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Ethanol release was tested in relation to seed vigour measured by seedling emergence and controlled deterioration tolerance in radish, pepper, watermelon, aubergine, leek and onion seed lots. Ethanol release, measured with six lots from each species after four and eight hours at 40°C and 30% seed moisture, was highly negatively correlated with controlled deterioration tolerance (45°C, 48 hours, 20% seed moisture) in radish (r = 0.05, P < 0.05), with seedling emergence in watermelon (r = 0.99, P < 0.01) and to both traits in pepper (r = 0.87 to 0.94, P < 0.05), but not with any variable for aubergine and leek. One single case was significant between ethanol release after eight hours and controlled deterioration for onion seeds. Results indicate that there is a relationship between ethanol release and seed vigour, but this appears to be species dependent.

Uniform seedling emergence is crucial for economically viable spinach (Spinacia oleracea) leaf production. However, non-uniform seed germination occurs due to variation in moisture sensitivity between and within spinach seed lots. To test moisture sensitivity, we developed a floating germination system with fixed distances between germination papers and the water table, so that moisture levels could be standardised. We tested germination performance of different cultivar seed lots, with one seed lot fractioned in different seed sizes, and of seeds with an intact, open or removed pericarp. At a high moisture level, smaller seeds germinated better than larger seeds, and seeds without a pericarp or with an open pericarp germinated better than intact seeds. Further, the pericarp of smaller seeds was thinner than the pericarp of larger seeds. A lower temperature or increased oxygen level resulted in higher germination rates for differently-sized seeds at high moisture levels. In conclusion, the sensitivity of spinach seed germination to moisture is influenced by seed size, hence pericarp thickness and intactness, and is driven by the oxygen availability to the seed embryo. To determine the full germination potential of spinach seed lots, we recommend a standardised low moisture level in addition to a temperature of 15 or 20°C.

Plant development is highly malleable, as evidenced by the ability of cultured cells, tissues and organs to regenerate into whole plants in vitro. The ability of plants to regenerate in vitro is influenced by many different factors, including the donor plant growth conditions and the type of explant. Empirical trial and error manipulation of these and other culture parameters is the basis for improving plant regeneration protocols, but the mechanisms underlying the effects of these parameters on plant regeneration are unknown. Somatic embryogenesis (SE) is a type of in vitro plant regeneration where somatic/vegetative cells are induced to form embryos. Here we show that seed maturation is one of the parameters that affects the ability of germinating embryos to undergo auxin-induced somatic embryogenesis in Arabidopsis thaliana. Late maturation stage seeds harvested from yellow siliques have a higher capacity for somatic embryogenesis than seeds harvested later from brown siliques, a process that can be mimicked by post-harvest storage. Physiological and genetic analyses suggest that an oxidizing environment and ABA metabolism enhance the rate at which germinating embryos lose capacity to reactivate embryogenic growth. Our data suggest that there is a narrow window during late seed maturation in which embryogenic competence is reduced, and that this process also takes place, albeit more slowly, during seed storage. This knowledge provides a framework for identifying new plant totipotency factors and for directing efficient SE in systems that make use of mature seed explants. The ability of germinating arabidopsis seeds to undergo 2,4-D-induced somatic embryogenesis is negatively influenced by in planta and post-harvest seed maturation.

Seed longevity (storability) is an important seed quality trait. High seed quality is important in agriculture, for the industry, and for safeguarding biodiversity as many species are stored as seeds in genebanks. To ensure ex-situ seed survival, seeds are mostly stored at low relative humidity and low temperature. Oxidation is the main cause of seed deterioration in these dry storage conditions. The molecular mechanisms underlying dry seed survival remain poorly understood. Research on seed longevity is hampered by the lack of an experimental ageing method that mimics dry ageing well. Here, we propose the Elevated Partial Pressure of Oxygen (EPPO) method as the best available method to mimic and accelerate dry seed ageing. We have tested seed germination in Arabidopsis thaliana after EPPO storage at two different relative humidity (RH) conditions and confirm the large effect of oxygen and the seed moisture content on ageing during dry storage. Comparative Quantitative trait locus (QTL) analysis shows that EPPO at 55 % RH mimics dry ageing better than the commonly used Artificial Ageing and Controlled Deterioration tests at higher moisture levels.

Storage of seeds under elevated partial pressure of oxygen mimics dry after-ripening at the genetic level, as indicated by identification of DELAY OF GERMINATION quantitative trait loci., Seed dormancy determines the timing of seed germination and may be released by dry storage, also referred to as after-ripening. Studies on dormancy-release mechanisms are often hampered by the long after-ripening requirements of seeds. After-ripening is thought to be mainly caused by oxidative processes during seed dry storage. These processes are also the main cause of seed ageing. Increasing partial oxygen pressure through the elevated partial pressure of oxygen (EPPO) system has been shown to mimic and accelerate dry seed ageing. In this study, we investigated whether the EPPO system may also release primary seed dormancy in Arabidopsis thaliana. EPPO mimics dry after-ripening at the genetic level, as quantitative trait locus (QTL) analysis after EPPO treatment identified the DELAY OF GERMINATION loci DOG1, DOG2, and DOG6 that were first described in a study using dry after-ripening to release seed dormancy. QTL analysis also showed that dormancy release by cold stratification (another common method to break seed dormancy) partly overlaps with release by after-ripening and EPPO treatment. We conclude that EPPO is an appropriate method to mimic and accelerate dormancy release and, as such, may have applications in both research and industry.

Somatic embryogenesis (SE) is the most striking and prominent example of plant plasticity upon severe stress. Inducing immature carrot seeds perform SE as substitute to germination by auxin treatment can be seen as switch between stress levels associated to morphophysiological plasticity. This experimental system is highly powerful to explore stress response factors that mediate the metabolic switch between cell and tissue identities. Developmental plasticity per se is an emerging trait for in vitro systems and crop improvement. It is supposed to underlie multi-stress tolerance. High plasticity can protect plants throughout life cycles against variable abiotic and biotic conditions. We provide proof of concepts for the existing hypothesis that alternative oxidase (AOX) can be relevant for developmental plasticity and be associated to yield stability. Our perspective on AOX as relevant coordinator of cell reprogramming is supported by real-time polymerase chain reaction (PCR) analyses and gross metabolism data from calorespirometry complemented by SHAM-inhibitor studies on primed, elevated partial pressure of oxygen (EPPO)–stressed, and endophyte-treated seeds. In silico studies on public experimental data from diverse species strengthen generality of our insights. Finally, we highlight readyto- use concepts for plant selection and optimizing in vivo and in vitro propagation that do not require further details on molecular physiology and metabolism. This is demonstrated by applying our research & technology concepts to pea genotypes with differential yield performance in multilocation fields and chickpea types known for differential robustness in the field. By using these concepts and tools appropriately, also other marker candidates than AOX and complex genomics data can be efficiently validated for prebreeding and seed vigor prediction.

Brassica oleracea plants can suffer from shoot apical meristem arrest, when sown at cold temperatures, giving rise to so-called blind seedlings that stop development and the formation of new leaves. We developed a seed treatment that strongly reduces the occurrence of this meristem arrest in kohlrabi and cabbage seedlings. The developed treatment involves soaking the seeds in water for one hour at 22 °C before sowing at 0–2 °C. The reduction of susceptibility to induction of shoot apical meristem arrest could be maintained when seeds were dried after the pre-soaking treatment. A strong reduction in susceptibility could also be obtained by incubating the seeds for four hours with a limited amount of water to 20% moisture content. However, when the hydration was performed at seed moisture levels between 30 and 50%, the sensitivity to shoot apical meristem arrest was regained upon drying. The developed treatment to prevent the occurrence of shoot apical meristem arrest can easily be upscaled for implementation at the commercial level.

