Impact of foliar application of kinetin and seaweed extract on some vegetative growth and physiological status of olive (Olea europaea L. cv. sourani) Saplings

Introduction 1. General Background, Economic Significance, and Health Implications

The olive tree (Olea europaea L.), a quintessential xerophytic species, constitutes the cornerstone of agro-ecosystems in the Mediterranean basin and the Middle East. Beyond its socio-economic pivotal role in sustainable food security, it represents a biological model for resilience against climatic harshness [Fratianni et al., 2021; Jimenez-Lopez et al., 2020] [1,2] . The global trajectory of olive cultivation is driven by the escalating demand for virgin olive oil, a key component of the Mediterranean Diet, recognized for its potent nutraceutical properties. These health benefits are due to a unique set of bioactive secondary metabolites, especially phenolic compounds (like oleuropein and hydroxytyrosol) and monounsaturated fatty acids (like oleic acid). These compounds have strong antioxidant properties and can help fight chronic diseases [Servili, 2014; Visioli et al., 2018] [3,4] . In Iraq, the 'Sourani' cultivar is valorized as a premier indigenous genetic resource. It displays high phenotypic plasticity and dual-purpose productivity (oil and table olive), positioning it as a strategic candidate for horticultural expansion amidst challenging environmental constraints [Al-Ruqaie et al., 2016] [5] . Despite the crop's hardiness, nursery propagation faces physiological bottlenecks that hamper initial seedling establishment. These challenges include retarded root system development and restricted nutrient uptake, significantly exacerbated by the calcareous nature of regional soils. Such edaphic conditions lead to high pH-induced nutrient fixation (precipitation) and low bioavailability of essential elements [Chatzistathis and Therios, 2013] [6] . Consequently, relying solely on soil fertilization is often insufficient to satisfy the accelerated metabolic demands of young, rapidly growing seedlings. To circumvent these limitations, foliar biofortification has emerged as an indispensable agronomic strategy. This approach facilitates the direct delivery of nutrients and bioactive molecules to sink tissues, bypassing soil fixation and root transport lag phases [Fernández and Eichert, 2009] [7] . The efficacy of foliar sprays depends on solute permeation through the cuticle and stomata. Although the thick, waxy olive cuticle acts as a strong barrier, the presence of trichomes once wetted by surfactants can serve as preferential entry points [Fernández and Brown., 2013] [8] . Cytokinins such as Kinetin (N⁶-furfuryladenine) are pivotal in regulating plant morphogenesis. Kinetin promotes cytokinesis and cellular differentiation, effectively breaking apical dominance to induce lateral bud development a prerequisite for a well-branched seedling architecture [Kieber & Schaller, 2014; Ramlal et al., 2024] [9,10] . At the cellular level, Kinetin delays senescence by inhibiting the degradation of chlorophyll and proteins. Crucially, it enhances "sink strength," creating a metabolic gradient that mobilizes photo-assimilates towards active growing points [Zwack & Rashotte, 2015; Koprna et al., 2016] [11,12] . Parallel to hormonal regulation, seaweed extracts, particularly from Ascophyllum nodosum, have been widely adopted as sustainable biostimulants. They act as metabolic enhancers rich in bioactive moieties, including laminarins, alginates, and phytohormones [Battacharyya et al., 2015] [13] . Foliar application of these extracts triggers physiological responses that maintain cell turgor and protect photosynthetic apparatuses, translating into superior biomass accumulation [Ali et al., 2021] [14] . While the individual effects of Kinetin and seaweed extracts are documented, a knowledge gap exists regarding their synergistic interplay on the 'Sourani' cultivar under nursery conditions. This study hypothesizes that the co-application of A. nodosum extract and Kinetin will elicit a synergistic physiological response, maximizing vegetative vigor and biomass accumulation. Therefore, this study aims to evaluate the vegetative, biomass, and physiological responses of olive saplings to this combined protocol to define an optimal strategy for high-quality nursery production.

