Enhanced symbiotic seed germination of Cypripedium macranthos var. rebunense following inoculation after cold treatment

Cypripedium macranthos var. rebunense is a well-known wild orchid in Japan, and is considered to be a symbol for rare plant conservation. A fungus isolated from roots of C. macranthos var. rebunense induced symbiotic germination of the species in vitro. Cold treatment of the seeds at 4°C prior to fungal inoculation was required for the symbiotic germination. Changing the timing of inoculation of the fungus to the seeds greatly improved germination frequency. Maximum germination was attained after seeds were inoculated just after the cold treatment for 12 weeks, and approximately 20% of the seeds developed into protocorms more than 1 mm long. These results suggest that fungal inoculation takes place at the beginning of spring in nature, and the tough impervious seed coat may preserve the seed from the infection during autumn and winter seasons. The lengthy culture period of more than 16 weeks at 20°C on the same medium with the fungus caused gradual browning and rot of the protocorms. By elimination of the fungus with a fungicide and by transfer to a nutrient rich medium, approximately 20% of the protocorms developed into healthy plantlets. The methods obtained here appear to be applicable to symbiotic germination of many other threatened Cypripedium spp.     

The dynamics of indole-3-acetic acid in Acer platanoides seeds during stratification and germination

During stratification at 5°C indole-3-acetic acid (IAA) levels in embryos of Acer platanoides decreased during the early stages but subsequently increased again throughout the remainder of a 144 day period. The reduction in IAA levels in embryos of fruits stored at 17°C was even more pronounced, and in addition, no increase was observed after longer storage periods at this temperature, the levels of IAA remaining very low. Germination in seeds maintained at 5°C was not observed until after 120 days or longer, but germination potential increased at an earlier stage, as shown by the fact that seeds transferred to 20°C gave appreciable increases in germination after much shorter chilling periods. Endogenous IAA levels in embryos from seeds transferred to 20°C after a chilling period, long enough to break dormancy, increased within 24 h, i.e. before visible germination, to levels similar to those observed in embryos from seeds chilled continuously for 144 days. Embryos from seeds chilled for 120 days, i.e. when the samples already showed visible germination and when the endogenous IAA content was already high, showed no further increase in endogenous IAA during a three day incubation at 20°C. None of the treatments employed was effective in inducing germination of seeds or embryos from fruits stored at 17°C.     

Soluble sugar content of white spruce (Picea glauca) seeds during and after germination

In white spruce ( Picea glauca [Moench.] Voss.) seeds, the raffinose family oligosaccharides (RFOs) provide carbon reserves for the early stages of germination prior to radicle protrusion. Some seedlots contain seeds that are dormant, failing to complete germination under optimal conditions. Since dormancy may be imposed through a metabolic block in reserve mobilization, the goal of this project was to identify any impediment to RFO mobilization in dormant relative to nondormant seeds. Desiccated seeds contain primarily, and in order of abundance on a molar basis, sucrose and the first 3 members of the RFOs, raffinose, stachyose and verbascose. Upon radicle protrusion at 25°C, the contents of RFOs decreased to low amounts in all seed parts, regardless of prior dormancy status and sucrose was metabolized to glucose and fructose, which increased in seed parts. During moist chilling at 4°C, RFO content initially decreased before stabilizing and then increasing. In seeds that did not complete germination, the synthesis of RFOs at 4°C favored verbascose, so that at the end of 14 (nondormant) or 35 (dormant) weeks, verbascose contents in megagametophytes exceeded the amount initially present in the desiccated seed. This was also true in the embryos of the dormant seedlot. In seed parts from both seedlots after months of moist chilling, stachyose amounts exceeded raffinose amounts. Upon radicle protrusion at 4°C, RFO contents decreased to amounts most similar to those present in seeds that completed germination at 25°C. Hence, the RFOs are utilized as a source of energy, regardless of the temperature at which white spruce seeds complete germination. Based on the similarity of sugar contents in seed parts between dormant and nondormant seeds that did not complete germination, differences in sugar metabolism are probably not the basis of dormancy in white spruce seeds.     

