Manfred Jusaitis

Habitat specificity, seed germination and experimental translocation of the endangered herb Brachycome muelleri (Asteraceae)

Abstract Brachycome muelleri Sonder (Corunna daisy) is an endangered annual herb, endemic to the upper Eyre Peninsula of South Australia. It is restricted to a single population occupying an area of approximately 3 ha on steep, south-facing cliff-foot slopes of the Baxter Hills. Its unique habitat, within an otherwise generally arid region, optimises soil moisture retention through the provision of concentrated water catchment, shade, and low evaporation rates during the growing season. Weeds had the potential to significantly disrupt recruitment, growth and reproductive output, but grazing did not constitute a direct threat to the population. Fresh seeds were innately dormant, requiring a period of after-ripening before they would germinate. Dormancy was artificially broken by gibberellic acid (GA3) treatment. Germination was optimal at 20 °C, and it was proposed that temperature plays an important role in regulating germination in situ. B. muelleri seeds did not respond to smoked water treatment, and GA3 (1000 mg l−1) was recommended as a routine seed treatment to stimulate germination. Trial translocations using seed as founder propagules resulted in low establishment, growth and flowering rates. Established seedlings provided several advantages over seed as founder propagules, including higher success rates, more vigorous growth, and improved seed yields in the first season. Successful regeneration, proliferation and expansion of a new translocated population was observed and recorded over four consecutive years. We suggest that conservation management of this species should include the establishment of several new populations by translocation to suitable, isolated, weed-free sites.

Distribution of mycorrhizal fungus associated with the endangered pink‐lipped spider orchid (Arachnorchis (syn. Caladenia) behrii) at Warren Conservation Park in South Australia

Abstract Ex situ seed baiting of a population of Arachnorchis (syn. Caladenia) behrii at Warren Conservation Park was conducted to determine the distribution of this orchids mycorrhizal fungus (or fungi) and whether it was limiting the distribution of the orchid. Forty‐five samples of topsoil and organic matter were collected systematically from within and outside the orchid population. All samples were baited with A. behrii seeds and incubated for 8 weeks to assess germination. Seeds from 24 samples out of 45 (53%), from both within and outside the orchid population, reached stage‐two germination (seed coat cracked, protocorms swelling), but a fungus later killed five samples. Seeds from three samples (7%) reached stage‐three germination (seed coat shed, rhizomes developing). Clearing and staining of seeds revealed that mycorrhizal fungus was present in all samples that had reached stage‐two or stage‐three germination. Mycorrhizal fungus was distributed independently of the orchids, and, thus, the distribution of A. behrii, which depends on mycorrhizal fungus, did not appear to be limited by the distribution of mycorrhizal fungus. Further research must focus on the inoculum potential and efficacy of the fungus/fungi in situ over a longer time period, as well as other ecological aspects of the orchids natural history, to identify the reason(s) for its rarity.

Rooting response of mung bean cuttings to 1-aminocyclopropane-1-carboxylic acid and inhibitors of ethylene biosynthesis

Cuttings taken from light-grown Vigna radiata (L.) R. Wilcz. cultivar ‘Berken’ seedlings were incubated for various periods in aminoethoxyvinylglycine (AVG) or α-aminoisobutyric acid (AIB) with or without auxin (indolebutyric acid). The inhibitors of ethylene biosynthesis reduced both the number of roots produced on cuttings and root growth rate. The inhibitory effect of AIB on root production was not reversed by 1-aminocyclopropane-1-carboxylic acid (ACC), while the inhibitory effect of AVG was partially reversed by ACC at high levels of auxin (10−4 M). Short treatments of ACC (up to 6 h) applied alone to cuttings did not affect rooting. However, prolonged exposure of cuttings to ACC (24 h) resulted in a significant decrease in root numbers, particularly when applied concurrently with auxin. The results support the hypothesis that a low threshold quantity of endogenous ethylene is required for optimal root production, and if tissue levels of ethylene fall below, or exceed, this threshold, root production and root growth rate may be inhibited.

