Seed germination of Lasia spinosa as a function of temperature,light, desiccation,and storage

Lasia spinosa seeds were not dormant at maturity in early spring. The most favorable temperatures for germination were between 25 and 30 °C, and final percentage and rate of germination decreased with an increase or decrease in temperature. When L. spinosa seeds were transferred to 25 °C, after 60 days at 10 °C (where none of the seeds germinated), final germination increased from 0% to 78%. Seeds germinated to high percentage both in light and in dark, although dark germination took more than twice as long as in the light. During desiccation of seeds at 15 °C and 45% relatively humidity, moisture loss decreased exponentially from 2.02 to 0.13 g H₂O g⁻¹ dry wt within 16 days, and only a few seeds (12%) survived 0.13 g H₂O g⁻¹ dry wt moisture content. Seeds stored at 0.58 g H₂O g⁻¹ dry wt moisture content at four constant temperatures (4, 10, 15, and −18 °C) for up to 6 months exhibited a well-defined trend of decreasing viability with decreasing temperature. Thus, we concluded that freshly harvested L. spinosa seeds are non-dormant and recalcitrant. Also, the seeds with 0.58 g H₂O g⁻¹ dry wt moisture content could be effectively stored for a few months between 10 and 15 °C although the most appropriate temperature for wet storage appears to be 10 °C, as it is close to the minimum temperature for germination and so there will be less pre-sprouting compared to 15 °C.     

Germination requirements inCarex kobomugi (Sea Isle)

Laboratory and field germination experiments inCarex kobomugi seeds were pursued to clarify their germination requirements and availability of the requirements in the field. In the laboratory experiments, more than 50% of the seeds ofC. kobomugi germinated under 35/30C or 25/20C when they were scarified with 98% H₂SO₄ after removal of their utricles, and chilled in moist condition for 28 to 42 d. Seeds with utricles or those without scarified with H₂SO₄ did not germinate. Seeds sown at 10-cm depth at the Kado-ori coast on 11 February 1991 after soaked in H₂SO₄ showed 40% germination by 29 April 1991, whereas those without H₂SO₄ treatment did not germinate. These results suggest that seed-coat impermeability and embryo immaturity are possible causes of the dormant state in seeds ofC. kobomugi ripen in summer. In the field, the moist-chilling condition is available in winter and the seeds can germinate in the following spring if the seed-coat impermeability is relaxed before winter.     

Suitable water temperature for seed storage of Zostera japonica for subtropical seagrass bed restoration

Restoration of Zostera japonica is needed. Laboratory culture experiments to know the germination characteristics might be helpful for implementation of actual restoration. As a part of germination experiments, we explored suitable water temperature for long-term storage of Z. japonica seeds. This work was based on earlier reports of Zostera marina, which presumably has similar physiological properties to Z. japonica. This study consisted of two experiments: (1) preservation experiments to investigate the fate of stored Z. japonica seeds and (2) germination experiments to investigate the germination potential of the stored seeds. The results of the preservation experiments suggested that seed condition, that is, germinated, degraded, unstable, stable, etc., showed variations between the seeds stored at 4 and 23 °C. The majority of the seeds stored at 4 °C were germinated, while those at 23 °C seemed to be degraded, presumably by bacteria and mold. The germination experiments suggested high germination potential of seeds stored at 4 °C even after 302 days had elapsed. In conclusion, including previously reported results on Z. marina, low temperature was suitable for the preservation of seeds to maintain germination potential.     

Dormancy removal of apple seeds by cold stratification is associated with fluctuation in H2O2, NO production and protein carbonylation level

Reactive oxygen (ROS) and nitrogen (RNS) species play a signaling role in seed dormancy alleviation and germination. Their action may be described by the oxidative/nitrosative “window/door”. ROS accumulation in embryos could lead to oxidative modification of protein through carbonylation. Mature apple (Malus domestica Borkh.) seeds are dormant and do not germinate. Their dormancy may be overcome by 70–90 days long cold stratification. The aim of this work was to analyze the relationship between germinability of embryos isolated from cold (5 °C) or warm (25 °C) stratified apple seeds and ROS or nitric oxide (NO) production and accumulation of protein carbonyl groups. A biphasic pattern of variation in H₂O₂ concentration in the embryos during cold stratification was detected. H₂O₂ content increased markedly after 7 days of seeds imbibition at 5 °C. After an additional two months of cold stratification, the H₂O₂ concentration in embryos reached the maximum. NO production by the embryos was low during entire period of stratification, but increased significantly in germination sensu stricto (i.e. phase II of the germination process). The highest content of protein carbonyl groups was detected after 6 weeks of cold stratification treatment. Fluctuation of H₂O₂ and protein carbonylation seems to play a pivotal role in seed dormancy alleviation by cold stratification, while NO appears to be necessary for seed germination.     