Dormancy and germination of olive embryos as affected by temperature

Olive seeds do not germinate promptly when placed under favourable conditions, which is a problem in raising young plants for breeding or experimental purposes. In a series of experiments an investigation of the role of temperature in the germination of olive embryos was conducted. Naked, unchilled olive embryos ( Olea europaea L. cv. Chalkidikis), cultured in vitro at 20°C, had a germination capacity of 73%, whereas that of embryos which had previously been chilled at 10°C for 2 or more weeks reached 96%. Intact seeds did not germinate at 20°C unless they had previously been subjected to 10°C for 3 or 4 weeks. Embryos chilled while in the intact seed and excised just before transfer to 20°C, reacted in a similar way to naked embryos, but reached their maximum germination capacity after 4 weeks at 10°C. Under constant temperature conditions the highest germination percentage of embryos was observed at 10 and 15°C and the highest germination rate at 15°C, while a moderate capacity and rate of germination occurred at 20°C, and a very low percentage and rate at 25 and 30°C. Prechilling at 10°C did not affect germination at 15°C, but improved the percentage and the rate of germination at 20, 25 and 30°C. The germination percentages of embryos chilled for 1 or 2 weeks at 10°C and then transferred to 25°C were lower than those of similarly chilled embryos transferred to 20°C. The chilling effect could not be reversed at 25°C when the embryos had been chilled for 3 or more weeks. The results show that olive seeds exhibit a state of dormancy that is caused by factors residing partly in the endosperm and partly within the embryo.     

The effects of phosphate supply on uptake of potassium ions, 2,4-D and atrazine by wheat and maize

The germination percentage of peach [ Prunus persica (L.) Batsch cv. Halford] seeds at 20°C was low (< 20%) after incubation at 5°C for as long as 35 days, but then increased considerably (> 40%) when the seeds were maintained at 5°C for longer than 42 days. Four zones of gibberellin-like activity were found in partially purified seed extracts. Gibberellin-like activity remained low in seeds incubated at 5°C for as long as 28 days, but increased significantly in three of these zones after 35 days, and in the fourth zone after 49 days. The increase in gibberellin-like activity was evident prior to the transfer of the seeds to 20°C. Moreover, seeds maintained at 5°C germinated at this temperature after 63 days. For seeds incubated and germinated at 20°C, both the germination percentage and the gibberellin-like activity remained low throughout the experimental period. Application of the growth retardant paclobutrazol to seeds after 28 days of a 49 day total incubation period at 5°C did not substantially reduce seed germination, although the increase in gibberellin-like activity was prevented. Seeds did, however, require a longer time to germinate after transfer to 20°C and were dwarfed in appearance. Application of GA₃ to seeds prior to stratification increased the percentage germination of seeds only when they had been incubated at 5°C for at least 35 days. The major changes in gibberellin-like activity are, therefore, associated not so much with the processes which allow germination to take place in peach, but more with those processes which allow normal growth and development of the seedling.     

Breakage of Pseudotsuga menziesii seed dormancy by cold treatment as related to changes in seed ABA sensitivity and ABA levels

The main aims of the present work were to investigate whether a chilling treatment which breaks dormancy of Douglas fir ( Pseudotsuga menziesii (Mirb.) Franco) seeds induces changes in the sensitivity of seeds to exogenous ABA or in ABA levels in the embryo and the megagametophyte, and whether these changes are related to the breaking of dormancy. Dormant seeds germinated very slowly within a narrow range of temperatures (20–30°C), the thermal optimum being approximately 25°C. The seeds were also very sensitive to oxygen deprivation. Treatment of dormant seeds at 5°C improved further germination, and resulted in a widening of the temperature range within which germination occurred and in better germination in low oxygen concentrations. In dry dormant seeds the embryo contained about one-third of the ABA in the megagametophyte. ABA content of both organs increased during the first 4 weeks of chilling. It then decreased sharply in the megagametophyte to the level in the embryo after 7–15 weeks of chilling. At 15°C, a temperature at which dormancy was expressed, the ABA level increased in the embryo and the megagametophyte of dormant unchilled seeds whereas it decreased in the organs of chilled seeds. The longer the chilling treatment, the faster the decrease in ABA after the transfer of seeds from 5°C to higher temperatures, and the decrease was faster at 25 than at 15°C. These results suggest that the breaking of dormancy by cold was associated with a lower capacity of ABA biosynthesis and/or a higher ABA catabolism in the seeds subsequently placed at 15 or 25°C. Moreover, the chilling treatment resulted in a progressive decrease in the sensitivity of seeds to exogenous ABA. However, seeds remained more sensitive to ABA at 15 than at 25°C. The possible involvement of ABA synthesis and of responsiveness of seeds to ABA in the breaking of dormancy by cold treatment is discussed.     

Germination uniformity of Fraxinus excelsior controlled by seed water content during cold treatment

The seeds of Fraxinus excelsior L. are dormant after harvest, since they need a period of chilling for germination. Moist treatment at 20°C for 2–3 months followed by stratification at 4°C for 7 months breaks dormancy. We observed that germination occurred during stratification and was spread over a period of 3 months. Germination at low temperature was temporarily inhibited by a moderate reduction of the seed water content initiated after the third month of stratification. This allowed the afterripening process to continue.
The following procedure was developed to suppress dormancy and to induce uniform germination:

• 1. 

