Preparation and Standardisation of Smoke-Water for Seed Germination and Plant Growth Stimulation

Smoke-water (SW) has a positive effect on seed germination in many plant species and its application in various fields of plant science has become popular. The method of preparing SW is relatively easy and inexpensive. However, many researchers working in the field of smoke biology are still unfamiliar with preparing SW and testing it for bioactivity. Thus, the aim of this study was to develop a low-cost efficient apparatus to produce SW using grasses, its standardisation and quantification of major active biomolecules. The prepared crude SW was diluted with distilled water (SW:DW) to 75:25, 50:50 and 25:75 (v/v) and these sub-dilutions were further diluted to 1:500; 1:1000; 1:1500; 1:2000; 1:2500; 1:3000 and 1:3500 (v/v). For the standardisation of SW, lettuce seeds (Lactuca sativa L. cv. Grand Rapids) were tested to determine the best working concentration. The ratio 1:2500 (v/v) from the sub-dilution 25:75 (v/v) of SW was the best for germination of lettuce seeds in the dark, achieving 91% germination against the water control that had only 7–10% germination. This is the first study which reports the levels of stimulatory (karrikinolide 1, KAR₁ and karrikinolide 2, KAR₂) and inhibitory (trimethylbutenolide, TMB) compounds present in SW using ultrahigh-performance liquid chromatography–electrospray positive ionisation tandem mass spectrometry (UHPLC-ESI(+)-MS/MS).

     

Induction of agricultural weed seed germination by smoke and smoke-derived karrikin (KAR<Subscript>1</Subscript>), with a particular reference to <Emphasis Type="Italic">Avena fatua</Emphasis> L.

Plant-derived smoke, its water extract—the smoke water (SW), and karrikin (KAR₁) present in the smoke stimulate seed germination in plants from fire-prone and fire-free areas, including weeds and cultivated plants. There are also plants, the seeds of which can respond only to smoke, but not to KAR₁, and vice versa. Smoke and/or KAR₁ can be applied in horticulture, agriculture, and revegetation. This review describes effects of smoke and KAR₁ on weed seed germination and focuses mainly on the recent knowledge about the physiological role of these factors in dormancy release and germination of Avena fatua caryopses. The involvement of gibberellins, ethylene, and abscisic acid (ABA) in the response to smoke or KAR₁ is discussed. Effects of smoke or KAR₁ on the contents of reactive oxygen species (ROS), non-enzymatic antioxidants, and activity of the enzymes participating in ROS removal are presented. Cell cycle activity in the response to SW and KAR₁ is also considered. Effects of KAR₁ on thermodormancy release in A. fatua caryopses are highlighted, as well.     

Effect of smoke derivatives on in vitro pollen germination and pollen tube elongation of species from different plant families

Plant‐derived smoke stimulates seed germination in numerous plant species. Smoke also has a positive stimulatory effect on pollen germination and pollen tube growth. The range of plant families affected my smoke still needs to be established since the initial study was restricted to only three species from the Amaryllidaceae. The effects of smoke‐water (SW) and the smoke‐derived compounds, karrikinolide (KAR₁) and trimethylbutenolide (TMB) on pollen growth characteristics were evaluated in seven different plant families. Smoke‐water (1:1000 and 1:2000 v:v) combined with either Brewbaker and Kwack's (BWK) medium or sucrose and boric acid (SB) medium significantly improved pollen germination and pollen tube growth in Aloe maculata All., Kniphofia uvaria Oken, Lachenalia aloides (L.f.) Engl. var. aloides and Tulbaghia simmleri P. Beauv. Karrikinolide (10^?6 and 10^?7 m ) treatment significantly improved pollen tube growth in A. maculata, K. uvaria, L. aloides and Nematanthus crassifolius (Schott) Wiehle compared to the controls. BWK or SB medium containing TMB (10^?3 m ) produced significantly longer pollen tubes in A. maculata, K. uvaria and N. crassifolius. These results indicate that plant‐derived smoke and the smoke‐isolated compounds may stimulate pollen growth in a wide range of plant species.     