Achene germination of the spring ephemeroid species Carex physodes in the Gurbantunggut Desert

Much of the seed germination research on Carex has focused on wetland species, and little is known about the species of arid habitats. Here, we investigated seed dormancy and germination of Carex physodes, which is an important component of the plant community of the Gurbantunggut Desert of the Junggar Basin in Xinjiang, China. Our studies included the effects of mechanical and chemical scarification, dry storage, treatment with GA₃, wet‐cold stratification, and burial in the field. No freshly matured achenes germinated over a range of temperature regimes after treatment with GA₃, 6 months of dry storage or removal of part of the endosperm. The mechanical scarification resulted in < 5% achene germination, however, higher percentage of achene germination occurred after removal of the pericarp (60%), H₂SO₄ scarification (30%) or scarification in 10% NaOH (85%). Six and nine months of wet‐cold stratification promoted < 40% achene germination. The optimal germination temperatures ranged from 25/10°C to 35/20°C. Maximum germination after 9 months of burial at a depth of 3 cm in the field was 36%. These results indicate that the seeds have non‐deep physiological dormancy (PD) and that the pericarp contains germination inhibitors and has strong mechanical resistance to germination.     

Mechanical Resistance of the Seed Coat and Endosperm during Germination of Capsicum annuum at Low Temperature

Decoated pepper (Capsicum annuum L. cv Early Calwonder) seeds germinated earlier at 25°C, but not at 15°C, compared to coated seeds. The seed coat did not appear to impose a mechanical restriction on pepper seed germination. Scarification of the endosperm material directly in front of the radicle reduced the time to germination at both 15°C and 25°C.

The amount of mechanical resistance imposed by the endosperm on radicle emergence before germination was measured using the Instron Universal Testing Machine. Endosperm strength decreased as imbibition time increased. The puncture force decreased faster when seeds were imbibed at 25°C than at 15°C. The reduction in puncture force corresponded with the ability of pepper seeds to germinate. Most radicle emergence occurred at 15°C and 25°C after the puncture force was reduced to between 0.3 and 0.4 newtons.

Application of gibberellic acid₄₊₇ (100 microliters per liter) resulted in earlier germination at 15°C and 25°C and decreased endosperm strength sooner than in untreated seeds. Similarly, high O₂ concentrations had similar effects on germination earliness and endosperm strength decline as did gibberellic acid₄₊₇, but only at 25°C. At 15°C, high O₂ concentrations slowed germination and endosperm strength decline.

     

Germination of Tagetes minuta L. II. Role of gibberellins

The effect of the application of gibberellins to Tagetes minuta L. achenes (seeds) was determined at both 25°C, the optimal germination temperature, and 35°C, at which temperature the achenes are thermoinhibited. Both GA₃ and GA₄₊₇ accelerated germination at 25°C. Seed germination at 25°C was inhibited by paclobutrazol, but on subsequent application of GA₄₊₇ rapid germination was induced. Following application of GA₃ or GA₄₊₇ to thermoinhibited seeds, a significantly higher final germination percentage was observed than in the distilled water control. However, endogenous gibberellin levels in germinating and thermoinhibited seeds did not differ significantly.     

Selection of somaclonal variants with low-temperature germinability in melon (Cucumis melo L.)

