Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Chemiluminescence during rice seed imbibition at different temperatures.

The chemiluminescence (CL) of rice (Oryza sativa L.) seeds at different temperatures and the CL spectra of rice seed, caryopses and seed coat were studied during early imbibition. Compared with the CL of barley (Hordeum vulgare L.) and soybean (Glycine max L. Merr.) seeds, the CL of rice seeds had a non-linear, logarithmic-like increase of intensity in the temperature range 30-50 degrees C. The Van't Hoff coefficient Q(10) = I(T+10)/I(T) was equal to 2. The emission spectrum of whole rice seed, rice and coat had a greater proportion of red light during early imbibition, which led to the conclusion that the CL of rice seed during early imbibition arises partially from enzyme-catalysed reactions.     

Exudation of an allelopathic substance lepidimoide from seeds during germination

Lepidimoide promotes growth of the cockscomb hypocotyl. It is exuded from germinating seeds of various plant species into their culture environment. The mode of exudation of lepidimoide from seeds into the culture solution, using sunflower and buckwheat seeds, was studied in relation to seed germination. In the dry seeds, about 75% of the lepidimoide was found in the seed coat and about 25% in the kernel. Upon water imbibition it was released into the culture solution. However, the quantity of lepidimoide detected in the seed exudate was more than three times the total amount in dry and imbibed seeds, suggesting that lepidimoide was also produced de novo in the seeds and subsequently released. When seed coats or kernels were imbibed separately, the quantity of lepidimoide released from the seed coats into the culture solution was much the same as that in the dry seeds, but the amount of lepidimoide detected in the exudate of kernels was about 16 times that in the dry kernels. These results suggest that lepidimoide, already present in dry seeds, as well as that newly produced in the kernels following imbibition, was released into the environment.     

Morphological analysis of seed shape in Arabidopsis thaliana reveals altered polarity in mutants of the ethylene signaling pathway

The shape of Arabidopsis thaliana dry seed is described here as a prolate spheroid. The accuracy of this approximation is discussed. Considering its limitations, it allows a geometric approximation to the analysis of changes occurring in seed shape during imbibition prior to seed germination as well as the differences in shape between genotypes and their changes during imbibition. The triple mutant ein2-1, ers1-2, etr1-7 presents notable alterations in seed shape. In addition, seeds of this and other mutants in the ethylene signaling pathway (ctr1-1, eto1-1, etr1-1, ein2-1) show different response to imbibition than the wild type. Imbibed seeds of the wild type increase their asymmetry compared with the dry seeds. This is detected by the relative changes in the curvature values in both poles. Thus, during imbibition of the wild-type seeds, the reduction in curvature values observed in the basal pole gives them an ovoid shape. In contrast, in the seeds of the ethylene mutants, reduction in curvature values occurs in both basal and apical poles, and its shape remains as a prolate spheroid. Our data indicate that the ethylene signaling pathway is involved, in general, in the complex regulation of seed shape and, in particular, in the establishment of polarity in seeds, controlling curvature values in the seed poles.     

Spectrophotometric characterization of phytochromes in dimorphic cocklebur seeds in relation to capacity for germination

Phytochrome was measured spectrophotometrically in different tissues of the upper (positively photoblastic) and lower (negatively photoblastic) seeds of the cocklebur (Xanthium pennsylvanicum Wallr.). Axial parts of the seeds, in particular parts of the radicle, contained high levels of phytochrome, while cotyledonary parts contained only low levels. These results were consistent with the distribution of the light-sensitive areas of the seeds that were associated with germination. Phytochrome levels in both types of dimorphic seeds increased gradually with increasing duration of dark imbibition for 4–8 h, then the rates of increase in levels of phytochrome accelerated. In both types of seed, some phytochrome was measurable even before imbibition. In the lower seeds, up to 20% of the phytochrome was occasionally observed as P_fr in samples imbibed in darkness for a short time (up to 12 h). A slight blue shift of the peak of P_T in the difference spectrum of phytochrome was observed in the case of lower seeds imbibed for 0–2 h. These results suggest that, to some extent, the lower axes contain dehydrated P_fr or intermediate(s) in the photoconversion of phytochrome. The dark reactions of P_fr were also examined in excised axes of both types of dimorphic seed after they had been pre-imbibed for 16 h in darkness. Dark destruction of P_fr was observed in both types of seed. In addition, net increases in levels of P_r were observed in the dark controls and in the samples irradiated with red light after the level of P_fr diminished. No ‘inverse’ dark reversion from P_r to P_fr was detected. Thus, after 16 h of imbibition, there were no differences in terms of properties of phytochrome between the two types of seed, and the different responses to light of upper and lower seeds might depend mainly on a difference in the physiological state of the two types of seed rather than the properties of phytochrome.     