Karrikinolide residues in grassland soils following fire: Implications on germination activity

Smoke plays a positive role in promoting seed germination and enhancing post-germination processes. The compound in smoke is 3-methyl-2H-furo[2,3-c]pyran-2-one (KAR₁). Recently a structurally related butenolide [3,4,5-trimethylfuran-2(5H)-one, (trimethylbutenolide, TMB)], which inhibits germination and reduces the effect of KAR₁, was isolated. The mechanisms of action and interaction of these karrikins are unknown. In addition, the ecological significance of fires in altering soil-smoke-chemistry and the spatial dimensions of the influence on burnt sites and neighbouring areas are undetermined. This study quantified KAR₁ and TMB residues in soils following fire and assessed the germination activity of burnt soil extracts. Soil samples from 0 to 2, 2 to 4, 4 to 6 and 6 to 8 cm depths were extracted using dichloromethane and bioassayed using Lactuca sativa L. achenes (seeds). At all soil depths, L. sativa seeds exhibited significantly greater percentage germination when treated with burnt soil extracts compared to the no-burn soil (control). The L. sativa seeds also showed significantly greater percentage germination when treated with soil extracts from the adjacent plots. Compared to the no-burn soil, higher concentrations of KAR₁ and TMB were detected in the surface layers of the burnt soils. Considerable concentrations of KAR₁ and TMB were also detected in no-burn soil indicating that sources other than fire may also generate karrikins. Findings of this study imply that post-fire increases in KAR₁ residues in the soil may influence soil seed bank stimulation of certain smoke-responsive plant communities in both burnt and adjacent non-burnt areas.     

Seed germination and dormancy traits of forbs and shrubs important for restoration of North American dryland ecosystems

• In degraded dryland systems, native plant community re‐establishment following disturbance is almost exclusively carried out using seeds, but these efforts commonly fail. Much of this failure can be attributed to the limited understanding of seed dormancy and germination traits.
• We undertook a systematic classification of seed dormancy of 26 species of annual and perennial forbs and shrubs that represent key, dominant genera used in restoration of the Great Basin ecosystem in the western United States. We examined germination across a wide thermal profile to depict species‐specific characteristics and assessed the potential of gibberellic acid (GA₃) and karrikinolide (KAR₁) to expand the thermal germination envelope of fresh seeds.
• Of the tested species, 81% produce seeds that are dormant at maturity. The largest proportion (62%) exhibited physiological (PD), followed by physical (PY, 8%), combinational (PY + PD, 8%) and morphophysiological (MPD, 4%) dormancy classes. The effects of chemical stimulants were temperature‐ and species‐mediated. In general, mean germination across the thermal profile was improved by GA₃ and KAR₁ for 11 and five species, respectively. We detected a strong germination response to temperature in freshly collected seeds of 20 species. Temperatures below 10 °C limited the germination of all except Agoseris heterophylla, suggesting that in their dormant state, the majority of these species are thermally restricted.
• Our findings demonstrate the utility of dormancy classification as a foundation for understanding the critical regenerative traits in these ecologically important species and highlight its importance in restoration planning.

     

Structure–activity relationships of <Emphasis Type="Italic">N</Emphasis>- and <Emphasis Type="Italic">S</Emphasis>-analogs of the seed germination inhibitor (3,4,5-trimethylfuran-2(5<Emphasis Type="Italic">H</Emphasis>)-one) for mode of action elucidation

Smoke-derived butenolides are important germination signaling molecules which may be useful in promoting or controlling germination in agriculture and horticulture. The butenolide 3,4,5-trimethylfuran-2(5H)-one (trimethylbutenolide; TMB) was previously isolated from plant-derived smoke and was found to significantly reduce the germination stimulatory activity of the highly active germination promoter, 3-methyl-2H-furo[2,3-c]pyran-2-one (karrikinolide; KAR₁), another smoke-derived butenolide, when applied together. In this study, the germination inhibitory activity of eight N-analogs and one S-analog of TMB, modified in position 1 were evaluated. All synthesized analogs significantly reduced the germination of ‘Grand Rapids’ lettuce (Lactuca sativa) seeds compared to the respective controls, when applied at 1000 µM alone, or in combination with 0.01 µM KAR₁. The S-analog was the most active of the test analogs in this study, with inhibitory activity ranging from 10 to 1000 µM. This was the only compound with activity similar to that of TMB. Thus, these results indicate that N-alkylation of the lactam ring reduces its germination inhibitory activity, while the activity of the S-analog is comparable to TMB. This provides valuable information toward understanding the mode of action of these compounds in regulating germination and for the design of related synthetic compounds with potential use in agriculture or horticulture.     

