Germination Stimulus Research Articles

Characterization of high-pressure-treated Bacillus subtilis spore populations using flow cytometry — Shedding light on spore superdormancy at 550 MPa

Mild spore inactivation can be challenging in industry because of the remarkable resistance of bacterial spores. High pressure (HP) can trigger spore germination, which reduces the spore's resistance, and thereby allows mild spore inactivation. However, spore germination is heterogenous. Some slowly germinating or non-germinating spores called superdormant spores remain resistant and can survive. Therefore, superdormant spores need to be characterized to understand the causes of their germination deficiency.Bacillus subtilis spores were pressurized for 50 s – 6 min at a very high pressure (vHP) level of 550 MPa and 60 °C in buffer to trigger germination. For a rapid quantification of the remaining ungerminated superdormant spores, flow cytometry (FCM) analysis was validated using single cell sorting and growth analysis. FCM based on propidium iodide (PI) and SYTO16 can be used for 550 MPa-superdormant spores after short vHP treatments of ≤1 min and post-HP incubation at 37 °C or 60 °C. The need for a post-HP incubation is particular for vHP treatments. The incubation was successful to separate FCM signals from superdormant and germinated spores, thus allowing superdormant spore quantification. The SYTO16 and PI fluorescence levels did not necessarily indicate superdormancy or apparent viability. This highlights the general need for FCM validation for different HP treatment conditions.The ∼7 % of ungerminated, i.e., superdormant, spores were isolated after a vHP treatment (550 MPa, 60 °C, 43–52 s). This allowed the characterization of vHP superdormant spores for the first time. The superdormant spores had a similar dipicolinic acid content as spores of the initial dormant population. Descendants of superdormant spores had a normal vHP germination capacity. The causes of vHP superdormancy were thus unlikely linked to the dipicolinic acid content or a permanent genetic change. Isolated superdormant spores germinated better in a second vHP treatment compared to the initial spore population. This has not been observed for other germination stimuli so far. In addition, the germination capacity of the initial spore population was time-dependent. A vHP germination deficiency can therefore be lost over time and seems to be caused by transient factors. Permanent cellular properties played a minor role as causes of superdormancy under chosen HP treatment conditions.The study gained new fundamental insights in vHP superdormancy which are of applied interest. Understanding superdormancy helps to efficiently develop a strategy to avoid superdormant spores and hence to inactivate all spores. The development of a mild HP spore germination-inactivation process aims at better preserving the food quality.

Mechanical scarification technique breaks seed coat-mediated dormancy in wild oat (Avena fatua)

AbstractWild oat is a herbicide resistance-prone global weed species that causes significant economic losses in dryland and horticultural agriculture. As a result, there has been a significant research effort to control this species. A major impediment to this research is the seed coat-mediated dormancy of wild oat, which requires a labor-intensive incision or puncturing of the seed coat to initiate seed germination. This study defines the most efficient settings of a mechanical thresher to overcome wild oat seed dormancy and then validates these settings using multiple populations collected from the Western Australian grain belt. We also compare the effects of rapid mechanical scarification and known germination stimulus tactics such as scarification with sulfuric acid (H2SO4), partial endosperm removal, sandpaper scarification of the seed coat, and immersion in sodium nitroprusside (NO donor SNP) solution on wild oat seedling growth rate. Threshing treatment of 1,500 rpm for 5 s provides equivalent germination compared with manually puncturing individual wild oat seeds, with no difference in seedling relative growth rate. The mechanical scarification of seeds using the thresher resulted in greater germination (66%) than H2SO4 scarification (0%), partial endosperm removal (10%), sandpaper seed coat scarification (25%), and exposure to NO donor SNP (34%). This study demonstrates that the physical dormancy of wild oat can be rapidly overcome using a commercially available mechanical thresher.

