Julie A. Plummer

Seed germination ecology in southwestern Western Australia

Germination responses of species from the native plant communities of southwestern Western Australia can be related to syndromes of life history, fire response, and seed storage, and also to factors related to environmental stress. The Mediterranean-type climate of the region with periodic drought and recurrent fires affects the production of viable seeds in plants of limited stature and rooting depth. Fire response ephemerals and species cued to flower by fire tend to produce viable, readily germinable seeds, but there are instances where seed production is aborted in these predominantly herbaceous life forms. Clonal, rhizomatous species often produce mainly inviable seeds. Production of viable seeds in woody species of these highly diverse communities may also be restricted by limitations to cross pollination. Obligate post-fire seeding species tend to produce a greater proportion of viable seeds than species which are capable of resprouting following fire. Serotinous species, whether post-fire re-seeders or post-fire resprouting species, produce mainly viable seeds, which germinate readily once freed from protective fruits. Species of the legume families and a few others of the soil seed bank produce innately dormant seeds which can be germinated following heat shock treatments which simulate the effects of fire. Heat shock in these species appears mainly as a mechanism to crack the hard seed coats, but the effect of heat to denature seed coat inhibitors has not been eliminated. Western Australian species do not seem to break dormancy when exposed to leachates from burned wood as has been observed in comparable habitats in California and South Africa, but further research is advised. Germination in many native southwestern Australian species is cued by temperatures that correspond to the winter rainfall period. There are also indications that an after-ripening period of warm, dry storage increases percentage of germinable seeds. Stimulation of germination by hormones is almost unresearched in Western Australia, but germination percentages have been increased in a small number of species of horticultural potential. Stimulation of germination by soil nutrient concentrations is almost unresearched in Western Australia, except for the inhibitory effect of excess sodium chloride levels inEucalyptus andMelaleuca. These species only germinate when osmotic effects are reduced to lower levels as would occur when winter rains dilute soil salts. Application of research on seed germination has already enhanced the establishment of seedlings in the restoration of mine sites and is becoming important in aspects of the breeding and selection of native plants for the cut flower, bedding plant and essential oil industries.AbstraktDas Keimverhalten von Arten der nativen Pflanzengesellschaften aus dem Südwesten Westaustraliens kann mit Syndromen ihrer Entwicklung, dem Verhalten gegen Waldbrände, der Speicherung in Samen und auch mit Umwelt-Sressfaktoren in Verbindung gebracht werden. Das mediterrane Klima dieser Region mit regelmäßiger Trockenheit und wiederholt auftretenden Bränden beeinflußt die Produktion keimfähiger Samen bei kleineren Pflanzen mit geringer Wurzeltiefe. Wadbrand-Ephemere und Arten, die nach Feuer blühen, produzieren im allgemeinen rasch keimende Samen, jedoch kann die Samenproduktion unter Umständen bei diesen vorwiegend krautigen Lebensformen fehlschlagen. Klonbildende Arten mit Rhizomen erzeugen im wesentlichen nicht keimfähige Samen. Bei holzigen Arten dieser enorm mannigfaltigen Pflanzengesellschaften kann die Produktion keimfähiger Samen auch durch Erfordernis einer Fremdbestäubung begrenzt sein. Obligat nach Feuer aussamende Arten tragen tendenziell einen größeren Anteil keimfähiger Samen als Arten, die nach Feuer regenerieren können. Serotine (bradykarpe) Arten mit langsam öffnenden Früchten, seien es nach Feuer aus Samen keimende ‘reseeders’ oder regenierende ‘resprouters,’ produzieren im wesentlichen keimfähige Samen, die rasch, sobald sie aus den schützenden Früchten befreit sind, keimen. Leguminosenarten und einige andere produzieren von Natur aus ruhende Samen, die nach einer die Wirkung von Feuer simulierenden Hitzeschockbehandlung zum Keimen gebracht weren können. Bei diesen Arten scheint Hitzeschock vor allem ein Mechanismus zu sein, der die harte Testa sprengt, aber eine Auswirkung von Hitze auf eine Denaturierung von Keimungshemmern konnte nicht ausgeschlossen werden. Bei westaustralischen Arten scheint die Keimruhe nicht durch eine Wirkung von Eluaten aus verbranntem Holz gebrochen zu werden, wie dies auf vergleichbaren Standorten in Kalifornien und Südafrika beobachtet wurde; weitere Untersuchungen sind freilich erforderlich. Die Samenkeimung vieler in Südwestaustralien einheimischer Arten wird durch Temperaturen ausgelöst, die der Winterregenperiode entsprechen. Es gibt auch Hinweise, daß eine Nachreifeperiode mit warmer, trockener Lagerung die prozentuale Keimfähigkeit erhöht. Eine hormoneile Keimförderung ist bislang fast nicht in Westaustralien untersucht worden, aber bei einer kleinen Anzahl von gartenbaulich interessierenden Arten konnte die Keimfähigkeit erhöht werden. Auch eine Keimförderung durch Bodennährstoffkonzentrationen ist in Westaustralien so gut wie nicht untersucht, abgesehen von hemmender Wirkung exzessiver Natriumchloridkonzentrationen beiEucalyptus undMelaleuca. Samen dieser Arten keimen nur, wenn die osmotischen Bedingungen soweit erniedrigt werden, wie es geschieht, wenn Winterregen Bodensalze verdünnen. Die Anwendung der Untersuchungen der Samenkeimung hat bereits das erfolgreiche Anwachsen von Sämlingen bei der Wiederbegrünung ehemaliger Gruben verbessert und wird ein wichtiger Aspekt bei Zucht und Auslese einheimischer Pflanzen zur Nutzung als Schnittblumen, Gartenpflanzen und beim Anbau für die Nutzung ätherischer Öle sein.