  Imbibition of the seeds and moist treatment at 20°C for 2–3 months;
• 2. 

  stratification for 3 months;
• 3. 

  treatment at low temperature and low water potential for at least 4 months, this treatment should not exceed 6 months;
• 4. 

  complete rehydration of the seeds at 16°C.

     

黄花杓兰种子无菌萌发的培养条件研究

授粉１５周后的黄花杓兰（Cypripedium flavum P.F.Hunt et Summerh.)种子经０．５％ＮaClO溶液处理后,无菌下播于培养基因表面,２０周后种子最高萌发率达到９０％。ＫＴ和ＢＡ促进兰花种子萌发的机理是作为一种萌发诱导物质直接起作用。而不仅仅是拮抗ＡＢＡ的抑制作用而促进种子的萌发,培养基,激素和种子预处理萌发率高低的关键,种皮是阻碍黄花杓兰种子萌发的主要原因之一。     

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.     

Seed dormancy patterns in Epipactis palustris (Orchidaceae): Requirements for germination and establishment of mycorrhiza

Some terrestrial orchid species, including Epipactis palustris (L.) Crantz, are considered extremely difficult to germinate and cultivate in vitro. Observations of orchids germinating in nature are very few, and the timing and requirements for seedling establishment are unknown for most species. Seeds of E. palustris were incubated in vitro with an appropriate fungus, but germination was poor unless several other conditions were also met: scarification of the testa in Ca(OCL)₂, an initial incubation for several weeks at 27°C, and a subsequent cold stratification for 8–12 weeks at 4–8°C, With these pretreatments, germnation responses exceeded 50% after incubation for 4 weeks at 20°C. Healthy protocorms with normal organ development were only produced by symbiotic culture following this lengthy seed preparation. The findings suggest that under natural conditions the seeds need some after-ripening, and the testa needs to be partially decomposed before germination. The requirement for chilling suggests that germination of seeds in situ occurs in spring.     

In vitro germination of some Western European orchids

Several germination factors were studied in 23 Western European orchids in order to obtain a germination percentage which approximates the maximum percentage of coloured embryos after an adaptation of the tetrazolium test. The duration of seed pretreatment with Ca(OCl)₂+ Tween-80 was one of the most important factors controlling the germination of Western European orchids. Best germination occurred in continuous darkness at 23°C on dilute media. The nitrogen source had a species related influence. The best results were obtained with casein hydrolysate, although it could nearly be replaced by one amino acid, L-glutamine. A cytokinin was required by Cypripedium calceolus and Epipactis hellborine but was not necessary for Lisiera ovata and Dactylorhiza maculata.
Experiments with 3 species have shown that the objective, to obtain a germination percentage, which approximated the maximum percentage of coloured embryos, was achieved. This has led to the elaboration of a basic sowing method for European orchids. The application of this method gave good germination responses with 21 of the 23 species tested.     

Germination Responses of Taraxacum platycarpum Seeds to Temperature

Abstract Germination responses of Taraxacum platycarpum seeds to temperature were examined under laboratory conditions to investigate the emergence-season choice mechanism of the seeds. Almost all the newly collected seeds were non-dormant. Under constant temperature conditions, maximum percentage germination (approximately 90%) was attained at temperatures 6–16°C, where simple linear relationships were observed between the temperature and the rates of germination, i.e. the reciprocals of the time taken to germinate by seed subpopulations with 10–80% germination. Thermal time required for germination of the subpopulations ranged from 600 Kh (degree Kelvin × hours) to 1500 Kh with a relatively constant base temperature of about 2.5°C. Lower limit temperature for germination was slightly below 6°C. Higher limit temperature for germination has the normal distribution with the mean ±SD of 19±2.5°C. Pre-exposure of imbibed seeds to temperatures higher than the higher limit temperature for germination, 25 and 30°C, had no effect on the germinability and the rate of germination at a circa-optimum temperature. Moist chilling treatment at 4°C caused an increase in the variation of germination rate within the seed population, but no evidence for dormancy-inducing or breaking effects was obtained.     