Endophytic fungi from Pecteilis susannae (L.) Rafin (Orchidaceae), a threatened terrestrial orchid in Thailand

Eight endophytic fungi were isolated from roots of the threatened terrestrial orchid, Pecteilis susannae (L.) Rafin. Phylogenetic analysis based on an alignment of internal transcribed spacer regions of nuclear rDNA indicated that seven isolates belonged to the genus Epulorhiza and one to Fusarium. All fungal isolates were cultured with orchid seeds collected from three field sites near Doi Suthep-Pui National Park, Chiang Mai, Thailand. Seed germination and protocorm development were evaluated up to 70 days after sowing. Percent symbiotic seed germination was highest (86.2%) when seeds were cultured with Epulorhiza (CMU-Aug 013). The protocorm development was the most advanced up to stage 2, continued embryo enlargement, or rupture of the testa, and the highest percentage was 17.8% when seeds were cultured with Epulorhiza (CMU-Aug 007). Without fungi, seed germination and protocorm development were 62.1% and 11.1%, respectively. The dependency of P. susannae on fungal symbionts for early seedling development is yet to be determined. Optimizing seed germination and seedling fitness will assist the conservation of this threatened orchid in Thailand.     

The fire ephemeral Tersonia cyathiflora (Gyrostemonaceae) germinates in response to smoke but not the butenolide 3-methyl-2H-furo[2,3-c]pyran-2-one

Background and Aims

Tersonia cyathiflora (Gyrostemonaceae) is a fire ephemeral with an obligate requirement for smoke to germinate. Whether it is stimulated to germinate by 3-methyl-2H-furo[2,3-c]pyran-2-one (karrikinolide, KAR₁), the butenolide isolated from smoke that stimulates the germination of many other smoke-responsive species, is tested.

Methods

Seeds of T. cyathiflora were buried in autumn following collection and were exhumed 1 year later, as this alleviates dormancy and enables seeds to germinate in response to smoke-water. Exhumed seeds were tested with smoke-water and KAR₁. Fresh preparations of these solutions were again tested on seeds exhumed 2 months later under a broader range of conditions. They were also tested on Grevillea eriostachya (Proteaceae) and Stylidium affine (Stylidiaceae) to confirm the activity of KAR₁.

Key Results

T. cyathiflora seeds germinated in response to smoke-water but not to KAR₁. In contrast, G. eriostachya and S. affine germinated in response to both smoke-water and KAR₁.

Conclusions

Although many smoke-responsive seeds germinate in the presence of KAR₁, this does not apply universally. This suggests that other chemical(s) in smoke-water may play an important role in stimulating the germination of certain species.     

Symbiotic seed germination of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid

The rapid loss of native orchid habitat throughout ecologically important areas (e.g., Florida) has prompted researchers to develop appropriate plans for the propagation and reintroduction of many native orchid species. Ideally, symbiotic orchid seed germination methods are utilized in the production of orchid seedlings to be used in plant reintroduction programs. In the current study we (1) describe an efficient symbiotic seed germination protocol to germinate seeds of the rare sub-tropical terrestrial orchid Habenaria macroceratitis; (2) discuss the in vitro fungal specificity demonstrated by this species; and (3) describe the effects of three photoperiods (0/24 h, 16/8 h, 24/0 h L/D) on in vitro symbiotic seed germination of H. macroceratitis. Six fungal mycobionts were isolated from both vegetative and flowering plants of H. macroceratitis from two geographically distinct sites. Symbiotic seed germination percent was highest (65.7%) and protocorm development was most advanced (Stage 2) when seeds were cultured with fungal mycobiont Hmac-310. Seeds of H. macroceratitis demonstrated a degree of specificity toward fungal mycobionts isolated from plants originating from the same site where seed was collected. Continual darkness (0/24 h L/D) inhibited initial seed germination (Stage 1; 17.1%), but stimulated subsequent protocorm development (Stage 2; 53.5%). These findings will aid in developing an efficient symbiotic seed germination protocol for the conservation of this rare Florida terrestrial orchid, and may prove useful in the conservation of other sub-tropical terrestrial orchid species.     

