Genotype-dependent induction of embryogenic callus and programmed cell death in Korean pine

The strong genotype dependence of embryogenic callus (EC) induction limits the high-frequency regeneration of Korean pine (Pinus koraiensis) via somatic embryogenesis (SE).

We examined the morphological and histocytological features of various callus types using external morphology, microstructure, and ultrastructure analyses. To assess their embryogenic potential, we conducted somatic embryogenesis (SE) experiments. Additionally, we investigated programmed cell death (PCD) events associated with genotype-dependent embryogenic callus (EC) formation in Korean pine by performing TUNEL assays, detecting caspase-3 and vacuolar processing enzyme (VPE) activity, and measuring H₂O₂ and O₂^•- levels.

Obvious cell death was observed in the cell ultrastructure of LEC and NEC induced by recalcitrant genotypes. In LEC cells, we found typical characteristics of autolytic PCD, such as vacuole fusion, vacuolation of cells, invagination and rupture of the plasma membrane, few cytoplasm and organelles. In the NEC, degrading nucleus, withered cytoplasm and invagination and rupture of the plasma membrane were detected in early stage, but vacuole fusion was not observed. In addition, we observed DNA fragmentation into small fragments in LEC and NEC. Significant changes in the activities of caspase-3 and VPE, reactive oxygen species (ROS) levels, especially for H₂O₂, have been identified in LEC. However, in NEC, only the significant changes in the O₂^•- level and the caspase-3 activity were observed.

PCD was present in NEC and LEC cells in recalcitrant genotypes of Korean pine, which may be responsible for the loss or reduction of embryonic capacity of induced Korean pine callus. Our novel findings on PCD help elucidate the underlying causes of the difficulty in inducing EC from recalcitrant genotypes of Korean pine from a novel perspective.