Materials and Methods

Study Site and Plant Material

The experiment was conducted at the Qamar Extension Farm in Kirkuk city (Coordinates: 35.678483, 44.568570) during the 2024 growing season. The study aimed to investigate the physiological response of two-year-old 'Sourani' olive saplings (Olea europaea L.) to foliar application of Kinetin and seaweed extract (Ascophyllum nodosum). Uniform saplings, grafted onto local olive seedling rootstocks, were obtained from the Al-Hawija Horticulture Station. To minimize experimental error arising from the genetic variability of the seedling rootstocks, saplings were strictly selected for uniformity in height, canopy size, and stem diameter. They were transplanted into 10-kg plastic pots filled with a potting substrate consisting of river soil (loam) and sand at a ratio of 2:1 (v/v). Standard agricultural practices, including basal fertilization, irrigation and periodic pest control, were applied uniformly to all saplings. Irrigation was applied manually based on the visual monitoring of the saplings' water requirements to prevent water stress throughout the experimental period. Samples of the potting soil were analyzed to determine their physical and chemical properties. The analysis (Table 1) indicated that the soil texture was Sandy Loam with a pH of 7.5, which is typical for the region, and moderate fertility levels.

Experimental Treatments and Design

The experiment was arranged as a factorial experiment based on a Randomized Complete Block Design (RCBD) with three replicates. The study comprised two factors:

1. Kinetin Levels: Four concentrations (0, 50, 100, and 150 mg L⁻¹). Kinetin was dissolved in minimal 1N

NaOH, and the pH was adjusted to 6.5-7.0 prior to final volume adjustment.

Seaweed Extract Levels: Four concentrations of

Ascophyllum nodosum extract (0, 2, 4, and 6 ml L⁻¹), prepared using distilled water.

The total number of saplings utilized was 48 (4 × 4 × 3 replicates)

Application Protocol

Treatments were applied in four intervals, commencing on March 15, 2024 (coinciding with sap flow and vegetative bud break), with a 21-day interval between applications.

Foliar spraying was conducted in the early morning until runoff using a 5-liter manual sprayer. A surfactant, (Tween-20 ® , 0.1%) from (Sigma-Aldrich) company, was added to all solutions, including the control, to ensure adhesion to the waxy cuticle.

Data Collection and Statistical Analysis

Measurements were recorded one month after the final application, Parameters included:

Vegetative Growth: Plant height increase (cm), Stem diameter increase (mm), Number of branches, Average branch length (cm), Number of leaves, and Leaf area (cm²) using ImageJ software.

Biomass: Fresh and Dry weights (g) for both leaves/shoots and roots. Samples were oven-dried at 70°C until constant weight.

Physiological Indices: Root-to-Shoot Ratio (calculated on a dry weight basis) and Total Chlorophyll Content (mg g⁻¹) using ethanol extraction (Knudson et al., 1977) [15] .

6: Leaf area (cm²)

Leaf expansion was significantly affected by Kinetin (Table 6). Interestingly, the 100 mg/L concentration not the highest dose recorded the maximum leaf area (4.54 cm²). Seaweed extract treatments significantly enlarged leaf surface area, with the 6 ml/L concentration yielding the highest value (4.81 cm²).

The interaction between the two biostimulants significantly maximized leaf area, reaching 5.6 cm² under the combination of 150 mg/L Kinetin and 6 ml/L Seaweed extract.

Kinetin application significantly enhanced fresh biomass accumulation in the shoot system (Table 7), with the 150 mg/L concentration producing the highest weight (76.31 g). A significant increase in shoot fresh weight was observed with Seaweed extract, peaking at 75.76 g under the 6 ml/L treatment.

