Seed Science Research

The seed reserves of sugar beet (Beta vulgaris L.) are starch, located exclusively in the perisperm, and lipids, proteins and a small quantity of soluble sugars, located mainly in the cotyledons. It is shown that lipids are the main respiratory substrate used during germination whereas starch and remaining lipids are only hydrolysed after root extrusion, to sustain root and hypocotyl growth. By removing the perisperm during imbibition, it was demonstrated that lipids and proteins alone are sufficient for viable seedling development, though such seedlings grew less than those developing in the presence of perisperm. The utilization of seed reserves was followed during seedling development in the dark in various organs. At 20°C, the reserves were sufficient for 8 d of growth in the dark, the hypocotyl attaining a length of 5 cm. Specific problems relating to field establishment of sugar beet are discussed in relation to these results.

After synthesis in the vegetative parts of the plant, assimilates are translocated to fruits through xylem and phloem. Research on factors controlling nutrient transport into developing seeds via the phloem has been stimulated by the development of the empty seed coat technique.

There is a consensus that as assimilates are transported from maternal tissues to filial tissues they are delivered to the extracellular space (the apoplast) separating the two generations, prior to uptake from the apoplast into the tissues of the embryo or endosperm. The empty seed coat technique has been used for the study of several aspects of nutrient transport into seeds, e.g. metabolic control and turgorsensitive transport. The osmotic environment of seed tissues has a strong effect on assimilate transport into empty seeds. Several lines of evidence suggest that one of the main ‘secrets’ of the high sink strength of developing seeds, at least in many taxonomic groups of dicotyledons, is that the sink end of the phloem pathway is ‘bathed’ in an apoplast solution with a high concentration of osmotically active solutes. Data on maize do not fit this pattern. A turgor homeostat mechanism may help to maintain high solute concentrations in the seed apoplast. The apoplast environment of seed tissues may also stimulate synthesis of storage proteins and be involved in the prevention of precocious germination. In addition to the osmotic environment, other factors influencing sink strength are discussed. Some aspects of solute transformation during transport through seed tissues are described.

To elucidate the molecular aspects of cereal endosperm development, a good understanding of the histo-differentiation process is necessary. Reviewing the extensive history of lightand electron-microscopic investigations has revealed similarities between cereals with respect to cell ontogeny. Thus, using barley as a model and taking into account many developmental mutants in this and other species, a model for endosperm differentiation is proposed. Based on this model, molecular studies on endosperm gene-regulation are discussed and the available techniques for further investigations are considered.

Somatic embryo quality is an important aspect of artificial or synthetic seed production and is dependent on proper morphological development and deposition of sufficient storage reserves to support early growth of the seedling. The abilities of a somatic embryo to survive desiccation and to establish a rapidly growing seedling were used as estimates of quality and were dependent on a number of factors during the elongation and maturation phases of somatic embryo development. Embryo quality was significantly enhanced when the sucrose content of the elongation and maturation media was increased to 50 g I−1 from 30 g I−1. This increased dry weight to >2 mg embryo−1 suggesting greater deposition of storage reserves. Higher sucrose concentrations were inhibitory and reduced survival after desiccation and seedling vigour. An optimum number of embryos can be supported on one Petri plate; the number was controlled by the plating density of the embryogenic cell clusters spread on the elongation medium. This optimum was dependent to some extent on the sucrose content of the medium, indicating that sucrose had a nutritive effect on embryo development and not solely an osmotic effect. Light intensity was another critical factor. Intensities > 75 μmol m−2 s−1 photosynthetic photon flux density (PPFD) at the elongation stage of development blocked globular-stage somatic embryo development. Maturing somatic embryos were far less sensitive to high light intensities and development was more rapid at 150 μmol m−2 s−1 PPFD. Light quality was manipulated by covering plates with filters that selectively transmitted certain regions of the visible spectrum. All filter treatments reduced embryo yield and quality compared with embryos developing under fullspectrum light; varying the light spectrum does not seem to be a viable method of enhancing somatic embryo quality.

