Induction of walnut callus and physiological and molecular differences among tissues with varying degrees of browning

Immature cotyledons from ‘Xiangling’ walnut ( Juglans regia L.) donor trees were used to screen callus induction conditions and characterize six browning grades (S1–S6). Single-factor experiments with three biological replicates per concentration identified 1 mg L⁻¹ α-naphthaleneacetic acid, 0.5 mg L⁻¹ 6-benzyladenine, and 200 mg L⁻¹ ascorbic acid as the concentrations yielding the highest mean induction rates in their respective screens (87.8%, 85.6%, and 88.9%). At these concentrations, approximately 11% of induced calli exhibited more severe browning (S4–S6), while 62%–64% showed some browning (S2–S6). Morphological observations, histology, a 40 min 2,3,5-triphenyltetrazolium chloride (TTC) reduction assay, and reverse transcription quantitative polymerase chain reaction provided complementary assessments of tissue condition. Mean apparent TTC reduction activity decreased from 1409.4 ± 42.8 in S1 to 275.6 ± 13.1 μg TTC equivalents g⁻¹ fresh weight h⁻¹ in S5. The eight candidate embryogenesis-related genes reached their highest mean expression in S2–S4: JrSERK1 and JrBBM1 in S2; JrBBM2 and JrLEC2 in S3; and JrLEC1 , JrAGL15 , JrFUS3 , and JrWOX2 in S4. JrLEC2 reached 9.86-fold relative to uncultured cotyledons. In S6, JrSERK1 and JrBBM1 expression was below control levels, whereas the other six genes remained elevated. Histology revealed variation in cell arrangement, staining, and tissue continuity. These findings identify favorable induction conditions and characterize browning-associated physiological and molecular variation, providing a basis for prioritizing S2–S4 for embryogenic competence assessment and subsequent regeneration studies.