Effect of alone & combined application of benzylaminopurine and gibberellic acid on in-vitro germination and growth rate of broccoli

Broccoli (Brassica oleraceae L. var. italica) is an important crop and faces challenges of further propagation due to genetic segregation and climate change. Efficient micropropagation protocols are necessary for the rapid production of true-to-type plantlets and are essential for this process. However, the synergistic effects of 6-benzylaminopurine (BAP) and gibberellic acid (GA₃) for this species have not been extensively studied. The study evaluated five pre-sowing seed-soaking treatments with various concentrations of BAP and GA₃ (Control: T₀; T₁: 25 + 25; T₂: 25 + 50; T₃: 50 + 25; T₄: 50 + 50 ppm) on Murashige and Skoog (MS) medium over a 16 h photoperiod, using a completely randomized design (CRD) with individual in vitro seedlings as experimental units. Morphological, physiological, biochemical, antioxidant, and gene expression (RT-qPCR) parameters were assessed.. All hormonal treatments significantly exhibited better response as compared to the control (T₀) treatment (p ≤ 0.05). The application of hormonal treatment (25 ppm BAP + 50 ppm GA3) (T2) significantly better response with an increase in shoot length and elevation in plant biomass, chlorophyll (a, b and T Chl), total phenolic content (TPC), flavonoids (TFC) and DPPH scavenging activity. Antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) and seedling vigor index (SVI-I, SVI-II) were significantly increased under T₂, markedly a mitigating enzyme activity. RT-qPCR analysis further confirmed these results by showing high levels of transcript abundance of antioxidant-related genes (BoSOD1, BoAPX1 and BoCAT) under T2 than in the control group with amplification specificity verified through melt curve analysis. Multivariate analyses, correlation heatmap, network analysis and principal component analysis indicated significant positive interrelationships among the growth traits, photosynthetic pigments, antioxidant capacity and vigor indices, implying the coordinated physiological regulation. Similarly, T2 as an efficient hormonal approach to enhance growth, antioxidant control, in vitro micropropagation efficiency in broccoli.