John Delpratt

In situ seed development and in vitro regeneration of three difficult-to-propagate Lepidosperma species (Cyperaceae)

Field studies of fruit production from Lepidosperma concavum R.Br., L. laterale R.Br. and L. longitudinale Labill.showedthatlargeproportions(21-77%)offruitswereunfilledandthat filledandunfilledfruitslookedalike.Bagging ofinflorescencesdemonstratedthat filledfruitstendedtobeshed,whileemptyfruitsremainedwithintheinflorescence.Time of collection was critical for obtaining viable seeds, with successful harvesting limited to a short period (weeks) after maturation.Thetimingof floweringandfruitmaturationwerefairlyconsistentbetweenspecies,populationsandyearsinour studyarea.InL.concavumfruitproductionwasincreasedincultivationcomparedwithwildpopulations.Inallthreespecies, verylittleornogerminationoffruitsoccurredundernurseryconditions.Invitrocultureinitiationwasattemptedusingintact fruits, nicked fruits and seeds on 1/2MS (Murashige and Skoog) medium with 1 mM zeatin and 0.5 mM gibberellic acid in darkness.Cultureofintactfruitresultedinnogermination,whilenickedfruitshowedsomegerminationresponse.Bestresults were achieved from seeds with germination occurring as early as 7 to 18 days depending on the species. Germination of L.concavum,L.lateraleandL.longitudinalewas86%,64%and83%respectivelywithin5weeks.Germinationresponsewas strongly influenced by seed maturity. Mature seeds germinated significantly faster than immature seeds. On a small proportion of cultured seeds, calli formed and differentiated into numerous plantlets on growth regulator-free medium. Given the promising results observed in this study, in vitro culture appears to be a practical means of mass propagating Lepidosperma species.

Does diversity influence soil nitrate, light availability and productivity in the establishment phase of Australian temperate grassland reconstruction?

Summary  The successful conservation and restoration of the temperate native grasslands of south‐eastern Australia is critical to reversing the decline in range and diversity of these threatened plant communities. Yet the goals of high native species diversity and weed management are difficult to achieve in grassland restoration projects. To increase our understanding of whether synergies exist between these goals (i.e. whether early introduction of a larger number of species might improve both outcomes in the reassembly of native grassland), we examined the relationships between plant species number, functional group number and resource use during the establishment phase of direct‐sown grassland. We did this by sowing a representative suite of species (at varying levels of species number and functional group number) into experimental plots and then measuring and analysing the extent to which the newly established assemblages captured available resources, i.e. used soil nitrate, absorbed light and produced biomass (vegetative cover). Statistically significant correlations were common between the predictor variables (species number, functional group number, percentage vegetative cover, plant number, presence of idiosyncratic (dominating) species) and responses (soil nitrate concentration, light reduction or ‘extinction’). Higher diversity was associated with lower soil nitrate, while percentage vegetative cover and the presence of idiosyncratic species best predicted light extinction. The relationship between diversity, and plant biomass (measured as vegetative cover) and plant number was positive in the first year of the study. The diversity/biomass relationship became negative in the second year due to the higher numbers and cover of ‘idiosyncratic’ species. The diversity/plant number relationship also became negative in the autumn of the second year and was reduced to a trend by the winter. We found that lower nitrate and increasing plant numbers and vegetative cover were most strongly linked to increasing species number in the early stages of this study. This suggests that introducing and maintaining high diversity early in a native grassland reassembly or enhancement project will improve the resistance (e.g. to weed) of these communities. At later stages of grassland development, this function may be provided by the more dominating idiosyncratic species. The maintenance of diversity, an important goal in its own right, will therefore necessitate managed disturbances to periodically reduce the vegetative dominance of idiosyncratic species, releasing resources for the diverse range of other species whose early introduction will have allowed them to persist in the soil seed bank or as suppressed rootstocks.

