In vitro regeneration and conservation of Ruta chalepensis L. through cell suspension cultures and plating method

Document Type : Research Paper

Author

Department of Biology, College of Education for Pure Sciences, University of Diyala, Iraq

Abstract

Objective
Ruta chalepensis L., commonly known as fringed rue, is a medicinal plant belonging to the Rutaceae family. It is widely recognized for its diverse pharmacological properties, including antimicrobial, antifungal, anti-inflammatory, and antioxidant activities. Rich in bioactive compounds such as alkaloids, flavonoids, coumarins, and essential oils, this plant holds significant value in both traditional medicine and modern pharmaceutical applications. However, natural populations of R. chalepensis face threats due to habitat destruction, overharvesting, and low seed viability, necessitating alternative propagation and conservation strategies. Therefore, this study aimed to optimize the in vitro regeneration of Ruta chalepensis L. by establishing cell suspension cultures and regenerating plants using a plating method.

Materials and Methods
Callus induction was achieved from R. chalepensis stem explants cultured on Murashige and Skoog (MS) medium supplemented with varying concentrations (0.0, 1.0, 2.0, and 3.0 mg/L) of 2,4-Dichlorophenoxyacetic acid (2,4-D) in combination with 0.5 mg/L Benzyl Adenine (BA). The highest fresh callus weight (2.036 g) was observed at 3.0 mg/L 2,4-D + 0.5 mg/L BA after four weeks of culture. The induced callus exhibited a friable texture and was subsequently used to initiate cell suspension cultures in liquid MS medium supplemented with the same concentration of plant growth regulators.

Results
The plating method was employed to culture cell suspensions at different densities in solid induction medium, promoting cell division and the formation of cellular colonies. These colonies progressed to callus primordia, which later developed into small callus segments. The highest average number of cellular colonies (84.61 per dish) was recorded at a cell density of 20.2 × 10^5 cells/mL, significantly surpassing the initial density of 18.47 colonies per dish. This high-density culture also resulted in an average of 70.24 callus primordia per dish after 27 days. Furthermore, shoot formation was observed, with 60 shoots successfully obtained after seven weeks from callus derived from cell suspensions using the plating method. These shoots were maintained for 30 days on MS medium containing the same concentrations of plant growth regulators. Rooting of regenerated shoots was achieved at a 100% success rate in full-strength MS medium, and the newly developed plants were acclimatized in vitro in pots by the eighth week.

Conclusions
The regenerated shoots exhibited high rooting efficiency and successful acclimatization, validating the effectiveness of this in vitro propagation approach. These findings present a valuable biotechnological method for the conservation and sustainable production of Ruta chalepensis, supporting its pharmaceutical and medicinal applications.

Keywords

References

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Agricultural Biotechnology Journal
Volume 17, Issue 1
March 2025
Pages 193-212

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