Totalling more than 3000 species, parasitic angiosperms are a most diverse group of organisms. Classified as either shoot or root parasites according to site of attachment, plant parasites are further classified, according to the degree of parasitism, into one of two divisions: holoparasitic or hemiparasitic. Within these divisions, angiosperm parasites are either obligate or facultative.

Obligate parasites cannot survive to maturity without attaching to a host plant, thus the need to germinate in close proximity to a host first characterizes the specialized germination and seed dispersal strategies of these plants. However, seeds of all species of the rootholoparasitic Orobanchaceae and four genera within the Scrophulariaceae have evolved very specialized germination strategies. Once the seeds have undergone an after-ripening period, they require a prolonged imbibition period at temperatures of approx. 30°C before they acquire the potential to germinate. Minute seeds with very little reserves, they must germinate within millimetres of the host root for successful host attachment and continued survival. To this end, germination only occurs in responseto specific chemical germination triggers released from the host root. In Striga hermonthica (Del.) Benth., the perception of the host-derived trigger results in stimulation of endogenous ethylene production and it is this ethylene that initiates germination. It is proposed that the chemical triggers released from the host be consideredas ‘elicitors’ which function primarily as allelochemicals and that the parasite seeds have become adapted in such a way as to have an absolute requirement for external germination triggers.

Wheat seeds were imbibed in solutions of polyethylene glycol (PEG) and NaCl, and embryo water content and incorporation of [35S]methionine into proteins were determined. Both osmotica reduced water uptake and protein synthesis, though these were immediately resumed upon removal of stress. Results of in vivo labelling of newly synthesized proteins showed differences in the synthesis of many polypeptides when embryos that had imbibed in water passed from the radicle pre-emergence phase to early growth. The synthesis of a group of proteins associated with radicle emergence was reduced during imbibition in PEG or NaCl. All the major proteins of the pre-germination phase were produced in PEG-treated embryos, while some new polypeptides (‘salt stress’ proteins?) were actively synthesized in salt-treated embryos. Upon removal of PEG or NaCl, synthesis of proteins common to the phase of early embryo growth increased, while specific saltimbibition proteins disappeared. These findings are consistent with the hypothesis that biochemical processes leading to radicle emergence can be affected by osmotic stress and are likely to be damaged by severe stress.

Seed development and changes in germination ability and longevity were monitored in two spring and two winter cultivars of each of barley (Hordeum vulgare L.) and wheat(Triticum aestivum L.). Physiological maturity (end of the seed-filling period) occurred between 31 and 41 d after 50% anthesis in the eight cultivars, at which time seed moisture contents had declined naturally to 40–49% (wet basis). Most seeds were able to germinate (in 28 d tests at 10°C) following enforced desiccation at this time (normal germination varied from 83% in one cultivar of winter wheat to 97% in one of spring wheat).Potential seed longevity continued to increase after physiological maturity. The intervalbetween physiological maturity and the time when seeds attained maximum potential longevity varied between 3 and 21 d; maturation drying had reduced seed moisture contents to 16–28% (wb) at this time. The results for all eight cultivars contradict the hypothesis that maximum seed quality is attained at physiological maturity and thereafter declines. The term physiological maturity is potentially misleading, therefore; mass maturity is more appropriate to describe this developmental stage. It is shown that the results of accelerated ageing or controlled deterioration tests may provide misleading conclusions in seed quality development studies if the single storage treatment chosen is inappropriate.

Nuclear DNA of wheat embryos was probed with a synthetic oligonucleotide (C3TA3C3TA3) to assess changes in the length of telomeres during storage and germination. Chromosomal DNA of dry embryos, especially those derived from long-term-stored caryopses, was considerably depleted of telomeric repeats, but, shortly after the start of imbibition, the chromosomal DNA became rich in sequences hybridizing to the telomeric probe. Simultaneously, newly synthesized telomere-related sequences appeared in an extrachromosomal form that could be separated from bulk nuclear DNA by velocity and isopycnic centrifugations. It is concluded that, in addition to typical repair events, rapid synthesis of telomeric repeats occurs in wheat embryos at the onset of germination.

