germinator: a software package for high‐throughput scoring and curve fitting of Arabidopsis seed germination

Over the past few decades seed physiology research has contributed to many important scientific discoveries and has provided valuable tools for the production of high quality seeds. An important instrument for this type of research is the accurate quantification of germination; however gathering cumulative germination data is a very laborious task that is often prohibitive to the execution of large experiments. In this paper we present the germinator package: a simple, highly cost‐efficient and flexible procedure for high‐throughput automatic scoring and evaluation of germination that can be implemented without the use of complex robotics. The germinator package contains three modules: (i) design of experimental setup with various options to replicate and randomize samples; (ii) automatic scoring of germination based on the color contrast between the protruding radicle and seed coat on a single image; and (iii) curve fitting of cumulative germination data and the extraction, recap and visualization of the various germination parameters. The curve‐fitting module enables analysis of general cumulative germination data and can be used for all plant species. We show that the automatic scoring system works for Arabidopsis thaliana and Brassica spp. seeds, but is likely to be applicable to other species, as well. In this paper we show the accuracy, reproducibility and flexibility of the germinator package. We have successfully applied it to evaluate natural variation for salt tolerance in a large population of recombinant inbred lines and were able to identify several quantitative trait loci for salt tolerance. germinator is a low‐cost package that allows the monitoring of several thousands of germination tests, several times a day by a single person.     

Seed germination traits of two plant functional groups in the saline deltaic ecosystems

Aims Salt stress resulting from soil salinization is one of the driving forces of the land degradation throughout the world. The modern Yellow River delta is one of the most saline areas in China. Phytoremediation can be an effective way to restore the salinized ecosystems, which requires selecting appropriate plant species. This study explored the germination responses of common plant species from contrasting habitats in the Yellow River delta to varying salinity, offering experimental information for ecosystem restoration in the Yellow River delta.Methods In this study, 15 common plant species from the Yellow River delta were divided into two groups (high-salinity and low-salinity groups) by their natural habitats using Canonical Correlation Analysis. Seeds of each species were treated with five salinity levels (0, 5, 10, 20 and 30 ppt), using a randomized complete block design, and germinated seeds were counted and removed daily for 28 days to calculate the final germination proportion and mean time to germination. The germination responses of seeds to salinity treatments were compared between the two groups.Important findings In relation to salinity, seed germination behavior of the test species was closely related to the salinity level of the habitats over which they were distributed. Species from the habitats with higher salinity had generally higher final germination proportion but shorter mean time to germination than those from the habitats with lower salinity in all of five salinity treatments used. The final germination proportion and mean time to germination of low-salinity group species were more sensitive to salinity than those of high-salinity group species. Selecting the species with high final germination proportion and short mean time to germination is important for restoration of salinized land.     

Transient kinetics of thermodynamic buffering

The transient response of mitochondrial ATP production towards perturbations was studied by analyzing the trajectories leading from arbitrary initial conditions of the adenine nucleotide pool to the final steady state. These trajectories were calculated from differential equations based on linear relations between flows and thermodynamic forces of the adenylate kinase system including oxidative phosphorylation. The motion of the system along the trajectories consists of two phases: (1) a rapid phase leading from initial states to a common relaxation curve; and (2) a slow phase leading along the relaxation curve to the final steady state. The first phase corresponds to a motion close to the loci of constant adenylic energy charge. In line with this observation is the finding that the energy charge is a constant of motion of the adenylate kinase reaction. The second phase corresponds to a motion along a relaxation curve characterized by minimal Lyapunov exponents in the concentration space of the adenine nucleotides. Thus, both phases of the transient kinetics can be approximated in terms of thermodynamic functions to a high degree of precision. Incubations with isolated rat liver mitochondria were in excellent agreement with the theoretical predictions. In summary, these studies show that the adenylate kinase system not only optimizes the efficiency of oxidative phosphorylation through thermodynamic buffering but, in addition, also deeply influences the transient response of the whole system.     

