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 uti...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 hyperaccumula-tor 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.展开更多
Background： TB （Tuberculosis） is the second leading killer infectious disease after HIV （human immunodeficiency virus）. Its incidence is worsened by development of multi-drag resistant and extensive drug resistan...Background： TB （Tuberculosis） is the second leading killer infectious disease after HIV （human immunodeficiency virus）. Its incidence is worsened by development of multi-drag resistant and extensive drug resistant TB stxains. Available treatment regimens are expensive, toxic and lengtjy resulting to problems of non-adherence and inadequate response. Medicinal plants on the other hand may offer hope for developing alternative medicine for treatment of TB. This study evaluated the anti-tuberculosis activity of Echinops amplexicaulis. Materials and methods： Total crude extracts ofE. amplexicaulis were tested for activity against a wild strain resistant to Rifampicin and Isoniazid （MDR）, a fully susceptible laboratory strain （H37Rv） and Mycobacwrium boris （BCG strain） using disk diffusion method. MIC （minimum inhibitory concentration） was determined using Middlebrook 7H9 broil1. The strains were sub-cultured on Middlebrook 7H10 medium and MBC （minimum bactericidal concentration） determined. Susceptibility was evaluated by measuring zones of inhibition; MIC was obtained as the lowest concentration with no significant growth as shown by clog formation ofMTB （Mycobacwria tuberculosis） cells on the walls of the macro broth tube and MBC was obtained as the lowest concentration that inhibited growth of MTB colonies on Middlebrook 7H10 medium. Results： The extract showed a significant effect at a concentration of 50 mg/mL against all the three test strains F （2, 18） = 437.7, p = 0.00. It exhibited a MIC of 0.0488 mg/mL against MDR-TB and M. boris. Its MBC was the same at 0.0977 mg/mL against both MDR TB and M. boris. The MIC was much lower （0.0122 mg/mL） for the H37Rv strain. Terpenoids, alkaloids and tannins were present in large amount in the extract while saponins were present in small amounts. Flavonoids were not detected in the extract. Conclusion： E. amplexicaulis has the potential to be developed into new anti-TB drug and outcome of tile study supports the folkloric claims of anti-tuberculosis activity of tile plant.展开更多
The trace element selenium is essential for human nutrition but is distributed unevenly in soils worldwide with exten-sive selenium-deficient regions and selenium-enriched(seleniferous)areas.Neptunia amplexicaulis is ...The trace element selenium is essential for human nutrition but is distributed unevenly in soils worldwide with exten-sive 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 biotech-nological and molecular tools for functional genetics.Therefore,this study aimed to develop a de novo shoot regen-eration 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 Med-icago 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 initia-tion.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 dem-onstrated 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.展开更多
文摘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 hyperaccumula-tor 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.
文摘Background： TB （Tuberculosis） is the second leading killer infectious disease after HIV （human immunodeficiency virus）. Its incidence is worsened by development of multi-drag resistant and extensive drug resistant TB stxains. Available treatment regimens are expensive, toxic and lengtjy resulting to problems of non-adherence and inadequate response. Medicinal plants on the other hand may offer hope for developing alternative medicine for treatment of TB. This study evaluated the anti-tuberculosis activity of Echinops amplexicaulis. Materials and methods： Total crude extracts ofE. amplexicaulis were tested for activity against a wild strain resistant to Rifampicin and Isoniazid （MDR）, a fully susceptible laboratory strain （H37Rv） and Mycobacwrium boris （BCG strain） using disk diffusion method. MIC （minimum inhibitory concentration） was determined using Middlebrook 7H9 broil1. The strains were sub-cultured on Middlebrook 7H10 medium and MBC （minimum bactericidal concentration） determined. Susceptibility was evaluated by measuring zones of inhibition; MIC was obtained as the lowest concentration with no significant growth as shown by clog formation ofMTB （Mycobacwria tuberculosis） cells on the walls of the macro broth tube and MBC was obtained as the lowest concentration that inhibited growth of MTB colonies on Middlebrook 7H10 medium. Results： The extract showed a significant effect at a concentration of 50 mg/mL against all the three test strains F （2, 18） = 437.7, p = 0.00. It exhibited a MIC of 0.0488 mg/mL against MDR-TB and M. boris. Its MBC was the same at 0.0977 mg/mL against both MDR TB and M. boris. The MIC was much lower （0.0122 mg/mL） for the H37Rv strain. Terpenoids, alkaloids and tannins were present in large amount in the extract while saponins were present in small amounts. Flavonoids were not detected in the extract. Conclusion： E. amplexicaulis has the potential to be developed into new anti-TB drug and outcome of tile study supports the folkloric claims of anti-tuberculosis activity of tile plant.
基金The International Office of Leibniz University Hannover(Germany)secured the Erasmus+“Mix IT”grant for the internship of Bennet BuhmannThis publication is part of the project“Living on the edge:unravelling the secrets of selenium hyperaccumulator plants”(with project number VI.Vidi.213.037)of the research programme ENW-VIDI which is(partly)financed by the Dutch Research Council(NWO).
文摘The trace element selenium is essential for human nutrition but is distributed unevenly in soils worldwide with exten-sive 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 biotech-nological and molecular tools for functional genetics.Therefore,this study aimed to develop a de novo shoot regen-eration 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 Med-icago 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 initia-tion.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 dem-onstrated 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.