Genebanks aim to optimize their storage conditions in order to postpone seed ageing as long as possible. As most genebanks have a relatively short life history, empirical data about seed longevity during ex situ storage are almost absent. Based on seed characteristics, theoretical predictions indicate that cereal seeds can be stored without substantial loss of viability for time periods exceeding 100 years, even under temperatures of a few degrees above zero. Here we present the results of a germination study in wheat and barley, comparing genebank seed samples maintained at different temperatures for 23–33 years. Wheat and barley seed samples stored at −20°C showed a mean germination of 94% and 90%, respectively, indicating no loss of the initial viability determined for the accessions prior to introduction in the collection. Seed samples maintained at 4°C showed a mean germination of 62% for wheat and 75% for barley. In addition to the observed loss of viability, the 4°C samples also showed a loss in vigour as the time period to reach their final germination was about twice as long compared to the −20°C samples. A subset of the wheat accessions tested in 2011 were retested in 2017, showing further reduction in mean germination to 35% for the 4°C samples, while the −20°C samples remained stable at 95%. Several 4°C samples were even close to a complete loss of viability. Considering that wheat and barley are generally regarded as good maintainers, the rapid loss of seed viability observed in the present study indicates that the ex situ seed storage of genetic resources at 4°C should be treated with caution by genebanks, particularly when used for long-term conservation.

Plant genetic resources are conserved by genebanks mainly in the form of seeds. In most of the cases, the dried seeds can be stored for a considerable period of time, but eventually seed deterioration results in the inability to generate healthy seedlings. Prolonging seed longevity during storage reduces the frequency of regeneration, which is beneficial from a genetic as well as a management point of view. To reduce the rate of deterioration, cool and dry storage conditions are usually practised for long-term seed storage. In spite of the growing body of evidence that seed deterioration is predominantly caused by oxidative processes, the importance of seed storage under anoxic conditions has received little attention from the genebank community. Herein, we report on the effects of anoxia on seed viability, the oxygen uptake by dry seeds in closed containers and the permeability for oxygen of various seed storage containers. Our results confirm that the ageing of dry seeds is accelerated by the presence of oxygen in the storage environment. Therefore, we recommend that genebanks store dry seeds under anoxic conditions to prolong their longevity during ex situ conservation. To reduce the initial rate of viability loss, we further recommend that the period of temporary storage after seed harvest be minimized and also that the seeds are kept during this period under controlled conditions, including anoxia.

Most of the above ground tissues in higher plants originate from stem cells located in the shoot apical meristem (SAM). Several plant species can suffer from spontaneous stem cell arrest resulting in lack of further shoot development. In Brassica oleracea this SAM arrest is known as blindness and occurs in an unpredictable manner leading to considerable economic losses for plant raisers and farmers. Detailed analyses of seedlings showed that stem cell arrest is triggered by low temperatures during germination. To induce this arrest reproducibly and to study the effect of the environment, an assay was developed. The role of genetic variation on the susceptibility to develop blind seedlings was analyzed by a quantitative genetic mapping approach, using seeds from a double haploid population from a cross between broccoli and Chinese kale, produced at three locations. The analysis revealed, besides an effect of the seed production location, a region on linkage group C3 associated with blindness sensitivity. A subsequent dynamic genome-wide transcriptome analysis resulted in the identification of around 3000 differentially expressed genes early after blindness induction. A large number of cell cycle genes were en masse induced early during the development of blindness, whereas shortly after, all were down-regulated. This miss-regulation of core cell cycle genes is accompanied with a strong reduction of cells reaching the DNA replication phase. From the differentially expressed genes, 90 were located in the QTL region C3. Among them are two genes belonging to the MINICHROMOSOMAL MAINTENANCE gene family, known to be involved in DNA replication, a RETINOBLASTOMA-RELATED gene, a key regulator for cell cycle initiation, and several MutS homologs genes, involved in DNA repair. These genes are potential candidates for being involved in the development of blindness in Brassica oleracea sensitive genotypes.

†Background and Aims Despite differences in physiology between dry and relative moist seeds, seed ageing tests most often use a temperature and seed moisture level that are higher than during dry storage used in commercial practice and gene banks. This study aimed to test whether seed ageing under dry conditions can be accelerated by storing under high-pressure oxygen. †Methods Dry barley (Hordeum vulgare), cabbage (Brassica oleracea), lettuce (Lactuca sativa) and soybean (Glycine max) seeds were stored between 2 and 7 weeks in steel tanks under 18 MPa partial pressure of oxygen. Storage under high-pressure nitrogen gas or under ambient air pressure served as controls. The method was compared with storage at 458C after equilibration at 85 % relative humidity and long-term storage at the laboratory bench. Germination behaviour, seedling morphology and tocopherol levels were assessed. †Key Results The ageing of the dry seeds was indeed accelerated by storing under high-pressure oxygen. The morphological ageing symptoms of the stored seeds resembled those observed after ageing under longterm dry storage conditions. Barley appeared more tolerant of this storage treatment compared with lettuce and soybean. Less-mature harvested cabbage seeds were more sensitive, as was the case for primed compared with non-primed lettuce seeds. Under high-pressure oxygen storage the tocopherol levels of dry seeds decreased, in a linear way with the decline in seed germination, but remained unchanged in seeds deteriorated during storage at 45 8C after equilibration at 85 % RH. †Conclusions Seed storage under high-pressure oxygen offers a novel and relatively fast method to study the physiology and biochemistry of seed ageing at different seed moisture levels and temperatures, including those that are representative of the dry storage conditions as used in gene banks and commercial practice.

The most common way to test seed quality is to use a simple and reliable but time- and space-consuming germination test. In this paper we present a fast and simple method to analyse cabbage seed deterioration by measuring ethanol production from partially imbibed seeds. The method uses a modified breath analyser and is simple compared to gas chromatographic or enzymatic procedures. A modified method using elevated temperatures (40°C instead of 20°C) shortened the assay time and improved its sensitivity. The analysis showed an inverse correlation between ethanol production and seed quality (e.g. the final percentages or speed of germination and the number of normal seedlings). The increase in ethanol production was observed when cabbage seeds were deteriorated by storage under ambient conditions or hot water treatments, both of which reduced the number of normal seedlings. Premature seeds produced more ethanol upon imbibition than mature seeds. Ethanol production occurred simultaneously with oxygen consumption, indicating that lack of oxygen is not the major trigger for ethanol production.