The aim of this work was to analyse the capacity for protein synthesis in embryonic axes from long-term-stored maize seeds, including the role of proline. Embryonic axes from seeds stored for 13 years (S) and non-stored seeds (NS) were incubated in nutrient media after application of [14C]proline. Transformation of [14C]proline into other amino acids was analysed by thin-layer chromatography. After 6 h of incubation, no other labelled amino acids were found. Incorporation of 14C into total soluble and cell-wall (proline-rich) proteins was assessed during this period. Incorporation of [14C]proline into specific cell-wall proteins was lower in S than in NS axes.

Studies using [35S]methionine showed that protein synthesis was slower in axes of S than in NS seeds. Analyses of these proteins by gel electrophoresis and fluorography revealed qualitative differences between the [35S]methionine proteins synthesized by both types of axes. The NS: S ratios for the [35S]proteins were larger than those from the [14C]proline assays. These data may be interpreted as an indication of differential deterioration of transcription or translation in the axes during long-term seed storage.

Changes in tomato (Lycopersicon esculentum Mill.) seed quality were monitored during seed development and maturation in glasshouse experiments in 2 years. The end of the seedfilling period (mass maturity) occurred 35–41 d after anthesis (differing among trusses) in 1989 and 42 d after anthesis in 1990. Seed moisture contents at this developmental stage were 53–72% (wet basis), while the onset of ability to germinate (during 21-d tests at 20°/30°C) and the onset of tolerance to rapid enforced desiccation occurred just before (1990) or just after (1989) mass maturity. In 1989, seed quality was assessed primarily by seedling size in a glasshouse experiment; maximum mean seedling dry weight 25 d after sowing was not achieved until 24–40 d after mass maturity. In 1990, seed quality was assessed primarily by germination following storage; maximum normal germination after 35 d in storage at 40 °C with 14 ± 0.5% moisture content was attained 23 d after mass maturity, but with little difference among seed lots harvested 10 d earlier or up to 30 d later. The results contradict the hypothesis that maximum seed quality is attained at the end of the seed-filling period and that seed viability and vigour begin to decline immediately thereafter.

Changes in germination and desiccation sensitivity were measured throughout the seed expansion phase of development in fruits of Quercus robur L. The onset of a reduction in sensitivity to desiccation during development on the tree coincided with acquisition of the capacity for seed germination on moist sand substrate. Tolerance of desiccation then increased throughout development to shedding, but viability was still lost at a relatively high moisture content, and seeds did not therefore pass through a fully desiccation-tolerant phase. These data suggest that desiccation sensitivity in Q. robur may have resulted from the premature termination of development.

No desiccation was required to initiate germination in prematurely harvested fruits orseeds. Germination rate of seeds on moist sand increased at successive harvests during development, and was also increased by presoaking seeds in water. Variation in germination rate following shedding was not related to seed size or moisture content, but was affected by shedding date. This effect was not observed when the pericarp was removed.

Partial drying of non-dormant seeds had little effect on germination behaviour compared with undried controls. In contrast, partial drying resulted in a marked increase in the germination response of freshly harvested (dormant) seeds of Z. palustris. Partial drying also resulted in a 100-fold reduction in the concentration of gibberellins (GA4+7) required for maximum germination. Although the concentration range of applied abscisic acid (ABA) that permitted germination was unaltered, partial drying increased the final germination percentage at all concentrations tested. The concentration of ABA above which germination was reduced in freshly harvested (dormant) seeds of Z. palustris was 3.16 × 10−7m compared with 10−4m in fresh (non-dormant) seeds of P. coarctata. Changes in the germination response of Z. palustris seeds to applied growth regulators, following partial drying or cold stratification might be explained by similar changes in hormonal status.