Effects of temperature, light and gibberellic acid on the germination of seeds of 43 species native to Western Australia

Species native to the southwest of Western Aus- tralia, representing a range of plant families, life-history strat- egies, fire-response syndromes, seed-store types and seed weights, were tested for viability using tetrazolium chloride and for germination under combinations of constant tempera- tures of 15 ?C or 23 ?C, constantly dark or 12 h diurnal white- light conditions, and with, or without, addition of gibberellic acid (GA3, 50 mg/1). Species previously known to require a heat-shock treatment to overcome dormancy due to an imper- vious testa were pre-treated prior to imposition of tempera- ture, light and GA3 conditions. The test environmental condi- tions related to differences between winter and autumn tem-

Sandalwood Fragrance Biosynthesis Involves Sesquiterpene Synthases of Both the Terpene Synthase (TPS)-a and TPS-b Subfamilies, including Santalene Synthases

Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of α-, β-, epi-β-santalene, and α-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). Recombinant SaSSy was additionally tested with (Z,Z)-farnesyl diphosphate (Z,Z-FPP) and remarkably, found to produce a mixture of α-endo-bergamotene, α-santalene, (Z)-β-farnesene, epi-β-santalene, and β-santalene. Additional cDNAs that encode bisabolene/bisabolol synthases were also cloned and functionally characterized from these three species. Both the santalene synthases and the bisabolene/bisabolol synthases reside in the TPS-b phylogenetic clade, which is more commonly associated with angiosperm monoterpene synthases. An orthologous set of TPS-a synthases responsible for formation of macrocyclic and bicyclic sesquiterpenes were characterized. Strict functionality and limited sequence divergence in the santalene and bisabolene synthases are in contrast to the TPS-a synthases, suggesting these compounds have played a significant role in the evolution of the Santalum genus.

Geographical patterns of genetic variation in the world collections of wild annual Cicer characterized by amplified fragment length polymorphisms

Cicer reticulatum, C. echinospermum, C. bijugum, C. judaicum, C. pinnatifidum, C. cuneatum and C. yamashitae are wild annual Cicer species and potential donors of valuable traits to improve chickpea (C. arietinum). As part of a large project to characterize and evaluate wild annual Cicer collections held in the world gene banks, AFLP markers were used to study genetic variation in these species. The main aim of this study was to characterize geographical patterns of genetic variation in wild annual Cicer germplasm. Phylogenetic analysis of 146 wild annual Cicer accessions (including two accessions in the perennial C. anatolicum and six cultivars of chickpea) revealed four distinct groups corresponding well to primary, secondary and tertiary gene pools of chickpea. Some possible misidentified or mislabelled accessions were identified, and ILWC 242 is proposed as a hybrid between C. reticulatum and C. echinospermum. The extent of genetic diversity varied considerably and was unbalanced between species with greatest genetic diversity found in C. judaicum. For the first time geographic patterns of genetic variation in C. reticulatum, C. echinospermum, C. bijugum, C. judaicum and C. pinnatifidum were established using AFLP markers. Based on the current collections the maximum genetic diversity of C. reticulatum, C. echinospermum, C. bijugum and C. pinnatifidum was found in southeastern Turkey, while Palestine was the centre of maximum genetic variation for C. judaicum. This information provides a solid basis for the design of future collections and in situ conservation programs for wild annual Cicer.