Greenhouse trials were carried out in order to test the efficacy of different seed treatments as alternatives to chemicals against Colletotrichum lindemuthianum cause of anthracnose on bean and Ascochyta spp. cause of Ascochyta blights on pea, respectively. Resistance inducers, commercially formulated microorganisms, non-formulated selected strains of different microorganisms (fungi, bacteria and yeasts) and plant extracts were applied as dry or liquid seed treatments on naturally infested seeds. Seedling emergence and disease incidence and/or severity were recorded. Almost all seed treatments turned out to be ineffective in controlling the Ascochyta infections, which is in line with the literature stating that these pathogens are difficult to control. The only alternative treatments that gave some control of Ascochyta spp. were thyme oil and a strain of Clonostachys rosea. The resistance inducers tested successfully controlled infections of bean by C. lindemuthianum. Among the formulated microorganisms, Bacillus subtilis-based formulations provided the best protection from anthracnose. Some strains of Pseudomonas putida, a disease-suppressive, saprophytic strain of Fusarium oxysporum and the mustard powder-based product Tillecur also proved to be effective against bean anthracnose. However, among the resistance inducers as well as among the other groups, certain agents caused a significant reduction of plant emergence. Different alternative seed treatments can therefore be used for the control of C. lindemuthianum on bean, while on pea only thyme oil and a strain of Clonostachys rosea showed some effectiveness against Ascochyta spp.

Fourteen old seeds of cabbage ( Brassica oleracea var. capitata ) were sorted on the basis of chlorophyll fluorescence (CF) signal. Seed fractions of 54.3% and 45.7% with low CF and high CF, respectively, were obtained. Unsorted seeds were considered as controls. The effects of CF sensor based sorting on quality parameters of aged cabbage seeds were examined. It was observed that the seeds with low CF resulted in significantly higher germination, vigour and germination rate over unsorted fraction. However, no significant differences were noticed in ethanol produced (μg/l) at 40 °C in unsorted, low and high CF seeds. Significantly less numbers of hard seeds were noticed in seeds with low CF, whereas numbers of abnormal seedlings and dead seeds were found at par with unsorted seeds. Numerically higher values over unsorted seeds for fresh weight, dry weight, root and total seedlings length were found in seed fraction with low CF. The sorting improved the germination of aged cabbage seeds by 16%. Therefore, this method of quality up-gradation could be used to obtain a better quality seeds even from the lots which otherwise become substandard during storage. The technique could also be commercially exploited for sorting seeds and upgrading the quality of all those crop seeds where chlorophyll is present in the seed coat and persist during storage.

During seed maturation and germination, major changes in physiological status, gene expression, and metabolic events take place. Using chlorophyll sorting, osmopriming, and different drying regimes, Brassica oleracea seed lots of different maturity, stress tolerance, and germination behavior were created. Through careful physiological analysis of these seed lots combined with gene expression analysis using a dedicated cDNA microarray, gene expression could be correlated to physiological processes that occurred within the seeds. In addition, gene expression was studied during early stages of seed germination, prior to radicle emergence, since very little detailed information of gene expression during this process is available. During seed maturation expression of many known seed maturation genes, such as late-embryogenesis abundant or storage-compound genes, was high. Notably, a small but distinct subgroup of the maturation genes was found to correlate to seed stress tolerance in osmoprimed and dried seeds. Expression of these genes rapidly declined during priming and/or germination in water. The majority of the genes on the microarray were up-regulated during osmopriming and during germination on water, confirming the hypothesis that during osmopriming, germination-related processes are initiated. Finally, a large group of genes was up-regulated during germination on water, but not during osmopriming. These represent genes that are specific to germination in water. Germination-related gene expression was found to be partially reversible by physiological treatments such as slow drying of osmoprimed seeds. This correlated to the ability of seeds to withstand stress.

Proteomics of Arabidopsis seeds revealed the differential accumulation during germination of two housekeeping enzymes. The first corresponded to methionine synthase that catalyses the last step in the plant methionine biosynthetic pathway. This protein was present at low level in dry mature seeds, and its level was increased strongly at 1-day imbibition, prior to radicle emergence. Its level was not increased further at 2-day imbibition, coincident with radicle emergence. However, its level in 1-day imbibed seeds strongly decreased upon subsequent drying of the imbibed seeds back to the original water content of the dry mature seeds. The second enzyme corresponded to S-adenosylmethionine synthetase that catalyses the synthesis of S-adenosylmethionine from methionine and ATP. In this case, this enzyme was detected in the form of two isozymes with different pI and Mr. Both proteins were absent in dry mature seeds and in 1-day imbibed seeds, but specifically accumulated at the moment of radicle protrusion. Arabidopsis seed germination was strongly delayed in the presence of dl-propargylglycine, a specific inhibitor of methionine synthesis. Furthermore, this compound totally inhibited seedling growth. These phenotypic effects were largely alleviated upon methionine supplementation in the germination medium. The results indicated that methionine synthase and S-adenosylmethionine synthetase are fundamental components controlling metabolism in the transition from a quiescent to a highly active state during seed germination. Moreover, the observed temporal patterns of accumulation of these proteins are consistent with an essential role of endogenous ethylene in Arabidopsis only after radicle protrusion.

Aerated hydration (AH) treatments of cauliflower seeds for 12 h (12AH) and 28 h (28AH) at 20 degrees C resulted in improved or reduced storage potential of low or high vigour seeds, respectively. Seeds were stored at their initial seed moisture content (mean 5.5% mc) or at 12% mc at 10 degrees C for 12 months and at 20 degrees C for 4 months. The improved longevity of low vigour seeds was associated with increased K(i) (initial seed viability) and a reduced rate of deterioration (1/sigma) whereas the K(i) of high vigour seeds fell after 28AH and the rate of deterioration increased such that the time to lose one probit of viability decreased from 28.7 to 5.3 months at 10 degrees C and from 10.4 to 1.2 months at 20 degrees C. The improved K(i) of low vigour seeds could be explained by the reduction in the extent of deterioration after AH, as indicated by the increase in germination after cotrolled deterioration (CD), and the possible activation of metabolic repair during treatment. In contrast the reduced germination after CD of AH-treated high vigour seeds was indicative of deterioration as a result of treatment. Both high and low vigour seeds contained constitutive levels of ss-tubulin which increased during AH treatment, the increase being greater in high vigour seeds. High vigour seeds also showed an increase in the proportion of nuclear DNA present as 4C DNA, from 3% (untreated seeds) to 26% (28AH), indicative of germination advancement from the G(1) to G(2) phase of the cell cycle during treatment. This higher proportion of 4C DNA is correlated with the increased sensitivity of seeds to drying and/or storage after AH, leading to their reduced K(i) and storage potential. In contrast, there was little change in %4C in low vigour seeds. Priming in polyethylene glycol (PEG, -1.0 MPa) for 5 d or 13 d also improved the longevity of low vigour seeds stored at their initial and 12% mc at 10 degrees C for 8 months, as reflected in their laboratory and CD germination. In this case, however, the improved longevity of the low vigour seeds following 13 d priming was associated with an increase in 4C DNA from 4% (dry control) to 56% after treatment. The germination of both untreated and primed high vigour seeds remained high throughout the storage period. Increases in the rate of germination (decreased mean germination time) observed after all AH and PEG treatments were not consistently associated with an increase in the proportion of nuclei containing 4C DNA.