SEED GERMINATION OF GUNDELIA TOURNEFORTII L. UNDER DIFFERENT DORMANCY BREAKING TREATMENTS

Gundelia tournefortii L. is adapted to mountains area climate, and the germination of this plant is not easily. This research conducted to study the effect of different seed treatments in various treatment durations in two experiments in petri dish in the laboratory and in seedling tray in the lath house to breaking seed dormancy and germination stimulus. Results indicated that there is significant enhancement of germination by all treatments and the maximum germination percentage was obtained by seed freezing 99.17% in the laboratory and 93.33% in the lath house for all treatment durations 12, 14, 48 and 72 hours. The other parameters of seed germination like germination speed, peak value, mean daily germination, mean germination time, germination value, radicle length, radicle elongation velocity, plumule length, plumule elongation velocity, seedling fresh and dry weight escalated by seed freezing and seed soaking in tap water at all soaking durations 12, 24, 48 and 72 hours. Gundelia tournefortii L. germinated seed was an anatomically examined by paraffin method and calculated diploid number of chromosomes 2n=2x=18 by aceto-carmine squash methods

ABA signaling components in Phelipanche aegyptiaca

Obligate root holoparasite Phelipanche aegyptiaca is an agricultural pest, which infests its hosts and feeds on the sap, subsequently damaging crop yield and quality. Its notoriously viable seed bank may serve as an ideal pest control target. The phytohormone abscisic acid (ABA) was shown to regulate P. aegyptiaca seed dormancy following strigolactones germination stimulus. Transcription analysis of signaling components revealed five ABA receptors and two co-receptors (PP2C). Transcription of lower ABA-affinity subfamily III receptors was absent in all tested stages of P. aegyptiaca development and parasitism stages. P. aegyptiaca ABA receptors interacted with the PP2Cs, and inhibited their activity in an ABA-dependent manner. Moreover, sequence analysis revealed multiple alleles in two P. aegyptiaca ABA receptors, with many non-synonymous mutations. Functional analysis of selected receptor alleles identified a variant with substantially decreased inhibitory effect of PP2Cs activity in-vitro. These results provide evidence that P. aegyptiaca is capable of biochemically perceiving ABA. In light of the possible involvement of ABA in parasitic activities, the discovery of active ABA receptors and PP2Cs could provide a new biochemical target for the agricultural management of P. aegyptiaca. Furthermore, the potential genetic loss of subfamily III receptors in this species, could position P. aegyptiaca as a valuable model in the ABA perception research field.

Seed size and burial depth influence Zostera marina L. (eelgrass) seed survival, seedling emergence and initial seedling biomass development.

Seed burial in the sediment is critical for successful seedling establishment in seagrasses because it protects from predation and dispersal into unsuitable sites, and it may enhance germination by exposing the seeds to suitable germination stimuli. However, relatively little is known about the fate of buried seeds and their ability to emerge from greater depths. The goal of this study was to determine seed survival in the sediment, seedling emergence success and initial seedling biomass of Zostera marina in relation to burial depth and to evaluate if large seeds, having larger energy reserves, are more tolerant to burial than small seeds. Seeds from a perennial Z. marina population were buried at 7 different sediment depths (0.1–8 cm), and seeds sorted by size (large and small) were buried at depths of 2, 4 and 6 cm in outdoor mesocosms. Total seedling emergence after 2 months was significantly affected by seed burial depth, with maximum values in the top 2 cm of the sediment (48.1–56.7% of planted seeds), and a marked decline below 4 cm depth to only 5% seedling emergence at the deepest burial depth of 8 cm. Moreover, seeds had shorter time to emergence from shallow compared to deep burial depths. At all burial depths, a small fraction of seeds (<10%) died after germination but before emerging, and 15–30% remained viable after 6 months. Seed mortality was the major limitation to seedling recruitment from the deeper burial depths. The effect of seed size on seedling emergence success and time was not clear, but heavier seeds displayed greater longevity and gave rise to seedlings of significantly higher biomass, indicating that the mobilization of metabolic reserves may be important during initial seedling development.

Superdormant Spores as a Hurdle for Gentle Germination-Inactivation Based Spore Control Strategies.

Bacterial spore control strategies based on the germination-inactivation principle can lower the thermal load needed to inactivate bacterial spores and thus preserve food quality better. However, the success of this strategy highly depends on the germination of spores, and a subpopulation of spores that fail to germinate or germinate extremely slowly hinders the application of this strategy. This subpopulation of spores is termed ‘superdormant (SD) spores.’ Depending on the source of the germination stimulus, SD spores are categorized as nutrient-SD spores, Ca2+-dipicolinic acid SD spores, dodecylamine-SD spores, and high pressure SD spores. In recent decades, research has been done to isolate these different groups of SD spores and unravel the cause of their germination deficiency as well as their germination capacities. This review summarizes the challenges caused by SD spores, their isolation and characterization, the underlying mechanisms of their germination deficiency, and the future research directions needed to tackle this topic in further depth.