The combined application of 150 mg/L Kinetin and 6 ml/L Seaweed extract exhibited a significant synergistic effect, yielding the maximum shoot fresh weight (92.73 g). Table 12 demonstrates that Kinetin significantly enhanced photosynthetic pigment content. The 150 mg/L concentration yielded the highest total chlorophyll content (1.93 mg/g). Foliar application of Seaweed extract significantly improved leaf greenness, with the 6 ml/L concentration recording the highest chlorophyll content (1.78 mg/g). A significant interaction was observed regarding biochemical attributes; the combined application of 150 mg/L Kinetin and 6 ml/L Seaweed extract resulted in the maximum total chlorophyll content (2.18 mg/g).

Discussion

The results obtained in this study demonstrate that the foliar biofortification of 'Sourani' olive saplings with Kinetin and Ascophyllum nodosum seaweed extract (SWE) elicits a significant synergistic response. This combined protocol effectively overcame the growth limitations typically associated with nursery establishment in semi-arid regions.

Enhancement of Vegetative Growth and Branching Architecture

The remarkable increase in plant height and, particularly, the number of branches (Table 4) driven by Kinetin application can be physiologically attributed to its role in modulating apical dominance. Exogenous cytokinins antagonize the inhibitory effect of auxins transported from the apical bud, thereby releasing lateral buds from dormancy and stimulating axillary shoot proliferation (Kieber and Schaller, 2014) [9] . Furthermore, Kinetin promotes cytokinesis in the Shoot Apical Meristem (SAM), translating into increased shoot length and leaf number. This hormonal signal was supported by the metabolic fuel provided by the Seaweed Extract. The extract's richness in macro-and micronutrients, alongside auxin-like compounds and betaines, likely maintained cell turgor pressure the biophysical force driving cell expansion (Battacharyya et al., 2015) [13] . The synergy observed (K3+S3) suggests that while Kinetin triggered the "signal" for branching, the nutrient-rich SWE provided the "substrate" to sustain this vigorous growth, preventing nutrient depletion.

Physiological Efficiency: Chlorophyll Content and Leaf Area

The significant rise in chlorophyll content (Table 13) is a classic manifestation of the "Richmond-Lang effect," where Kinetin delays senescence by inhibiting chlorophyllase activity and maintaining chloroplast ultrastructure (Zwack and Rashotte, 2015) [11] . Simultaneously, SWE contributed to this "greening effect" by supplying Magnesium (central to chlorophyll) and Iron (a biosynthesis cofactor). Additionally, the antioxidants in seaweed extracts likely protected the photosynthetic apparatus from photooxidation, a common stressor in the study region. The consequent increase in Leaf Area Index (LAI) and chlorophyll density maximized the Net Assimilation Rate (NAR), leading to the observed surge in biomass.

Biomass Accumulation and Resource Partitioning

A critical analysis of the Root-to-Shoot (R/S) ratio (Table 12) reveals an adaptive physiological adjustment. Although the R/S ratio slightly decreased in the combined treatments, the absolute root biomass more than doubled compared to the control (22.77 g vs. 9.83 g). This result aligns with the "Functional Equilibrium" hypothesis. When plants receive abundant nutrition and hormonal signals via foliar biofortification, the pressure on roots to forage for nutrients is alleviated. Consequently, the plant allocates more photoassimilates to shoot expansion to maximize light interception (Fernández and Brown, 2013) [8] However, the crucial finding is that the root system did grow vigorously enough (due to the auxin-like compounds in SWE) to mechanically and hydraulically support the larger canopy, ensuring a balanced and resilient sapling. (Arioli et al., 2015) [16] .

Conclusion

This study provides empirical evidence that combining Kinetin (150 mg L⁻¹) with Ascophyllum nodosum Seaweed Extract (6 ml L⁻¹) constitutes an optimal foliar protocol for 'Sourani' olive nurseries. This treatment acts synergistically to:  Overcome physiological bottlenecks: By enhancing sink strength and nutrient availability.  Optimize architecture: By promoting lateral branching and stem thickening.  Ensure quality: By producing saplings with a robust root system (high absolute mass) and a vigorous canopy.