Cell cycle activity was studied in apical and axillary buds of growing plant material, whereas in dormant buds the accumuNorway maple (Acer platanoides L.), apple (Malus ‘M9’), lation was much lower or below detection level. It was obpedunculate oak (Quercus robur L.), Scots pine (Pinus syl7es - served for all species that during dormancy induction the tris L.) and rose (Rosa corymbifera ‘Laxa’) during dormancy amount of beta-tubulin decreased, while during dormancy release a fast accumulation of beta-tubulin occurred. The induction and release. Flow cytometric analyses revealed that in dormant buds, cells mainly were quiescent at the G 0/G1 dynamics of the beta-tubulin accumulation reflected the dormancy status of tree buds of the five species studied suggesting phase, while in non-dormant buds, a significantly higher frequency of G 2 cells was found in all species. In western blots that the beta-tubulin level might be useful as a marker for the dormancy status in buds of temperate woody species. accumulation of 55 kDa beta-tubulin was found in active

Resumption of DNA synthetic activities and β-tubulin accumulation was studied in embryo organs of germinating cucumber (Cucumis sativus L.) seeds. Flow-cytometric analysis indicated the existence of 2C, 4C, and 8C nuclei in the radicle of mature embryos, whereas in cotyledons most of the cells contained nuclei with 2C DNA content. Upon imbibition of water, nuclear DNA replication was initiated in the radicle within 15 h, subsequently spreading towards the cotyledons. Bromodeoxyuridine incorporation preceded detectable changes in the relative amounts of DNA, implying the occurrence of putative DNA repair. Organellar DNA synthesis occurred independently of the nuclear DNA synthetic cycle. Western blotting and immunohistochemical localization demonstrated that the constitutive level of β-tubulin originated from preserved β-tubulin granules. During imbibition, disappearance of fluorescent tubulin granules, accumulation of β-tubulin, and formation of microtubular cytoskeleton were found in the radicle, but not in the cotyledon areas. Mitosis only occurred after radicle protrusion at 21 h of imbibition. It is concluded that the differences in the initiation and progress of these cellular and molecular events are associated with the discrete behaviors of the radicle and the cotyledons upon imbibition. The formation of cortical microtubular cytoskeleton and the accumulation of tubulins are important features in preparation of radicle protrusion, whereas DNA synthesis may contribute to postgerminative growth.

Using flow cytometric analyses of the nuclear DNA content, we studied the effects of various conditions of osmopriming on the activation of the cell cycle in embryo root tips of tomato (Lycopersicon esculentum‘Elko’) seeds. In dry untreated seeds, 90.7% of the nuclei revealed 2C signals. Priming of seeds in polyethylene glycol-8000 (PEG) improved the germination rate of seeds transferred onto water at 15 °C. This was associated with an increase in 4C signals when priming was carried out at −1.0 and −1.5 MPa. Priming at −2.0 MPa enhanced subsequent germination but had no effect on DNA replication. For temperatures during priming up to 25 °C, a positive linear correlation existed between the efficiency of the treatment, evaluated by the reciprocal of time to obtain 50% germination at 15 °C, and the frequency of 4C nuclei or the 4C/2C values. Such a correlation did not exist when priming was performed at higher temperatures. At least 5% oxygen in the atmosphere was required during priming for the induction of DNA replication and for the enhancement of subsequent germination. In the presence of 5×10−4m and 10−3m NaN3during priming, most of the cells were maintained with 2C DNA levels and the treatment had no stimulatory effect on germination. The results show a positive linear relationship between the frequency of 4C DNA nuclei or the 4C/2C ratio and the improving effect of priming. However, in suboptimal conditions of priming (−2.0 MPa or temperatures higher than 25 °C), the improvement of seed germination was not associated with the onset of DNA replication.Copyright 1999 Annals of Botany Company

Cell cycle activity in dry and germinating untreated and treated (soaked in water and subsequently in fungicide) seeds of two sugarbeet cultivars, collected at commercial harvest time (late mature seeds) and about 2 weeks before this (immature seeds), was investigated by flow cytometry, and by immuno-detection of β-tubulin and the B-subunit of the 11 S globulin. Germination capacity and field emergence were tested. With dry seeds of both cultivars, higher G2/ G1ratios were observed in the radicle tips of late mature seeds, as compared with those from immature seeds. The late mature seeds contained more partly degraded (soluble) B-subunit of 11 S globulin, typical of germinating or primed sugarbeet seeds. Thus events associated with the onset of germination had occurred in the seed lots collected at commercial harvest time. The cytoskeleton protein β-tubulin was not detectable in dry seeds from either harvest. Western blotting revealed an accumulation of β-tubulin during germination and this was faster in the late mature harvested seeds which was correlated with the onset of DNA replication. Soaking enhanced the rate of cell cycle activation during germination as well as vigour, germination capacity, and field emergence. There was positive correlation between the G2/ G1ratio and the traits examined in laboratory and field tests. It is concluded that a combined analysis of proteins and cell-cycle-related events can be used in understanding and predicting sugarbeet seed quality.

Flow cytometric analyses of nuclear DNA levels were carried out during development, stratification and germination of dormant seeds from three tree species with contrasting characteristics. Norway maple (Acer platanoides) and sycamore (Acer pseudoplatanus) have orthodox (desiccation-tolerant) and recalcitrant (desiccation-sensitive) storage behaviours, respectively, and require only a period of cold to break dormancy, whereas, orthodox cherry (Prunus avium) seeds require an initial warm period before cold stratification to fully stimulate germination. Whole embryos and radicle tips of both Norway maple and sycamore were found to have stable high levels of 4C DNA during the latter stages of development and both contained nuclei arrested at the 2C and 4C levels at maturity. Mature cherry embryos had nuclei predominantly arrested at the 2C level. This suggests that the acquisition of desiccation tolerance is not correlated with the arrest of the cell cycle at any particular nuclear DNA level. Neither DNA replication in radicle cells nor germination occurred when seeds were maintained moist at a constant 20 °C. However, in the late stages of cold treatment during stratification, nuclear DNA levels in radicle cells changed in advance of radicle emergence in the orthodox Norway maple and cherry, whereas in the recalcitrant sycamore, change was not recorded until after radicle emergence. These results show that DNA replication has potential use as an indicator of the progress of tree seeds through stratification treatments used to break some types of dormancy. The ways in which this indicator could be exploited for seed quality and performance testing are discussed.

Using X-ray photography and flow cytometry, the internal morphology and DNA replication activity of wild type (wt), GA- (gib-1) and ABA-deficient (sitw) tomato (Lycopersicon esculentumMill. cv. Moneymaker) mutant seeds were studied. During seed formation, from 30 to 45 d after pollination (DAP) the endosperm becomes solid and the seed starts to gain desiccation tolerance. At this time significant changes occur in the amounts of DNA in radicle tip cells. At 30 DAP, radicle tip cells of the three genotypes manifest about 60% of 2C, 30% of 4C and 10% of 8C amounts of DNA. Upon maturation (45 DAP onwards), most cells in the seeds of the three genotypes arrest in the G1phase of the cell-cycle with 2C amounts of DNA. However, a relatively high proportion of cells with 4C amounts of DNA was detected in the radicle tip cells ofsitwcompared with wild type andgib-1. At the well-matured stage (60 DAP), there were about 2% of seeds with free space in wild type andgib-1, and about 13% insitw. At the over-matured stage (75 DAP), even more seeds with free space were found insitw, whereas no increase in the proportion of the seeds with free space was detected in the other two genotypes. In −1.0 MPa PEG-6000 with or without 10 μMGA4+7, no germination occurred in well-matured wild type andgib-1seeds, whether or not they were dried after harvest. However,sitwseeds were able to germinate both in over-mature fruit and in −1.0 MPa PEG-6000. Priming of dried seeds in −1.0 MPa PEG induced a large amount of free space in almost all seeds of the three genotypes, and nuclear DNA synthesis in the radicle tip cells of wild type andsitwseeds. However, PEG priming of fresh (non-dried) seeds had no effect on the amount of free space and 2C/4C DNA ratios in wild type orgib-1seeds, but did induce free space in about 20–25% ofsitwseeds and provoked 4C signals insitwseeds. Removal of the endosperm and testa opposite the radicle tip of seeds resulted in root protrusion, the induction of free space and an increase of 4C DNA signals in the three genotypes. It is concluded that ABA is crucial for the efficient arrest of tomato embryo radicle tip cells in G1phase upon maturation, whereas GAs play an important role in re-initiating 4C DNA levels upon germination.