Summary Plants were regenerated from adventitious buds and somatic embryos (R₀) of melon (Cucumis melo L.), the cultivar Andes. Somaclonal variants of melon with low temperature germinability were selected from the progenies (R₁) of R₀ plants. Among 5,618 R₁ seeds harvested from 23 R₀ plants that were regenerated from adventitious buds 4 seeds germinated after 5 days of culture at 15 °C (selection rate; 0.07%). However, among 374 R₂ seeds harvested from 2 R₁ plants no seed germinated after 7 days of culture at 14 °C. Among 9,181 R₁ seeds harvested from 50 R₀ plants regenerated from somatic embryos 110 seeds germinated after 5 days of culture at 15 °C (selection rate; 1.20%). Among 3,717 R₂ seeds harvested from 17 R₁ plants 113 seeds germinated after 7 days of culture at 14 °C (selection rate; 3.04%). R₃ seeds were collected from these R₂ plants following self-pollination. Forty-five of the 47 lines (R₃) originated from 10 R₀ plants showed higher germination rates than that of the original cultivar. Selected lines with low-temperature germinability showed greater fruit growth rate than the original cultivar during the middle stage when they were cultivated in a greenhouse under low-temperature conditions. Of fruits harvested from 31 lines, 15 lines showed greater fruit volume than the original cultivar.     

Relationships between position on the parent plant and germination characteristics of seeds of parsley (Petroselinium crispum Nym.)

The percentage germination of seeds of parsley cv. Imperial Curled was higher in the light than in the dark, the high temperature limits for germination being 30 and 28°C for light and dark respectively. At the higher temperatures, the germination rate was slower in the dark. At 30°C, treatment with a gibberellin A_4/7 mixture at 2 × 10^–4 M partially alleviated the inhibiting effect of darkness on the germination percentage. Pre-incubation of parsley seeds at 35°C in the dark for 30 h increased the rate, but decreased the percentage, of germination of seeds incubated at 15°C in the light. Germination and seedling emergence studies were made on seed harvested from four different umbel positions. Although heavier seeds were produced from primary umbels than from other umbel orders, they were less viable as measured by seedling emergence in the glasshouse. The rate of emergence was decreased with increasing umbel order i.e. with later seed development: this was reflected in subsequent seedling weights, with seedlings from quarternary umbel seeds being about half the weight of those from primary umbel seeds. The upper temperature limit for dark germination was only slightly affected by umbel order, with quarternary umbel seeds being the most thermo-inhibited.Abbreviations BA N⁶-benzyladenine - GA_4/7 a mixture of gibberellins A₄ and A₇ - SD8339 6-benzyl-amino-9-(tetrahydropyran-2-yl)-9H-purine     

Kinetics of dormancy release and the high temperature germination response in Aesculus hippocastanum seeds

The kinetics of primary dormancy loss were investigated in seeds of horse chestnut (Aesculus hippocastanum L.) harvested in four different years. Freshly collected seeds from 1991 held for up to 1 year at temperatures between 2C and 42C exhibited two peaks in germination (radicle growth), representing a low temperature (2-8°C) and a high temperature response (31-36°C). Germination at 36°C generally occurred within 1 month of sowing, but was never fully expressed in the seedlots investigated. At low temperatures (2-8°C), germination started after around 4 months. Generally, very low levels of termination were observed at intermediate temperatures (11-26°C). Stratification at 6°C prior to germination at warmer temperatures increased the proportion of seeds that germinated, and the rate of germination for all seedlots. Within a harvest, germination percentage (on a probit scale) increased linearly with stratification time and this relationship was independent of germination temperature (16-26°C). However, inter-seasonal differences in the increases in germination capacity following chilling were observed, varying from 0.044 to 0.07 probits d^-1 of chilling at 6°C. Increased sensitivity to chilling was associated with warmer temperatures during the period of seed filling. The estimated base temperature for germination, Tb, for newly harvested seeds varied slightly between collection years but was close to 25°C. For all seedlots, Tb decreased by 1°C every 6 d of chilling at 6°C. This systematic reduction in Tb with chilling ultimately facilitated germination at 6°C after dormancy release.     

Reversal of ethylene action on cocklebur seed germination in relation to duration of pre-treatment soaking and temperature

