Delineation of the impacts of varying 6-benzylaminopurine concentrations on physiological, biochemical and genetic traits of different olive cultivars under in vitro conditions

Abstract

Abstract. The plant growth regulator 6-benzylaminopurine (BAP) is an important component of plant nutrient medium with tendency to accelerate physiological, biochemical and molecular processes in woody plants such as olive. To date, limited knowledge is available on the role of BAP in mediating physiological, biochemical and genetic activities in olives under in vitro conditions. To cover this research gap, the current study was conducted with the objective of studying the role of BAP in regulating physiological traits (chlorophyll, CO₂ assimilation), antioxidant enzymes (superoxide dismutase, catalase and peroxidase), metabolic contents (starch, sucrose and flavonoids) and gene expression (OeRbcl, OePOD10, OeSOD10, OeCAT7, OeSS4, OeSuSY7, OeF3GT and OeChlH) under varying concentrations (0, 0.5, 1.5 and 2.5 mg L^-1) within the provided in vitro conditions. The explants obtained from different olive cultivars ('Leccino', 'Gemlik', 'Moraiolo', 'Arbosana') were cultured on olive medium (OM) provided with different BAP concentrations using a two-factorial design, and data were analysed statistically. All traits increased significantly under in vitro conditions due to increasing concentrations of BAP; however, this increase was more dramatic at 2.5 mg L^-1 and the least dramatic at 0.5 mg L^-1. Moreover, correlation, principal component analysis and heatmap cluster analysis confirmed significant changes in the paired association and expression of traits with changing BAP concentration and type of olive cultivars. Likewise, the expression of all genes varied due to changes in BAP concentration in all cultivars, corresponding to variations in physiological and biochemical traits. Moreover, the spectrographs generated via scanning electron microscopy further indicated the variations in the distribution of elements in olive leaf samples due to varying BAP concentrations. Although all cultivars showed a significant response to in vitro varying concentrations of BAP, the response of Arbosana was statistically more significant. In conclusion, the current study proved the dynamic impact of the varying BAP concentrations on regulating the physiological, biochemical, and molecular attributes of olive cultivars.

Keywords: Antioxidant; PCA; SEM; gene expression; metabolites.

Figure 1.

The PCA scattered plot deciphering the magnitude and extent of association of traits as explicated by the length and angle of vectors respectively. Besides, the differential dispersion of eclipses in the plot indicates the differential effect of BAP concentrations (0, 0.5 mg L⁻¹, 1.5 mg L⁻¹and 2.5 mg L⁻¹) on the degree of traits association.

Figure 2.

The PCA biplot indicates the differential extent of relative gene expression in olive cultivars under varying concentrations of BAP. The gene lying in the same quadrant of biplot are more closely associated with respect to their expression. Besides, the response of all four olive cultivars varied due to varying concentrations of BAP as revealed by the varying position of concentration with respect to biplot origin.

Figure 3.

Relative expression of different genes associated with studied traits in different in vitro grown olive cultivars within olive medium (OM) supplemented with different concentrations of BAP (0, 0.5 mg L⁻¹, 1.5 mg L⁻¹and 2.5 mg L⁻¹). Bar graphs indicate mean gene expression (P ≤ 0.05) and error bars represent the standard deviations when each treatment was tri-replicated.

Figure 4.

The four clusters (Right to left) of heatmap dendrogram illustrate the differential level of traits expression in different olive cultivars under varying supplementations of BAP (0, 0.5 mg L⁻¹, 1.5 mg L⁻¹and 2.5 mg L⁻¹) in OM, with the highest expression in Arbosana and the lowest expression in Leccino under all levels of BAP supplementations. POD, peroxidase; SOD, superoxide dismutase; CAT, catalase; ${\text{A}}_{{\text{CO}}_2}$ CO₂ assimilation.

Figure 5.

The four clusters (Right to left) of heatmap dendrogram illustrate the differential level of genes expression in different olive cultivars under varying supplementations of BAP (0, 0.5 mg L⁻¹, 1.5 mg L⁻¹and 2.5 mg L⁻¹) in OM, with the highest expression in Arbosana and the lowest expression in Leccino under all levels of BAP supplementations.

Figure 6.

A general model describing how BAP triggers the expression of various genes in olive that determine the expression of physiological and biochemical traits in olive.

Figure 7.

Scanning electron micrographs representing differential elemental distribution in leaf samples of in vitro grown olive cultivars at BAP concentration of 2.5 mg L⁻¹.