Plant-derived smoke: Old technology with possibilities for economic applications in agriculture and horticulture

Fire and smoke have been used in traditional agricultural systems for centuries. In recent years, biologically active compounds have been isolated from smoke with potential uses in agriculture and horticulture. This article highlights the possibilities of using smoke-water or smoke-derived butenolide (3-methyl-2H-furo[2,3-c]pyran-2-one, termed karrikinolide, KAR₁) for the cultivation of agricultural and horticultural crops. Treatments with smoke-water show promising results for improving seed germination, seedling growth and crop productivity. In certain cases, even under adverse conditions, such as low or high temperatures and low osmotic potentials, smoke-water or a KAR₁ solution can promote seed germination and seedling growth. This phenomenon is of great significance when seeds are sown under drought conditions. Smoke-technology, therefore, has potential for use in arid and semi-arid regions. Possibilities may also exist for controlling some plant diseases and managing weeds with the use of smoke or KAR₁ solutions. In addition, smoke-technology can possibly economize the use of commercial chemical fertilizers, pesticides and herbicides, making it a feasible technology for organic farming and for resource-poor farmers in developing nations. The positive role of smoke-water in flowering and fruiting of crops cannot be overlooked as the karrikins found in smoke are now recognized as potential new plant growth regulators. Very low concentrations of smoke-water or a KAR₁ solution are effective in promoting germination and post-germination growth. Thus, early harvesting and increasing the productivity of crops using smoke-technology may be possible. Here we review some of the effects of smoke and KAR₁ on various crop species and discuss the potential uses of smoke technology in agriculture and horticulture.     

Transcriptome analysis of germinating maize kernels exposed to smoke-water and the active compound KAR<Subscript>1</Subscript>

Background  

Smoke released from burning vegetation functions as an important environmental signal promoting the germination of many plant species following a fire. It not only promotes the germination of species from fire-prone habitats, but several species from non-fire-prone areas also respond, including some crops. The germination stimulatory activity can largely be attributed to the presence of a highly active butenolide compound, 3-methyl-2H-furo[2,3-c]pyran-2-one (referred to as karrikin 1 or KAR₁), that has previously been isolated from plant-derived smoke. Several hypotheses have arisen regarding the molecular background of smoke and KAR₁ action.     

Parental environment changes the dormancy state and karrikinolide response of Brassica tournefortii seeds

Background and Aims

The smoke-derived chemical karrikinolide (KAR₁) shows potential as a tool to synchronize the germination of seeds for weed management and restoration. To assess its feasibility we need to understand why seeds from different populations of a species exhibit distinct responses to KAR₁. Environmental conditions during seed development, known as the parental environment, influence seed dormancy so we predicted that parental environment would also drive the KAR₁-responses of seeds. Specifically, we hypothesized that (a) a common environment will unify the KAR₁-responses of different populations, (b) a single population grown under different environmental conditions will exhibit different KAR₁-responses, and (c) drought stress, as a particular feature of the parental environment, will make seeds less dormant and more responsive to KAR₁.

Methods

Seeds of the weed Brassica tournefortii were collected from four locations in Western Australia and were sown in common gardens at two field sites, to test whether their KAR₁-responses could be unified by a common environment. To test the effects of drought on KAR₁-response, plants were grown in a glasshouse and subjected to water stress. For each trial, the germination responses of the next generation of seeds were assessed.

