Abstract
This study described a protocol for genetically stable synseed production of Azadirachta indica using shoot tips of in vitro grown plantlets. For this, the shoot tips from in vivo grown tree were cultured on Murashige and Skoog (MS) media. The highest shoot response, number of buds per shoot and longest shoot were 94.44%, 5.07 and 3.73 cm, respectively, with 0.5 mg L−1 benzyladenine (BA) and 0.1 mg L−1 α-naphthaleneacetic acid in combination. These shoots were further enhanced by subculturing and the second subculture yielded both maximum shoots per micro shoot (6.08) and longer shoot length (4.64 cm) on the same media composition. The shoot tips of this subculture were employed for encapsulation. The 3% sodium alginate and 75 mM calcium chloride with an anion exchange period of 20 min was found to be best for synseed production which displayed a quick response (3.17 days) for germination with 100% germination frequency. The maximum in vitro root response, the highest number of roots per shoot and longest root length were 91.67%, 4.78 and 3.18 cm, respectively, in 1/2 strength MS media with 0.5 mg L−1 indole-3-butyric acid and 0.1 mg L−1 BA in combination. The 24 °C stored synseeds showed more germination frequency and regeneration potentiality in all storage periods as compared to 4 °C stored synseeds. The soil: organic manure (1:1) exhibited the best result of plant survival (91.18%) for primary hardening after 1 month and it displayed 85.9% after two months in the vermiculite-soil mixture (2:1) after secondary hardening. This investigation certified the genetic fidelity of synseed-derived plantlets after storage at 4 °C and 24 °C using ISSR markers. None of the ISSR markers exposed polymorphism for hardened plants.
Key message
This study reported first time the development of synseed of neem. The genetic fidelity of regenerants were performed after storage at 4°C and 24°C for different period of times (Days) using ISSR markers.
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Acknowledgements
The authors are thankful to the head of the department of Botany, the University of Kalyani for providing DST-FIST central equipment facility.
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The authors declare that no funds, grants, or other support were received during performing the experiments and the preparation of this manuscript.
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AK performed the research and wrote the manuscript. PG provided the laboratory and SNS analyzed the data.
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Communicated by Ranjith Pathirana.
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Kader, A., Sinha, S.N. & Ghosh, P. A strategy for development of genetically stable synseeds of Azadirachta indica A. Juss. (Neem) suitable for in vitro storage . Plant Cell Tiss Organ Cult 151, 47–58 (2022). https://doi.org/10.1007/s11240-022-02329-x
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DOI: https://doi.org/10.1007/s11240-022-02329-x