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<p>Ashwagandha (<i>Withania somnifera</i> L. Dunal) is a medicinally important plant known for its high-value bioactive compounds and their widespread use in Ayurveda and other indigenous medicinal systems. Micropropagation offers an effective alternative for large-scale production of true-to-type plants with a uniform metabolic profile. However, prolonged in vitro culture can impose physiological and oxidative stresses that may lead to biochemical, morphological, and genetic alterations. In the present study, we investigated the effects of long-term subculturing on growth, oxidative stress responses, and withanolide A accumulation in callus- and shoot-derived regenerants. Efficient protocols were established for both direct and indirect regeneration using Murashige and Skoog (MS) medium. Shoot tip explants regenerated directly on MS medium supplemented with 5.0&#xa0;mg/L 6-benzylaminopurine (BAP), whereas callus cultures were induced on MS medium containing 0.5&#xa0;mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Complete plantlets were developed on MS medium containing 2.0&#xa0;mg/L kinetin (KIN) and 0.1&#xa0;mg/L indole-3-acetic acid (IAA). Biochemical analysis revealed elevated total protein and flavonoid contents during early subcultures, whereas H₂O₂ levels declined progressively with successive passages. Activities of key antioxidant enzymes, including superoxide dismutase (SOD) and ascorbate peroxidase (APX) increased significantly in long-term culture. Regenerated plants exhibited distinct morphological leaf variations, which were supported by genetic fidelity analysis. Transcript profiling of oxidative and abiotic stress-related genes revealed pronounced upregulation of HSP70 (6.93-fold) in advanced subcultures. Additionally, short-term exposure to elevated temperature significantly enhanced withanolide A accumulation in callus tissues. Overall, the study demonstrates that prolonged in vitro culture induces coordinated biochemical, molecular and metabolic adjustments in <i>W. somnifera.</i> These findings highlight the need for monitoring biochemical and genetic stability during the micropropagation of medicinal plants.</p>

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Prolonged in vitro culture alters biochemical, morphological, and genetic stability in ashwagandha (Withania somnifera L. Dunal)

  • 期刊论文

  • 发表日期：

    2026年1月9日

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  • Adrija Banerjee,
  • Titiryu Chakraborty,
  • Alakesh Pal,
  • Dipak Manna,
  • Kishor Kumar

摘要

Ashwagandha (Withania somnifera L. Dunal) is a medicinally important plant known for its high-value bioactive compounds and their widespread use in Ayurveda and other indigenous medicinal systems. Micropropagation offers an effective alternative for large-scale production of true-to-type plants with a uniform metabolic profile. However, prolonged in vitro culture can impose physiological and oxidative stresses that may lead to biochemical, morphological, and genetic alterations. In the present study, we investigated the effects of long-term subculturing on growth, oxidative stress responses, and withanolide A accumulation in callus- and shoot-derived regenerants. Efficient protocols were established for both direct and indirect regeneration using Murashige and Skoog (MS) medium. Shoot tip explants regenerated directly on MS medium supplemented with 5.0 mg/L 6-benzylaminopurine (BAP), whereas callus cultures were induced on MS medium containing 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Complete plantlets were developed on MS medium containing 2.0 mg/L kinetin (KIN) and 0.1 mg/L indole-3-acetic acid (IAA). Biochemical analysis revealed elevated total protein and flavonoid contents during early subcultures, whereas H₂O₂ levels declined progressively with successive passages. Activities of key antioxidant enzymes, including superoxide dismutase (SOD) and ascorbate peroxidase (APX) increased significantly in long-term culture. Regenerated plants exhibited distinct morphological leaf variations, which were supported by genetic fidelity analysis. Transcript profiling of oxidative and abiotic stress-related genes revealed pronounced upregulation of HSP70 (6.93-fold) in advanced subcultures. Additionally, short-term exposure to elevated temperature significantly enhanced withanolide A accumulation in callus tissues. Overall, the study demonstrates that prolonged in vitro culture induces coordinated biochemical, molecular and metabolic adjustments in W. somnifera. These findings highlight the need for monitoring biochemical and genetic stability during the micropropagation of medicinal plants.