Effects of temperature and gibberellic acid on 4-desmethylsterol composition of Avena sativa stem segments

Abstract Of the three morphological subunits of Avena sativa stem segments (node, leaf-sheath and internode) examined, internodes constituted the richest source of phospholipids and sterols, yielding almost double the concentration of lipid found in the leaf-sheath. The phospholipid compositions of nodes and internodes were similar, comprising mostly phosphatidylcholine (PC) and phosphatidylethanolamine (PE), with linoleic, linolenic and palmitic acids as the predominant component fatty acids. Leaf-sheath tissue contained mainly PE, with equally high amounts of palmitic, linoleic and linolenic acids. β-Sitosterol and cholesterol were the major 4-desmethylsterols of the internode, while β-sitosterol was predominant in the node and leaf-sheath tissues. The growth temperature of segments prior to isolation produced its major effect on the concentration of stigmasterol, which decreased markedly with temperature. The sitosterol/stigmasterol ratio increased significantly as temperature decreased. Stem segments isolated from plants treated with gibberellic acid (GA 3 ) for 3 weeks, showed a significant reduction in the amounts of 4-desmethyl sterols on a dry wt basis when compared with control segments. However, when stem segments were incubated with GA 3 for 20 hr, no change in 4-desmethylsterol composition or concentration was observed, even though significant growth in response to GA 3 occurred.

Conservation implications of clonality and limited sexual reproduction in the endangered shrub Acanthocladium dockeri (Asteraceae)

Allozyme electrophoresis was used to determine the extent of clonality in four naturally occurring populations of Acanthocladium dockeri F.Muell. from the mid-north of South Australia. A total of 33 loci was scored for at least 16 plants from each population. The four known populations of A. dockeri represent four quite distinct genetic clones. Each natural population consists of a single genet, and no indication of any correlation between geographical proximity and genetic similarity was found. Seed set in flowers was very low (0.6% of florets), probably owing to low pollen germinability. Seedlings raised from A. dockeri seed collected at Hart displayed obvious genetic affinities to their parent, but were nevertheless genetically distinct. Differences were consistent with the derivation of this seed by selfing of the Hart genet. A. dockeri is considered at high risk of extinction because of its low genetic diversity, poor seed production, lack of seedling recruitment and population growth by clonal reproduction alone. On the basis of the outcomes of this research, the following recommendations for conservation management of A. dockeri are made: (1) all four extant genets should be preserved in their respective habitats; (2) at least one ex situ collection of all four extant genets should be maintained at a secure site as an insurance against population loss; (3) further surveys are required to potentially locate new populations with superior fertility; and (4) mixing of clones or seed provenances into a single population should be avoided until appropriately designed experimental translocations have evaluated the possible consequences of such mixing on competitive, reproductive and genetic responses of populations.

Asymbiotic in vitro germination and seed quality assessment of Australian terrestrial orchids

Determining the seed quality and germination requirements for threatened orchid species in storage is vital for future conservation efforts. Seeds of many Australian terrestrial orchid species are held in conservation collections around the country, but few have been germinated in vitro, fuelling concerns over their long-term viability. This study tested three methods of assessing orchid seed quality; asymbiotic germination was compared with vital staining using triphenyltetrazolium chloride or fluorescein diacetate. Six culture media were examined for efficacy in promoting asymbiotic seed germination of four Australian terrestrial orchid species (Pterostylis nutans, Microtis arenaria, Thelymitra pauciflora and Prasophyllum pruinosum). Germination occurred on all media but germination rates were consistently highest on BM1 and development was most advanced on BM1, P723 and Malmgren media. Subsequent trials tested the efficacy of BM1 for asymbiotic germination of additional genera (Caladenia, Calochilus and Diuris), several congeneric species, and two species collected from several different provenances within each of their ranges. The results indicate that asymbiotic germination on BM1 medium is an effective technique for testing the performance of Australian terrestrial orchid seeds. The efficacy of vital stains to determine seed viability, however, remains uncertain, as significant disagreement between degree of staining and germinability was observed for some species.

Effect of pollen load, self-pollination and plant size on seeds and germination in the endangered pink-lipped spider orchid, Caladenia behrii

Caladenia behrii Schltdl. (Orchidaceae) (syn. Arachnorchis behrii) is a sexually deceptive, endangered orchid that produces aggregated pollen as pollinia. It is pollinated by a thynnine wasp, and may also be pollinated incidentally by other insects. Pollinator effectiveness may depend on the number of pollinia that pollinators carry and deposit, and on whether they mediate cross-pollination or self-pollination. To understand the role of pollinators and guide conservation programs, we determined the effect of pollen load (one pollinium v. two pollinia) and self-pollination on seed number, seed (embryo) size and germination at 35 days. We also examined the effect of plant size on seed size and seed number. By using partial correlations with leaf width, seed size, seed number, capsule volume and stem length, we found that leaf width was a good predictor for seed number, and that seed size was not correlated with any of the variables examined. Flowers pollinated with one pollinium and two pollinia did not produce seeds that differed in size or number. Cross-pollinated flowers produced fewer but larger seeds, which germinated faster than did seeds from self-pollinated flowers. We conclude that seed production in the field may be estimated from leaf size, that pollinators carrying one pollinium are as effective as those carrying two pollinia and that selfing affects germination negatively, partly because of the smaller size of selfed seeds. Conservation programs aiming to perform hand-pollination of this species should use crossing with a single pollinium.

