The impact of chemical and hormonal treatments to improve seed germination and seedling growth of Juniperus procera Hochst. ex Endi

Seed samples and collection processing

J. procera Hochst. ex Endi. cones were collected from Baljurashi, Al-Baha, Saudi Arabia (19°51′18.72″N, 41°33′33.29″E). Cones were collected from at least 10 randomly selected mother trees. The collected cones were transported to the laboratory within 4 to 5 days after collection, where the seeds were extracted by manual depulping, cleaned, and air dried to approximately 5%–8% moisture content. The air-dried seeds were stored in the refrigerator until use.

Imposition of treatments and germination experiment

J. procera seeds were superficially sterilized with sodium hypochlorite (1%) for approximately 5 min, then rinsed carefully with distilled water, and divided into nine groups. The first group soaked in distilled water served as the control group. The second and third groups were soaked in 90% sulfuric acid and 20% acetic acid, respectively, for 6 min and then washed many times with tap water. The other six groups were soaked in either 50 or 100 ppm indole acetic acid (IAA), GA₃, and kinetin for 72 h. Three replicates of 20 seeds from each treatment were placed into 11-mm Petri dishes on sterilized filter paper that was kept continuously moist with distilled water. All Petri dishes for all treatments of the experiment were placed in an incubator that was kept at room temperature 24 °C  ± 1 °C with alternating light and dark conditions. The germination experiment was continued for 6 weeks, and when germinated seeds formed 3-mm radicles, the germination percentage for all treatments was calculated. The germinated seeds from each treatment were removed from the Petri dishes for the seedling growth experiment.

Seedling growth experiment

Germinated seeds from each treatment were planted in 10-cm plastic pots filled with a homogenate of sterilized peat moss and sand at a ratio of (1:1, v/v). All were retained in a greenhouse with a randomized complete block design with three replicates for each treatment and were irrigated with distilled water for 6 weeks. Two weeks after planting, each hormone-treated seedling was sprayed with its own hormone concentration.

Methods

Statistical analysis

Data are expressed as mean values ± standard error. Statistical analysis was performed using the one-way analysis of variance test followed by Dunkin’s test accompanied by the least significant difference test at P value < 0.05 (Snedecor & Cochran, 1990).

Effect of growth regulators on seed germination and seedling growth in Juniperus procera

Recently, numerous techniques have been performed to achieve seed priming, such as hydropriming, osmopriming, and thermopriming (Ashraf & Foolad, 2005). The treatments could have different effects according to the priming agent level/dose and its incubation period and depending on the species and stage of plant development. Moreover, various studies on seed germination have shown the useful effects of seed priming using many techniques such as soaking, temperature, and scarification (Ahmed et al., 2006).

Exogenous application of growth regulators improved the seed germination process and seedling growth of plants, which induced various physiological responses (Ries, 1991; Terzi & Kocaçalışkan, 2010). It plays an effective role in the upregulation of various biochemical and physiological aspects in plants (Ivanov & Angelov, 1997; Chen et al., 2003).

The results obtained in this study indicate a significant change in the main growth attributes and physiological activities of the developed J. procera seedlings in response to soaking grains in either IAA, gibberellin (GA₃), or kinetin with relatively low (50 ppm) or high (100 ppm) concentrations. Thus, in response to soaking J. procera seeds in either relatively low or high concentrations, the emergence of radicles was noticeable. This was accompanied by a concomitant significant increase in the germination percentage, lengths of shoots and roots, fresh and dry weights of seedlings, and leaf contents of chlorophylls a and b and carotenoids (Fig. 1 and Tables 1 and 2).

Among all treatments used in this study, when juniper seeds were treated with 50 ppm GA₃, a maximum percentage germination (40%) was recorded, followed by 100 ppm GA₃ and 50 ppm kinetin, which recorded 30% of germination. Then, 100 ppm kinetin and 50 and 100 ppm IAA showed the lowest percentage of germination as growth regulators. However, the juniper seeds that were chemically scarified with either 90% sulfuric acid or 20% acetic acid significantly recorded a minimum percentage of germination (10%) over the control value (H₂O), which recorded 0% germinated seeds (Fig. 1). Our results are in accordance with those of Banerji (1998), who reported that the response of GA₃ had a better performance than IAA on the germination of Melia azedarach seeds.