Modelling the germination of Stellaria media using the concept of hydrothermal time

The germination characteristics of Stellaria media (common chickweed) were investigated over a range of constant temperatures and degrees of moisture stress in order to assess the suitability of hydrothermal time as a basis for modelling germination under field conditions. Maximum percentage germination occurred over a much narrower temperature range around the optimum temperature than previously seen for cultivated crop seed. The entire final percentage germination response to temperature in water was well described by two probit curves, and this model was extended to describe the data at all water potentials at a temperature close to the optimum. The implications of the reduction in germination at nonoptimal temperatures are discussed with respect to the interpretation of germination progress curves and conditional dormancy. After adjusting for maximum percentage germination, a hydrothermal time model was found to fit the data set well within the conditions normally encountered in horticultural seedbeds. This separation of the final percentage germination presents a flexible modelling approach that allows for the different levels of dormancy typically expressed within weed populations. By contrast with many previously reported species, S. media had a synchronous germination rate within the population at any given temperature/water potential combination. This synchronous germination of at least a proportion of the population over a wide range of temperature and water potentials might have ecological significance for the opportunistic germination behaviour of this weed species.     

Germination and establishment of Sahelian rangeland species

Summary In the context of a study on the dynamics of Sahelian rangelands, seed characteristics of about 30 species have been described, with particular attention to germination characteristics. Data on both form and weight of the seeds are presented. Germination was studied under controlled conditions in a climate room. Optimum conditions for germination were established, and the species were characterized by induction time, slope of germination vs. time curve and final germination percentage. The results obtained under climate room conditions appear to be correlated satisfactorily with the behaviour of the different species in the field.     

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.     

Effect of Light and Moisture Conditions and Seed Age on Germination of Three Closely Related Myosotis Species

Germination of three closely related species from the Myosotis palustris group (M. nemorosa, M. palustris subsp. laxiflora, M. caespitosa) differing in their habitats and capacity for clonal growth, was compared in two greenhouse experiments. To evaluate both inter- and intraspecific variation, each species was represented by seeds from several populations. Final germination percentage and germination rates T₅₀ were compared both between species and populations within species. In the first experiment, we studied the influence of two external factors, moisture and light. Four moisture levels (dry, wet, periodically flooded and permanently flooded soil) and three types of shading (without shading, shaded with green foil, shaded with solid paper sheet) were combined in a complete factorial design. In all three species, total germination percentage was the same in the three wettest treatments, and decreased in the dry treatment. Germination in the treatments shaded with green foil (simulating vegetation cover, which changed light quality) was significantly slower than in treatments without shading and treatments shaded with a solid paper sheet. There were significant differences among species, but we also found very pronounced differences among populations within a species. M. caespitosa had the uniformly highest germination percentage (reaching in some cases 100%) and also fastest germination. Germination of M. palustris subsp. laxiflora populations was slower and reached lower final proportions, and medium variability among populations. Inter-population variability in the final germination percentage was highest, and the final germination the lowest in M. nemorosa. In addition, M. nemorosa, a species typical for permanent meadow communities was delayed by permanent flooding. In the second experiment, we studied the effects of seed age and storage conditions. Three combinations of seed age and storage were used: younger seeds (half year old) with no-chilling, younger seeds with chilling and old seeds (three years old) with chilling. M. caespitosa had again the highest final germination percentage and fastest germination rates T₅₀. In addition, final germination percentage of this species slightly increased with seed age, whereas it decreased in the other two species. The germination behaviour corresponded well to expectation based on species life histories and habitat preferences. Remarkably stable and high germination percentages and fastest germination rates T₅₀ were ascertained in M. caespitosa, a species of disturbed habitats, with lowest capacity for clonal spread. M. palustris subsp. laxiflora (species with highest clonal capacity) and M. nemorosa (species with medium clonal potential) achieved lower, but still very high final germination percentage. In addition, M. nemorosa showed the highest inter-populations variability in our experiments.     