Hypoxia and Imbibition Injuries to Aging Seeds

The development of hypoxia and primary injuries were examined during the imbibition of aging pea seeds (Pisum sativum L., cv. Nemchinovskii). The distribution of air-dry pea seeds by their room-temperature phosphorescence revealed the presence of two fractions (I and II) in a seed lot with 72% germinability and three fractions (I, II, and III) in a seed lot with 50% germinability. The water uptake during imbibition was slower in the fraction I seeds than in the fraction-II seeds. The fraction-I seeds produced normal seedlings, whereas the fraction-II seeds either produced seedlings with morphological defects (abnormal) or did not germinate at all. The fraction-III seeds were all dead. The phosphorescence of endogenous porphyrins, emitted only at low O₂ content, was measured after 20-h seed imbibition. The fraction-I seeds emitted no discernible phosphorescence. The fraction-II comprised highly phosphorescent seeds incapable of radicle protrusion and moderately phosphorescent seeds producing abnormal seedlings. The fraction-II seeds experienced hypoxia during the imbibition because of rapid oxygen consumption by the embryo and restrictions to O₂ diffusion imposed by the seed coat. In the fraction-I seeds, the rate of oxygen consumption by the embryo was slower and the seed coat resistance to oxygen diffusion was lower than in the fraction-II seeds. Therefore, hypoxia did not arise in the fraction-I seeds. The submergence of seeds in water caused lethal injuries. The imbibition of seeds without any contact with water caused no lethal damages but did not reduce the percentage of seeds dying of hypoxia. A slow imbibition of seeds in the media containing either an osmoticum (PEG) or an inhibitor of aquaporin channels (p-chloromercuribenzoate) prevented the lethal injuries at early stages of seed hydration and retarded the appearance of oxygen deficiency in fraction-II seeds. Different rates of water uptake by fraction-I and fraction-II seeds were controlled by permeability of cell membranes rather than by permeability of seed coat. It is proposed that low permeability of plasma membranes to water in fraction-I seeds results from the predominantly closed aquaporin channels, whereas a higher permeability of weak seeds (fraction II) is due to open channels.     

Dynamic analysis of Arabidopsis seed shape reveals differences in cellulose mutants

In a previous work, the shape of Arabidopsis seed was described as a cardioid modified by a factor of Phi. In addition, J index was defined as the similarity of the seed (in an orthogonal, bi-dimensional image) to a cardioid, thus allowing the quantitative comparison of seed shape in seeds of varieties and mutants at different stages of development. Here, J index is used for modeling changes in seed morphology during the dynamic process of seed imbibition before germination. The analysis was carried out by means of a general linear model with two fixed factors (genotype and time) applied to two Arabidopsis varieties: Columbia and Wassilewskija and two mutants in cellulose synthesis: prc1-1 (CESA6 in Columbia) and kor1-1 (in Wassilewskija). Equations representing the changes in seed form during imbibition are given. The analysis of changes in seed shape by this procedure provides (1) a quantitative method to record changes in seed shape and to compare between genotypes or treatments showing the time points with maximum differences, and (2) the observation of remarkable differences between wild-type seeds and mutants in cellulose biosynthesis, indicating new phenotypic characteristics previously unknown in the latter. While wild-type seeds increase their J index values during imbibition, in the cellulose mutants J index values decrease. In addition, shape comparisons were done with other mutants. Seeds of ga1-1 mutants behave like cellulose mutants, whereas different ethylene mutants present varied responses. Quantitative analysis of seed morphology is a new basis for the record of differences between wild-type and mutants as well as for phenotypic characterization.     

菜用大豆种子活力与DNA,RNA及蛋白质合成的关系

菜用大豆种子随着其活力的下降,对DNA,RNA和蛋白质前体的吸收,以及合成这些大分子的能力都明显下降,已丧失合成DNA和蛋白质能力的失活种子,仍能进行微弱的RNA合成。高活力种子在吸胀初期DNA合成速率较低,然后增加,至16h达高峰;RNA的合成速率在吸胀一开始就很高,在整个吸胀过程中均保持较高水平;蛋白质的合成速率则在开始较高,并随着吸胀过程呈增强趋势。     

Starch Mobilization in Ultradried Seed of Maize (Zea mays L.) During Germination

The effects of ultradry storage on the starch mobilization in maize (Zea mays L.) seed after aging were investigated. The results indicated that there were no significant differences in the content of ATP,starch, and soluble sugar, as well as the activity of amylase, between ultradried seeds and seeds stored at -20 ℃ during germination. These results were consistent with the higher level of vigor of the ultradried seed. Sieve tube introduction of a fluorescence dye (carboxyl fluoresceindiacetate) and laser confocal microscopy were used to study the development of plasmodesmata in the ultradried seeds. The results indicated that plasmodesmata developed well in ultradried seeds. Fluorescence analysis also showed that the fluorescence intensity in the radicle of ultradried seeds was stronger than that in seeds with a higher moisture content. This suggests that ultradry treatment has no adverse effects on the seeds. After seed imbibition, cell orgaelles could be resumed. It is concluded that ultradry seed storage is beneficial for maintaining seed vigor and that starchy mobilization proceeds regularly during germination.     