Key Results

The KAR₁-responses of seeds differed among populations, but this variation was reduced when seeds developed in a common environment. The KAR₁-responses of each population changed when seeds developed in different environments. Different parental environments affected germination responses of the populations differently, showing that parental environment interacts with genetics to determine KAR₁-responses. Seeds from droughted plants were 5 % more responsive to KAR₁ and 5 % less dormant than seeds from well-watered plants, but KAR₁-responses and dormancy state were not intrinsically linked in all experiments.

Conclusions

The parental environment in which seeds develop is one of the key drivers of the KAR₁-responses of seeds.     

The role of after‐ripening in promoting germination of arid zone seeds: a study on six Australian species

The effects of after‐ripening (storage under warm, dry conditions) on seed germination was examined in six plant species from the arid zone of Western Australia with the aim of improving germination and germination rate for rehabilitation objectives. Study species (Acanthocarpus preissii, Anthocercis littorea, Dioscorea hastifolia, Eremophila oldfieldii, Thryptomene baeckeacea and Zygophyllum fruticulosum) were selected based on diverse plant habits, seed types and requirements for rehabilitation. After‐ripening was investigated by adjusting seed moisture content to 13 and 50 equilibrium relative humidity (eRH) at 23 °C and storing seeds at two temperatures (30 and 45 °C) from 1 to 18 months. Following storage, seeds were incubated in water, gibberellic acid (GA₃) or karrikinolide (KAR₁; the butenolide, 3‐methyl‐2H‐furo[2,3‐c]pyran‐2‐one). All after‐ripening conditions increased germination percentage and rate of A. littorea and D. hastifolia, with A. littorea only germinating when treated with GA₃ or KAR₁. The germination of Z. fruticulosum was dependent on after‐ripening temperature and seed moisture content. After‐ripening had a limited effect on the remaining three species. The restoration implications of the findings are discussed. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161 , 411–421.     

Enhancing Phytoremediation Potential of Pennisetum clandestinum Hochst in Cadmium-Contaminated Soil Using Smoke-Water and Smoke-Isolated Karrikinolide

The use of plant growth regulators (PGRs) and biostimulants to enhance phytoextraction is gaining popularity in phytoremediation technology. This study investigated the stimulatory effects of smoke-water (SW), a smoke-derived compound karrikinolide (KAR₁) and other known plant growth regulators (PGRs) [gibberellic acid (GA₃), kinetin (Kin) and indole-3-butyric acid (IBA)] to enhance the phytoextraction potential of Pennisetum clandestinum. Pennisetum clandestinum seedlings were grown for 10 weeks in vermiculite using Hoagland's nutrient solution and were treated with cadmium (Cd) (2, 5, and 10 mg L^?1) and SW, KAR₁ and PGRs. KAR₁ exhibited positive effects on shoot and root dry weight (140 and 137 mg respectively) at the highest concentration of Cd (10 mg L^?1) compared to all the other treatments. KAR₁ and SW treatments used in the present study significantly improved the phytoextraction potential of P. clandestinum (602 and 575 mg kg^?1 respectively) compared to the other tested PGRs. This is the first report on the use of SW and KAR₁ to enhance phytoremediation potential in P. clandestinum. Further studies are needed to elucidate the exact mechanisms of smoke constituents involved in phytoextraction potential of plant species.     

Prior hydration of Brassica tournefortii seeds reduces the stimulatory effect of karrikinolide on germination and increases seed sensitivity to abscisic acid

Background and Aims

The smoke-derived compound karrikinolide (KAR₁) shows significant potential as a trigger for the synchronous germination of seeds in a variety of plant-management contexts, from weed seeds in paddocks, to native seeds when restoring degraded lands. Understanding how KAR₁ interacts with seed physiology is a necessary precursor to the development of the compound as an efficient and effective management tool. This study tested the ability of KAR₁ to stimulate germination of seeds of the global agronomic weed Brassica tournefortii, at different hydration states, to gain insight into how the timing of KAR₁ applications in the field should be managed relative to rain events.

Methods

Seeds of B. tournefortii were brought to five different hydration states [equilibrated at 15 % relative humidity (RH), 47 % RH, 96 % RH, fully imbibed, or re-dried to 15 % RH following maximum imbibition] then exposed to 1 nm or 1 µm KAR₁ for one of five durations (3 min, 1 h, 24 h, 14 d or no exposure).