Ploidy stability of somatic embryo-derived plants in two ecological keystone sedge species (Lepidosperma laterale and L. concavum, Cyperaceae)

We report on the development of a somatic embryogenesis system for Lepidosperma concavum R.Br. and L. laterale R.Br. and the determination of ploidy stability of plants derived from somatic embryos. These keystone Lepidosperma species cannot currently be returned to restoration sites because of propagation difficulties (i.e. seed dormancy, low seed fill and recalcitrance to vegetative propagation). Three explant types (in vitro-germinated seedlings, immature seed and immature inflorescences) were used for the assessment of callus production potential. Embryogenic callus was induced and multiplied on 1/2MS medium with 2,4-D either alone, or in combination with zeatin. Over 90% of seedling explants of L. laterale produced regenerative calli after 6 weeks and 53% of seedling explants of L. concavum produced calli after 16 weeks on media containing 2,4-D and zeatin. Inflorescence material appeared to be least responsive. High rates of conversion to plants were achieved on medium containing activated charcoal, followed by thidiazuron medium. Acclimatisation success of plants ranged from 86% to 95%. Acclimatised plants grew vigorously under standard nursery conditions. The DNA ploidy level of 486 somatic embryogenesis-derived plantlets was analysed by flow cytometry. Only one plant (=0.2% of all plantlets tested) was found mixoploid. All other plants showed a stable ploidy level and stable C-values within the species. There was a small but significant C-value difference between the two Lepidosperma species. Five variegated plants (=0.3%) were observed among a total of ~1600 plants acclimatised. The application of tissue culture techniques such as somatic embryogenesis brings large-scale production of Lepidosperma plants for revegetation and horticultural purposes closer to commercial feasibility.

Large-scale micropropagation of the Australian key species Gahnia radula (Cyperaceae) and its return to revegetation sites

We report on the successful propagation of the sedge Gahnia radula (R.Br.) Benth. from seed by using plant tissue culture, and its successful establishment in the field. This keystone species, although common along parts of the eastern coast of Australia, is currently not available for revegetation because of a lack of efficient propagation methods, leading to the use of substitute species in many restoration programs. Even though seed quality is a common problem for G. radula, one population bearing filled seed was located in the near-east of Melbourne and after harvest of fruit in December 2011, seeds were successfully germinated in vitro after removal of the pericarp. Overnight soaking in sterile 10% (v/v) smoke water before culturing enhanced in vitro germination from 29.2% to 66.7%. In vitro-grown seedlings were then used as starting material for tissue-culture propagation via shoot culture. A micropropagation rate of about six new plantlets per cycle was achieved within 5–6 weeks with liquid half-strength Murashige–Skoog medium and a pulse treatment with 10 µM 6-benzylaminopurine (BAP) and 2 µM naphthalene acetic acid (NAA). Plants rooted after receiving a pulse treatment with 5 µM kinetin and were successfully acclimatised into potting mix and were ready for field planting after 5–6 months. Tube stock was planted into two field sites with minimal weed control. Survival was 98% in both cases 1 month after planting and 54% and 74% after the summer. Division of in vitro-derived plants in the nursery was very successful, with 93–96% establishment of divisions. This research highlights the important role of plant tissue culture in conserving biodiversity of native flora.

Genetic structure of Gahnia radula (Cyperaceae), a key sedge for revegetation

Genetic studies can help guide effective ecological restoration by identifying potential source populations that contain the genetic variation necessary for adaptive potential, based on past landscape processes. Here we investigate genetic patterns in Gahnia radula (R.Br.) Benth., a sedge from south-eastern Australia that has potential for revegetation of disturbed areas. We developed microsatellite markers for this species and used them to show that it propagates mostly in a clonal manner. Levels of genetic variability differed between populations and the spatial scale of this variability within these populations is identified. A population used in recent restoration efforts and which sets seed has a particularly high level of variability. Recommendations are developed for sourcing material when using this sedge for revegetation.