Abstract At 23°C, both C₂H₄ and CO₂ stimulated the germination of freshly imbibed upper cocklebur (Xanthium pennsylvanicum Wallr.) seeds, but C₂H₄, unlike CO₂, changed to an inhibitor of germination under some soaking conditions. However, when seeds were pre-soaked for more than several hours at 23 °C prior to treatment, C₂H₄ strongly inhibited their germination at 33 °C, the degree of inhibition increasing with the duration of pre-soaking. Maximum inhibition occurred at 1–3 cm³ m^?3 C₂H₄ when seeds were pre-soaked for 1 week; further increases of C₂H₄ concentration and pre-soaking period decreased the inhibitory effect. C₂H₄ was synergistic with CO₂ when C₂H₄ promoted germination, whereas it was antagonistic when inhibitory. Such a transition of the C₂H₄ action occurred at ca. 27 °C. Also 1-andnocyclopropane-1-carboxylic acid, a C₂H₄ precursor, inhibited the germination of pre-soaked seeds at 33 °C, although it promoted the germination at 23 °C. When pre-soaked seeds were prepared for germination by chilling at 8 °C for 3 d, the inhibitory effect of C₂H₄ on the subsequent germination was manifested even at 23 °C. The reversal of the C₂H₄ action from promotion to inhibition in cocklebur seed germination is discussed in relation to the engagement of two respiratory pathways in the imbibed seeds.     

Responses of florence fennel (Foeniculum vulgare azoricum) seeds to light,temperature and gibberellin A4/7

The percentage and the rate of germination of seeds of three varieties of Florence fennel was higher in the dark than in the light, the high temperature cut-off points being between 27.2 and 29.4°C. The optimum temperature for germination was between 20 and 25°C. Seeds of all three varieties responded to incubation in solutions containing gibberellin A_4/7 mixture (GA_4/7; Regulex), giving higher germination in the light at temperatures from 20 to 30°C. Seeds steeped for 4 h at 25°C or for 24 h at 5°C in GA_4/7 solutions gave a higher percentage and increased rate of emergence as compared with untreated dry seeds, when sown in compost at 25°C; steeping in water alone was also beneficial. In general, drying the treated seeds before sowing reduced the rate but sometimes increased the percentage of germination as compared with seeds sown when still moist. Seeds harvested from secondary umbels of var. Zefa fino germinated better both in the light and dark than those taken from primary umbels.Abbreviations GA_4/7 a commercial mixture of the gibberellins A₄ and A₇ (trade name Regulex)     

Morphological characteristics of annual Zostera marina shoots at various germination temperatures

The timing of the transition from seed, seedlings and development into flowering is paramount importance in annual-type Zostera marina, because flowering is the first step of sexual reproduction. A majority of plants use environmental cues to regulate the transition to their developmental stages because plants must flower synchronously for successful outcrossing and must complete their sexual reproduction under favorable external conditions. The morphological characteristics (seeds and lateral shoot production, branch number, and inflorescence length) of reproductive shoots of Z. marina L. were examined in outdoor mesocosms to better understand the reproductive strategies of annual populations. Seeds in the germination experiment were divided into two groups: those exposed to cold (7 °C; vernalized group) and those left untreated (25-21 °C; non-vernalized group). All 600 seeds (300 from each group) were cultured for 2 months at 7, 10, 15, 20, and 25 °C in an indoor incubator. In the vernalized group, the germination rates were almost significantly higher than in the non-vernalized group. However, germination rates were not significantly affected by germination temperature. In outdoor mesocosms, production of vegetative shoots was observed in plants germinated at 15 and 20 °C in the vernalized group and at 10, 15 and 20 °C in the non-vernalized group. The highest number of vegetative shoots produced (35) was observed in plants germinated at 20 °C in the vernalized group, whereas seeds of either group failed to produce vegetative shoots when germinated at a low temperature (7 °C).In the flowering phase, the number of branches per shoot in the vernalized group was significantly higher than in the non-vernalized group. The total number of spadices on the 1st branches of plants in the vernalized group (germination at 20 °C) was significantly lower than that in the non-vernalized group at the same germination temperature. The total number of spadices per reproductive shoot in the vernalized group (germination at 10 °C) was also higher than in the non-vernalized group. Thus, both low temperature (vernalization) and seed germination temperature have implications for the sexual and asexual propagation of annual Z. marina populations.     

