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.Nicole Dowling and Manfred Jusaiti

Gibberellic acid-induced changes in the response of Avena sativa stem segments to temperature

xvii, 255 leaves : photos, tables, graphs ; 30 cm.Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Physiology, 197

The Influence of Gibberellic Acid and Temperature on the Growth Rate of Avena sativa Stem Segments

A micro-growth measuring technique was used to determine the growth response of stem segments of Avena sativa cv. Avon to a variety of gibberellic acid (GA(3)) concentrations over a range of incubation temperatures. Growth rate varied with GA(3) concentration, the temperature at which the rate was measured, and the growth temperature of the plants prior to excision of the segments. The curves relating segment extension rates to temperature were affected by GA(3) such that the linear portion of the curve was shifted to higher rates as GA(3) concentration was increased. The results seem to be analogous to the GA(3)-induced shifts of thermally induced phase transitions in glucose leakage from liposomes, observed earlier (Wood, Paleg 1974 Aust J Plant Physiol 1: 31-40)

Correlation between the Lipid Composition and the Responsiveness of Avena sativa Stem Segments to Gibberellic Acid

The lipid composition of Avena sativa stem segments was manipulated using BASF 13-338 (formerly Sandoz 9785) and growth temperature, in order to establish whether there were correlations between responsiveness of the tissue to gibberellic acid (GA(3)) and the presence, before hormone treatment, of specific lipid components. High correlations were obtained between GA(3)-induced growth and total phospholipid, individual phospholipids, and fatty acids (except for linolenic acid), total saturated fatty acids, stigmasterol content, and the unsaturated/saturated fatty acid ratio. It was concluded that, although the lipid composition, and particularly the total saturated fatty acid content, seem to be important contributory determinants of the GA(3)-induced growth response in this system, they may not be obligatory prerequisites, nor the only endogenous factors capable of influencing the response. However, the results are consistent with the hypothesis that membranes are involved in the hormonal mechanism and/or very early stages of the mode of GA(3) action in this tissue

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.Manfred Jusaitis and Mark Adam

Micropropagation of adult Swainsona formosa (Leguminosae: Papilionoideae: Galegeae)

Explants of axillary buds excised from mature adult stems of Swainsona formosa (G. Don) J. Thompson (syn. Clianthus formosus) were cultured on Mureshige and Skoog medium supplemented with a range of auxins, cytokinins, and sucrose concentrations. Auxins did not increase shoot or bud numbers above controls, and 2,4-dichlorophenoxyacetic acid was the only auxin to significantly increase callus production. Benzyladenine or thidiazuron incorporated into the medium at 0.1 μM stimulated shoot and bud production, and shoot growth occurred following removal of cytokinins from the medium after 4 wk. Shoot number increased linearly with sucrose concentration up to 40 g 1-1, but shoot height and the number of cytokinin-induced buds were optimal at sucrose levels of 20-30 g 1-1. Roots were initiated in vitro following treatment of cuttings with 0.1% indole-3-butyric acid and 0.1% α-naphthaleneacetic acid. Plantlets were successfully established in soil but were plagiotropic and exhibited distichous phyllotaxy.Manfred Jusaiti

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.Sophie Petit, Manfred Jusaitis and Doug Bickerto