In vitro induction of tetraploids and their morphological and phenolic acid content in Cnidium officinale

Cnidium officinale (Makino) Kitag (C. officinale; 2n = 2x = 22) is a medicinal plant that exhibits species-wide infertility due to meiotic instability and abnormal embryo sac development, resulting in exclusive vegetative propagation. This reproductive limitation restricts genetic diversity and breeding opportunities. Polyploidization is an effective breeding strategy for generating novel germplasm with altered morphology and enhanced metabolite production. This study aimed to induce tetraploids in C. officinale and evaluate their morphological and phytochemical characteristics.

In vitro shoots were treated with colchicine or oryzalin at various concentrations using liquid and solid culture systems. Ploidy levels were determined by flow cytometry and confirmed through somatic chromosome counting. Morphological traits, stomatal characteristics, and phenolic acid contents were compared between diploids and tetraploids.

Colchicine successfully induced tetraploids, whereas oryzalin was ineffective under the tested conditions. The highest induction rate (40%) was achieved by treating shoots with 20 mg/L colchicine for 48 h in liquid Murashige and Skoog medium. A total of 28 tetraploid plants were identified from 300 colchicine treated explants. Subsequent ploidy monitoring during repeated subcultures confirmed 11 plants as stable tetraploids. Chromosome counting of a representative stable tetraploid verified chromosome doubling from 2n = 22 to 2n = 44. Tetraploid exhibited typical polyploid characteristics, including significantly increased stomatal length and reduced stomatal density. Under in vitro conditions, tetraploids developed significantly thicker petioles than diploids. Following acclimatization, tetraploid plants showed significantly greater leaf length and area than diploids. Quantitative analysis of phenolic acid content revealed a significantly higher ferulic acid content in tetraploid fibrous roots (3.49 ± 0.14 µg/mL) compared with diploids.

This study provides the first report of colchicine-mediated tetraploid induction in C. officinale. Future research should focus on evaluating the agronomic performance of tetraploid plants under greenhouse and field conditions, examining the long-term stability of metabolite production, and clarifying the molecular basis of metabolite biosynthesis associated with polyploidization.