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Ex situ conservation of the endangered species Androcalva perlaria (Malvaceae) by micropropagation and cryopreservation

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Abstract

Micropropagation and cryopreservation protocols were developed for the threatened Australian species Androcalva perlaria. Vegetative shoots were brought into culture using a simplified surface sterilisation process with between 26 and 100 % of shoots successfully initiated across all genotypes. Shoots were multiplied on ½ MS basal salts medium (BM) with 1.25 µM 6-furfurylaminopurine (K) + 0.125 µM 6-benzylaminopurine (BAP). Cryopreservation was then developed for a single genotype to facilitate long-term ex situ storage for conservation purposes. Highest survival (>80 %) of shoot tips was achieved by preculture on 1.2 M glycerol for 48 h, incubation in PVS2 solution at 0 °C for 30 min, followed by rapid LN immersion then recovery. Application of this cryogenic approach to shoot tips from a range of genotypes gave variable post-cryopreservation regeneration results; survival for one genotype was only 3 %, while for four other genotypes survival varied between 60 and 80 % which compared favourably with post-cryopreservation regeneration (85 %) of the genotype used to develop the protocol. Callus production was achieved by culturing stem segments on ½ MS BM with 2.5 µM α-naphthaleneacetic acid + 2.5 µM BAP. Adventitious shoots were best regenerated from callus through incubation on BM only. Small callus pieces were successfully cryopreserved from 16 genotypes (1–88 % regeneration). Using a callus tissue pathway plant material was placed into LN storage after 6–8 weeks from the time of collection (compared to ~6 months using shoot tips). Plants derived from cryogenically preserved callus tissues were re-established in soil 28 weeks after removal from LN. This study demonstrates how biotechnology can be effectively utilised for the rapid ex situ conservation of endangered flora while ensuring that a significant range of genetically diverse samples can be conserved for long-term biosecurity.

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Acknowledgments

The authors wish to thank Grange Resources for providing financial and logistical support for this study, and the Department of Parks and Wildlife (in particular Dr Sarah Barrett) for their ongoing guidance and assistance with this research program. Thanks also goes to Dr Anja Kaczmarczyk and Dr Bryn Funnekotter for their cryogenic expertise and advice and finally to Pui Ye Phang for initially establishing the feasibility of using cryopreservation approaches for the conservation of vegetative tissues from Androcalva perlaria.

Author’s contribution

Susan E. Whiteley was involved in the acquisition, analysis, and interpretation (statistical assessment) of the cryogenic and callus data for the study. Susan E. Whiteley was also the primary person responsible for the initial drafting of the work (including construction of five figures) and for revising different drafts after feedback from co authors and has given approval for the work to be published and agrees to be accountable for all aspects of the work. Eric Bunn was heavily involved in the design of the surface sterilization experiment, and both callus induction experiments and was also involved in the interpretation of data (surface sterilization, callus induction and cryogenic experiments). Eric Bunn was also a major contributor providing detailed comments, additions and major text modifications for drafts as the manuscript developed and has given approval for the work to be published and agrees to be accountable for all aspects of the work. Akshay Menon was involved in the design of the cryogenic experiments, their implementation; and the acquisition of data and data management to align with the original concept of the work. Akshay Menon was also involved in the final editing of the manuscript and has given approval for the work to be published and agrees to be accountable for all aspects of the work. Ricardo L. Mancera was heavily involved in the original conception of the work and the interpretation of cryogenic data. Ricardo L. Mancera was also involved in the final editing of the manuscript and has given approval for the work to be published and agrees to be accountable for all aspects of the work. Shane R. Turner was heavily involved in sourcing funding to support the project, the collection, initiation and maintenance of all A. perlaria genotypes and the conception and design of the surface sterilization experiments, cryogenic experiments and the callus induction experiments and was also heavily involved in the acquisition of some of the data sets (surface sterilization, callus induction experiments and one cryogenic experiment), and the interpretation of data and its presentation in the manuscript. Shane R. Turner was also a major contributor providing detailed comments, additions and text modifications for different drafts as the manuscript developed (including the construction of all tables and Fig. 5) and has given approval for the work to be published and agrees to be accountable for all aspects of the work.

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Authors and Affiliations

  1. School of Plant Biology, The University of Western Australia, Crawley, WA, 6009, Australia

    Susan E. Whiteley, Eric Bunn & Shane R. Turner

  2. Botanic Gardens and Parks Authority, Kings Park, WA, 6005, Australia

    Susan E. Whiteley, Eric Bunn, Akshay Menon & Shane R. Turner

  3. School of Biomedical Sciences, CHIRI Biosciences, Curtin University, GPO Box U1987, Perth, WA, 6845, Australia

    Akshay Menon & Ricardo L. Mancera

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  1. Susan E. Whiteley
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  2. Eric Bunn
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  5. Shane R. Turner
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Corresponding author

Correspondence to Shane R. Turner.

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Whiteley, S.E., Bunn, E., Menon, A. et al. Ex situ conservation of the endangered species Androcalva perlaria (Malvaceae) by micropropagation and cryopreservation. Plant Cell Tiss Organ Cult 125, 341–352 (2016). https://doi.org/10.1007/s11240-016-0955-z

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