Modeling of Fungal and Bacterial Spore Germination under Static and Dynamic Conditions

Isothermal germination curves, sigmoid and nonsigmoid, can be described by a variety of models reminiscent of growth models. Two of these, which are consistent with the percent of germinated spores being initially zero, were selected: one, Weibullian (or “stretched exponential”), for more or less symmetric curves, and the other, introduced by Dantigny''s group, for asymmetric curves (P. Dantigny, S. P.-M. Nanguy, D. Judet-Correia, and M. Bensoussan, Int. J. Food Microbiol. 146:176–181, 2011). These static models were converted into differential rate models to simulate dynamic germination patterns, which passed a test for consistency. In principle, these and similar models, if validated experimentally, could be used to predict dynamic germination from isothermal data. The procedures to generate both isothermal and dynamic germination curves have been automated and posted as freeware on the Internet in the form of interactive Wolfram demonstrations. A fully stochastic model of individual and small groups of spores, developed in parallel, shows that when the germination probability is constant from the start, the germination curve is nonsigmoid. It becomes sigmoid if the probability monotonically rises from zero. If the probability rate function rises and then falls, the germination reaches an asymptotic level determined by the peak''s location and height. As the number of individual spores rises, the germination curve of their assemblies becomes smoother. It also becomes more deterministic and can be described by the empirical phenomenological models.     

不同性别河北柴鸡早期生长规律及其生长曲线拟合

本研究运用Logistic、Gompertz和Bertalanffy三种非线性模型对不同性别河北柴鸡早期生长规律和生长曲线进行分析及拟合比较.结果表明,3种曲线模型拟合度均达到0.99以上,但Gompertz曲线模型在拟合度和预测极限生长量、拐点周龄和最大周增重等方面相对较好.进一步分析表明,河北柴鸡公鸡的极限体重和拐点体重均高于母鸡,拐点周龄性别间差异不大,公鸡最大周增重与实际观测值接近.本文有助于了解不同性别河北柴鸡各自的生长模式及其对营养、环境的需求,为开展规模化饲养提供参考.     

Sequence of events during germination of putrefactive anaerobe 3679 spores

The sequence of changes during germination of putrefactive anaerobe 3679h spores was studied under aerobic conditions in a solution containing l-alanine and sodium pyrophosphate. Evidence that specific changes occurred in two distinct regions of the spore is given by data on several criteria that were used to measure germination. During the initial stage of germination, the absorbancy decreased, dipicolinic acid was released, the spores lost their resistance to heat and toxic chemicals, and the spore periphery (cortex) darkened gradually under phase-contrast microscopy. The final stage of germination was characterized by changes in the central spore region (core), notably phase darkening of the spore center and stainability with mercurochrome, and by a slight additional absorbancy decrease.     

Effect of Water Stress, Seed Coat Restraint, and Abscisic Acid upon Different Germination Capabilities of Two Tomato Lines at Low Temperature

Two tomato (Lycopersicon esculentum Mill.) lines with greatly different capabilities to germinate at 10°C were compared with respect to sensitivity to experimental treatments which affect the water status of the embryo. Germination rates and final percentages could be drastically changed (at 25°C) by (a) removing the mechanical constraint from the radicle tip, (b) imposing water stress by an osmoticum, (c) stress hardening of the seeds through osmotic pretreatment, and (d) inhibiting embryo expansion by abscisic acid (ABA). All treatments showed a similar difference in germination vigor between the two lines indicating that cold sensitivity is in fact a matter of water relations rather than of phase transitions in membrane lipids. Inhibition of germination by ABA was completely abolished by removing the mechanical constraint from the radicle tip. Osmotic stress of −3 bar which quantitatively replaced this constraint in inhibiting germination also restored the sensitivity to ABA. It is concluded that all these treatments act on the balance between the hydraulic extension force of the embryo radicle and the opposing force of the seed layers covering the radicle tip. The difference in cold sensitivity between the two seed lines resides either in the osmotic potential or the pressure potential of the germinating embryo.     

