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