Possible involvement of aquaporins in water uptake by pea seeds differing in quality

Using the method of room temperature phosphorescence (RTP), we divided air-dry pea (Pisum sativum L.) seeds subjected to accelerated ageing (40°C, 85% relative humidity) into three fractions: (I) high-quality seeds, (II) weakened seeds, and (III) dead seeds. In the process of ageing, seed germinability firstly decreased and then increased due to so-called “improved” seeds of fraction II, which returned to fraction I as judged from the RTP level; the germinability of these seeds became equal to that of fraction I seeds. Seeds capable of germination (fractions I and II) differed in the rates of imbibition, which depended on plasma membrane permeability (opened or closed water channels) but not on the presence of the seed coat. A low activation energy of seed imbibition in fraction II (less than 5 kcal/mol) indicates that water channels are open. A mercury-containing compound (5 μM p-chloromercuribenzoate (PCMB) reduced the rate of water uptake by these seeds, and dithiothreitol restored it. A high activation energy of fraction I seed imbibition (more than 12 kcal/mol) corresponded to the water uptake mainly across the lipid bilayer when water channels are closed. PCMB did not affect the rate of fraction I seed imbibition. We supposed that mature air-dry pea seeds had open water channels. During the first stages of fraction I seed imbibition, these channels were closed, limiting water uptake. NaF (100 μM), an inhibitor of phosphatase, prevented channel closing and accelerated the imbibition of fraction I seeds. It did not affect the imbibition rate of fraction II seeds, indicating their water channels to be opened. However, NaF did not affect the water uptake of “improved” fraction II seeds as well. It seems likely that their channels were closed during accelerated ageing but otherwise than via dephosphorylation. The results obtained indicate the possibility of water inflow regulation in the weakened seeds via the state of aquaporins, which form water channels in the membranes.     

Starch Mobilization in Ultradried Seed of Maize （Zea mays L.） During Germination

The effects of ultradry storage on the starch mobilization in maize (Zea mays L.) seed after aging were investigated. The results indicated that there were no significant differences in the content of ATP, starch, and soluble sugar, as well as the activity of amylase, between ultradried seeds and seeds stored at -20℃ during germination. These results were consistent with the higher level of vigor of the ultradried seed. Sieve tube introduction of a fluorescence dye (carboxyl fluoresceindiacetate) and laser confocal microscopy were used to study the development of plasmodesmata in the ultradried seeds. The results indicated that plasmodesmata developed well in ultradried seeds. Fluorescence analysis also showed that the fluorescence intensity in the radicle of ultradried seeds was stronger than that in seeds with a higher moisture content. This suggests that ultradry treatment has no adverse effects on the seeds. After seed imbibition, cell orgaelles could be resumed. It is concluded that ultradry seed storage is beneficial for maintaining seed vigor and that starchy mobilization proceeds regularly during germination.     

beta]-Tubulin Accumulation and DNA Replication in Imbibing Tomato Seeds

The activation of the cell cycle in embryo root tips of imbibing tomato (Lycopersicon esculentum Mill. cv Lerica) seeds was studied by flow cytometric analyses of the nuclear DNA content and by immunodelection of [beta]-tubulin. With dry seeds, flow cytometric profiles indicated that the majority of the cells were arrested at the G1 phase of the cell cycle. In addition, [beta]-tubulin was not detectable on western blots. Upon imbibition of water, the number of cells in G2 started to increase after 24 h, and a 55-kD [beta]-tubulin signal was detected between 24 and 48 h. Two-dimensional immunoblots revealed at least three different [beta]-tubulin isotypes. Thus, [beta]-tubulin accumulation and DNA replication were induced during osmotic priming. These processes, as well as seed germination rate, were enhanced upon subsequent imbibition of water, compared with control seeds that imbibed but were not primed. By contrast, when aged seeds imbibed, DNA replication, [beta]-tubulin accumulation, and germination were delayed. In all cases studied, both DNA replication and [beta]-tubulin accumulation preceded visible germination. We suggest that activation of these cell-cycle-related processes is a prerequisite for tomato seed germination. Furthermore, [beta]-tubulin expression can be used as a parameter for following the initial processes that are activated during seed imbibition.     

