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<p>The present study investigates the effect of in vitro Ethyl Methane Sulfonate (EMS) on somatic embryogenesis, aiming to generate high secondary metabolite-producing variant lines of <i>Tylophora indica</i>. Internodal segments were cultured on Murashige and Skoog’s (MS) (1962) medium along with four different concentrations (0.5, 2.5, 5.0, 7.5 µM) of auxins such as 2,4-Dicholoro phenoxy acetic acid (2, 4-D), Naphthalene acetic acid (NAA) and Indole butyric acid (IBA). Among tested auxins, 5.0 µM 2, 4-D was proved to be optimum to induce embryogenic calli. Best embryogenic calli were treated with five different, EMS concentrations (0.1%–0.5%) to determine the optimal mutagenic dose. Treated and non-treated callus were transferred to somatic embryo (SE) induction medium. The highest somatic embryoid induction frequency and average number of somatic embryoids per 100&#xa0;mg of callus were recorded 72 ± 1.22% and 91 ± 0.89 embryoids/culture, respectively on MS + 5.0 µM 2,4-D + 0.3% EMS. Induced somatic embryo then transferred to maturation and germination medium, which comprised of MS + 0.5 µM (gibberellic acid) GA<sub>3</sub> + 0.5 µM IBA and with 3 different concentrations (2.5, 5.0 7.5 µM) of cytokinin’s (6- benzyl adenine (BA), meta-Toplin (mT), Kinetin (Kn)). Among the tested combinations, BA 5.0 µM along with GA<sub>3</sub> 0.5 µM and IBA 0.5 µM was found to be best for somatic embryo proliferation and maturation. For germination of somatic embryos ½ MS, 1µM BA, and 0.5 µM GA<sub>3</sub> were utilised. Among all treatments, including the control, the highest germination percentage was observed with 0.3% EMS treatment, achieving a germination rate of 62.6 ± 0.24% and a plantlet conversion rate of 59.0 ± 0.31%. EMS also affected secondary metabolite profiles, as analysed through Gas chromatography Mass spectrometry, and induced genetic polymorphism, as shown by start codon-targeted markers. The highest number of compounds was recorded in 0.3% of EMS treated while the lowest number of compounds was found in 0.5% of EMS-treated plants. This study highlights induced in vitro EMS variation as a viable method for accelerating genetic variation and enhancing secondary metabolite production in <i>Tylophora indica</i>.</p>

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Study of EMS induced somatic embryogenesis and genetic variation in Tylophora indica through, morphological, phytochemical and molecular approach

  • 期刊论文

  • 发表日期：

    2026年6月2日

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  • Adla Wasi,
  • Anwar Shahzad,
  • Sabaha Tahseen

摘要

The present study investigates the effect of in vitro Ethyl Methane Sulfonate (EMS) on somatic embryogenesis, aiming to generate high secondary metabolite-producing variant lines of Tylophora indica. Internodal segments were cultured on Murashige and Skoog’s (MS) (1962) medium along with four different concentrations (0.5, 2.5, 5.0, 7.5 µM) of auxins such as 2,4-Dicholoro phenoxy acetic acid (2, 4-D), Naphthalene acetic acid (NAA) and Indole butyric acid (IBA). Among tested auxins, 5.0 µM 2, 4-D was proved to be optimum to induce embryogenic calli. Best embryogenic calli were treated with five different, EMS concentrations (0.1%–0.5%) to determine the optimal mutagenic dose. Treated and non-treated callus were transferred to somatic embryo (SE) induction medium. The highest somatic embryoid induction frequency and average number of somatic embryoids per 100 mg of callus were recorded 72 ± 1.22% and 91 ± 0.89 embryoids/culture, respectively on MS + 5.0 µM 2,4-D + 0.3% EMS. Induced somatic embryo then transferred to maturation and germination medium, which comprised of MS + 0.5 µM (gibberellic acid) GA3 + 0.5 µM IBA and with 3 different concentrations (2.5, 5.0 7.5 µM) of cytokinin’s (6- benzyl adenine (BA), meta-Toplin (mT), Kinetin (Kn)). Among the tested combinations, BA 5.0 µM along with GA3 0.5 µM and IBA 0.5 µM was found to be best for somatic embryo proliferation and maturation. For germination of somatic embryos ½ MS, 1µM BA, and 0.5 µM GA3 were utilised. Among all treatments, including the control, the highest germination percentage was observed with 0.3% EMS treatment, achieving a germination rate of 62.6 ± 0.24% and a plantlet conversion rate of 59.0 ± 0.31%. EMS also affected secondary metabolite profiles, as analysed through Gas chromatography Mass spectrometry, and induced genetic polymorphism, as shown by start codon-targeted markers. The highest number of compounds was recorded in 0.3% of EMS treated while the lowest number of compounds was found in 0.5% of EMS-treated plants. This study highlights induced in vitro EMS variation as a viable method for accelerating genetic variation and enhancing secondary metabolite production in Tylophora indica.