Habituation in callus culture refers to:
Normal plant cell proliferation requires exogenous auxin to activate S-phase genes and cytokinin to induce mitotic cyclins. Habituation denotes acquired ability to divide without these supplements due to stable metabolic reprogramming. Prolonged culture selects variants overproducing cytokinins via isopentenyl transferase genes or auxins via YUCCA flavin monooxygenases, or mutations conferring constitutive signaling through type-B response regulators and auxin receptors TIR1. Epigenetic changes in promoter methylation lock these biosynthetic genes in active state, making trait heritable through mitosis but occasionally reversible. Habituated callus appears vigorous, proliferates on basal medium lacking hormones, yet often shows reduced capacity to form organized primordia because internal hormone ratio is unbalanced. Recognition of hormone independence as habituation explains why some callus lines become recalcitrant to regeneration despite optimal external hormone supplementation and require rejuvenation strategies. Transcript profiling of habituated lines reveals constitutive expression of cytokinin response regulators ARR and auxin biosynthesis genes, circumventing requirement for external supply. Such autotrophy often accompanied by loss of regenerative competence because high internal hormone levels inhibit organized meristem formation. Reversal attempted by culture on hormone-free medium with activated charcoal to adsorb residual regulators.
Ref: Gautheret 1955 habituation; Meins & Binns PNAS 1979 hormone autotrophy; Taiz Plant Physiology Ch 21 cytokinin autonomy; NCBI NBK218342 habituation concept review.