Seed dormancy and germination in Dodonaea viscosa (Sapindaceae) from south-western Saudi Arabia

Dodonaea viscosa (Sapindaceae) is widespread in the mountainous highlands of the southwestern part of Kingdom of Saudi Arabia, where it is a medicinally important species for the people in Saudi Arabia. Seeds of this species were collected from Mount Atharb in Al-Baha region, at an altitude of 2100 m. The aims of this study were to determine if the seeds of D. viscosa have physical dormancy (i.e. a water-impermeable seed coat) and, if so, what treatments would break dormancy, and what conditions promote germination after dormancy has been broken. The dormancy-breaking treatments included: soaking of seeds in concentrated sulfuric acid (H₂SO₄) for 10 min, immersion in boiling water for 10 min and exposure to 50 °C for 1 min. After seeds had been pre-treated with H₂SO₄, to break dormancy, they were incubated at constant temperatures from 5 to 35 °C, under 12-h photoperiods or in continuous darkness, and germination recorded. Salinity tolerance was investigated by incubating acid-scarified seeds in different concentrations of mM NaCl in the light at 25 °C.Untreated seeds had low final germination 30%. Seeds that had been acid-scarified, immersed in boiling water or exposed to 50 °C all achieved 91% subsequently when incubated at 25 °C. Thus, seeds of this species in Saudi Arabia have physical dormancy, which can be broken by all three treatments designed to increase the permeability of the testa. After pre-treatment, there was a broad optimum constant temperature for germination that ranged between 5 and 25 °C but germination was inhibited by higher temperatures (30 and 35 °C). Light had little effect on this germination response. Scarified seeds were also sensitive to salinity, with the highest germination in distilled water and complete inhibition in 400 mM NaCl. Seeds that failed to germinate in saline treatments were mostly able to germinate on transfer to distilled water, suggesting osmotic inhibition.     

Maturation and germination of walnut somatic embryos

Walnut somatic embryos were multiplied by repetitive embryogenesis on a solid basal DKW medium at 25°C in the dark. When the embryos were isolated at early cotyledonary stage (1–2 mm long) from the primary embryos and cultured on the medium for 3 weeks, they developed into mature embryos showing white, enlarged cotyledons and shoot and root apex. After transfer to light on solid germination medium, however, few mature embryos (0–5%) germinated. Germination percentage increased to about 10% when the mature embryos were pretreated by a storage at 4°C in the dark for 2 months, or by desiccation at 25°C in the dark for 3 or 5 days under an air-humidity conditioned by saturated salt solutions (Mg(NO₃)₂.6H₂O, or ZnSO₄.7H₂O). Similar results were obtained by the addition of gibberellic acid (GA₃) to the germination medium. When mature embryos were desiccated and then placed on medical cotton compresses in liquid germination medium, 45% of the embryos germinated into complete plantlets. These plantlets continued their growth after transplanting to a mixture of peat and vermiculite in pots.Abbreviations GA₃ gibberellic acid - DKW medium Driver & Kuniyuki Walnut medium     

Oxygen-dependent aging of seeds

When seeds of soybeans (Glycine max Amsoy var.) or safflower were stored under high O₂ concentrations, their per cent germination declined rapidly. For example, soybean seeds stored under 7.7 atmospheres O₂ pressure at 25°C and 17% moisture lost all viability within 22 days, whereas under 7.7 atmospheres N₂, the per cent germination remained greater than 80%. Germination decreased continually in O₂ pressures ranging from 0 to 7.7 atmospheres. High levels of O₂, moisture, or temperature each acted independently to cause losses of germination, but when applied simultaneously, these factors acted synergistically. Soybean seeds were also aged under conditions of high temperature (44°C) and humidity (100% RH), which have been routinely used to accelerate aging. Under these conditions, no O₂ dependence of seed death was observable.

Increased lipid oxidation was not detected in seeds that had lost germination ability due to high O₂ treatment. Seeds of two safflower varieties that contained either high oleic or high linoleic fatty acid compositions were subjected to high O₂ treatment. Although the lipid of the high oleic variety is markedly more stable to oxidative degradation, we detected no significant difference in the O₂ tolerance of these seeds.

     

Induction of secondary dormancy in Amaranthus caudatus seeds

Nondormant A. caudatus seeds germinated in the darkat temperatures between 20 and 35° but not at 45 °C.Incubation at this temperature for at least 10 h inhibited seedgermination over the temperature range 20 to 35 °C,temperatures previously suitable for germination. Thus incubation at 45°C induced secondary dormancy. Mechanical or chemicalscarification or exposure to pure oxygen caused complete or almost completegermination of dormant seeds although more slowly in comparison to nondormantseeds. Secondary dormant scarified seeds required a lower concentration of ABAthan nondormant seeds to inhibit germination. The high temperature, whichinduced dormancy, 45 °C, caused the seed coat to be partiallyresponsible for secondary dormancy. Involvement of ABA (synthesis orsensitivity) in the induction and/or maintenance of this dormancy should beconsidered.     