新疆干旱区植物藜的种子异型性及其萌发机理

新疆干旱区分布的植物藜(Chenopodium album)的种子有黑色和褐色两种类型。对藜的异型性种子从形态结构、不同环境因素及激素或化学物质对萌发的影响以及同工酶谱等方面进行了研究,并对其萌发及适应异质环境的机理进行了讨论。结果表明:(1)藜的异型性种子在形态结构、萌发休眠特性等方面都存在明显差异:黑色种子种皮厚且硬,休眠,萌发慢,萌发率低;褐色种子种皮薄而软,不休眠,萌发快且萌发率高;(2)黑色种子的休眠可通过切除胚根外缘种皮得以完全解除;(3)赤霉素、乙烯利对黑色种子的萌发无明显促进作用;KNO3可较显著促进黑色种子的萌发;协同使用乙烯利和KNO3时,可显著提高黑种子萌发率,完全打破休眠;(4)黑色种子和褐色种子的酯酶、过氧化物酶及过氧化氢酶同工酶谱带存在差异;(5)黑色种子的萌发需要光照,而褐色种子则对光不敏感;低温贮藏对二者的萌发均无显著影响,尽管黑色种子的萌发率有波动。研究结果初步显示黑色种子的休眠是内源(胚)和外源(种皮)因素共同所致。藜的种子异型性及其萌发机理的形成是其对新疆干旱区异质化环境的高度适应。     

The use of image analysis to monitor the germination of seeds of broccoli (Brassica oleracea) and radish (Raphanus sativus)

To study broccoli and radish seed germination under different temperature regimes the germination test has been used to assess final germination percentage, start and rate. This method has been integrated with a computer‐aided image analysis test which is more accurate in monitoring the extent of imbibition phases through the assessment of seed area increase and timing of radicle emergence detected on single seeds. In addition, seed area increase has been used also to establish a close relationship with radicle elongation rate in the time range when ‘visible germination’ is scored by a classical germination test. The results suggest that this image analysis parameter may be considered as a reliable seed imbibition marker to integrate the germination parameters obtained by a germination test.     

Effect of exogenous cytokinins on dynamics of development and differentiation of infectious structures of the pathogen of wheat powdery mildew

The infectious structures for the development and differentiation of Erysiphe graminis DC. f. sp. tritici March., the pathogen of wheat powdery mildew, under the effects of exogenous zeatin was studied by methods of light and electron scanning microscopy. It was shown for the first time that physiologically active substances, specifically phytohormones of the cytokinin type, can affect dimensions of the halo revealed in the pathogen penetration site at cytochemical staining. Treatment with zeatin affected conidia germination and pathogen growth in the ectophytic stage. The concentration curve of the action of zeatin for the number of mature pathogen colonies (6 days after infection) was represented by a multiphase curve with two maxima (1 and 3 μM) and one minimum (1.5 μM). Similar curves have been obtained for the number of normal appressoria and for large halo diameters, which possibly indicates the existence of the factors affecting these both parameters, as well as the final number of pathogen colonies. The obtained data indicate that the origin of the multiphase dose response curve for effect of cytokinins on the development of powdery mildew pathogen is connected with factors that are active at the early stages of pathogenesis.     

The effect of parent plant nutrition on seed size, viability and vigour and on germination of wheat and triticale at different temperatures

Seed was obtained from two adjacent trials of wheat (cv. Avalon) and triticale (cv. Lasko) harvested in 1990. The treatments consisted of factorial combinations of five levels of spring nitrogen (50, 100, 150, 200 and 250 kg N ha^-1) with fungicide treatments designed to control foliar diseases. Seed was graded by size into different fractions. Application of foliar fungicides to the parent plant increased the proportion of large seed in wheat but not in triticale. Fungicide application had no effect on seed vigour (quantified as the value of K; after controlled deterioration), final germination, median germination time (t₅₀) or rate of germination. Parent plant nutrition altered the proportions of seed in different size categories in triticale and wheat. In laboratory germination tests, final germination of wheat increased with seed size. Large (> 3.0 mm diameter) wheat seed from plots receiving 50 kg N ha“¹ had a lower final germination than large seed from plots which had received greater amounts of nitrogen, but t₅₀ and K; were similar for all nitrogen levels. In triticale, the higher the seed weight, the higher the final germination, the faster the rate and the higher the Ki. Final germination, t₅₀ and K;, were all positively correlated with the amount of nitrogen received by the parent plants. Parent plant nutrition had no effect on the response to temperature of final germination, t₅₀, or rate of germination for large or small seeds of both wheat and triticale.     

