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<p>Kinnow mandarin needs an improved somatic embryogenesis protocol in the context of embryogenic stability and genetic fidelity for trait-specific varietal improvement. The present investigation was undertaken using plant bio-regulator (PBR) autotrophy, and a habituation-mediated regeneration technique. Results revealed that non-desiccated fully matured seeds (inoculated after vertical incision at chalazal end) during their conversion to the desiccation stage induced embryogenic callus from the outer integument (21.33%) on medium containing Murashige and Skoog (MS) basal salts containing 50.0&#xa0;g L<sup>−1</sup> sucrose, 200.0&#xa0;mg L<sup>−1</sup> activated charcoal, and 2.5&#xa0;g L<sup>−1</sup> gelrite. Further efficient habituation of embryogenic callus was observed when inoculated on the same medium without activated charcoal, followed by its activation through frequent subculturing (20-day (d) interval) twice on the same medium with and without activated charcoal. Subsequent transfer of activated calluses to a liquid suspension system (MS with 50.0&#xa0;g L<sup>−1</sup> sucrose) resulted in synchronized somatic embryogenesis (92.00%) within 20.81 d after the second subculture. Although maturation and germination of somatic embryos took a longer time (108.97 d), nearly 88.67% germination was achieved. Plantlet establishment and acclimatization frequency were 87.00% and 73.00% on the liquid paper bridge system and P<sub>1</sub> (cocopeat:vermiculite:perlite (2:1:1)) potting medium. Histological and microscopic studies revealed the single-cell origin of embryos and a high frequency of cytoplasmic single embryogenic cell occurrence 7 d after the second subculture. The inter simple sequence repeat (ISSR)–based genetic fidelity testing resulted in the identification of markers that can distinguish somaclonal variation. Hence, this high-frequency system can be a beneficial tool for the induction of mutations using random (physical and chemical) and site-specific (genome editing) mutagenesis.</p>

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Hormone autotrophy and habituation-mediated culture technique on embryogenic stability, synchrony, and somaclonal variation in Kinnow mandarin

  • 期刊论文

  • 发表日期：

    2025年10月8日

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  • Theivanai Murugan,
  • Om Prakash Awasthi,
  • Zakir Hussain,
  • Amina Shukoor

摘要

Kinnow mandarin needs an improved somatic embryogenesis protocol in the context of embryogenic stability and genetic fidelity for trait-specific varietal improvement. The present investigation was undertaken using plant bio-regulator (PBR) autotrophy, and a habituation-mediated regeneration technique. Results revealed that non-desiccated fully matured seeds (inoculated after vertical incision at chalazal end) during their conversion to the desiccation stage induced embryogenic callus from the outer integument (21.33%) on medium containing Murashige and Skoog (MS) basal salts containing 50.0 g L−1 sucrose, 200.0 mg L−1 activated charcoal, and 2.5 g L−1 gelrite. Further efficient habituation of embryogenic callus was observed when inoculated on the same medium without activated charcoal, followed by its activation through frequent subculturing (20-day (d) interval) twice on the same medium with and without activated charcoal. Subsequent transfer of activated calluses to a liquid suspension system (MS with 50.0 g L−1 sucrose) resulted in synchronized somatic embryogenesis (92.00%) within 20.81 d after the second subculture. Although maturation and germination of somatic embryos took a longer time (108.97 d), nearly 88.67% germination was achieved. Plantlet establishment and acclimatization frequency were 87.00% and 73.00% on the liquid paper bridge system and P1 (cocopeat:vermiculite:perlite (2:1:1)) potting medium. Histological and microscopic studies revealed the single-cell origin of embryos and a high frequency of cytoplasmic single embryogenic cell occurrence 7 d after the second subculture. The inter simple sequence repeat (ISSR)–based genetic fidelity testing resulted in the identification of markers that can distinguish somaclonal variation. Hence, this high-frequency system can be a beneficial tool for the induction of mutations using random (physical and chemical) and site-specific (genome editing) mutagenesis.