The effects of development of leek seeds at 20/10°, 25/15° and 30/20°C (day/night) and drying of seed harvested at different developmental stages on subsequent performance were examined in each of 3 years. An increase in temperature from 20/10° to 30/20°C reduced mean seed weight from 2.90 to 2.55 mg as a result of a reduction in the duration of seed growth from 80 to 55 days; seed growth rate was unaffected. Seed moisture content reached a minimum, up to 35 days after the attainment of maximum seed dry weight and 115, 90 and 70 days after anthesis at 20/10°, 25/15° and 30/20°C, respectively. The curves relating seed moisture to time for each temperature regime were mapped onto a single line accounting for >90% of the variation in moisture content, using accumulated day-degrees >6°C instead of chronological time. Seeds were capable of germinating when seed moisture contents were >60% (fresh weight basis), but maximum viability and minimum mean time to germination were not attained until seed moisture contents at harvest had fallen to 20–30%. Germination was little affected by temperature of seed development. Drying immature seeds increased percentage germination. Growing seeds at 30/20°C and drying at 35°C and 30% RH raised the upper temperature limit of germination compared with growing at 20/10°C and drying at 15°C and 30% RH.

Profiles of proteins synthesized in vivo in radicle tips were compared among unaged, artificially aged (controlled deterioration: 10% moisture content at 42°C for 3 weeks), osmoprimed (−1.5 MPa polyethylene glycol 6000 at 20°C for 1 week), and artificially aged and subsequently osmoprimed cauliflower (Brassica oleracea L.) seeds. Germination and initiation of incorporation of 35S-methionine into radicle tips were delayed by controlled deterioration. Osmopriming accelerated these processes and also alleviated the delays. The labelled proteins were analysed by two-dimensional gel electrophoresis and fluorography. Proteins were found whose expression correlated with the rate of germination, and was reduced by controlled deterioration but enhanced by osmopriming. Subsequent osmopriming reversed the effect of controlled deterioration and even caused an enhancement up to the level of unaged osmoprimed seeds. These proteins appeared to be related to processes preceding visible germination. In unaged seeds, formation of the proteins in the radicle tip began upon imbibition but decreased towards visible germination. On silver-stained gels, one of the proteins was shown to be mobilized by the time of radicle protrusion, while all were found in dry seeds. Proteins whose pronounced expression was observed only after both treatments were also found.

A survey of Pisum genotypes for seed trypsin inhibitors revealed a tenfold range in the extent of inhibition. Approximately 90% of trypsin inhibitory activity was associated with two albumin fractions in selected variant lines. The differences among extreme variants were consistent in three environments, between two sources of trypsin tested and whether expressed on a unit protein or dry weight basis.

A study of the appearance of trypsin inhibitors during seed development in selected highand low-inhibitor lines showed differences in the accumulation pattern of active inhibitors. An endogenous protease was identified in Pisum seed protein preparations, whose in vitro trypsin-like activity was predominant in protein from early stages of seed development, when little or no trypsin inhibitor was present. However, there was no correlation between the amount of this protease and the extent of trypsin inhibitory activity in lines that varied for inhibitor content.

Submerging Phaseolus vulgaris cv. Top Crop seeds in air-saturated water for 16 h markedly depresses subsequent germination. This is termed soaking injury. Soaking injury does not occur in seeds soaked in CO2-saturated water. Previous studies have shown that soaking injury can be alleviated by drying seeds or removing seed coats. Submergence therefore leads to a situation in bean seeds which is similar to secondary dormancy.

As with dormant seeds, C6/C1 ratios of embryonic axes of seeds soaked in air-saturated water remained high (0.8–1.0) during and after soaking. This was paralleled by low activities of glucose-6-phosphate dehydrogenase (EC.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44). In axes of seeds soaked in CO2-saturated water and in unsoaked seeds C6/C1 ratios declined steadily during soaking/imbibition and reached values of around 0.3 after germination. Slight increases ofglucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities occurred in the pre-germination phase. This was followed by a massive increase after radicle emergence. Synthesis of the plastid isoenzymes was a post-germinative event.

