Articles
Cryopreservation of the endangered Hawaiian fern Asplenium peruvianum var. insulare using green globular bodies
Article number
1421_12
Pages
87 – 94
Language
English
Abstract
Many fern species can be cryopreserved fairly simply using spores, but when spores are not available for or amenable to cryopreservation, green globular bodies (GGB) from in vitro propagated sporophyte tissue can be an effective alternative.
GGBs are tight bundles of sporophyte tissue capable of regrowth and are easy to isolate and manipulate for cryopreservation. Asplenium peruvianum var. insulare is an endangered fern endemic to the Hawaiian Islands and is maintained in tissue culture as part of the species’ recovery plan.
The species occasionally produces GGBs in vitro and initial cryopreservation tests using GGBs showed up to 100% survival following a droplet vitrification cryopreservation protocol, but GGB production was sporadic and sparse.
In order to increase the efficiency of GGB production in preparation for banking, we designed a media experiment to test the effects of auxin and cytokinin in tissue culture media on GGB growth and proliferation.
More specifically, we tested 13 media containing 0-1 mg L‑1 of the auxin 1-napthaleneacetic acid (NAA) and/or 0-1 mg L‑1 of the cytokinins 6-benzylaminopurine (BAP) or thiadiazuron (TDZ) on four genotypes of A. peruvianum var. insulare. Overall, NAA significantly increased the number of GGBs after one month while BAP significantly decreased the number of GGBs.
The highest mean number of GGBs (16.33) were produced on medium containing 0.1 mg L‑1 NAA and 0.5 mg L‑1 TDZ, with one genotype producing 45 GGBs on this medium.
Following one month of proliferation, inclusion of any cytokinin in the media was associated with significantly less growth of GGBs over the next two months.
GGBs of nine genotypes of A. peruvianum var. insulare were subsequently cryopreserved and banked using droplet vitrification, with an average survival of 64%. This study presents a straightforward and efficient method for the production of propagules for cryopreservation and their successful preservation.
GGBs are tight bundles of sporophyte tissue capable of regrowth and are easy to isolate and manipulate for cryopreservation. Asplenium peruvianum var. insulare is an endangered fern endemic to the Hawaiian Islands and is maintained in tissue culture as part of the species’ recovery plan.
The species occasionally produces GGBs in vitro and initial cryopreservation tests using GGBs showed up to 100% survival following a droplet vitrification cryopreservation protocol, but GGB production was sporadic and sparse.
In order to increase the efficiency of GGB production in preparation for banking, we designed a media experiment to test the effects of auxin and cytokinin in tissue culture media on GGB growth and proliferation.
More specifically, we tested 13 media containing 0-1 mg L‑1 of the auxin 1-napthaleneacetic acid (NAA) and/or 0-1 mg L‑1 of the cytokinins 6-benzylaminopurine (BAP) or thiadiazuron (TDZ) on four genotypes of A. peruvianum var. insulare. Overall, NAA significantly increased the number of GGBs after one month while BAP significantly decreased the number of GGBs.
The highest mean number of GGBs (16.33) were produced on medium containing 0.1 mg L‑1 NAA and 0.5 mg L‑1 TDZ, with one genotype producing 45 GGBs on this medium.
Following one month of proliferation, inclusion of any cytokinin in the media was associated with significantly less growth of GGBs over the next two months.
GGBs of nine genotypes of A. peruvianum var. insulare were subsequently cryopreserved and banked using droplet vitrification, with an average survival of 64%. This study presents a straightforward and efficient method for the production of propagules for cryopreservation and their successful preservation.
Authors
M. Philpott, N. Sugii, V.C. Pence
Keywords
exceptional species, plant tissue culture, droplet vitrification, banking, ex situ conservation
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