Trigenomic bridges for Brassica improvement

We introduce and review Brassica crop improvement via trigenomic bridges. Six economically important Brassica species share three major genomes (A, B, and C), which are arranged in diploid (AA, BB, and CC) and allotetraploid (AABB, AACC, and BBCC) species in the classical triangle of U. Trigenomic bridges are Brassica interspecific hybrid plants that contain the three genomes in various combinations, either triploid (ABC), unbalanced tetraploid (e.g., AABC), pentaploid (e.g., AABCC) or hexaploid (AABBCC). Through trigenomic bridges, Brassica breeders can access all the genetic resources in the triangle of U for genetic improvement of existing species and development of new agricultural species. Each of the three Brassica genomes occurs in several species, where they are distinguished as subgenomes with a tag to identify the species of origin. For example, the A subgenome in B. juncea (2n = AABB) is denoted as Aj and the A subgenome in B. napus (2n = AACC) as An. Trigenomic bridges have been used to increase genetic diversity in allopolyploid Brassica crop species, such as a new-type B. napus with subgenomes from B. rapa (Ar) and B. carinata (Cc). Recently, trigenomic bridges from several sources have been crossed together as the ‘founders’ of a potentially new allohexaploid Brassica species (AABBCC). During meiosis in a trigenomic bridge, crossovers are expected to form between homologous chromosomes of related subgenomes (for example Ar and An), but cross-overs may also occur between non-homologous chromosomes (for example between A and C genome chromosomes). Irregular meiosis is a common feature of new polyploids, and any new allotetraploid or allohexaploid Brassica genotypes derived from a trigenomic bridge must achieve meiotic stability through a process of diploidisation. New sequencing technologies, at the genomic and epigenomic level, may reveal the genetic and molecular basis of diploidization, and accelerate selection of stable allotetraploids or allohexaploids. Armed with new genetic resources from trigenomic bridges, Brassica breeders will be able to improve yield and broaden adaptation of Brassica crops to meet human demands for food and biofuel, particularly in the face of abiotic constraints caused by climate change.

Isolation of cDNAs and functional characterisation of two multi-product terpene synthase enzymes from sandalwood, Santalum album L

Sandalwood, Santalum album (Santalaceae) is a small hemi-parasitic tropical tree of great economic value. Sandalwood timber contains resins and essential oils, particularly the santalols, santalenes and dozens of other minor sesquiterpenoids. These sesquiterpenoids provide the unique sandalwood fragrance. The research described in this paper set out to identify genes involved in essential oil biosynthesis, particularly terpene synthases (TPS) in S. album, with the long-term aim of better understanding heartwood oil production. Degenerate TPS primers amplified two genomic TPS fragments from S. album, one of which enabled the isolation of two TPS cDNAs, SamonoTPS1 (1731bp) and SasesquiTPS1 (1680bp). Both translated protein sequences shared highest similarity with known TPS from grapevine (Vitis vinifera). Heterologous expression in Escherichia coli produced catalytically active proteins. SamonoTPS1 was identified as a monoterpene synthase which produced a mixture of (+)-alpha-terpineol and (-)-limonene, along with small quantities of linalool, myrcene, (-)-alpha-pinene, (+)-sabinene and geraniol when assayed with geranyl diphosphate. Sesquiterpene synthase SasesquiTPS1 produced the monocyclic sesquiterpene alcohol germacrene D-4-ol and helminthogermacrene, when incubated with farnesyl diphosphate. Also present were alpha-bulnesene, gamma-muurolene, alpha- and beta-selinenes, as well as several other minor bicyclic compounds. Although these sesquiterpenes are present in only minute quantities in the distilled sandalwood oil, the genes and their encoded enzymes described here represent the first TPS isolated and characterised from a member of the Santalaceae plant family and they may enable the future discovery of additional TPS genes in sandalwood.

Biosynthesis of Sandalwood Oil: Santalum album CYP76F cytochromes P450 produce santalols and bergamotol.

Abstract Sandalwood oil is one of the world’s most highly prized essential oils, appearing in many high-end perfumes and fragrances. Extracted from the mature heartwood of several Santalum species, sandalwood oil is comprised mainly of sesquiterpene olefins and alcohols. Four sesquiterpenols, α-, β-, and epi-β-santalol and α-exo-bergamotol, make up approximately 90% of the oil of Santalum album. These compounds are the hydroxylated analogues of α-, β-, and epi-β-santalene and α-exo-bergamotene. By mining a transcriptome database of S. album for candidate cytochrome P450 genes, we cloned and characterized cDNAs encoding a small family of ten cytochrome P450-dependent monooxygenases annotated as SaCYP76F37v1, SaCYP76F37v2, SaCYP76F38v1, SaCYP76F38v2, SaCYP76F39v1, SaCYP76F39v2, SaCYP76F40, SaCYP76F41, SaCYP76F42, and SaCYP76F43. Nine of these genes were functionally characterized using in vitro assays and yeast in vivo assays to encode santalene/bergamotene oxidases and bergamotene oxidases. These results provide a foundation for production of sandalwood oil for the fragrance industry by means of metabolic engineering, as demonstrated with proof-of-concept formation of santalols and bergamotol in engineered yeast cells, simultaneously addressing conservation challenges by reducing pressure on supply of sandalwood from native forests.