Osmopriming-Regulated Changes of Plasma Membrane Composition and Function were Inhibited by Phenylarsine Oxide in Soybean Seeds

The aim of the present work was to investigate the effects of osmoconditioning on chilling injury in chilling-sensitive soybean ( Glycine max (L.) Merr. Zhonghuang No. 22) seeds during imbibition. Low temperatures reduced the germination rate and no seed germinated at 1 °C. Osmoconditioning of seeds at 20 °C with a polyethylene glycol-8000 (PEG8000) solution at 1.5 MPa for 72 h followed by drying back to their initial moisture content (MC) reduced their chilling sensitivity. The phenylarsine oxide (PAO), an inhibitor of protein tyrosinephosphatases, was used to investigate the possible involvement of phosphorylation-dephosphorylation of Tyr residues in the plasma membrane composition and function when seeds were osmoconditioned. The results showed the germination of osmoconditioned seeds decreased significantly when PAO was added in PEG solution after chilling treatment. PAO inhibited changes in composition of plasma membrane phospholipids and fatty acid induced by osmocondition, indicated that tyrosine protein phosphorylation is involved in the regulatory mechanisms of osmocondition-responsive chilling in soybean seeds. Western blot result further indicated that osmocondition treatment improved the activity of plasma membrane H⁺-ATPase after chilling treatment, but this effect was abolished by PAO. The possible regulation mechanism by Tyr protein phosphorylation is discussed.     

Differential effects of camptothecin and interactions with plant hormones on seed germination and seedling growth

Effects of camptothecin, a naturally occurring alkaloid, on seed germination varied from promotive to inhibitory, depending on the species used. It markedly inhibited seedling root growth but its inhibition of hypocotyl growth varied among species. Camptothecin inhibited GA₃-induced dark germination of lettuce (Lactuca sativa L.) seeds and hypocotyl elongation of seedlings. In contrast to ABA, the camptothecin inhibition of GA₃-induced germination could not be overcome by cytokinin. When seeds were germinated at 29C with a 0.5 h light treatment, little or no germination occurred in the camptothecin treatment, but addition of cytokinin overcame this inhibition.     

Antagonistic effects of high and low temperature pretreatments on the germination and pregermination ethylene synthesis of lettuce seeds

Red light-induced germination of Grand Rapids lettuce seeds (Lactuca sativa L.) incubated at 20 C was inhibited if the seeds were first imbibed at 30 C for 36 hours. This effect was counteracted by exogenous ethylene and associated with a reduction in the rate at which the seeds produced ethylene throughout the pregermination period. A chilling treatment reversed the effect of a prior imbibition at 30 C on both germination and ethylene production. The possibility that the pretreatments influence germination through their effects on ethylene production is discussed.     

The Effect of Sodium Hypochlorite on Germination of Lettuce Seed at High Temperature

Investigations into the effects of treatment with sodium hypochloritesolution on high-temperature germination of lettuce seed aredescribed. A 2 h treatment in a solution with 10% availablechlorine enabled up to c. 70% of seeds to germinate at 35 °C,without impairing germination and normal seedling developmentat 20 °C. A 10 min rinse in 0.01 N HCI following such treatmentenhanced its effect and treated seeds retained their abilityto germinate at high temperature for at least 18 months. Seedlingdevelopment at 20 °C following treatment was influencedby treatment temperature and batch of hypochlorite solution.Emergence from compost in a cycling (30/15 °C) temperatureregime was improved in three out of five cultivars tested, andthere were indications that hypochlorite treatment enabled oneof the other cultivars to escape induced thermodormancy. Key words: Lactuca sativa L., Sodium hypochlorite, Thermo-inhibition     

Temperature control of germination and its possible role in the survival of a non-dormant population of Avena fatua

Germination of freshly harvested seeds of a non-dormant (ND) line (Stonehouse 319) of wild oats ( Avena fatua L.) was inhibited by incubation of the seeds at relatively high temperatures of 25 and 30°C. The germination inhibition in these seeds appeared to be a case of thermo-inhibition which was the direct effect of hightemperature treatment (HIT), since it did not persist after transferring the seeds to an optimum germination temperature of 20°C. Even a prolonged HTT of 30°C for over 5 weeks did not prevent germination of about 80% of the seeds transferred to 20°C. However, in a significant proportion of the seeds, thermo-dormancy was induced by 10 days of HTT at 30°C if the seeds were then incubated at sub-optimal temperatures of 5 to 15°C. This thermo-dormancy would appear to be 'restrictive' in form, since its expression was restricted to very specific conditions. Relatively low inclubation temperaturs of 5 and 10°C markedly slowed germination whether HTT was applied or not. The results suggest that thermo-inhibition and thermo-dormancy, induced during seasonal temperature fluctuations, may provide a survival mechanism for seeds of such ND lines as Stonehouse 319.     