Effects of temperature and gibberellic acid on phospholipid composition of Avena sativa stem segments

Abstract-Stem segments taken from Auena satiua plants grown at lo”, 20” or 30” varied in their phospholipid composition depending on the growth temperature; as temperature was lowered, there was a shift towards a greater proportion of unsaturated fatty acids. A significant increase was observed in the concentration of linolenic acid (18: 3) as growth temperature was lowered. Although prolonged treatment of oat plants with GA, produced marked changes in phospholipid composition of stem segments, these changes did not always accompany the GA,-induced growth response of segments, Treatment of stem segments with GA, for only 20 hr produced a significant growth response with little or no effect on phospholipid composition over this time. The data support the hypothesis that GAS-induced growth in Avena stem segments can occur without a concomitant change in phospholipid composition. INTRODUCTION The mechanism of action of gibberellic acid (GA,) in plant tissue is still unclear. Some evidence has suggested that the hormone may act by altering either membrane composition [l, 21 or membrane structure [3, 41. Compositional alteration may be brought about by changing the ratios ofphospholipid: sterol: protein, or by replacing individual phospholipids, sterols and proteins with new or different species (e.g. by changing the fatty acid structure of phospholipids, or by changing sterol composition). Work on the barley aleurone system has indicated a possible effect of GA, on enzymes of phospholipid synthesis [5, 61, although more recent studies on wheat aleurone tissue failed to show any control of phospholipid synthesis by GA, [7, 8-j. The

Reproductive biology of the endangered Brachycome muelleri (Asteraceae), an endemic herb of Eyre Peninsula, South Australia

The reproductive biology of Brachycome muelleri Sonder (Asteraceae) was studied from 1995 to 1997 by using nursery and field experiments. Reproductive development and seed dispersal occupied approximately half of the 4-month growth cycle. Flowers of B. muelleri did not have any obvious self-incompatibility systems and were readily selfed to produce viable seed. It appeared that B. muelleri may be preferentially cross-pollinated under ideal conditions of pollinator visitation, but could revert to self-pollination in the event of outcross failure. Seed dispersal was assisted by the epinastic curvature of peduncles as fruit matured, bringing capitula into contact with the soil a short distance away from the parent plant. Slight wind-stimulated movement of the capitulum was sufficient to dislodge seed directly onto the soil surface. Seed was shed in the immediate vicinity of parent plants and although some short-range movement facilitated by rain splash, water flow and gravity was observed, no long-distance dispersal mechanisms were apparent. Annual seed production of the single extant population of B. muelleri in South Australia was estimated at about 5 million seeds. Immediately after dispersal, the soil seed bank in the vicinity of B. muelleri plants contained, on average, over 1700 germinable seeds m -2 . Many of these seeds germinated or died within a year, the remainder persisting into a second or third year. Seed viability under field-burial conditions declined to less than 10% over that time. Population size did not appear to be limited by seed production, but rather by seed dispersal syndrome.

Influence of seed dimorphism and provenance on seed morphology, dispersal, germination and seedling growth of Brachyscome ciliaris (Asteraceae)

Brachyscome ciliaris is a floriferous Australian native daisy, with potential for use as a horticultural species. The species is hardy and seeds are relatively easy to germinate, but it is unique within the Brachyscome genus in that seeds are distinctly dimorphic. Within a fruiting capitulum, ray seeds are smooth and narrow with a minute pappus, whereas disc seeds have broad flat wings with curled hairs and a longer pappus than that of ray seeds. Both seed morphs, collected from five populations of the species, were tested to determine differences in their morphology, germination speed and percentage, seedling growth and wind-dispersal characteristics. Ray seeds were generally lighter and smaller than disc seeds and their length varied significantly with provenance. Dormancy levels of the two seed morphs and growth of ray- and disc-derived seedlings did not differ significantly, but differences were significant among the five populations tested. Seeds germinated readily, and germination was optimal under winter or summer conditions and lower in spring or autumn. Seed production by plants raised from ray or disc seeds was identical, but Noora-sourced plants yielded more seed than did plants sourced from the other provenances tested. Seed size, germination and plant growth of B. ciliaris varied significantly among populations. Winged disc seeds were dispersed slightly further by wind than were wingless ray seeds. We concluded that dormancy, germination and seed-yield characteristics of B. ciliaris were all influenced more by seed provenance than by seed morph (ray or disc).