Freshly harvested tomato (Lycopersicon esculentumMill, cv. Moneymaker) seeds were osmotically primed for 8 d in −1.0 MPa PEG-6000 solution and dried to about 6% water content for storage. Such so–called ‘fresh PEG priming’ enhanced seed germination and improved seedling performance as compared with the untreated control. Fresh PEG priming neither alleviated seed dormancy nor promoted DNA replication as was the case when seeds were dried upon harvest and subsequently primed in PEG (normal PEG priming). However, the addition of 10 μM GA4+7to the osmotic priming solution triggered replicative DNA synthesis of fresh-priming seeds and further enhanced the germination process. After 5 months of storage in ambient temperature conditions, fresh PEG-primed seeds maintained more positive effects gained from priming, whereas, normal PEG-primed seeds had lost the promoting effects on germination. Normal PEG-primed seeds were much more susceptible to controlled deterioration than fresh PEG-primed seeds. It is suggested that the advancement of germination is negatively correlated with seed storability. The mechanisms of seed priming in relation to nuclear replication activities and physical changes are discussed.

Storage of tomato seeds under deteriorating conditions reduced the rate of germination, the uniformity of germination, total germination and the proportion of normal seedlings. Pre-storage humidification and hydropriming of seeds resulted in a significant increase in resistance to deterioration. In contrast, osmoprimed seeds were more deterioration sensitive. Humidification or hydropriming for one day did not allow nuclei to enter the S phase of the cell cycle. Osmopriming strongly increased the percentage of nuclei with replicated DNA in the embryonic root tip, indicating initiation of the cell cycle and progression towards the G2 phase. The interaction between the prestorage treatments, cell cycle progression and deterioration resistance, is discussed. Based on the changes in moisture content equilibrium and cell cycle activity it is hypothesized that the beneficial effects of pre-storage humidification and hydropriming were related to metabolic activities induced by the partial hydration and that the adverse effects of osmopriming were caused by a decrease in DNA repair activity due to progression in the cell cycle.

We studied axillary meristem formation of the lateral suppressor (ls) mutant of tomato after elevating the endogenous cytokinin levels through introduction of the isopentenyltransferase (ipt) gene from Agrobacterium tumefaciens. Growth and development of several transformants were examined during in vitro culture. Transformants exhibited phenotypes varying in severity and were divided into four classes. A number of the ipt transformants had a normal phenotype, as non-transformed plants. Others showed a mild to severe ‘cytokinin-like’ phenotype. Transformants with a mild phenotype exhibited reduced internode length and reduced root development. Transformants with a severe phenotype showed even shorter internodes, loss of apical dominance, reduction of leaf size, production of callus at the basis of the shoots and absence of root development or development of green non-branching roots. The severity of the phenotype correlated well with the level of ipt gene expression, as measured by northern analysis. Transformants with a severe phenotype also exhibited increased levels of zeatin riboside, but zeatin levels were not elevated. The increase in endogenous zeatin riboside levels in the ls mutant did not restore axillary meristem formation, but sometimes bulbous structures were formed in the initially ‘empty’ leaf axils. Several adventitious meristems and shoots developed from below the surface of these structures. It is concluded that a reduced level of cytokinins in the ls mutant shoots is not responsible for the absence of axillary meristem formation.

The lateral suppressor mutant (ls) of tomato, mainly characterised by the suppression of axillary shoot development, exhibits reduced fruit growth, thereby limiting its use in tomato production. In this study male and female fertility, seed and fruit set have been investigated in detail. Mutant flowers produced less pollen. Pollinations with wild type pollen showed that mutant flowers exhibit reduced female fertility. With the ls mutant, reduced fruit weight due to decreased seed numbers was partly compensated by an additional stimulus of fruit growth, since mutant fruits were heavier than wild type fruits, when fruits with similar amounts of seeds were compared.

Variation for growth and production of the grafted cucumber shoot as a result of genotypic differences in the root system was analyzed for 24 cucumber accessions used as rootstock. In a first grafting experiment with five rootstocks and three scions different rootstocks led to differences in growth and production of the scion. No interaction was found between scion and rootstock effects with respect to growth and production. Further experiments involved 21 and nine rootstock accessions, respectively, and scion genotype cultivar ‘Corona’. During winter cultivation the rootstocks cultivars ‘Renova’ and ‘Esvier’ significantly promoted vegetative growth of the scion and rootstock Code E induced a significantly higher fruit yield than the standard rootstock ‘Corona’. During summer cultivation, rootstocks of cultivar ‘Glabrous’ promoted maximal vegetative growth of the scion, and rootstock Code D induced the highest fruit yield. During winter cultivation early fruit production correlated well with vegetative growth induced by the rootstock, but this correlation was almost absent during cultivation in the summer. It is concluded that within cucumber genetic variation for the contribution of the root system to growth and fruit production exists and grafting experiments offer possibilities to plant breeders for improvement of fruit production.

Due to the lack of foliar fungicide use, the organic production of Brassica seeds free of Alternaria spp. is difficult. Therefore, effective seed treatments certified for use in organic farming are needed to eradicate or at least effectively reduce the seed-borne inoculum. We here report results of greenhouse and field experiments in which non-chemical seed treatments were tested for control of A. brassicicola on cabbage seeds naturally infested with the pathogen. In greenhouse experiments, significant improvements were obtained by seed treatment with some commercialised and experimental microbial biocontrol agents, an emulsion of thyme oil in water (0.1%) and by the tested physical seed treatments methods ( i.e. hot water, aerated steam and electron seed treatment). Resistance inducers tended to increase the percentage of healthy plants, but the effects were statistically not significant. Generally the combination of physical treatments with the effective agents did not result in improved performance. Positive effects on crop establishment and yield by the same treatments were also observed in field tests. Overall the results indicate that several options for non-chemical control of A. brassicicola on Brassica seeds exist that are comparable in efficacy to the chemical standard Aatiram (active ingredient thiram) used in this study. german version

The current study was initiated to evaluate the efficacy of physical methods (hot water, aerated steam, electron treatment) and agents of natural origin (resistance inducers, plant derived products, micro-organisms) as seed treatments of carrots for control of Alternaria dauci and A. radicina. Control of both Alternaria species by seed treatment with the resistance inducers was generally poor. Results were also not satisfactory with most of the formulated commercial micro-organism preparations. Based on the average of five field trials, one of these, BA 2552 (Pseudomonas chlororaphis), provided a low but significant increase in plant stand. Among the experimental micro-organisms, the best results were obtained with Pseudomonas sp. strain MF 416 and Clonostachys rosea strain IK726. A similar level of efficacy was provided by seed treatment with an emulsion (1%) of thyme oil in water. Good and consistent control was generally achieved with the physical methods aerated steam, hot water and electron treatment. Aerated steam treatment was, apart from the thiram-containing chemical standard, the best single treatment, and its performance may at least partially be due to extensive pre-testing, resulting in dosages optimally adapted to the respective seed lot. In some of the experiments the effect of the hot water treatment, which was tested at a fixed, not specifically adapted dosage, was significantly improved when combined with a Pseudomonas sp. MF 416 or C. rosea IK726 treatment. The results are discussed in relation to the outcome of experiments in which the same seed treatment methods and agents were tested in other seed-borne vegetable pathosystems.