Dormancy release in Australian fire ephemeral seeds during burial increases germination response to smoke water or heat

Fire ephemerals are short-lived plants that primarily germinate after fire. Fresh and laboratory-stored seeds are difficult to germinate ex situ, even in response to fire-related cues such as heat and smoke. Seeds of eight Australian fire ephemeral species were buried in unburnt and recently burnt sites of natural bushland during autumn. Seeds were exhumed after 6 and 12 months and incubated in water and smoke water, either with or without a heat treatment at 70 degrees C for 1 h. Generally, germination did not increase after 6 months of burial, but after 12 months of burial germination was enhanced in seven of the eight species. Actinotus leucocephalus produced higher germination following 12 months of burial without any further treatment, and smoke water and heat further improved germination. The four Gyrostemonaceae species, Codonocarpus cotinifolius, Gyrostemon racemiger, Gyrostemon ramulosus and Tersonia cyathiflora, only germinated in the presence of smoke water, and their germination was enhanced by burial. Burial improved germination in response to a heat treatment in Grevillea scapigera and Alyogyne huegelii seeds, but did not enhance Alyogyne hakeifolia germination. During concurrent dry laboratory storage of seeds at 15 degrees C, only Actinotus leucocephalus produced increased germination in response to smoke water and heat over time. In summary, soil burial can alter the dormancy status of a number of Australian fire ephemeral seeds, rendering them more responsive to germination cues such as smoke water and heat. The requirement for a period of burial before seeds become responsive to smoke and/or heat would ensure that seeds persist in the soil until a subsequent fire, when there is an increase in nutrients available for growth and reduced competition from other plants.

Germination of Four Species of Native Western Australian Plants using Plant-derived Smoke

Plant-derived smoke was investigated in this study as a pre-germination treatment for seed of four native plant species from south-western Western Australia. Many Australian native species demonstrate low germination responses using conventional nursery propagation methods. Smoke water, prepared from burning 6 kg of fresh and dry foliage of Eucalyptus, Adenanthos and Banksia species and bubbling the smoke through water (20 L) for 1 h was found to substantially improve germination of Stylidium affine Sonder., Stylidium brunonianum Benth. (Stylidiaceae) and Conostylis setigera R.Br. (Haemodoraceae), whereas Actinotus leucocephalus Benth. (Apiaceae) retained a high level of dormancy despite prolonged exposure to smoke water. Diluted and full strength smoke water was found to significantly increase germination of S. brunonianum, C. setigera and A. leucocephalus over water alone. Seeds retained the smoke cue after imbibition in smoke water, whether incubated continuously with smoke water or imbibed in smoke water and dehydrated or dehydrated, stored for 3 weeks. Species varied in their germination response to changes in imbibing time in diluted smoke water. A. leucocephalus required longer periods of smoke water imbibition (24-48 h) to elevate germination irrespective of the three treatments following imbibition. All imbibition periods were effective in enhancing germinability of S. affine and S. brunonianum regardless of subsequent treatments. However, S. affine required shorter imbibition exposure times (3Œ12 h) for optimal germination for the dehydrated and stored treated seeds. C. setigera required shorter exposure to smoke water of 3 and 6 h, when germinated immediately, whereas longer exposure to smoke water was necessary for effective germination in dehydrated (24-48 h) and dehydrated and stored (48 h) treated seeds. The site of action of smoke water in seed was investigated and found to reside in part in the seed coat in S. affine, and the embryo and/or endosperm in A. leucocephalus. The smoke chemical(s) overcame multiple dormancy mechanisms in S. affine and A. leucocephalus whereas gibberellic acid (GA) and zeatin were unable to break dormancy. Mechanism of dormancy relief by smoke water was not the same as GA and zeatin. These data indicate that there are good prospects using imbibition with smoke water as a pre-treatment for seeds in the horticulture and land restoration activities.

Fingerprinting of cauliflower cultivars using RAPD markers

Randomly amplified polymorphic DNA (RAPD) was used to investigate genetic relationships among 25 cultivars of cauliflower (Brassica oleracea var. botrytis L.). Forty decamer primers were examined, among which 15 primers produced polymorphism. Twenty-five polymorphic bands were observed, ranging in size from 428 to 1646 bp. A fingerprinting key was generated using these polymorphic bands. A dendogram was constructed using neighbour-joining analysis based on phylogenetic analysis using parsimony (PAUP). Results indicate that RAPD markers can be used for the routine identification of cauliflower cultivars within B. oleracea var. botrytis L.