The Importance of Genetically Determined Seed Coat Characteristics to Seed Quality in Grain Legumes

Comparisons of five pairs of isogenk lines of peas, differingonly in the A gene for seed coat colour showed that white seeds(genotype aa) imbibed more rapidly than coloured seeds (AA),suffered greater imbibition damage revealed by dead tissue onthe cotyledons, and higher solute leakage. Seed-coat pigmentationwas closely associated with slow water uptake, since when expressionof the A gene was suppressed by the recessive pollens gene,the resulting white seeds {palpal AA) imbibed rapidly. The slowwater uptake by coloured seeds was not due to the restrictionof water entry by the seed coat since the differences in imbibitionrate were maintained when a portion of the seed coat was removedand seeds were imbibed with the exposed cotyledon in contactwith moist filter paper. Imbibition of similarly treated seedsby immersion in polyethylene glycol solutions (1–4%) whichincreased the seed/solution wettability, had little effect onthe water uptake of coloured seeds compared to imbibition inwater whereas that of white seeds increased in the first 10mins imbibition. Poor wettability of the inner surface of colouredseed coats did not therefore explain the slow imbibition ofthese seeds. The white seed coats loosened rapidly during imbibitionwhilst the coloured seed coats remained closely associated withthe cotyledons suggesting that the adherence of the seed coatto the cotyledons and therefore the ease of access of waterbetween the testa and cotyledons determines the rate of imbibition.The rapid water uptake by white-coated seeds and the subsequentimbibition damage may explain the high incidence of infectionof these seeds by the soil-bome fungus Pythhan after 2 d insoil. Improved seed quality and emergence may therefore be achievedby breeding for seed coat characteristics leading to reducedrates of imbibition Pisum sativum, isogenic lines, A gene, seed coat colour, imbibition, imbibition damage, wettability, pollens gene, seed quality, grain legumes     

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.     

The significance of structure for imbibition in seeds of the Norway spruce,Picea abies (L.) Karst.

Since the observations of those regularly handling Norway spruce [Picea abies (L.) Karst.] seeds with regard to their imbibition frequently disagree with earlier opinions that this process is markedly inhibited by the seed coat, we decided to examine the morphological factors influencing imbibition in seeds of different colour and different provenances. The seed coat, consisting of the sarcotesta, sclerotesta and endotesta, was found to have little influence on the passage of water, despite the presence of sclereids full of wax lamellae. No differences in seed coat structure were observed between provenances or colours of seeds. The cells of the endotesta were lignified in the area of the micropyle, however, and stood out lip-like on the outer surface of the micropyle after imbibition. An opening in the sclerotesta filled with parenchyma cells was also seen at the chalazal end of the seed. Neither of these openings, which were covered by accumulations of wax, served as the main route for the passage of water, though the micropyle opened up slightly after only 24 h incubation, when the lignified cells bordering it swelled differently from the rest of the endotesta. The progress of water into the seed soon discontinued, however, as the tip of the nucellar cap, covered with wax and crystals, effectively plugged the micropyle. This opening of the micropyle may be the reason why the IDS method does not always succeed in separating viable from non-viable spruce seeds sufficiently well by their density. Imbibition was mostly regulated by the lipophilic layers surrounding the endosperm, which are mainly of nucellar origin, and particularly the megaspore membranes, the outer and inner exine. Imbibition was further hampered by the impermeable nucellar cap, which covered about 3/4 of the length of the endosperm and had merged with the outer exine at its edges. Deposits of wax were observed both between the exines and between the endotesta and the nucellar layers at the edges of the nucellar cap. Waxes may serve as a defence against diseases at the sites of water penetration, while simultaneously increasing the significance of the nucellar endosperm covers as regulators of imbibition.     

The use of an ELISA to quantitate the extent of 11S globulin mobilization in untreated and primed sugar beet seed lots

Seed priming (controlled imbibition) is a widely used technique for improving crop establishment, because it allows a reduction of the time to radicle emergence following seed imbibition and synchronization of individual seeds within seed lots with respect to germination timing. The major problem encountered in seed priming is the control of seed imbibition to a level permitting pre-germinative processes to proceed but that blocks radicle emergence. If not, the consequence of drying back the seeds to initial moisture content for storage purposes could be a total loss of the treated batch. This is because, as long as radicle growth has not begun, seeds may be re-dried without any permanent deleterious effects upon subsequent germination or growth. Recently, we reported the discovery of a molecular marker of sugar beet seed priming, corresponding to the basic B-subunit of the seed storage protein 11S globulin. An ELISA based upon this molecular marker has been used to analyse how different sugar beet seed lots respond to a priming treatment. The results demonstrate that this ELISA allows us to readily distinguish between the primed seeds and the corresponding untreated seeds.