The role of abscisic acid (ABA) in the dormancy induction of tomato (Lycopersicon esculentum) seeds was studied by comparison of the germination behavior of the ABA-deficient sitiens mutant with that of the isogenic wild-type genotype. Freshly harvested mutant seeds, in contrast to wild-type seeds, always readily germinate and even exhibit viviparous germination in overripe fruits. Crosses between mutant and wild-type and self-pollination of heterozygous plants show that in particular the ABA fraction of embryo and endosperm is decisive for the induction of dormancy. After-ripened wild-type seeds fully germinate in water but are more sensitive toward osmotic inhibition than mutant seeds. Germination of both wild-type and mutant seeds is equally sensitive toward inhibition by exogenous ABA. ABA content of mature wild-type seeds is about 10-fold the level found in mutant seeds. Nevertheless, it is argued that the differences in dormancy between the seeds of both genotypes are not a result of actual ABA levels in the mature seeds or fruits but a result of differences in ABA levels during seed development. It is hypothesized that the high levels of ABA that occur during seed development in wild-type seeds induce an inhibition of cell elongation of the radicle that can still be observed after long periods of dry storage.

Powdery mildew-resistant (PMR) cultivars of cucumber (Cucumis sativus L.) grown in greenhouses in the Netherlands during winter show chlorosis on the full-grown leaves of the main stem. The symptoms are yellowing and occasional necrosis between the main veins of the leaves, resembling symptoms of P toxicity. Severity of the chlorosis may vary from one winter to another. Variation is also observed in the severity of the symptoms between cultivars and individual plants of a cultivar. High P nutrition results in an increase of the severity of chlorosis and provides a better discriminating environment for the selection of PMR genotypes that are less susceptible for leaf chlorosis.

The current powdery mildew (Sphaerotheca fuligninea) resistant cucumber varieties suffer from leaf chlorosis during autumn, winter and early spring cultivation in the Netherlands. Therefore screening was carried out for novel powdery mildew resistance genes. From 177 accessions, derived from different sources, 108 accessions proved to be partially resistant to S. fuliginea. Crosses were made with 53 resistant accessions to distinguish the presence of novel genes. It is likely that the accessions C. sativus 2145, C. sativus LV 41, PI 188807, Vladivostokij, White and Yellow 1 have one or more recessive powdery mildew resistance genes, different from powdery mildew resistance genes of the line NPI, which was used for variety breeding. Powdery mildew resistance tests with S. fuliginea give similar results in different regions of the world.

The aim of the present study was to identify seed treatment methods for eradicating Phoma valerianellae from lamb’s lettuce seeds in organic vegetable production. Using seeds naturally infested with the pathogen, the effect of three physical methods (hot water, aerated steam, electron treatment) and different agents of natural origin (micro-organisms, plant derived products, resistance inducers) was tested on moist filter paper, in seed trays under controlled conditions and in the field. in an initial screening, none of the tested putative resistance inducers prevented infection by P. valerianellae, while two out of seven formulated micro-organism preparations and six out of 16 experimental microbial strains were effective. When selected agents and the three physical seed treatment methods were compared in blotter and seed tray tests, the physical methods were generally the most effective treatments, while the micro-organism treatments were clearly less efficacious. However, in field experiments with the same seed lots and the same treatments, a statistically significant increase in plant stand was not obtained with any of the treatments. Combinations of the three physical treatment methods with selected non-chemical agents did not perform better than the physical treatments alone. The most effective alternative seed treatments identified in the present study, aerated steam, hot water, electron treatment and thyme oil (0.1%), can be recommended for eradication of P. valerianellae from lamb’s lettuce seeds in organic farming. Because their efficacy was generally as high as that of the chemical fungicide Aatiram (active ingredient thiram), they are also potentially suited for use in conventional vegetable production.

The role of endogenous abscisic acid (ABA) in seed development was studied with the use of the AB A-deficient sit"' {sitiens) mutant of tomato {Lycopersieon esculentum Mill. cv. Moneymaker). The sit"' mutation causes a strong reduction of the endogenous ABA level in the developing seed. Reciprocal crosses of wild-type and the sit" mutant show a dual origin of ABA. The genotype of the mother plant regulates the ABA content present in the testa, which shows a peak half-way through seed development. The genotype of the embryo and endosperm is responsible for a second ABA fraction, present in these tissues. This second fraction re.aches its peak during the second half of seed development. The strong reduction of endogenous ABA level in the developing sit'lsit"' seed does not change the final fresh and dry weights of the seed nor the accumulation and composition of storage proteins.

A variety of mechanisms have been proposed to account for the extension of life span in seeds (seed longevity). In this work, we used Arabidopsis (Arabidopsis thaliana) seeds as a model and carried out differential proteomics to investigate this trait, which is of both ecological and agricultural importance. In our system based on a controlled deterioration treatment (CDT), we compared seed samples treated for different periods of time up to 7 d. Germination tests showed a progressive decrease of germination vigor depending on the duration of CDT. Proteomic analyses revealed that this loss in seed vigor can be accounted for by protein changes in the dry seeds and by an inability of the low-vigor seeds to display a normal proteome during germination. Furthermore, CDT strongly increased the extent of protein oxidation (carbonylation), which might induce a loss of functional properties of seed proteins and enzymes and/or enhance their susceptibility toward proteolysis. These results revealed essential mechanisms for seed vigor, such as translational capacity, mobilization of seed storage reserves, and detoxification efficiency. Finally, this work shows that similar molecular events accompany artificial and natural seed aging.

Background and Aims: The chemical composition of green coffee and thus the final coffee quality are specifically determined by the mode of post-harvest treatment, i.e. the wet and dry processing. Recently, it was shown that metabolic processes, i.e. germination and, a slightly delayed stress-related metabolism are executed during the course of processing. The specific ambient conditions of either post-harvest treatment may influence differentially the extent and time course of these metabolic reactions; therefore, the incidence and intensity of germination processes in coffee seeds were analysed during processing. Methods: Expression of the germination-specific isocitrate lyase was monitored using competitive RT-PCRs analyses. Resumption of cell cycle activity and cell division were determined by flow cytometry, as well as by the abundance of ß-tubulin quantified by Western blot analyses. Key Results: The extent and the time courses of germination processes in coffee seeds differed significantly between wet and dry processed beans. The highest germination activity occurred 2 d after the onset of wet processing, whereas the corresponding maximum in the course of dry processing appeared about 1 week after the start of post harvest treatment. Conclusions: As recently shown, there are specific differences in the chemical composition of differentially processed coffee beans. It is concluded that these substantial differences are the consequence of the differential expression of germination processes, i.e. they are the result of differences in the corresponding metabolic activities. The coherence of germination-related metabolism and of expression-specific coffee qualities establishes the basis for a novel approach in coffee research. Key words: Coffea arabica, coffee processing

During maturation seeds acquire stress tolerance and increase in longevity, which are lost completely or partially during germination and priming. Slow drying can partly restore the longevity of primed seeds. Cabbage (Brassica oleracea L.) seeds were used as a model to analyse expression of genes during these processes. Six genes for which the expression profiles correlated with the longevity level were identified. Two of these genes are discussed in more detail, EM6 and RAB18, next to the kinetics of two cell cycle genes, historic H1-1 and histone H3. These genes, and the proteins they encode, might serve as markers for optimization of seed processing protocols to produce seeds with a high level of longevity.

