# Initial shoot regeneration in the selenium hyperaccumulator Neptunia amplexicaulis and in vitro test system for selenium tolerance and accumulation

## TL;DR

This study develops a method to grow shoots from the selenium hyperaccumulator plant Neptunia amplexicaulis in the lab, enabling future research into its selenium accumulation mechanisms.

## Contribution

The first successful protocol for initial shoot regeneration in Neptunia amplexicaulis, a key step for genetic studies.

## Key findings

- A shoot regeneration protocol was developed using hypocotyl and root explants of Neptunia amplexicaulis.
- Neptunia amplexicaulis efficiently accumulates selenium in vitro, reaching concentrations over 300 µg Se g−1 DM.
- Medicago truncatula was identified as a secondary selenium accumulator.

## 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.

The online version contains supplementary material available at 10.1007/s13659-025-00532-9.