The highest percentage of germination recorded with GA₃ treatments might be attributed to GA₃ acting on the embryo and induced the synthesis of hydrolytic enzymes involved in seed germination especially α-amylase and protease, which consequently hydrolyzed seed storage substance reserves such as insoluble sugar and proteins into simple mobilizing soluble sugar and proteins, which are consumed for embryo growth and consequently enriched germination and expansion processes (Paleg, 1965; Bewley & Black, 1994; Kaneko et al., 2002). Moreover, the growth regulators GA₃ and IAA could inhibit these inhibitors because ethylene exists in seeds. Similar findings were confirmed by Suryakanth, Mukunda & Raghavendraprasad (2005) and Babu et al. (2010) in guava and papaya plants, respectively. Furthermore, the soaking of juniper seeds in either sulfuric or acetic acid for approximately 6 min stimulated the percentage of germination above the control value; water treatment (0%), but still the lowest values of germination compared with the investigated growth regulators (Fig. 1). The reduction in germination percentage in both acids may be due to the toxic influence of ions present in the acids, particularly sulfate; moreover, these acids may cause damage to the seed coat and food reserves.

The significantly variable changes in the shoot, root length, and fresh and dry weights of J. procera seedlings treated with the three investigated growth regulators are recorded in Table 1. The highest shoot length was measured in seedlings treated with 100 ppm IAA (3.6 cm), followed by 50 ppm IAA (3.4 cm), 50 ppm GA₃ (3.1 cm), 100 ppm GA₃ (2.9 cm), 50 ppm kinetin (2.9 cm), and 100 ppm kinetin (2.8 cm). Moreover, pretreatment of juniper seeds with either sulfuric or acetic acid induced the lowest increase in shoot, root length, and fresh and dry weights compared with other treatments. The most pronounced growth parameters were recorded after IAA treatment. The significant increase in shoot and root lengths and fresh and dry weights of J. procera seedlings obtained in this study (Table 1) could presumably be ascribed to the positive effects of exogenously applied growth regulators on cell elongation, division, and extensibility (Taiz & Zeiger, 2006). The discrete changes in different growth attributes are hypothesized to be associated with or the result of discrete changes in the metabolic and hormonal activities of the developed seedlings. Thus, the appreciable increase recorded in the growth parameters of juniper seedlings in response to either relatively low (50 ppm) or high (100 ppm) concentrations of the investigated growth regulators could reflect relatively high measurable metabolic activities that are responsible for successfully growing seedlings. The rate of metabolism in seeds during germination is controlled by signals originating from the embryo, which are mostly coherently related to its sink capacity (Taiz & Zeiger, 2006).

After 90 days of age, the root length was highest in IAA (100 ppm; 7.6 cm), with a seedling fresh weight of 0.19 g. The exogenous application of growth regulators may improve growth by increasing cell multiplication and elongation, resulting in rapid plant growth and development (Anjanawe et al., 2013). Consequently, the fresh and dry weights of juniper seedlings were significantly pronounced under IAA (50 and 100 ppm) and GA₃ (50 ppm) treatments, which might be because of mobilized water and the translocation of nutrients at higher levels, which stimulated the excess synthesis of photosynthetic products. Additionally, the stimulating effect of IAA may be attributed to cell enlargement and increased photosynthetic processes (Naeem et al., 2004), with cell division accompanied by augmentation of building units such as polysaccharides and total sugar content (Sadak et al., 2013). The results obtained in this study are in agreement with those of Atteya et al. (2018), who confirmed the stimulating role of GA₃ on the growth parameters of Simmondsia chinensis.

