Multiple shoot induction from embryonic axes of mature seeds supports a novel regeneration platform for biolistic DNA delivery in Theobroma cacao L.

Abstract

Theobroma cacao L. genetic transformation still relies predominantly on somatic embryogenesis-based systems, which are often genotype-dependent, labor-intensive, and associated with prolonged culture periods. In this study, we developed an alternative regeneration platform based on mature seed-derived embryonic axes and evaluated its suitability for biolistic DNA delivery. Embryonic axes from the cacao cultivars ‘TSH1188’ and ‘Parazinho’ were cultured on media supplemented with different cytokinins for multiple shoot induction. Among the plant growth regulators tested, 6-benzylaminopurine (BAP) was the most effective, promoting direct organogenesis from the apical dome and producing the highest shoot regeneration frequency and number of shoots per explant at 0.75 mg L⁻¹ for ‘TSH1188’ and 1.0 mg L⁻¹ for ‘Parazinho’. Sensitivity assays using antibiotics and herbicides identified genotype-dependent responses to kanamycin and hygromycin, whereas ammonium glufosinate, imazapyr, and glyphosate caused severe phytotoxic effects and completely inhibited shoot regeneration at relatively low concentrations. Hygromycin at 2.5 mg L⁻¹ (‘Parazinho’) and 5.0 mg L⁻¹ (‘TSH1188’) provided effective inhibition of shoot regeneration while maintaining the recovery of regenerating tissues, supporting its use in future selection protocols. For rooting, indole-3-acetic acid (IAA) at 0.5 mg L⁻¹ promoted the highest rooting efficiency, generating elongated roots and vigorous plantlets without intermediate callus formation. Preliminary biolistic assays using the pCAMBIA2301 vector resulted in localized transient GUS expression in the apical dome, demonstrating that this meristematic region is competent for exogenous DNA delivery. Collectively, these findings identify mature seed-derived embryonic axes as a promising explant for future stable genetic transformation and genome editing in cacao. A novel embryonic axis-based regeneration system enables rapid shoot recovery, rooting, and biolistic gene delivery in cacao, substantially shortening the timeline for plant transformation.