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Alginate Encapsulation of Shoot Tips and Their Regeneration for Enhanced Mass Propagation and Germplasm Exchange of Genetically Stable Stevia rebaudiana Bert.

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Abstract

An accelerated, unique and efficient alginate-encapsulation (synthetic seed)-based genetically true-to-type mass propagation system in stevia (Stevia rebaudiana Bert.) was successfully developed. Multiple shoot cultures in stevia were initially established from shoot tip explants cultured on Murashige and Skoog (MS) semi-solid medium supplemented with 0.5 mg/l N6-benzyladenine. Synthetic seeds were developed from individual shoot tips (obtained from in vitro multiple shoots) using 3% (w/v) sodium alginate and 75 mM calcium chloride solutions. In comparison with non-encapsulated in vitro shoot tips, alginate-encapsulated ones (i.e. synthetic seeds) exhibited accelerated germination (in the form of fresh shoot emergence) (~ 2 days), higher shoot proliferation rate and elongation (9.47 shoots with 10.94 mm length, and 64.33 leaves) as well as quicker root initiation (~ 9 days), multiplication and elongation (~ 13 roots with 38 mm length) after 30 days of inoculation in filter paper (M-bridge)-immersed half-strength MS liquid medium without plant growth regulator. Non-encapsulated in vitro shoot tips failed to initiate roots and showed reduced shoot growth. Synthetic seed-regenerated complete plantlets were successfully acclimatized in cocopeat. Clonal fidelity analysis of synthetic seed-regenerated plantlets was performed using ten inter-simple sequence repeats primers that exhibited monomorphic banding pattern, ensuring no genetic variations among the plantlets as well as with their mother plant. The present protocol will be beneficial not only for the smooth exchange of germplasms but also as an alternative approach for accelerated-cum-enhanced in vitro mass propagation of stevia.

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All data generated or analysed during this study are included in this published article.

Abbreviations

BA:

N6-benzyladenine

MS:

Murashige and Skoog (1962)

MSL:

MS liquid medium

ISSR:

Inter-simple sequence repeats

RAPD:

Random amplified polymorphic DNA

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Acknowledgements

The authors acknowledge the experimental assistance from Plant Tissue Culture laboratory at Regional Nuclear Agricultural Research Centre, Bidhan Chandra Krishi Viswavidyalaya, West Bengal, India, and Miss Manisha Mahanta for her assistance during genetic fidelity assessment.

Funding

This research was funded by Board of Research in Nuclear Sciences, BARC, Trombay, Mumbai, India (Sanction No. 55/14/09/2021-BRNS).

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Authors and Affiliations

  1. Crop Research Unit (Genetics and Plant Breeding), Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal, 741252, India

    Tsama Subrahmanyeswari, Saroj Laha, Somnath Bhattacharyya & Saikat Gantait

  2. Plant Biotechnology and Secondary Metabolites Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai, 400 085, India

    Suchita N. Kamble & Sudhir Singh

  3. Homi Bhabha National Institute, Anushaktinagar, Mumbai, 400094, India

    Sudhir Singh

Authors
  1. Tsama Subrahmanyeswari
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  2. Saroj Laha
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  3. Suchita N. Kamble
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  4. Sudhir Singh
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  5. Somnath Bhattacharyya
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  6. Saikat Gantait
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Contributions

TS was involved in methodology, data curation, formal analysis and original manuscript draft. SL was involved in methodology and data curation. SNK was involved in manuscript review and editing. SS was involved in manuscript review and editing. SB was involved in supervision, manuscript review and editing. SG was involved in conceptualization, funding acquisition, methodology, data curation, formal analysis, supervision, original manuscript draft, review and editing. All the authors read and approved the final version of the manuscript prior to its submission.

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Correspondence to Saikat Gantait.

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Subrahmanyeswari, T., Laha, S., Kamble, S.N. et al. Alginate Encapsulation of Shoot Tips and Their Regeneration for Enhanced Mass Propagation and Germplasm Exchange of Genetically Stable Stevia rebaudiana Bert.. Sugar Tech 25, 542–551 (2023). https://doi.org/10.1007/s12355-022-01194-4

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