A Parametric ROC Model‐Based Approach for Evaluating the Predictiveness of Continuous Markers in Case–Control Studies

Summary The predictiveness curve shows the population distribution of risk endowed by a marker or risk prediction model. It provides a means for assessing the model's capacity for stratifying the population according to risk. Methods for making inference about the predictiveness curve have been developed using cross‐sectional or cohort data. Here we consider inference based on case–control studies, which are far more common in practice. We investigate the relationship between the ROC curve and the predictiveness curve. Insights about their relationship provide alternative ROC interpretations for the predictiveness curve and for a previously proposed summary index of it. Next the relationship motivates ROC based methods for estimating the predictiveness curve. An important advantage of these methods over previously proposed methods is that they are rank invariant. In addition they provide a way of combining information across populations that have similar ROC curves but varying prevalence of the outcome. We apply the methods to prostate‐specific antigen (PSA), a marker for predicting risk of prostate cancer.     

Effect of phylogeny, life history and habitat correlates on seed germination of 69 arid and semi-arid zone species from northwest China

We tested the effects of phylogeny, life history and habitat conditions for seedling establishment on seed germination of 69 arid/semi-arid zone species from northwest China. Final germination percentages had a bimodal distribution, whereas, days to first germination were skewed toward short periods. Stepwise multi-way ANOVAs showed that the amount of variance in final germination percentage among species for seeds incubated in light was explained by phylogeny (13.6%), dispersal mode (11.9%), seed mass (3.6%), and habitat (0.2%); in darkness by dispersal mode (17.5%), phylogeny (12.7%), seed mass (5.7%), and habitat (0%). A series of ANOVAs revealed that the variance in final germination percentages among species is largely dependent upon phylogeny and dispersal mode but that it is also influenced by seed mass and habitat. The effects of dispersal mode and seed mass on final germination percentage among species were phylogenetic group- and habitat-specific. Wind-dispersed seeds had higher germination percentages than unassisted and vertebrate-dispersed seeds. Compared to xerophytes, mesophytes tended to have smaller seed mass, higher mean germination percentages and a greater effect of seed mass on final germination fractions. Our results suggest that phylogeny and life history should be taken into account in studies on the role of natural selection in regulation of seed germination.     

Accurate mathematical models to describe the lactation curve of Lacaune dairy sheep under intensive management

Although the intensive production system of Lacaune dairy sheep is the only profitable method for producers outside of the French Roquefort area, little is known about this type of systems. This study evaluated yield records of 3677 Lacaune sheep under intensive management between 2005 and 2010 in order to describe the lactation curve of this breed and to investigate the suitability of different mathematical functions for modeling this curve. A total of 7873 complete lactations during a 40-week lactation period corresponding to 201 281 pieces of weekly yield data were used. First, five mathematical functions were evaluated on the basis of the residual mean square, determination coefficient, Durbin Watson and Runs Test values. The two better models were found to be Pollott Additive and fractional polynomial (FP). In the second part of the study, the milk yield, peak of milk yield, day of peak and persistency of the lactations were calculated with Pollot Additive and FP models and compared with the real data. The results indicate that both models gave an extremely accurate fit to Lacaune lactation curves in order to predict milk yields (P = 0.871), with the FP model being the best choice to provide a good fit to an extensive amount of real data and applicable on farm without specific statistical software. On the other hand, the interpretation of the parameters of the Pollott Additive function helps to understand the biology of the udder of the Lacaune sheep. The characteristics of the Lacaune lactation curve and milk yield are affected by lactation number and length. The lactation curves obtained in the present study allow the early identification of ewes with low milk yield potential, which will help to optimize farm profitability.