Control processes in the induction and relief of thermoinhibition of lettuce seed germination : actions of phytochrome and endogenous ethylene

Germination of lettuce seeds (Lactuca sativa L. cv Grand Rapids) in the dark was nearly 100% at 20°C but was inhibited at 27°C and higher temperatures (thermoinhibition). A single 5-minute exposure to red light completely overcame the inhibition at temperatures up to 28°C, above which the effectiveness of single light exposures gradually declined to reach a negligible level at 32°C. However, the promotive effect of light could be extended to 34°C by repeated irradiations. At any one temperature, increased frequency of irradiations increased germination percentage, and with each degree increase in temperature, increasingly frequent irradiations were necessary to elicit maximal germination. Loss of the effectiveness of single irradiations with increase in temperature may result either from acceleration of the thermal reversion of the far red-absorbing form of phytochrome or decrease in seed sensitivity toward a given percentage of the far red-absorbing form of phytochrome. Using continuous red light to induce germination, the role of endogenous C₂H₄ in germination at 32°C was studied. Ethylene evolution from irradiated seeds began to increase 2 hours prior to radicle protrusion, whereas the dark-incubated (nongerminating) seeds produced a low, constant amount of C₂H₄ throughout the 24 hour incubation period. Inhibition of C₂H₄ synthesis with 2-aminoethoxyvinyl glycine and/or inhibition of C₂H₄ action with 2,5-norbornadiene blocked the promotive effect of light. Exogenous C₂H₄ overcame these blockages. The results showed that participation by endogenous C₂H₄ was essential for the light-induced relief of thermoinhibition of lettuce seed germination. However, light did not act exclusively via C₂H₄ since exogenous C₂H₄ alone in darkness did not promote germination.     

Profile of Plant Hormones and their Metabolites in Germinated and Ungerminated Canola (Brassica napus) Seeds Imbibed at 8°C in either GA4+7, ABA, or a Saline Solution

Abscisic acid (ABA) and gibberellins (GAs) are two major phytohormones that regulate seed germination in response to internal and external factors. In this study we used HPLC-ESI/MS/MS to investigate hormone profiles in canola (Brassica napus) seeds that were 25, 50, and 75% germinated and their ungerminated counterparts imbibed at 8°C in either water, 25 μM GA₄₊₇, a 80 mM saline solution, or 50 μM ABA, respectively. During germination, ABA levels declined while GA₄ levels increased. Higher ABA levels appeared in ungerminated seeds compared to germinated seeds. GA₄ levels were lower in seeds imbibed in the saline solution compared to seeds imbibed in water. Ungerminated seeds imbibed in ABA had lower GA₄ levels compared to ungerminated seeds imbibed in water; however, the levels of GA₄ were similar for germinated seeds imbibed in either water or ABA. The ABA metabolites PA and DPA increased in seeds imbibed in either water, the saline solution, or ABA, but decreased in GA₄₊₇-imbibed seeds. In addition, ABA inhibited GA₄ accumulation, whereas GA had no effect on ABA accumulation but altered the ABA catabolism pathway. Information from our studies strongly supports the concept that the balance of ABA and GA is a major factor controlling germination.     

Effects of ethylene and carbon dioxide on the germination of osmotically inhibited lettuce seed

Lettuce seeds (Lactuca sativa L.) used in this study germinated 98% at 25 C in light or dark. Their germination was completely inhibited by 0.20 m NaCl, 0.35 m mannitol, or polyethylene glycol 6000 (−7 bars) under continuous light when germination tests were made in Petri dishes. Approximately 50% germination occurred in sealed flasks due to endogenously produced C₂H₄ and CO₂. Removal of either or both gases prevented germination. In the presence of endogenous CO₂, addition of C₂H₄ (0.5 to 16 microliters/liter) stimulated 95 to 100% germination (after 5 days) only in the light, but the rate of germination was dependent on C₂H₄ concentration. At 16 microliters/liter C₂H₄, full germination occurred within 72 hours. Addition of up to 3.2% CO₂ had no adverse effect on the C₂H₄ action. Higher concentrations or the complete absence of CO₂ reduced both rate and total germination. CO₂ alone was ineffective.