Morphological dormancy in seeds of the autumn-germinating shrub Lonicera caerulea var. emphyllocalyx (Caprifoliaceae)

To better understand the germination ecophysiology of the genus Lonicera , the dormancy class, temperature requirements for embryo growth and radicle emergence and phenology of seedling emergence were determined for Lonicera caerulea var. emphyllocalyx . At maturity, seeds have an underdeveloped embryo (approximately 28% of the length of full-grown embryos). Embryos in fresh seeds grew to full length at 15, 20, 20/10 and 25/15°C within 3 weeks, but failed to grow at ≤ 10°C and at 30°C. Radicles emerged from 86–100% of freshly matured seeds in light at 15, 20, 20/10 and 25/15°C within 28 days, but failed to emerge at 10°C. Radicles emerged equally well in a 12 h photoperiod and in continuous darkness at 25/15°C. Rapid embryo growth and germination over a range of conditions indicate that seeds of this taxon have morphological dormancy (MD); this is the first report of MD in a species of Lonicera . Seeds are dispersed in summer, at which time high temperatures promote embryo growth. Embryos grow to the critical length for germination in approximately 1 month; the peak of seedling emergence occurs in early autumn. Radicles emerged within 2 months from 98% of seeds buried at soil depths of 2 cm and 10 cm in the field in August in Sapporo, Japan; thus, seeds have no potential to form a persistent soil seed bank. However, seeds sown too late in autumn for embryos to grow remained viable and germinated the following summer when temperatures were high enough to promote embryo growth.     

The effect of stratification on the endogenous levels of gibberellins and cytokinins in seeds of douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and sugar pine (Pinus lambertiana Dougl.)

Abstract Seeds of Douglas-fir and sugar pine which had been exposed to various periods of moist chilling (stratification) were extracted and bioassayed for gibberellin and cytokinin activity. In Douglas-fir a peak of gibberellin activity increased progressively during stratification and was seven times higher after 7 weeks, but declined subsequently. No change in the level of cytokinin activity was observed during stratification. However, a slight increase (1.7-fold) occurred in stratified seeds placed at 22°C for 3 days. In sugar pine a high level of gibberellin activity, approximately 20-fold higher than in unstratified controls, was present in extracts of seeds which had been stratified for 8 weeks. After 10 weeks of stratification cytokinin levels had increased approximately 200-fold. Four cytokinin-like substances were detected in seeds. Three of the substances are similar in their chromatographic properties to zeatin glucoside, zeatin riboside and zeatin, respectively. Both gibberellin and cytokinin activity declined rapidly after reaching maximum levels. In seeds with the coats removed, germination was rapid and complete in the absence of stratification. Prior to radicle emergence in coatless seeds cytokinin activity increased to a maximum (approximately a 1-7-fold increase) within 24 h. Significant levels of gibberellin-like substances were present after 8 h. Gibberellin activity could not be detected in coatless seeds which were imbibed in AMO-1618, and germination was inhibited significantly.     

Endo-β-mannanase activity during dormancy alleviation and germination of white spruce (Picea glauca) seeds

It is not known how embryos of seeds of the Pinaceae protrude from their enclosing tissues to complete germination. Prior to protrusion of the radicle there is an increase in endo-β-1,4-mannanase (EC 3.2.1.78) activity associated with weakening of the micropylar megagametophyte/nucellus from seeds of white spruce ( Picea glauca [Moench.] Voss). Mannanase activity is present as three isoforms (pI values 5.0, 4.8, 4.7) in both the embryo and surrounding structures (megagametophyte and nucellus) prior to and during imbibition. Activity of all the isoforms increases in the chalazal and micropylar megagametophyte during germination. Activity then declines after the testa splits, typically 1 day prior to radicle protrusion, due partially to its leaching from the seed into the surrounding water. Activity increases in the cotyledons and axis as the embryo commences elongation. Seeds from dormant seedlots exhibit a lower germination percentage, relative to seeds from nondormant seedlots, and the force necessary for the embryo to puncture the surrounding structures tends to be greater. Although similar mannanase activities are present in unimbibed seeds of dormant and nondormant seedlots, during germination, enzyme activity in seeds of dormant seedlots is lower. Moist chilling alleviates dormancy in the seeds of the Pinaceae and, during 3 weeks of this treatment, mannanase activity slowly increases. After 3 weeks of moist chilling and regardless of whether the seedlot was dormant or not prior to moist chilling, the force necessary to puncture the micropylar megagametophyte and nucellus is lower, and the speed of germination greater. Seeds from previously dormant seedlots also complete germination to a greater percentage, relative to unchilled seeds from dormant seedlots. Upon transfer to 25°C, mannanase activity in moist-chilled seeds decreases during germination of all seedlots regardless of their previous dormancy status.