Comparing germination stimuli of two alien invasive species and a native analogue: Towards sustainable management of invasives

Invasive alien plants that have seeds with innate dormancy, and thus large, persistent soil-stored seed banks, are particularly difficult to manage. Dormant seeds typically require specific stimuli as cues for germination, resulting in mass recruitment. Sound understanding of germination stimuli may provide options to aid the management of invasives. We investigated the response of two invasive trees, Acacia mearnsii and A. melanoxylon, and an indigenous ecological analogue, Virgilia divaricata, to different germination stimuli (chipped (manually), hot water exposure, dry heat pulse, smoke, and no treatment) at two temperature regimes (winter ~12°C and summer ~20°C) to explore how their germination requirements may be used to favour V. divaricata in areas cleared of the invasives. Chipped and hot water exposure resulted in the highest germination in all three species. Virgilia divaricata responded marginally better than the invasives to the dry heat pulse, particularly at 20°C. However, the three species mostly responded comparably to the stimulus–temperature combinations, suggesting that manipulation of stimuli is not a feasible option for management of the invasives at a large scale. In the absence of any stimulus (~akin to conditions associated with aboveground clearing of alien vegetation cover), germination was poorer in V. divaricata than in the invasive species especially at 20°C, providing motivation for focusing Acacia management operations in cooler seasons in areas where V. divaricata seed banks occur. Another option is to sow V. divaricata seeds that have been chipped or exposed to hot water in areas cleared of the invasives of which the seeds would not have received stimuli.

Karrikins from plant smoke modulate bacterial quorum sensing

The discovery that plant smoke contains germination stimuli has led to the identification of a new class of signaling molecules named karrikins. Here we report a potential second role for these molecules: in various bacterial species -A. tumefaciens, P. aeruginosa and V. harveyi- they modulate bacterial quorum-sensing (QS), with very different outcomes.

Does cereal crop agriculture in dry swamps damage aquatic plant communities?

The response of swamp vegetation to cropping was measured by analysis of the seed bank present (as a measure of swamp resilience) and a survey of the extant vegetation (as a measure of swamp integrity). Samples of seed bank were exposed to a germination stimulus, the plants that germinated and established were identified, and the density of emergent vegetation was assessed. Surveys of the extant vegetation after flooding in cropped and uncropped swamps indicated that the plant communities establishing after the different treatments were different. Cropping results in a reduced diversity and density of plants, although swamp plant communities retain some resilience to such disturbances. Cropping also affects germination from the seed bank of these wetlands. The degree to which cropping is a threatening process to swamp plant communities and their dependent fauna will depend on whether vulnerable elements can return to swamps, which in turn depends on swamp management, connectivity and landscape level processes. As the climate changes and wetlands become more temporary and flooding less reliable, recognition and conservation of the processes that maintain biodiversity in the landscape will become more important.

Comparison the Effect of Different Treatments for Breaking Seed Dormancy of Citrullus colocynthis

Citrullus colocynthis is one of the major medic plants and it has many medical values. This plant adopts in desert regions and specifically in sand dunes. Forasmuch as seeds of this plant germinate not easily, so in this research tried to study about effect of different treatments for breaking seed dormancy, germination stimulus and growth of Citrullus colocynthis seeds. The treatments were include sulfuric acid 98% in 20 and 40 minutes time intervals, potassium nitrate 0.2% within 72 hours, hot water in 90 centigrade degree during 10 minutes and scratching by sand paper. For comparing this treatments and normal germination is used distilled water for control. Experiments were performed in a completely randomized design with four repetitions and six treatments. Results of variance analysis and mean comparison showed that there are significant statistical differences (0.01 levels) between treatments for percentage and velocity of germination, length of the Root and Shoot. The maximum percentage and velocity of germination and length of the Shoot obtain in scratching by sand paper treatment and results of other treatments were in lower level than this one. According to got results determined that scratching is the most suitable method for dominance on seed dormancy of Citrullus colocynthis species.