Differential display analysis using dormant and non-dormant Arabidopsis thaliana (L.) Heynh seeds resulted in a set of genes that were associated with either dormancy or germination. Expression of the germination-associated genes AtRPL36B and AtRPL27B, encoding two ribosomal proteins, was undetectable in the dry seed, low in dormant seed, and high under conditions that allowed completion of germination. Expression of these genes was also found to be light-regulated and to correlate with germination speed. Expression of the dormancy-associated genes ATS2 and ATS4, encoding a caleosin-like protein and a protein similar to a low-temperature-induced protein respectively, was high in the dry seed and decreased during germination. Expression of ATS2 and ATS4 was high in primary and secondary dormant seed but low in after-ripened or chilled seed. The expression of both genes was also light-regulated, but no relationship with temperature-dependent germination speed was found. Wetenschappelijke publicatie over genen die tot expressie komen tijdens zaadkieming. Arabidopsis zaden zijn als model gekozen.

Seeds of carrot, groundnut, lettuce, oilseed rape and onion were stored hermetically in laminated aluminium foil packets in four environments (dry or ultra-dry moisture contents combined factorially with temperatures of 20 degrees C or -20 degrees C), replicated at several sites. After ten years' hermetic storage, seed moisture content, equilibrium relative humidity, viability (assessed by ability to germinate normally in standard germination tests) and vigour were determined. After a decade, the change in seed moisture content of samples stored at -20 degrees C was small or nil. Except for groundnut and lettuce (where loss in viability was about 8 and 3%, respectively), no loss in viability was detected after 10 years' hermetic storage at -20 degrees C. In all cases, there was no difference in seed survival between moisture contents at this temperature (P > 0.25). Comparison of seed vigour (root length and rate of germination) also confirmed that drying to moisture contents in equilibrium with 10-12% r.h. had no detrimental effect to longevity when stored at -20 degrees C: the only significant (P

Cell cycle activity is required for plant growth and development, but its involvement in the early events that initiate seedling development remains to be clarified. We performed experiments aimed at understanding when cell cycle progression is activated during seed germination, and what its contribution is for proper seedling establishment. To this end, the spatial and temporal expression profiles of a large set of cell cycle control genes in germinating seeds of Arabidopsis (Arabidopsis thaliana) and white cabbage (Brassica oleracea) were analyzed. The in vivo behavior of the microtubular cytoskeleton was monitored during Arabidopsis seed germination. Flow cytometry of Arabidopsis germinating seeds indicated that DNA replication was mainly initiated at the onset of root protrusion, when germination reached its end. Expression analysis of cell cycle genes with mRNA in situ localization, β-glucuronidase assays, and semiquantitative reverse transcription-polymerase chain reaction showed that transcription of most cell cycle genes was detected only after completion of germination. In vivo green fluorescent protein analysis of the microtubule cytoskeleton demonstrated that mitosis-specific microtubule arrays occurred only when the radicle had started to protrude, although the assembly of the microtubular cytoskeleton was promptly activated once germination was initiated. Thus, seed germination involves the synthesis and/or activation of a reduced number of core cell cycle proteins, which only trigger DNA replication, but is not sufficient to drive cells into mitosis. Mitotic divisions are observed only after the radicle has protruded and presumably rely on the de novo production of other cell cycle regulators.

Quantitative trait loci (QTL) mapping was used to identify loci controlling various aspects of seed longevity during storage and germination. Similar locations for QTLs controlling different traits might be an indication for a common genetic control of such traits. For this analysis we used a new recombinant inbred line population derived from a cross between the accessions Landsberg erecta (Ler) and Shakdara (Sha). A set of 114 F9 recombinant inbred lines was genotyped with 65 polymerase chain reaction-based markers and the phenotypic marker erecta. The traits analyzed were dormancy, speed of germination, seed sugar content, seed germination after a controlled deterioration test, hydrogen peroxide (H2O2) treatment, and on abscisic acid. Furthermore, the effects of heat stress, salt (NaCl) stress, osmotic (mannitol) stress, and natural aging were analyzed. For all traits one or more QTLs were identified, with some QTLs for different traits colocating. The relevance of colocation for mechanisms underlying the various traits is discussed.

Seeds gradually lose their viability during dry storage. The damage that occurs at the biochemical level can alter the seed physiological status and is affected by the storage conditions of the seeds. Although these environmental conditions controlling loss of viability have been investigated frequently, little information is available on the genetics of seed longevity. Using Arabidopsis mutants in defined developmental or biochemical pathways such as those affected in seed coat composition, seed dormancy, hormone function and control of oxidative stress, we tried to gain insight into the genes and mechanisms controlling viability of stored seeds. Mutations like abscisic acid insensitive3 (abi3) as well as abscisic acid deficient1 (aba1) show reduced longevity, which may be partially related to the seed dormancy phenotype of these mutants. Mutants with seed coat alterations, especially aberrant tests shape (ats), showed a stronger reduction in germination percentage after storage, indicating the importance of a 'functional' seed coat for seed longevity. A specific emphasis was placed on mutants affected in dealing with Reactive Oxygen Species (ROS). Because several pathways are involved in protection against ROS and because gene redundancy is a common feature in Arabidopsis, 'double' mutants were generated. These 'double' mutants and the corresponding single mutants were subjected to a controlled deterioration test (CDT) and a germination assay on hydrogen peroxide (H2O2) after prolonged storage at two relative humidities. CDT and germination on H2O2 affected all genotypes, although it appears that other effects like genetic background are more important than the deficiencies in the ROS scavenging pathway. Explanations for this limited effect of mutations affecting ROS scavenging are discussed

A study was conducted to investigate the hypothesis that the expression of ribosomal protein genes is correlated with germination performance. Cabbage cv. Bartolo seeds were germinated under 25 or 35°C in darkness or in red light (652 nm, 26 W/m2), and imbibed either in water or in polyethylene glycol (PEG). The expression of the AtRPL27B and AtRPL36B genes were studied in relation to germination performance, using Arabidopsis thaliana genes as heterologous probes in Northern analysis. Both genes showed marginal expression in dry seeds, and increased expression was observed during germination in water. The expression decreased with increasing osmotic stress. The expression of both genes after 18 h in either water or PEG correlated with maximum germination and inversely with the period to reach 50% germination. The cabbage clone BoRPL27 showed high nucleotide similarity to putative A. thaliana RPL27 homologues located at chromosome 3.

