Effect of potent inhibitors of phenylalanine ammonia-lyase and PVP on in vitro morphogenesis of Fagopyrum tataricum

Background:
Fagopyrum tataricum (Tartary buckwheat) is known for its high phenolic content, particularly rutin. High concentrations of these compounds secreted in the tissue culture medium can lead to its darkening and the eventual death of explants in in vitro cultures. This study aims to enhance the morphogenesis of F. tataricum callus cultures by utilising phenylalanine ammonia-lyase (PAL) inhibitors and polyvinylpyrrolidone (PVP) to mitigate oxidative browning and improve tissue viability.

Results:
We analysed the response of protoplasts isolated from morphogenic callus to media supplemented with varying concentrations of PAL inhibitors (AIP, AOPP, OBHA) and PVP. The flow cytometry results revealed that 10 µM AIP and 1% PVP yielded exclusively diploid plants, whereas higher concentrations (100 µM AIP and 3% PVP) failed to regenerate plants. Moreover, AOPP and OBHA addition resulted in the regeneration of tetraploid plants. Further analysis of proembryogenic cell complexes (PECCs) isolated from Tartary buckwheat morphogenic calli responses to AIP and PVP indicated that 100 µM AIP was most effective for plant regeneration. Metabolomic analysis showed that AIP treatments reduced phenolic compounds, notably rutin, and increased the GSH/GSSG ratio, indicating reduced oxidative stress. Gene expression analysis highlighted elevated expression of somatic embryogenesis-related genes (LEC2, BBM) and WUSCHEL in AIP-treated callus.

Conclusions:
This study demonstrates that AIP enhances the regeneration potential of F. tataricum callus cultures, offering valuable insights for optimising tissue culture techniques for industrial crops. Additionally, we have detailed the metabolomic changes in calli treated with PVP and AIP, highlighting their impact on metabolism.