Seed ultrastructure and water absorption pathway of the root-parasitic plant Phelipanche aegyptiaca (Orobanchaceae)

Obligate root parasitic plants of the Orobanchaceae do not germinate unless they chemically detect a host plant nearby. Members of this family, like Orobanche, Phelipanche and Striga, are noxious weeds that cause heavy damage to agriculture. In spite of their economic impact, only a few light microscopical studies of their minute seeds have been published, and there is no knowledge of their ultrastructure and of the role each tissue plays during the steps preceding germination. This paper describes the ultrastructure of Phelipanche seeds and contributes to our understanding of seed tissue function. Seeds of P. aegyptiaca were examined under light, scanning electron, transmission electron and fluorescence microscopy following various fixations and staining protocols. The results were interpreted with physiological data regarding mode of water absorption and germination stimulation. The endothelium, which is the inner layer of the testa, rapidly absorbs water. Its interconnected cells are filled with mucilage and contain labyrinthine walls, facilitating water accumulation for germination that starts after receiving germination stimuli. Swelling of the endothelium leads to opening of the micropyle. The perisperm cells underneath this opening mediate between the rhizosphere and the embryo and are likely to be the location for the receptors of germination stimuli. The other perisperm cells are loaded with lipids and protein bodies, as are the endosperm and parts of the embryo. In the endosperm, the oil bodies fuse with each other while they are intact in the embryo and perisperm. Plasmodesmata connect the perisperm cells to each other, and the cells near the micropyle tightly surround the emerging seedling. These perisperm cells, and also the proximal embryo cells, have dense cytoplasmic contents, and they seem to represent the two seed components that are actively involved in transfer of reserve nutrients to the developing seedling during germination.

Seasonal Changes in Germination Responses of Seeds of the Winter Annual Weed Littleseed Canarygrass (Phalaris minor) to Light

Photocontrol of weeds requires knowledge about the response of weeds to light and its changes over time. Thus, littleseed canarygrass germination, as an important weed in winter crops, in response to the light environment was evaluated in seeds retrieved from different burial (10, 20, and 40 cm, under irrigated or nonirrigated conditions) or storage (room temperature 25 C and cold 3 C) conditions for 1 yr. Seeds buried in the soil showed a cyclical germination behavior when tested at 20 C, with high germination percentages (68%) occurring in August, October, and December and low percentages (12%) in February and April, with another late germination in June. Germination percentages were mostly higher for seeds incubated in light than in darkness and seeds were more likely to positively respond to light in June than at the other retrieval dates, with differences as great as 60% having been observed under irrigated conditions and at depths of 20 and 40 cm. The most outstanding effect of light as a germination stimulus was observed for seeds stored at room temperature where germination in light was always 20 to 35% higher than that in darkness. The viability of seeds did not change over time in seeds kept at room or cold temperature. However, the proportion of surviving seeds was reduced by 35 to 65% when buried in the soil. Littleseed canarygrass seeds tended to survive more when buried 40 cm deep and the differences between irrigated and nonirrigated conditions were only detectable at 10 cm deep, with higher seed mortalities under irrigated conditions. Information gained in this study would be useful in developing weed control programs for this species.

Is seed conditioning essential for Orobanche germination?

Parasitic Orobanchaceae germinate only after receiving a chemical stimulus from roots of potential host plants. A preparatory phase of several days that follows seed imbibition, termed conditioning, is known to be required; thereafter the seeds can respond to germination stimulants. The aim of this study was to examine whether conditioning is essential for stimulant receptivity. Non-conditioned seeds of both Orobanche cumana Wallr. and O. aegyptiaca Pers. [syn. Phelipanche aegyptiaca (Pers.) Pomel] were able to germinate in response to chemical stimulation by GR24 even without prior conditioning. Stimulated seeds reached maximal germination rates about 2 weeks after the onset of imbibition, no matter whether the seeds had or had not been conditioned before stimulation. Whereas the lag time between stimulation and germination response of non-conditioned seeds was longer than for conditioned seeds, the total time between imbibition and germination was shorter for the non-conditioned seeds. Unlike the above two species, O. crenata Forsk. was found to require conditioning prior to stimulation. Seeds of O. cumana and O. aegyptiaca are already receptive before conditioning. Thus, conditioning is not involved in stimulant receptivity. A hypothesis is put forward, suggesting that conditioning includes (a) a parasite-specific early phase that allows the imbibed seeds to overcome the stress caused by failing to receive an immediate germination stimulus, and (b) a non-specific later phase that is identical to the pregermination phase between seed imbibition and actual germination that is typical for all higher plants.

