In Vitro Callus Induction and Morphogenesis in Soybean (Glycine max L.) Using Optimized Plant Growth Regulators

The present investigation established an efficient in vitro regeneration protocol for Glycine max using cotyledonary node, hypocotyl, and leaf explants cultured on MS medium supplemented with different concentrations of plant growth regulators. Among the auxin treatments evaluated, 2.0 mg L⁻¹ 2,4-D proved to be the most effective for callus induction, producing rapidly proliferating, friable callus with high morphogenic competence. Similar observations have been reported in soybean and other legumes, where 2,4-D promotes cellular dedifferentiation by activating auxin-responsive transcription factors involved in cell cycle re-entry and acquisition of pluripotency (Ikeuchi et al., 2013; Xu et al., 2022). The superior regenerative response of cotyledonary node explants compared with hypocotyl and leaf tissues may be attributed to their greater meristematic activity and higher endogenous hormonal balance. Juvenile tissues generally possess enhanced regenerative competence due to the presence of actively dividing cells, making them more responsive to exogenous growth regulators. Similar findings have been reported by Joyner et al. (2010), who identified cotyledonary nodes as the most suitable explant for soybean regeneration. Callus characteristics varied considerably with auxin concentration. Cream-coloured, friable calli obtained on 2.0 mg L⁻¹ 2,4-D exhibited excellent regenerative potential, whereas higher auxin concentrations induced tissue browning and reduced morphogenesis. Browning is commonly associated with phenolic oxidation and loss of cellular viability, which adversely affects regeneration efficiency in soybean tissue culture. Successful shoot regeneration was achieved after transferring proliferating calli to regeneration medium containing 2.0 mg L⁻¹ BAP and 0.5 mg L⁻¹ NAA. The synergistic interaction between cytokinin and auxin promoted the transition from undifferentiated callus to organized shoot meristems. Cytokinins stimulate shoot apical meristem formation, while low concentrations of auxin maintain cellular competence during organogenesis. The present findings are consistent with recent reports demonstrating enhanced shoot regeneration on BAP-NAA combinations in soybean tissue culture. Recent molecular studies have further revealed that auxin-mediated activation of ARF7, ARF19, LBD16, LBD17, and LBD18 genes regulates cellular reprogramming and callus formation, whereas cytokinin signalling controls shoot meristem development through the activation of WUSCHEL and SHOOT MERISTEMLESS pathways (Frontiers in Plant Science, 2023; Alok et al., 2025). The regeneration efficiency obtained in the present study therefore supports current molecular evidence explaining hormone-regulated morphogenesis in soybean. the optimized regeneration system developed in this investigation provides a reliable platform for rapid clonal propagation, genetic transformation, genome editing, germplasm conservation, and soybean improvement programmes, thereby contributing to future applications in plant biotechnology and sustainable crop enhancement.

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