It appears that soaking injury depresses protein synthesis. Lack of oxidative pentose phosphate pathway activity appears to be a causative factor in soaking injury.

Protein disulfide isomerase (PDI E.C. 5.3.4.1) catalyses the formation of disulfide bonds in secretory or cell-surface proteins during their biosynthesis. PDI activity is associated with the microsomal fraction of the cell and has been found in several mammalian tissues and in developing wheat endosperm.

The embryo and aleurone layer of germinating seeds of the wheat cultivar Galahad showed PDI activity associated with a microsome-enriched fraction of cell homogenates PDI activity in microsome-enriched fractions from aleurone layers of normally germinating seeds was significantly higher than in their abnormally germinating counterparts and diminished as germination proceeded.

Dry embryos and germinating embryos showed less PDI activity than aleurone layers. There was no significant difference in PDI activity between microsome-enriched fractions from embryos of normally and abnormally germinating seeds at 3 or 6 days of germination.

Wheat aleurone PDI was partially purified using gel filtration and had a molecular mass of 110–130 kDa and a monomeric molecular mass of ca. 57 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

The effects of exogenous phaseic acid (PA) on germination and protein accumulation of cultured immature barley embryos were examined. Chemically synthesized PA was racemic, 87% pure and stable over the course of the experiment. Germination was observable in >90% of the untreated embryos after 3 days of incubation, whereas embryos treated with 10μm abscisic acid (ABA) or PA showed no evidence of germination. Buffer extracts from embryos treated with ABA or PA had similar protein profiles when examined by single and two-dimensional electrophoresis. The profiles differed significantly from those of embryos incubated in the absence of the two compounds. Concentrations of α-amylase inhibitor and barley-germ agglutinin (BGA) increased upon treatment of immature embryos with ABA or PA. This was due to de novo synthesis as there was increased incorporation of radioactivity from 35S-labelled amino acids into the proteins in treated embryos. Endogenous ABA content in PA-treated embryos was not significantly different from that in untreated embryos. An analogue of ABA, 2′, 3′-dihydroabscisic acid, which cannot be metabolized to phaseic acid, inhibited germination and caused increased synthesis of α-amylase inhibitor and germ agglutinin. ABA and PA may both be active in promoting responses associated with ABA in barley embryos, but the embryos are more sensitive to ABA.

Seeds of watermelon (Citrullus lanatus cv. Sugar Baby) germinated optimally in the dark, throughout the range 20–40°C. Germination was inhibited by continuous (c) irradiation with farred (FR), blue or white incandescent light; short pulses were ineffective. Intermittent FR could fully substitute for cFR; the effect of alternating red and FR pulses during 30-min dark cycles depended upon the timing of the illuminations within each cycle. It is concluded that germination in watermelon is controlled by the low-energy reaction of phytochrome. However, continuous and intermittent red light resulted in partial reductions in germinability. Opening the seed coat at the radicle end enhanced germination in the dark and reduced photosensitivity towards FR. The outer, lignified part of the testa exerted a mechanically restrictive force upon the expanding radicle; this force was estimated to be equivalent to 0.3 MPa. The kinetics, at 25°C in the dark, of the time course of germination and the escape from the inhibitory actions of cFR and osmoticum (0.5m mannitol) were all sigmoid curves, which, upon transformation to normal distributions, had different means, but statistically similar variances. The cFR-irreversible activation of germination by phytochrome and the mannitol-irreversible onset of radicle elongation preceded radicle protrusion (mean at 32 h, 25°C) by 8 and 6 h, respectively. From the results and data on imbition, it is concluded that activation of germination in watermelon takes place during the second, stationary phase of imbibition (20–30 h after sowing at 25°C).