Effect of growth regulators on the photosynthetic pigments of Juniperus procera seedlings

Data in Table 2 show that the exogenous application of the three investigated phytohormones significantly increased the contents of chlorophylls a and b as well as carotenoids in the leaves of J. procera. After 90 days of application, the highest levels of chlorophylls a and b and carotenoids in Juniperus communis leaves were observed at 50 ppm GA₃, followed by 100 ppm GA₃. The value of chl a reached approximately 401.6 and 380.5 µg/g FW at 50 and 100 ppm GA₃, respectively (Table 2). Moreover, presoaking of J. procera seeds in either 90% H₂SO₄ or 20% CH₃COOH resulted in lower chl a and b and carotenoid contents of J. procera leaves compared with other investigated treatments. The lowest values of chl a and b and carotenoids were noticeable in the acetic acid treatment, which reached 172.1, 45.3, and 100.8 µg/g FW, respectively (Table 2).

The significant increase obtained in this study in fresh and dry weights of J. procera seedlings in response to relatively low or high concentrations of GA₃, IAA, and kinetin with a concomitant increase in chlorophyll a and b and carotenoid contents of leaves could indicate that these growth regulators may stimulate the photosynthetic efficiency of J. procera seedlings and improve the rate of assimilation in leaves with accumulation of photosynthates. A significant increase in photosynthesis has previously been described as an important response to growth regulators (El Karamany, Sadak & Bakry, 2019). The promoted effect of either GA₃, IAA, or kinetin on photosynthetic pigments is presumably due to the action of phytohormones as coenzymes in the metabolism of plants, which play a vital role in the formation and/or retardation of photosynthetic pigments (Jacobs, 1979). The increments in photosynthetic pigments in J. procera leaves in this study were consistent with the findings of 49 Simmondsia chinensis, which confirmed that growth regulators, particularly gibberellic acid, enhanced the production of photosynthetic pigments. Moreover, this increase in pigments in response to GA₃, IAA, and kinetin was ascribed to their action in increasing the number and size of chloroplasts (Ivanov & Angelov, 1997; Chen et al., 2003; Muthuchelian, Meenakshi & Nedunchezhian, 2003). Similarly, the presoaking treatment of J. procera seeds with either GA₃, IAA, or kinetin at a relatively low or high concentration significantly increased all growth attributes (shoot and root lengths, fresh and dry weights, chlorophylls a and b, and carotenoids content, which indicate the role of these hormones to improve photosynthetic efficiency. The data in this study are in accordance with those of Mousa, El-Sallami & Ali (2001), who found that the application of gibberellic acid increased the carotenoid content in Nigella sativa leaves compared with cytokinin application.

Effect of growth regulators on total soluble sugar, protein, and oil content of Juniperus procera seedlings

Application of GA₃, IAA, or kinetin with relatively low (50 ppm) or high (100 ppm) concentrations caused a significant increase in total soluble sugar, protein, and oil content in J. procera seedlings compared with 90% sulfuric acid or 20% acetic acid (Table 3). The highest values of total soluble carbohydrate, protein, and oil content were verified in 50 ppm GA₃ 7.6, 10.5 mg/g FW, and 68.5%, respectively (Table 3).

The results of this study agree with many reports by Ibrahim et al. (2007), Sadak et al. (2013), and Choudhury et al. (2013) on faba bean and tomato plants, which confirmed the role of these hormones in the improvement of plant growth and development due to the excessive synthesis of Lemna minor starch (Prajapati et al., 2015). Moreover, an increase in the total soluble sugar level may be useful in producing vigorous plants. The positive role of sugar in plant growth and development is because it provides the energy required for the synthesis of new cells and cell division and enlargement. Additionally, pretreatments with phytohormones generally enhanced the total soluble carbohydrate content, which was mainly accompanied by enhanced chlorophyll content (Alsokari, 2011).

The cytokinin-stimulating effect may be related to protein synthesis because of the postulation that their activity is associated with putative receptor systems of specific proteins located in the nucleus and cytosol. Additionally, numerous studies have revealed that IAA augmented the levels of nitrogenous compounds, primarily total soluble proteins, in many plant types (Sarkar, Haque & Karim, 2002; Prajapati et al., 2015). Moreover, it was noticeable that the total soluble protein content in J. procera seedlings increased significantly with GA₃ application. In our study, the increase in these primary metabolites by pretreatments with GA₃, IAA, and kinetin could have contributed to the acceleration of the photosynthesis process because it was associated with an increase in photosynthetic pigments (Table 2).

