± 0.13bc
2,4-D × 10−3 M	5.30 ± 0.15c	5.50 ± 0.16c	5.80 ± 0.20c
2,4-D × 10−5 M	5.10 ± 0.17c	5.50 ± 0.16c	4.80 ± 0.20d

	Vessel diameter (μm), at
	Middle of the leaf	Near base of the leaf
Control	36.16 ± 1.48c	52.37 ± 2.87a
GA3 × 10−3 M	42.61 ± 1.07ab	42.57 ± 2.06b
GA3 × 10−5 M	46.96 ± 1.27a	45.68 ± 3.18ab
2,4-D × 10−3 M	35.67 ± 1.83c	39.29 ± 1.64b
2,4-D × 10−5 M	41.84 ± 2.05b	40.00 ± 1.57b

Foliar application of GA₃ (10⁻⁵M) enhanced the diameter of vessels at middle of the leaf (Table 1). However, 2,4-D × 10⁻⁵ was also found effective for vessel diameter at the middle of leaf. Both concentrations (10⁻⁵ and 10⁻³) of GA₃ and 2,4-D significantly decreased the diameter of vessels at near base of the leaf.

Ground tissue

Parenchymatous cells contain chloroplasts; they were not significantly affected by either low or high concentrations of GA₃ and 2,4-D treatments (Figs. 1 and 2(1–6,12)).

Lower epidermis

Both cuticle and epidermal cells were not significantly affected by the low or high concentration of GA₃ and 2,4-D treatments (Figs. 1 and 2(6 and 12).

Midrib (rib)

It was present in the leaf of the control and GA₃ treatments. It was prominent and contained about 9–21 large and small VBs in the control (Fig. 1); this midrib increased in size and contained about 63 VBs with high concentration of GA₃ treatment (Fig. 1(5)); however, its size was not much affected by low concentrations of GA₃ treatment, but contained 14–25 VBs (Fig. 1(4)). Midrib size was prominently affected by low and high concentration of 2,4-D treatment, it decreased in size to contain only one large vascular bundle in low concentrations of 2,4D treatment (Figs. 1 and 2(2 and 8)), and also decreased to contain two large vascular bundles in high concentration of 2,4-D treatment (Figs. 1 and 2(3 and 9)).

Discussion

The present study indicated that the anatomical features of the first emerging leaf after the beginning of spray were affected by GA₃ and 2,4-D treatments in contrast with those of the control. Thus, high concentration (10⁻³ M) of GA₃ increased the size and number of VBs of the midrib and the number of VBs in 1 mm length of the leaf blade (Table 1). These findings are in agreement with the generalization of Ammar and Hadad (1982). Treatment by 10⁻⁵ M of GA₃ increased the number of vascular bundles in the midrib but without a significant effect on its size (Fig. 1(4); Table 1). This increase could be explained by the general role of GA₃ in cell division and enlargement (Cleland, 1969; Stefanini et al., 2002). The significant increase in the vessel diameter in middle of the leaf treated by 2,4-D 10⁻⁵ M (Table 1) is in agreement with the conclusion of another investigation on secondary xylem differentiation in Xanthium by auxins and gibberellins that auxin has its direct effect on the induction of xylem differentiation rather than the induction of divisions prerequisite to differentiation (Shininger, 1971). In addition, 10⁻³ M of GA₃ and 2,4-D (10⁻³ M and 10⁻⁵ M) decreased the number of vessels in protoxylem and metaxylem of major veins, they also decreased vessel diameters (Table 1). This decrease is in accordance with the findings of Yamamoto et al. (1987) that xylem increment was increased following treatment with 0.4 or 1.6% ethrel (an ethylene-releasing compound) and reduced by 6.2 or 10.8% ethrel.

This study indicated that 10⁻³ M of 2,4-D increased the cell size and number of Multilayer hypodermal cells (Figs. 1 and 3(3 and 15)) while the concentration of 10⁻³ M of GA₃ increased the cell size of hypodermis under the upper epidermis. Low concentration of both 2,4-D (10⁻⁵ M) and GA₃ (10⁻⁵ M) did not affect the Multilayer hypodermal cells (Figs. 1 and 3(2,4,14,17), respectively). However, phloem tissue was not studied in detail in this work, but phloem elements showed wide lumina in some treatments, a note that supports the findings of Çavuşoğlu et al. (2008). Finally, an indication of differences in response to spraying growth regulators between leaf tissues and root tissues could be perceived from the results of this study and the results of another study on roots (Doaigey and Al-Whaibi, 1995). For example, it was found in this investigation that GA₃ (10⁻³ M) increased the number of vessels in the leaf but decreased them in the root; and 2,4-D (10⁻³ M) gave the opposite results; i.e. decreased the number of vessels in the leaf but increased them in the root. These differences are probably due to different plant organs as found in tissue culture reports where the variable responses of different plant species to the growth regulators are because of the requirement of exogenous plant growth regulators that depends on their endogenous levels which might differ in different plant species and also in different plant materials (Rai, 2007). Moreover, responses to auxinic compounds are known to vary by species, by tissue within species, and even by relative maturity of a given tissue (Beal, 1951). From the results of our study, it can be concluded that foliar application of GA₃ and 2,4-D may affect some anatomical characteristics of the leaves of date palm.