Initial shoot regeneration in the selenium hyperaccumulator Neptunia amplexicaulis and in vitro test system for selenium tolerance and accumulation
Abstract The trace element selenium is essential for human nutrition but is distributed unevenly in soils worldwide with extensive selenium-deficient regions and selenium-enriched (seleniferous) areas. Neptunia amplexicaulis is one of the strongest selenium hyperaccumulator plants known and native t...
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SpringerOpen
2025-08-01
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| Series: | Natural Products and Bioprospecting |
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| Online Access: | https://doi.org/10.1007/s13659-025-00532-9 |
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| author | Bennet Buhmann Jeroen van der Woude Traud Winkelmann Antony van der Ent |
| author_facet | Bennet Buhmann Jeroen van der Woude Traud Winkelmann Antony van der Ent |
| author_sort | Bennet Buhmann |
| collection | DOAJ |
| description | Abstract The trace element selenium is essential for human nutrition but is distributed unevenly in soils worldwide with extensive selenium-deficient regions and selenium-enriched (seleniferous) areas. Neptunia amplexicaulis is one of the strongest selenium hyperaccumulator plants known and native to Australian seleniferous soils. Research in the genetic background of the selenium accumulation and tolerance mechanisms of this species lacks biotechnological and molecular tools for functional genetics. Therefore, this study aimed to develop a de novo shoot regeneration protocol for N. amplexicaulis and validate an selenium accumulation test system. Callus was induced on root and hypocotyl explants excised from 5-day old seedlings and cultured on an adjusted MS medium (SIM9) containing 4.5 µM Thidiazuron (TDZ) for two weeks in darkness. After this period, the TDZ concentration was reduced to 0.45 µM, and the explants were transferred to light conditions. In addition, seedlings of N. amplexicaulis, N. heliophila and Medicago truncatula were placed on vertical MS agar plates containing 1.5 mM (standard) or 0.1 mM (low) magnesium sulphate with 0, 30, 90 µM sodium selenate. Initial shoot differentiation was observed 6 weeks after culture initiation. This regeneration response was successfully repeated in a second experiment. The outgrow of the shoot buds into complete shoots was not yet achieved but requires additional media optimization. Additionally, spontaneous shoot regeneration from a root was observed, highlighting potential for further studies. In vitro grown seedlings demonstrated efficient, selective selenium uptake in N. amplexicaulis and identified M. truncatula as a secondary selenium accumulator with selenium concentrations of > 300 µg Se g−1 DM. This project presents the first protocol for inducing early stages of development of indirect shoot organogenesis in N. amplexicaulis from hypocotyl and root explants as prerequisite for genetic transformation, though completing the regeneration cycle remains challenging. Neptunia amplexicaulis hyperaccumulates selenium also under in vitro conditions. Graphical Abstract |
| format | Article |
| id | doaj-art-7fb2f828cb8e4c55bafca11fc89e0e13 |
| institution | DOAJ |
| issn | 2192-2195 2192-2209 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | SpringerOpen |
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| series | Natural Products and Bioprospecting |
| spelling | doaj-art-7fb2f828cb8e4c55bafca11fc89e0e132025-08-20T03:06:13ZengSpringerOpenNatural Products and Bioprospecting2192-21952192-22092025-08-0115112010.1007/s13659-025-00532-9Initial shoot regeneration in the selenium hyperaccumulator Neptunia amplexicaulis and in vitro test system for selenium tolerance and accumulationBennet Buhmann0Jeroen van der Woude1Traud Winkelmann2Antony van der Ent3Institute of Plant Genetics, Section Reproduction and Development, Leibniz University HannoverLaboratory of Genetics, Wageningen University and ResearchInstitute of Plant Genetics, Section Reproduction and Development, Leibniz University HannoverLaboratory of Genetics, Wageningen University and ResearchAbstract The trace element selenium is essential for human nutrition but is distributed unevenly in soils worldwide with extensive selenium-deficient regions and selenium-enriched (seleniferous) areas. Neptunia amplexicaulis is one of the strongest selenium hyperaccumulator plants known and native to Australian seleniferous soils. Research in the genetic background of the selenium accumulation and tolerance mechanisms of this species lacks biotechnological and molecular tools for functional genetics. Therefore, this study aimed to develop a de novo shoot regeneration protocol for N. amplexicaulis and validate an selenium accumulation test system. Callus was induced on root and hypocotyl explants excised from 5-day old seedlings and cultured on an adjusted MS medium (SIM9) containing 4.5 µM Thidiazuron (TDZ) for two weeks in darkness. After this period, the TDZ concentration was reduced to 0.45 µM, and the explants were transferred to light conditions. In addition, seedlings of N. amplexicaulis, N. heliophila and Medicago truncatula were placed on vertical MS agar plates containing 1.5 mM (standard) or 0.1 mM (low) magnesium sulphate with 0, 30, 90 µM sodium selenate. Initial shoot differentiation was observed 6 weeks after culture initiation. This regeneration response was successfully repeated in a second experiment. The outgrow of the shoot buds into complete shoots was not yet achieved but requires additional media optimization. Additionally, spontaneous shoot regeneration from a root was observed, highlighting potential for further studies. In vitro grown seedlings demonstrated efficient, selective selenium uptake in N. amplexicaulis and identified M. truncatula as a secondary selenium accumulator with selenium concentrations of > 300 µg Se g−1 DM. This project presents the first protocol for inducing early stages of development of indirect shoot organogenesis in N. amplexicaulis from hypocotyl and root explants as prerequisite for genetic transformation, though completing the regeneration cycle remains challenging. Neptunia amplexicaulis hyperaccumulates selenium also under in vitro conditions. Graphical Abstracthttps://doi.org/10.1007/s13659-025-00532-9HyperaccumulatorNeptunia amplexicaulisNeptunia heliophilaMedicago truncatulaSeleniumSelenate |
| spellingShingle | Bennet Buhmann Jeroen van der Woude Traud Winkelmann Antony van der Ent Initial shoot regeneration in the selenium hyperaccumulator Neptunia amplexicaulis and in vitro test system for selenium tolerance and accumulation Natural Products and Bioprospecting Hyperaccumulator Neptunia amplexicaulis Neptunia heliophila Medicago truncatula Selenium Selenate |
| title | Initial shoot regeneration in the selenium hyperaccumulator Neptunia amplexicaulis and in vitro test system for selenium tolerance and accumulation |
| title_full | Initial shoot regeneration in the selenium hyperaccumulator Neptunia amplexicaulis and in vitro test system for selenium tolerance and accumulation |
| title_fullStr | Initial shoot regeneration in the selenium hyperaccumulator Neptunia amplexicaulis and in vitro test system for selenium tolerance and accumulation |
| title_full_unstemmed | Initial shoot regeneration in the selenium hyperaccumulator Neptunia amplexicaulis and in vitro test system for selenium tolerance and accumulation |
| title_short | Initial shoot regeneration in the selenium hyperaccumulator Neptunia amplexicaulis and in vitro test system for selenium tolerance and accumulation |
| title_sort | initial shoot regeneration in the selenium hyperaccumulator neptunia amplexicaulis and in vitro test system for selenium tolerance and accumulation |
| topic | Hyperaccumulator Neptunia amplexicaulis Neptunia heliophila Medicago truncatula Selenium Selenate |
| url | https://doi.org/10.1007/s13659-025-00532-9 |
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