Response of Chelidonium majus in vitro cultures and their alkaloid content under heavy metal stress Iva Doycheva1, Stefan Philipov2 1Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences 2Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences Introduction Heavy metal contamination is becoming an increasing environmental problem worldwide. Heavy metals (HM) retard plant growth, yield and affect the secondary metabolism which results in fluctuations in plant metabolites, such as the alkaloids. The aim of the study was to investigate the influence of HM on the growth of in vitro cultivated plants of Chelidonium majus L. (Fig. 1) and their alkaloid content. Results and discussion Plants cultivated at the lower concentration of Cd2+ formed roots and developed normally with a gradual reduction in their growth and rooting as the concentration of the supplemented HM increased (Fig. 2). There were not any differences in the development of the plants cultivated on Cd2+according to their type of origin (Fig. 2; 3). Epinasty and browning of the leaves were observed among the plants cultivated on the medium supplemented with Pb2+. These side effects got worse as the concentration of the HM increased, and were also accompanied by a strong reduction in the rooting (Fig. 4). The plants which were obtained from seeds developed normally and did not show any of the signs of the ones which were propagated through AS (Fig. 3; 4). None of the plants cultivated on a medium supplemented with Zn2+ survived, regardless of their origin and HM concentration. A phytochemical study showed that the plants cultivated at the highest concentrations of Cd2+ and Pb2+ and also obtained through AS had the highest RAM. However, the plants cultivated at the same concentrations but obtained from seeds had lower RAM similar to that of the plants cultivated at lower concentrations of HM and propagated through AS (Fig. 5). Materials and methods The plants were cultivated on a medium B5 with double the amount of macrosalts (2B5), 0.5 g/l activated charcoal and supplemented with different concentrations of HM – Cd2+ (0.5; 1 mg/l); Pb2+ (100; 250 mg/l); Zn2+ (100; 250 mg/l). Plants were obtained in vitro from seeds or were propagated through adventitious shooting (AS). The raw alkaloid mixtures (RAM) were obtained by usual chemical procedure. For contacts: idoycheva@gmail.com Conclusions In conclusion, Ch. majus was able to cope with the influence of Cd2+ and Pb2+ in the culture medium, but not with that of Zn2+. Quantity of RAM was influenced by the HM concentration and the origin of the plants. Acknowledgements: This research was supported by the Bulgarian National Science Fund, Bulgarian Ministry of Education and Science (Project КП-06-М26/4 from 01.12.2018). Fig. 1. Chelidonium majus L. Fig. 5. RAM (%) in native plant sample and in vitro cultivated plant samples obtained through AS or seed germination. 0,92 1,54 1,51 1,39 3,56 3,78 1,94 1,01 0,81 Reference sample 2B5 - Control AS 2B5 + 0.5 mg/l Cd (AS) 2B5 + 100 mg/l Pb (AS) 2B5 + 1.0 mg/l Cd (AS) 2B5 + 250 mg/l Pb (AS) 2B5 - Control Seeds 2B5 + 1.0 mg/l Cd (S) 2B5 + 250 mg/l Pb (S) Plant material from native plants Plants material from plants propagated through adventitious shooting Plant material from plants obtained from in vitro germinated seeds BalkanBio’2021 Fig. 2. Plants propagated through AS and cultured on 2B5 supplemented with Cd2+. Fig. 4. Plants obtained through AS and cultured on 2B5 supplemented with different concentrations of Pb2+. Fig. 3. Plants obtained from seeds and cultured on 2B5 supplemented with Cd2+ or Pb2+.