Key Results

Dry seeds with no history of imbibition were the most sensitive to KAR₁; sensitivity was lower in seeds that were fully imbibed or fully imbibed then re-dried. In addition, reduced sensitivity to KAR₁ was associated with an increased sensitivity to exogenously applied abscisic acid (ABA).

Conclusions

Seed water content and history of imbibition were found to significantly influence whether seeds germinate in response to KAR₁. To optimize the germination response of seeds, KAR₁ should be applied to dry seeds, when sensitivity to ABA is minimized.     

X-ray crystallographic structure determination of the smoke-derived karrikin KAR3

The discovery of karrikins as the chemical constituents responsible for the germination stimulatory effects of smoke has met with widespread interest from the botanical/scientific community. Although such beneficial properties of smoke in agriculture and environmental management have been known for several centuries, it is only in the last decade that this significant discovery has been made. The karrikins comprising KAR₁–KAR₆ have been shown to be potent promoters of germination in numerous model studies. KAR₁, the most abundant of the karrikins, has been revealed as the key germination cue present in smoke with activities as low as 100 ppt. Given the vast economic potential of these molecules as well as their low natural abundance, several strategies have been devised towards the synthesis of karrikins with the goal of elucidating structure–activity relationships. However, the targets of these structures as well as their mode of action are yet to be determined. To this end, we herein detail the first single crystal X-ray structure of KAR₃ with the aim that it may provide further insights to the molecular mechanism behind this group of compounds.     

Effects of smoke water and karrikin on seed germination of 13 species growing in China

Plant-derived smoke water (SW), derived from combusted plant material, has been shown to stimulate seed germination and improve seedling vigor of a number of plant species from fire-dependent Mediterranean-type climate areas. The effects of SW on seed germination of 13 plant species from southern tropical and subtropical monsoon climate regions of South China are reported for the first time in this study using laboratory and pot trials. Among the 13 species tested, only Aristolochia debilis showed a significant positive response to commercial SW when diluted 1:10. Seed germination of A. debilis was also stimulated by 1–100 nM 3-methyl-2H-furo [2, 3-c] pyran-2-one (karrikin 1 or KAR₁) and by 10–1000 µM gibberellic acid (GA₃). GA₃ stimulated seed germination of Santalum album and significantly elongated the radicles of A. debilis while SW could not. The functions and/or metabolic pathways of Kar₁ and GA₃ are likely to be separate and/or distinct.     

Seeds of Brassicaceae weeds have an inherent or inducible response to the germination stimulant karrikinolide

Background and Aims

Karrikinolide (KAR₁) is a smoke-derived chemical that can trigger seeds to germinate. A potential application for KAR₁ is for synchronizing the germination of weed seeds, thereby enhancing the efficiency of weed control efforts. Yet not all species germinate readily with KAR₁, and it is not known whether seemingly non-responsive species can be induced to respond. Here a major agronomic weed family, the Brassicaceae, is used to test the hypothesis that a stimulatory response to KAR₁ may be present in physiologically dormant seeds but may not be expressed under all circumstances.

Methods

Seeds of eight Brassicaceae weed species (Brassica tournefortii, Raphanus raphanistrum, Sisymbrium orientale, S. erysimoides, Rapistrum rugosum, Lepidium africanum, Heliophila pusilla and Carrichtera annua) were tested for their response to 1 µm KAR₁ when freshly collected and following simulated and natural dormancy alleviation, which included wet–dry cycling, dry after-ripening, cold and warm stratification and a 2 year seed burial trial.

Key Results

Seven of the eight Brassicaceae species tested were stimulated to germinate with KAR₁ when the seeds were fresh, and the remaining species became responsive to KAR₁ following wet–dry cycling and dry after-ripening. Light influenced the germination response of seeds to KAR₁, with the majority of species germinating better in darkness. Germination with and without KAR₁ fluctuated seasonally throughout the seed burial trial.

