Developmental progression toward embryogenic competence in Theobroma cacao L.: an integrated cytohistological and molecular analysis

Somatic embryogenesis is a platform for clonal propagation and genetic improvement of cacao, but its application is limited by genotype-dependent recalcitrance and restricted acquisition of embryogenic competence. This study integrated morphological, cytohistological, and gene expression analyses to identify features associated with embryogenic competence of Theobroma cacao L. clone CNCH13. Embryogenic callus frequency in petal-derived calli increased from 5–8% at 30 to 28–30% at 60 days, with no new embryogenic callus formed at 90 days. Embryogenic callus was identified by its light brown-yellowish color, granular appearance, and soft consistency. Histological analysis showed a continuum from parenchyma cells to competent cells, embryogenic cell clusters, proembryos, and somatic embryos. Phenolic accumulation was positively associated with embryogenic competence, whereas starch reserves occurred broadly in both embryogenic and non-embryogenic tissues. RT-qPCR across six tissues showed that BBM , ABI3 , AGL15 , and IAA20 were expressed in the induction-phase callus (hormone-containing medium) and in the expression-phase callus during the first 30 days of hormone-free culture; their expression declined after 60 days in expression medium, once embryogenic and non-embryogenic calli had become morphologically distinguishable and reached maximum levels in globular embryos. LEC2 and WUS followed a similar pattern at lower expression levels, whereas PIN1 , SERK1 , and SERK2 showed lower or broadly distributed expression across tissues, suggesting roles in additional developmental processes rather than in embryogenic competence. Phenolic accumulation and this early transcriptional signal emerge as the most reliable correlates of embryogenic competence, providing criteria for early selection of embryogenic tissue and improving somatic embryogenesis in recalcitrant cacao genotypes.