Vegetos
(An International Journal of Plant Research & Biotechnology)
(eISSN: 2229-4473)
Optimization of in vitro culture in Plantago lanceolata using plant growth regulators and evaluation of biocompatibility and toxicity against Artemia salina
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Research Articles | Published: 12 August, 2026
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Keywords: Brine shrimp, Callus formation, Hemolysis degree, Regeneration, Shoot organogenesis, n Plantago lanceolatan
Abstract
Plantago lanceolata L., a medicinal plant of the family Plantaginaceae, possesses considerable therapeutic potential. This study aimed to establish an efficient in vitro regeneration system for P. lanceolata and to evaluate the toxicity of methanolic extracts obtained from regenerated plantlets. Callus induction and shoot organogenesis were investigated using half-strength Murashige and Skoog (½MS) medium supplemented with different combinations of cytokinins, including Kinetin (Kin), 6-(γ,γ-Dimethylallylamino) Purine (2iP), and 6-Benzylaminopurine (BAP) in combination with auxins such as Indole-3-acetic acid (IAA), α-Naphthaleneacetic acid (NAA), and 2,4-Dichlorophenoxyacetic acid (2,4-D). Hemolysis and toxicity assays of the regenerated plantlets were evaluated. Maximum callus formation (100%) was simultaneously obtained by 2iP (1.0 or 2.0 mg/L) plus NAA (1.5 mg/L) and BAP (0.0 or 1.0 mg/L) with 2,4-D (0.5 or 1.5 mg/L) in shoot and root explants. The highest shoot organogenesis was observed with 2.0 mg/L 2iP and 0.5 mg/L NAA (62% and 100%), 2.0 mg/L Kin + 1.0 mg/L IAA (54% and 100%) and 1.5 mg/L BAP alone (83.33% and 100%) in leaf and root explants. The maximum number of shoots was recorded with 2 mg/L 2iP and 0.5 mg/L NAA (22.33 and 15.67 shoots), and 1.5 mg/L BAP (21.33 and 25.33 shoots) in leaf and root explants, respectively. Regenerated shoots were successfully rooted either without or with 0.5 mg/L indole-3-butyric acid, and plantlets were hardened with 100% survival. Toxicity assays showed that methanolic extracts of regenerated plantlets induced less than 1% hemolysis and no lethality in Artemia salina nauplii at 100 µg/mL, indicating no detectable toxicity. The results demonstrated optimized regeneration protocols and confirmed the bioactivity of regenerated plantlets, providing a foundation for future research on the pharmaceutical applications of P. lanceolata derived from tissue culture.
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Author Information
Agricultural Research, Education and Extension Organization (AREEO), Mashhad, Iran