Conclusions

KAR₁ responses are more complex than simply stating whether a species is responsive or non-responsive; light and temperature conditions, dormancy state and seed lot all influence the sensitivity of seeds to KAR₁, and a response to KAR₁ can be induced. Three response types for generalizing KAR₁ responses are proposed, namely inherent, inducible and undetected. Given that responses to KAR₁ were either inherent or inducible in all 15 seed lots included in this study, the Brassicaceae may be an ideal target for future application of KAR₁ in weed management.     

Comparison of germination responses of Anigozanthos flavidus (Haemodoraceae), Gyrostemon racemiger and Gyrostemon ramulosus (Gyrostemonaceae) to smoke-water and the smoke-derived compounds karrikinolide (KAR1) and glyceronitrile

Background and Aims

A major germination-promoting chemical in smoke-water is 3-methyl-2H-furo[2,3-c]pyran-2-one (karrikinolide, KAR₁). However, not all species that germinate in response to smoke-water are responsive to KAR₁, such as Tersonia cyathiflora (Gyrostemonaceae). In this study, a test was made of whether two Gyrostemon species (Gyrostemonaceae) that have previously been shown to respond to smoke-water, respond to KAR₁. If not, then the smoke-derived chemical that stimulates germination of these species is currently unknown. Recently, glyceronitrile was isolated from smoke-water and promoted the germination of certain Anigozanthos species (Haemodoraceae). Whether this chemical promotes Gyrostemon racemiger germination is also examined. Furthermore, an investigation was carried out into whether these species germinate in response to smoke-water derived from burning cellulose alone.

Methods Gyrostemon racemiger

and G. ramulosus seeds were buried after collection and retrieved in autumn the following year when dormancy was alleviated and seeds had become responsive to smoke-water. Anigozanthos flavidus seeds were after-ripened at 35 °C to alleviate dormancy. Gyrostemon and Anigozanthos seeds were then tested with ‘Seed Starter’ smoke-water, KAR₁, glyceronitrile and cellulose-derived smoke-water.

Key Results

Although Gyrostemon racemiger, G. ramulosus and A. flavidus were all stimulated to germinate by ‘Seed Starter’ smoke-water, none of these species responded to KAR₁. Gyrostemon racemiger germination was not promoted by glyceronitrile. This is in contrast to A. flavidus, where glyceronitrile, at concentrations of 1–500 µm, promoted germination, although seedling growth was inhibited at ≥400 µm. Maximum A. flavidus germination occurred at glyceronitrile concentrations of 25–300 µm. Some Gyrostemon germination was promoted by cellulose-derived smoke-water.

Conclusions

KAR₁ and glyceronitrile, chemicals in smoke-water that are known to stimulate germination in other species, did not promote the germination of G. racemiger. This suggests that other chemical(s) which promote germination are present in smoke, and may be derived from burning cellulose alone.     

Tools for conservation of Balsamorhiza deltoidea and Balsamorhiza sagittata: Karrikin and thidiazuron-induced growth

Wildfires are having both devastating and regenerative impacts on the ecosystems in the Pacific Northwest of North America. Balsamorhiza sagittata and B. deltoidea (balsamroot) are ecologically important species in this region, and B. sagittata populations are increasing, while B. deltoidea is critically imperiled. The aim of this research was to establish in vitro protocols for germination and regeneration of Balsamorhiza spp. to enable conservation efforts. It was hypothesized that karrikins, which are plant growth regulators released from burning plants during wildfires, would induce seed germination in Balsamorhiza spp. Three karrikins (KAR₁, KAR₂, and KAR₁₁) were tested for the ability to enhance germination in these species at two levels (5 or 10 μM). KAR₂ had the strongest positive effect on germination and induced 47% and 60% germination, respectively, in B. sagittata seeds compared to 14% germination obtained in the control (water agar media). In B. deltoidea, KAR₂ treatment resulted in a germination rate of 73.1% and 100%, compared to 69% in the control. A germplasm collection of seedlings of both species was established for conservation and regeneration experiments. Thidiazuron treatment (10 μM) induced formation of embryo-like structures in seedlings of both B. sagittata and B. deltoidea, with regenerants originating from the crown of seedlings. The present study provides in vitro methods for conservation and mass propagation of Balsamorhiza species.