Effect of growth regulators on endogenous phytohormones in Juniperus procera seedlings

At the hormonal level, J. procera seedlings resulted from presoaked seeds in the relatively low and high concentrations of the three investigated growth regulators, which induced a marked increase in their IAA, GA₃, and cytokinin contents, whereas ABA content was decreased compared with H₂SO₄ or CH₃COOH presoaked seedlings (Table 4).

Such hormonal imbalance is likely to impair the process of seed germination either directly via interference with their role in the growth requirements of cell elongation, expansion, etc., or indirectly by affecting the metabolic machinery of the seedlings, as growth hormone-inducing growth requires continuous metabolic input.

Auxins are implicated in the stimulation of cell elongation by increasing cell wall extensibility or plasticity, which is mediated by stimulating H+-ATPase, which is concomitant with proton extrusion and loosening of cell wall cellulose fiber, synthesis of expansions, etc. (Taiz & Zeiger, 2006). The significant increase in the endogenous content of auxins in response to the relatively low or high concentrations of phytohormones used in this investigation resulted in enhanced growth of seedlings expressed in terms of a significant increase in the lengths of shoots and roots and fresh and dry weights of J. procera seedlings (Table 1). The most pronounced increments in IAA, GA₃, and kinetin levels were recorded in 100 ppm IAA-, 100 ppm GA₃-, and 100 ppm kinetin-soaking seedlings.

Additionally, GA₃ plays an important role in seed germination and growth. Low GA₃ content affects metabolic activities via certain mechanisms, which could contribute to retardation of the efficiency of maize in the conversion of fats into sugars via the glyoxylate cycle because GA₃ plays a crucial role in this pathway (Heldt, Piechulla & Heldt, 2011). The role of GA₃ in the synthesis and/or stimulation of hydrolases in the mobilization of seed reserves has been established (Jacobsen, Gubler & Chandler, 1995).

The effective role of either GA₃ or cytokinins in the use of reserves, a prerequisite step for providing respiratory substrates, is well established. Therefore, the accumulation of endogenous GA₃ and cytokinins is expected to increase the respiratory intensity of J. procera seedlings. Consequently, an observed increase in respiration, a sharp increment in the growth requirements of energy and different building blocks involved in different growth aspects. Such increases in different endogenous growth bioregulatory pathways could improve their biosynthesis and/or decrease their degradation (Taiz & Zeiger, 2006). GA₃ initiates proteolytic enzymes that release tryptophan, a common precursor of IAA (El-Saeid, Abou-Hussein & El-Tohamy, 2010). An increase in endogenous hormones is associated with the generation of some metabolites, particularly sugar (Shah, 2011). Moreover, high GA₃ levels induced an increase in the availability of water uptake, which led to a rapid increase in the fresh weight of seedlings. Furthermore, kinetin-soaking treatment elevated cytokinin levels in J. procera seedlings, leading to an increase in seed division, enhanced seed germination, and improved growth performance (Vamil, Haq & Agnihotri, 2010).

The marked decrease in the ABA content of J. procera seedlings in response to relatively low or high concentrations of GA₃, IAA, or kinetin plays a role in improving growth because ABA interferes with the mode of action of GA₃ and suppresses GA₃-mediated genes encoding hydrolytic enzymes in germinating seeds and seedlings (Walker-Simmons et al., 2000). Moreover, ABA, which appears to delay DNA replication and inhibit DNA metabolism, inhibits seed germination and plant growth (Vazquez-Ramos, 1993). The data of this study are in agreement with those obtained by El-Saeid, Abou-Hussein & El-Tohamy (2010), Barakat Nasser (2011), and Sadak et al. (2013) on cowpea, wheat, and faba bean plants.

The increase in endogenous phytohormone levels due to the application of the three hormones under investigation may be attributed to a reduction in the activity of their degrading enzymes (Soda et al., 2021). Meanwhile, the decrease in the ABA content observed in J. procera seedlings treated with the investigated hormones could be attributed to the transfer of isopentenyl pyrophosphate, which is the main precursor of cytokinin and/or GA₃ biosynthesis, in place of ABA (Hopkins & Hünter, 2004).