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<p>Propagation of terrestrial orchids for ex situ or in situ conservation has relied on symbiotic propagation methods as a means of establishing the obligate mycorrhizal symbiosis. However, for critically endangered species, securing sufficient seed and fungal material for large scale propagation can be very difficult. Mass propagation by asymbiotic means is used commercially in the floriculture industry to generate large numbers of orchids. However, its application in the conservation of temperate terrestrial species as a means of supporting more traditional symbiotic propagation pathways remains largely unexplored. This study develops a propagation protocol for the critically endangered terrestrial orchid, <i>Thelymitra variegata</i> (Lindl.) F. Muell., that maximizes use of the limited primary ex situ propagative material available. Using plant growth regulators 2,4-Dichlorophenoxyacetic acid (2,4-D), 1-Naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA), light and dark treatments, and media with activated charcoal, we investigated the induction, proliferation, re-induction and optimisation of tuber production to create <i>T. variegata</i> plantlets. The use of 10 µM 2,4-D resulted in 100% of primary protocorms producing secondary protocorm-like bodies (PLBs). The addition of activated charcoal (AC) to half-strength Murashige and Skoog (HMS) media resulted in the conversion of secondary protocorms to plantlets, however, greater conversion was achieved with 3µM BA + 0.1µM 2, 4-D and 5µM NAA + 5µMBA in light. Reinduction of PLBs from PLB derived plantlets was optimised on basal media with 10µM 2,4-D. Tuberisation of plantlets was optimised by repeated transfer to commercial proprietary W3 media. This study shows for the first time that <i>T.variegata</i> plantlets can be produced using secondary PLBs, revealing a new in vitro propagation pathway for this critically endangered species with very limited seed and fungal collections. The method developed in this study holds significant promise in offsetting extinction risk by supporting ex situ conservation in this and other threatened terrestrial orchid species.</p>

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Protocorm-like body induction and proliferation as a conservation tool for the threatened Queen of Sheba orchid, Thelymitra variegata (Lindl.) F. Muell.

  • 期刊论文

  • 发表日期：

    2025年9月29日

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  • Belinda J. Davis,
  • Digby Growns,
  • Jason C. Stevens,
  • Anthony A. Scalzo

摘要

Propagation of terrestrial orchids for ex situ or in situ conservation has relied on symbiotic propagation methods as a means of establishing the obligate mycorrhizal symbiosis. However, for critically endangered species, securing sufficient seed and fungal material for large scale propagation can be very difficult. Mass propagation by asymbiotic means is used commercially in the floriculture industry to generate large numbers of orchids. However, its application in the conservation of temperate terrestrial species as a means of supporting more traditional symbiotic propagation pathways remains largely unexplored. This study develops a propagation protocol for the critically endangered terrestrial orchid, Thelymitra variegata (Lindl.) F. Muell., that maximizes use of the limited primary ex situ propagative material available. Using plant growth regulators 2,4-Dichlorophenoxyacetic acid (2,4-D), 1-Naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA), light and dark treatments, and media with activated charcoal, we investigated the induction, proliferation, re-induction and optimisation of tuber production to create T. variegata plantlets. The use of 10 µM 2,4-D resulted in 100% of primary protocorms producing secondary protocorm-like bodies (PLBs). The addition of activated charcoal (AC) to half-strength Murashige and Skoog (HMS) media resulted in the conversion of secondary protocorms to plantlets, however, greater conversion was achieved with 3µM BA + 0.1µM 2, 4-D and 5µM NAA + 5µMBA in light. Reinduction of PLBs from PLB derived plantlets was optimised on basal media with 10µM 2,4-D. Tuberisation of plantlets was optimised by repeated transfer to commercial proprietary W3 media. This study shows for the first time that T.variegata plantlets can be produced using secondary PLBs, revealing a new in vitro propagation pathway for this critically endangered species with very limited seed and fungal collections. The method developed in this study holds significant promise in offsetting extinction risk by supporting ex situ conservation in this and other threatened terrestrial orchid species.