Borya mirabilis: steps in the recovery of a critically endangered Australian native plant
Creators: name: Reiter, Noushka
Publisher: name: RMIT University
Year: 2024

description: Borya mirabilis is one of the world's most critically endangered plants. The research in this thesis has illuminated key aspects of: its reproductive biology; interspecies and intraspecies molecular relationships, mycorrhizal status, tissue culture potential and disease threats. Each of these aspects has fundamental management implications for the active management of B. mirabilis.<br><br>Floral observations of B. mirabilis and related species affirmed the uniqueness of the Boryaceae amongst the Asparagales. B. mirabilis had an unusually high number of floral abnormalities compared with other species of Borya observed. B. mirabilis is fly-pollinated. Pollen of Borya species showed little difference in the characteristics of mature pollen between species, with viable pollen being prolate and unicolpate with a single colpa-style aperture and a unique patterning of the pila. The structural immaturity of B. mirabilis pollen correlated with evidence from pollen growth experiments, where B. mirabilis pollen had extremely low germination rates, with those grains that did germinate being slow to do so and with slow-growing pollen tubes compared to those of fertile Borya species. Examination of the ovules of B. mirabilis showed that morphologically they were viable compared to viable Borya species. The field population of B. mirabilis was crossed, with one seed produced (the first recorded seed for th is species). Cross-pollination using the pollen of the closely related B. constricta and B. sphaerocephala with B. mirabilis ovules proved unsuccessful. Examination of the chromosome number of B. mirabilis showed that it had approximately 66 chromosomes and is probably hexaploid, relative to the diploid number of 26 in B. constricta. This may explain its low fertility. <br><br>Interspecies and intraspecies relationships of the Boryaceae and Borya mirabilis were investigated using sequences of chloroplast and nuclear DNA. The closest similarities to B. mirabilis were B. constricta and B. sphaerocephala. B. mirabilis may have emerged from alloploidy of these species in the past. Because of the consistent similarities of B. mirabilis and B. constricta chloroplast sequences, it is proposed that both shared a common ancestor with a chromosome number of 2n=22. A malfunction n meiosis may have resulted in ovules with 2n=44. The high similarity of the nuclear ribosomal ITS region DNA suggests that the nuclear DNA was derived from B. sphaerocephela. B. mirabilis may be an allopolyploid, from fertilisation of a diploid ovule of B. constricta with haploid pollen of B. sphaerocephala, resulting in a reproductively isolated polyploidy of low fertility. The wild population of B. mirabilis was determined to have a small amount of genetic variation. The genetic variation in the field population w as not fully reflected in the ex-situ population. <br><br>An effective means of micro-propagation of B. nitida for use in B. mirabilis has been established, providing an effective means of mass production of the species. The research has determined: a suitable explant (shoot tips) for regeneration; an effective means of reducing contamination in tissue culture (PPM); what medium is required to micro-propagate the species (LMHM); an appropriate gelling agent (Phytagel); and a practical method for inducing roots on the shoots grown in tissue culture. <br><br>B. mirabilis has been established as mycorrhizal. The predominant mycorrhizal association is a nodular arbuscular mycorrhiza, present in the form of coils in root nodules over wetter months and as spores in these nodules over dryer months. A significant increase in the health of the ex-situ population of B. mirabilis was recorded after addition of soil containing fine roots of the wild population. Of the plants associated with the wild population, Callitris rhomboidea had the most morphologically similar vesicular arbuscular mycorrhizal relationship. But molecular identification was not achieved due to recalcitrance of DNA in PCR attempts. <br><br>Potential translocation sites for some of the ex-situ population of B. mirabilis were examined for Phytophthora infestation. Reid's Lookout and Mackey's Peak were infected with P. cinnamomi. Vegetation at Mackey's Peak displayed characteristic infection symptoms, resulted in isolates of P. cinnamomi from baiting and would directly receive runoff from both the walking track and the existing infested B .mirabilis site. At the Reid's Lookout site, both walking track and proposed translocation site were infested with P. cinnamomi, yet did not display the associated symptoms in the vegetation. The Pine Plantation translocation site was uninfected at the level of sampling undertaken. Its vegetation did not display any characteristic infection symptoms and was not isolated when soil samples were baited. It was therefore chosen for translocation and so far the plants are healthy and actively growing. <br><br>This research has provided critical knowledge to aid the recovery team in its current and future endeavours to manage this species and bring it back from the brink of extinction.