To better understand seed germination, a complex developmental process, we developed a proteome analysis of the model plant Arabidopsis for which complete genome sequence is now available. Among about 1,300 total seed proteins resolved in two-dimensional gels, changes in the abundance (up- and down-regulation) of 74 proteins were observed during germination sensu stricto (i.e. prior to radicle emergence) and the radicle protrusion step. This approach was also used to analyze protein changes occurring during industrial seed pretreatments such as priming that accelerate seed germination and improve seedling uniformity. Several proteins were identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Some of them had previously been shown to play a role during germination and/or priming in several plant species, a finding that underlines the usefulness of using Arabidopsis as a model system for molecular analysis of seed quality. Furthermore, the present study, carried out at the protein level, validates previous results obtained at the level of gene expression (e.g. from quantitation of differentially expressed mRNAs or analyses of promoter/reporter constructs). Finally, this approach revealed new proteins associated with the different phases of seed germination and priming. Some of them are involved either in the imbibition process of the seeds (such as an actin isoform or a WD-40 repeat protein) or in the seed dehydration process (e.g. cytosolic glyceraldehyde-3-phosphate dehydrogenase). These facts highlight the power of proteomics to unravel specific features of complex developmental processes such as germination and to detect protein markers that can be used to characterize seed vigor of commercial seed lots and to develop and monitor priming treatments

This chapter discusses the gene expression under stress tolerance and in the absence of stress tolerance at seed priming and drying. An experimental model set up using cabbage seeds of different physiological qualities is described. The topics discussed include: effects of priming on germination rate and storability; sensitivity to ultra-drying; seed maturity; and gene expression studies using cDNA microarrays.

Seeds are usually stored in physiological conditions in which they gradually lose their viability and vigor depending on storage conditions, storage time, and genotype. Very little is known about the underlying genetics of seed storability and seed deterioration. We analyzed a mutant in Arabidopsis disturbed in seed storability. This mutant was isolated as a grs (green-seeded) mutant in an abi3-1 (abscisic acid 3) mutant background. Genetic and physiological characterization showed that the monogenic grs mutant was not visibly green seeded and mapped on chromosome 4. This enhancer mutation did not affect the ABA sensitivity of seed germination or seed dormancy but was found to affect seed storability and seedling vigor. Seed storability was assessed in a controlled deterioration test, in which the germination capacity of the mutant decreased with the duration of the treatment. The decrease in viability and vigor was confirmed by storing the seeds in two relative humidities (RHs) for a prolonged period. At 60% RH, the mutant lost germinability, but storage at 32% RH showed no decrease of germination although seed vigor decreased. The decrease in viability and vigor could be related to an increase in conductivity, suggesting membrane deterioration. This was not affected by light conditions during imbibition, expected to influence the generation of active oxygen species. During seed maturation, ABI3 regulates several processes: acquiring dormancy and long-term storability and loss of chlorophyll. Our results indicate that GRS is a common regulator in the latter two but not of dormancy/germinationSeeds are usually stored in physiological conditions in which they gradually lose their viability and vigor depending on storage conditions, storage time, and genotype. Very little is known about the underlying genetics of seed storability and seed deterioration. We analyzed a mutant in Arabidopsis disturbed in seed storability. This mutant was isolated as a grs (green-seeded) mutant in an abi3-1 (abscisic acid 3) mutant background. Genetic and physiological characterization showed that the monogenic grs mutant was not visibly green seeded and mapped on chromosome 4. This enhancer mutation did not affect the ABA sensitivity of seed germination or seed dormancy but was found to affect seed storability and seedling vigor. Seed storability was assessed in a controlled deterioration test, in which the germination capacity of the mutant decreased with the duration of the treatment. The decrease in viability and vigor was confirmed by storing the seeds in two relative humidities (RHs) for a prolonged period. At 60% RH, the mutant lost germinability, but storage at 32% RH showed no decrease of germination although seed vigor decreased. The decrease in viability and vigor could be related to an increase in conductivity, suggesting membrane deterioration. This was not affected by light conditions during imbibition, expected to influence the generation of active oxygen species. During seed maturation, ABI3 regulates several processes: acquiring dormancy and long-term storability and loss of chlorophyll. Our results indicate that GRS is a common regulator in the latter two but not of dormancy/germination.

We examined the role of gibberellins (GAs) in germination of Arabidopsis seeds by a proteomic approach. For that purpose, we used two systems. The first system consisted of seeds of the GA-deficient ga1 mutant, and the second corresponded to wild-type seeds incubated in paclobutrazol, a specific GA biosynthesis inhibitor. With both systems, radicle protrusion was strictly dependent on exogenous GAs. The proteomic analysis indicated that GAs do not participate in many processes involved in germination sensu stricto (prior to radicle protrusion), as, for example, the initial mobilization of seed protein and lipid reserves. Out of 46 protein changes detected during germination sensu stricto (1 d of incubation on water), only one, corresponding to the cytoskeleton component α-2,4 tubulin, appeared to depend on the action of GAs. An increase in this protein spot was noted for the wild-type seeds but not for thega1 seeds incubated for 1 d on water. In contrast, GAs appeared to be involved, directly or indirectly, in controlling the abundance of several proteins associated with radicle protrusion. This is the case for two isoforms of S-adenosyl-methionine (Ado-Met) synthetase, which catalyzes the formation of Ado-Met from Met and ATP. Owing to the housekeeping functions of Ado-Met, this event is presumably required for germination and seedling establishment, and might represent a major metabolic control of seedling establishment. GAs can also play a role in controlling the abundance of a β-glucosidase, which might be involved in the embryo cell wall loosening needed for cell elongation and radicle extension.

To better understand seed germination, a complex developmental process, we developed a proteome analysis of the model plant Arabidopsis for which complete genome sequence is now available. Among about 1,300 total seed proteins resolved in two-dimensional gels, changes in the abundance (up- and down-regulation) of 74 proteins were observed during germination sensu stricto (i.e. prior to radicle emergence) and the radicle protrusion step. This approach was also used to analyze protein changes occurring during industrial seed pretreatments such as priming that accelerate seed germination and improve seedling uniformity. Several proteins were identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Some of them had previously been shown to play a role during germination and/or priming in several plant species, a finding that underlines the usefulness of using Arabidopsis as a model system for molecular analysis of seed quality. Furthermore, the present study, carried out at the protein level, validates previous results obtained at the level of gene expression (e.g. from quantitation of differentially expressed mRNAs or analyses of promoter/reporter constructs). Finally, this approach revealed new proteins associated with the different phases of seed germination and priming. Some of them are involved either in the imbibition process of the seeds (such as an actin isoform or a WD-40 repeat protein) or in the seed dehydration process (e.g. cytosolic glyceraldehyde-3-phosphate dehydrogenase). These facts highlight the power of proteomics to unravel specific features of complex developmental processes such as germination and to detect protein markers that can be used to characterize seed vigor of commercial seed lots and to develop and monitor priming treatments.

The performance of cucumber (Cucumis sativusL.) seeds in relation to ovary and ovule position was monitored during seed production. Seeds from three (first, seventh and tenth nodes) fruit positions and three (stylar, intermediate and peduncular) ovule positions were harvested serially during development and characterized with physiological and cellular markers. Seed moisture contents declined to 30%approx. 35 d after pollination (DAP) and remained constant thereafter. At 42 DAP the maximum dry weight was acquired in seeds except those from peduncular segments, whose dry weight accumulation lagged behind. The onset of germinability and desiccation tolerance occurred before the attainment of maximum dry weight, whereas seed performance, as shown by laboratory germination and greenhouse emergence, was largely improved after the completion of seed development. Cell cycle activities (DNA per nucleus and β-tubulin content) ceased by 28 DAP, whereas the degradation of seed chlorophyll continued during the entire maturation period. Seeds from the top fruit and from the peduncular fruit segments were delayed in reaching maximum quality compared with seeds from other positions, and this was correlated with a slower decline of chlorophyll fluorescence.