Therefore, adopting this protocol is recommended to accelerate the production cycle and ensure the availability of resilient planting material suitable for orchard establishment in calcareous, semi-arid soils.

Recommendation

Based on these findings, it is recommended to adopt this combined foliar protocol (Kinetin 150 mg L⁻¹ + SWE 6 ml L⁻¹) in olive nurseries. This strategy not only accelerates the production cycle but also ensures the production of highquality, resilient saplings suitable for orchard establishment in semi-arid regions. Future research should investigate the long-term field performance of these biofortified saplings and the cost-benefit analysis of this protocol on a commercial scale.

References

1. Fratianni F, d'Acierno A, Albanese D, Matteo MD, Coppola R, Nazzaro F. Biochemical characterization of traditional varieties of apricots (Prunus armeniaca L.) of the Campania region, Southern Italy. Foods. 2021;11(1):100.
2. Jimenez-Lopez C, Carpena M, Lourenço-Lopes C, Gallardo-Gomez M, Lorenzo JM, Barba FJ, et al. Bioactive and quality of extra virgin olive oil. Foods. 2020;9(8):1014.
3. Servili M. The phenolic compounds: a commercial argument in the economic war to come on the quality of olive oil? OCL. 2014;21(5):D509.
4. Visioli F, Franco M, Toledo E, Luchsinger J, Willett WC, Hu FB, Martinez-Gonzalez MA. Olive oil and prevention of chronic diseases: summary of an international conference. Nutr Metab Cardiovasc Dis. 2018;28(7):649-656.
5. Al-Ruqaie I, Al-Khalifah NS, Shanavaskhan AE. Morphological cladistic analysis of eight popular olive (Olea europaea L.) cultivars grown in Saudi Arabia using numerical taxonomic system for personal computer to detect phyletic relationship and their proximate fruit composition. Saudi J Biol Sci. 2016;23(1):115-121.
6. Chatzistathis T, Therios I. How soil nutrient availability influences plant biomass and how biomass stimulation alleviates heavy metal toxicity in soils: the cases of nutrient use efficient genotypes and phytoremediators, respectively. In: Biomass Now -Cultivation and Utilization. IntechOpen; 2013.
7. Fernández V, Eichert T. Uptake of hydrophilic solutes through plant leaves: current state of knowledge and perspectives of foliar fertilization. Crit Rev Plant Sci. 2009;28(1-2):36-68.
8. Fernández V, Brown PH. From plant surface to plant metabolism: the uncertain fate of foliar-applied nutrients. Front Plant Sci. 2013;4:289.
9. Kieber JJ, Schaller GE. Cytokinins. Arabidopsis Book. 2014;12:e0168.
10. Ramlal A, Rajendran RA, Raju D, Singh VP. Introduction to phytohormones. In: Phytohormones in Abiotic Stress. CRC Press; 2024. p. 3-14.
11. Zwack PJ, Rashotte AM. Interactions between cytokinin signalling and abiotic stress responses. J Exp Bot. 2015;66(16):4863-4871.
12. Koprna R, De Diego N, Dundálková L, Spíchal L. Use of cytokinins as agrochemicals. Bioorg Med Chem. 2016;24(3):484-492.
13. Battacharyya D, Babgohari MZ, Rathor P, Prithiviraj B. Seaweed extracts as biostimulants in horticulture. Sci Hortic. 2015;196:39-48.
14. Ali O, Ramsubhag A, Jayaraman J. Biostimulant properties of seaweed extracts in plants: implications towards sustainable crop production. Plants. 2021;10(3):531.
15. Knudson LL, Tibbitts TW, Edwards GE. Measurement of ozone injury by determination of chlorophyll concentration. Plant Physiol. 1977;60:606-608.
16. Arioli T, Mattner SW, Winberg PC. Applications of seaweed extracts in Australian agriculture: past, present and future. J Appl Phycol. 2015;27(5):2007-2015.