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    A cryopreservation protocol for ex situ conservation of terrestrial orchids using asymbiotic primary and secondary (adventitious) protocorms

    Access Status
    Fulltext not available
    Authors
    Bustam, B.
    Dixon, Kingsley
    Bunn, E.
    Date
    2016
    Type
    Journal Article
    
    Metadata
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    Citation
    Bustam, B. and Dixon, K. and Bunn, E. 2016. A cryopreservation protocol for ex situ conservation of terrestrial orchids using asymbiotic primary and secondary (adventitious) protocorms. In Vitro Cellular and Developmental Biology - Plant. 52 (2): pp. 185-195.
    Source Title
    In Vitro Cellular and Developmental Biology - Plant
    DOI
    10.1007/s11627-015-9732-7
    ISSN
    1054-5476
    School
    Department of Environment and Agriculture
    URI
    http://hdl.handle.net/20.500.11937/48299
    Collection
    • Curtin Research Publications
    Abstract

    © 2015, The Society for In Vitro Biology. In a bid to better conserve endangered terrestrial orchids, we detail cryogenic research using a widely distributed terrestrial orchid, Caladenia latifolia, as a model species for development of cryopreservation for primary (seed generated) and secondary (adventitious) protocorms. Primary protocorm cryopreservation (using droplet vitrification) involved a number of experimental lines of inquiry: investigation of a suitable plant vitrification solution (PVS) by comparing three variants of a standard PVS (2, 3 and 4), determining the most suitable primary protocorm developmental stage for successful cryopreservation, testing the effectiveness of a preculture medium treatment prior to cryopreservation, and investigating temperature preconditioning at the preculture stage as well as different components of the recovery medium. Primary protocorms were generated using asymbiotic in vitro germination media developed by the authors specifically for the test species (half-strength MS macroelements and microelements with 5% (v/v) fresh filter sterilized coconut water). Secondary protocorms were propagated using an in vitro proliferation medium (½ MS with 5 µM a-naphthaleneacetic acid + 2 µM 6-benzylaminopurine). A modified preconditioning step was developed, involving incubation on ½ MS with 0.2 M raffinose for 48 h at 15°C instead of 20°C. The standard recovery medium (½ MS 1 µM zeatin + 0.5 µM gibberellic acid) was replaced after the first week following warming from liquid nitrogen (LN), with asymbiotic germination medium (½ MS + 5% (v/v) coconut water) for the remainder of the recovery phase. This new step increased the survival of primary protocorms from 68 to 85%. The average post-cryostorage regeneration of plants from primary protocorms increased from 17 to 48% after a 6-wk incubation. A similar protocol increased the survival of secondary protocorms from 63 to 84%. Regeneration of plants from secondary cryostored protocorms increased from 11 to 26% after 14 wk. The protocols developed here provide a useful template for advancing cryoconservation of other orchid taxa, particularly rare and threatened species.

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    • Ex situ germplasm preservation and plant regeneration of a threatened terrestrial orchid, Caladenia huegelii, through micropropagation and cryopreservation
      Bustam, B.; Dixon, Kingsley; Bunn, E. (2016)
      Caladenia huegelii Rchb.f. is a threatened obligate mycotrophic orchid species with limited distribution in its remaining habitat, hence, this study to develop ex situ conservation protocols. An asymbiotic seed germination ...
    • Cryopreservation of secondary Protocorms, an alternative pathway for conservation of Western Australian terrestrial orchids
      Bustam, B.; Dixon, Kingsley; Bunn, E. (2015)
      Orchidaceae contains many species worldwide with a high extinction risk. Efforts to overcome this problem include ex situ approaches such as seed banking and in vitro germination of orchid seed symbiotically or asymbiotically. ...
    • Cryopreservation of invitro-propagated protocorms of Caladenia for terrestrial orchid conservation in Western Australia
      Watanawikkit, P.; Tantiwiwat, S.; Bunn, E.; Dixon, Kingsley; Chayanarit, K. (2012)
      Cryopreservation is an important tool for the exsitu preservation of endangered plants. In this article, we describe the development of a cryopreservation protocol for orchid protocorms using the terrestrial Australian ...
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