Tissue culture tools for selenium hyperaccumulator Neptunia amplexicaulis for development in phytoextraction

Abstract

Neptunia amplexicaulis is an herbaceous legume endemic to the Richmond area in central Queensland, Australia and is one of the strongest known Selenium hyperaccumulators on earth, showing significant potential to be utilised in Se phytoextraction applications. Here a protocol was established for in vitro micropropagation of Se hyperaccumulator N. amplexicaulis using nodal segments from in vitro-germinated seedlings. Shoot multiplication was achieved on Murashige and Skoog (MS) basal media supplemented with various concentrations of 6-Benzylaminopurine (BA) (1.0, 2.0, 3.0 mg L^-1) alone or in combination with low levels of Naphthaleneacetic acid (NAA) (0.1, 0.2, 0.3 mg L^-1), with 2.0 mg L^-1 BA + 0.2 mg L^-1 NAA found to be most effective. Elongated shoots were rooted in vitro using NAA, with highest root induction rate of 30% observed at 0.2 mg L^-1 NAA. About 95% of the in vitro rooted shoots survived acclimatization. Clonally propagated plantlets were dosed with selenate/selenite solution and assessed for Se tissue concentrations using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) and found to retain their ability to hyperaccumulate. The protocol developed for this study has potential to be optimised for generating clonal plants of N. amplexicaulis for use in research and phytoextraction industry applications.

Keywords: Hyperaccumulation; Micropropagation; Neptunia amplexicaulis; Phytoextraction; Selenium; Tissue culture.

Fig. 1

Neptunia amplexicaulis established in its natural habitat near Richmond in Central Queensland, Australia

Fig. 2

Se dosed clonal plantlets after being removed from soil after 4–5 weeks and washed prior to ICP-AES analysis. Well-developed root with root branching can be seen

Fig. 3

Percent germination of sterilised Neptunia amplexicaulis seeds cultured on various media profiles; Full strength MS with addition of 40 ppm Se in the form of selenate, selenite, or a mix of both at a ratio of 20:20, half strength MS and plain full strength MS. Columns with same letter indicate no significant difference at P < 0.05. (n ≤ 15 per treatment)

Fig. 4

Images of the various distinct stages in Neptunia amplexicaulis nodal micropropagation. A Seeds in media for germination, B seedlings after 4 weeks of growth, C Nodes from 4-week-old seedlings in multiplication media, D. Node displaying multiple shoots, E Elongated shoots in rooting media, F Rooted shoots in media, G Rooted shoot removed from media, H Acclimatised plantlet

Fig. 5

Selenium biomass concentration for clonal propagated control plants and plants grown in soil spiked to 100 ppm Se with selenate and selenite at a ratio of 50:50. Two tailed t test showed a significant difference at P < 0.05 level. (P ≤ 0.001, n ≤ 9)

Fig. 6

Flow chart presenting main steps for the nodal culture micropropagation of N. amplexicaulis. Micropropagation from seed shown on the left side and mature nodes are on the right. Steps in which the process is the same for both types of plant material are in the middle of the diagram