Global Gene Expression Analysis during Germination in the Chytridiomycete Blastocladiella emersonii

Blastocladiella emersonii is an aquatic fungus of the Chytridiomycete class. During germination, the zoospore, a motile nongrowing cell, goes through a cascade of morphological changes that culminates with its differentiation into the germling cell, capable of coenocytic vegetative growth. Transcriptome analyses of B. emersonii cells were carried out during germination induced under various environmental conditions. Microarray data analyzing 3,563 distinct B. emersonii genes revealed that 26% of them are differentially expressed during germination in nutrient medium at at least one of the time points investigated. Over 500 genes are upregulated during the time course of germination under those conditions, most being related to cell growth, including genes involved in protein biosynthesis, DNA transcription, energetic metabolism, carbohydrate and oligopeptide transport, and cell cycle control. On the other hand, several transcripts stored in the zoospores are downregulated during germination in nutrient medium, such as genes involved in signal transduction, amino acid transport, and chromosome organization. In addition, germination induced in the presence of nutrients was compared with that triggered either by adenine or potassium ions in inorganic salt solution. Several genes involved in cell growth, induced during germination in nutrient medium, do not show increased expression when B. emersonii zoospores germinate in inorganic solution, suggesting that nutrients exert a positive effect on gene transcription. The transcriptome data also revealed that most genes involved in cell signaling show the same expression pattern irrespective of the initial germination stimulus.

Manganês e germinação de esporos de fungos micorrízicos arbusculares in vitro

A alta disponibilidade de íons metálicos no solo, dentre eles o Mn2+, pode inibir os fungos micorrízicos arbusculares (FMA), retardando a germinação dos esporos e, conseqüentemente, a formação de micorriza, o que reduz a eficiência simbiótica. O objetivo deste trabalho foi avaliar o efeito do íon Mn2+ sobre a germinação de esporos de seis espécies de FMA dos gêneros Glomus, Acaulospora, Gigaspora e Scutellospora em experimento in vitro Em substrato constituído por areia lavada, adicionaram-se 15; 30 e 75 mg kg-1 de Mn2+, na forma de MnCl2.4H2O, mantendo-se um controle sem adição de Mn. Acondicionaram-se os esporos em envelopes membranosos, introduzindo-os entre duas camadas de areia com diferentes níveis de Mn, em placas de Petri. Após 30 dias de incubação, avaliou-se a germinação dos esporos. Houve decréscimo médio de 32, 49 e 75 % na germinação dos esporos, à medida que se aumentaram as doses de Mn, em comparação ao controle. A germinação de esporos do gênero Glomus foi totalmente inibida na maior dose. O gênero Acaulospora sofreu decréscimos de até 50 % já na dose de 15 mg kg-1 de Mn2+, enquanto os gêneros Scutellospora e Gigaspora apresentaram os maiores índices de germinação de esporos, tendo havido tolerância no caso de Gigaspora, mesmo na maior dose de Mn2+, e estímulo à germinação no caso de Scutellospora até à dose 30 mg kg-1 de Mn2+.

Germination response of seven east Australian Grevillea species (Proteaceae) to smoke, heat exposure and scarification

Seeds of plant species from fire-prone vegetation often show dormancy, and germinate in response to fire-related cues. Seeds of some east Australian Grevillea species were known from earlier studies to germinate in response to fires, but gave mixed results when heat shock was tested as a germination stimulus. In the current study, seeds of seven east Australian Grevillea species were tested for their germination response to smoke, to short heat exposure, and to scarification. Seeds were smoked in a chamber by using a mixture of native woodland vegetation as fuel. The smoking and heating treatments were administered to seeds in bulk: this meant that subsequent germination data for these two treatments were not independent. Cumulative germination in the laboratory was scored for up to 60 days. All three germination treatments, when administered singly, increased germination: smoke increased germination in all seven species, heat in four species, and scarification in a different four species. Pairwise combinations of the three treatments also increased germination in some species e.g. smoke and heat treatments combined led to the highest germination observed in four species, and to increased germination compared with either treatment singly, in another species. Some treatment combinations appeared to interact: heat and scarification combined led to decreased germination in three species. The role of heat exposure and scarification in increasing germination was not due to the breaking of an impermeable seed coat: water uptake of both scarified and unscarified seeds was rapid and equal (after 24 h) in all seven species. The results indicate that seeds of the Grevillea species investigated are responsive to one or more fire-related germination cues.

Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle

Saccharomyces cerevisiae spore germination is a process in which quiescent, non-dividing spores become competent for mitotic cell division. Using a novel assay for spore uncoating, we found that spore germination was a multi-step process whose nutritional requirements differed from those for mitotic division. Although both processes were controlled by nutrient availability, efficient spore germination occurred in conditions that did not support cell division. In addition, germination did not require many key regulators of cell cycle progression including the cyclin-dependent kinase, Cdc28p. However, two processes essential for cell growth, protein synthesis and signaling through the Ras protein pathway, were required for spore germination. Moreover, increasing Ras protein activity in spores resulted in an accelerated rate of germination and suggested that activation of the Ras pathway was rate-limiting for entry into the germination program. An early step in germination, commitment, was identified as the point at which spores became irreversibly destined to complete the uncoating process even if the original stimulus for germination was removed. Spore commitment to germination required protein synthesis and Ras protein activity; in contrast, post-commitment events did not require ongoing protein synthesis. Altogether, these data suggested a model for Ras function during transitions between periods of quiescence and cell cycle progression.

Examination of sclerotial germination in Sclerotinia minor with an in vitro model

An in vitro model was developed to examine the effect of surface microflora and exogenous nutrient on sclerotial germination of Sclerotinia minor. Germinated sclerotia were identified by the presence of appressoria on the plastic Petri dish. Surface sterilised field sclerotia germinated at maximum rates on water agar, but unsterilised sclerotia failed to germinate, even after drying and rewetting, indicating that sclerotia of S. minor are unlikely to germinate spontaneously in the presence of soil microflora. Partial surface sterilisation gave intermediate germination rates, which were enhanced two- to four-fold by treatment with sunflower root sap. There was no significant difference in the germination response to root sap from susceptible and resistant sunflower lines, but germination rates were highest with root nutrient obtained from plants during vegetative growth. A positive germination response to peptone and an amino acid mixture implicated amino acids as germination stimuli. Germination of field sclerotia required incubation with root sap for more than 3 days, suggesting that prolonged exposure of sclerotia to root exudates may be required for the onset of stem rot in sunflowers. This is discussed in relation to the dynamics of root growth. Keywords: exogenous nutrient, field sclerotia, germination stimulus.

In vitro and in vivo germination of Nosema locustae (Microspora: Nosematidae) spores

Spores of Nosema locustae (E. U. Canning, Parasitology, 43, 287–290, 1953) were induced to germinate in vitro by a two-step procedure. Spores were first activated by drying in air, dehydrated in hyperosmotic solutions of sucrose or polyethylene glycol 400 (PEG), or by extended treatments in low concentrations of PEG. Germination occurred after transfer of the activated spores to an alkaline salt solution, with a pH optimum of 9.0–10.0. Percentage germination in vitro varied from 2 to 50% among different production batches of spores. Germination in the midgut of Melanoplus sanguinipes was consistently near 50% in all batches and dehydration prior to ingestion was not necessary. The in vitro germination stimulus described here is probably not the physiologically normal one.

The Effect of Ultraviolet Radiation on the Germination of Nosema algerae Vávra and Undeen (Microsporida: Nosematidae) Spores1

Spores of Nosema algerae Vávra and Undeen were subjected to various dosages of 254 nm ultraviolet radiation (UV). Very high dosages of UV were required to block germination. Germination was normal immediately after UV dosages of 0.2 to 1.0 J/cm2, followed by a delayed effect in which both percentage germination and the intrasporal concentration of trehalose decreased with time after UV exposure. Although a few spores were germinated, most of them were inactivated (rendered temporarily unable to germinate) by exposure to UV of 1.1 J/cm2. Ultraviolet radiation between 1.1 and 3.4 J/cm2 stimulated spores to germinate. However, spores were completely unable to germinate immediately after exposure to dosages above 3.8 J/cm2. Ammonia had little effect on stimulation by UV but was inhibitory to germination after stimulation had occurred. These results demonstrate that UV behaves like a germination stimulus and are discussed in terms of the hypothesis that germination is initiated by the breakdown of barriers between trehalose and trehalase.