Effects of osmotic priming and ageing on the germination and emergence of carrot and leek seed

Carrot and leek seed was osmotically primed in polyethylene glycol solution (273 g/kg water and 342 g/kg water respectively) for 10, 14 or 17 days before accelerated ageing for 0, 24, 48, 72 or 96 h. Priming reduced the germination time compared with non-primed seed. Accelerated ageing increased germination and emergence times and decreased percentage germination and emergence to a greater extent for the primed seeds than for non-primed seeds in both species. Primed and dried but non-aged seed from both species was stored at 10°C for 12 months. There was no loss of viability and improvements in germination time due to priming were maintained throughout the storage period for all the priming treatments in leek, and for the 10 and 14 day priming treatments in carrot. Carrot seed primed for 17 days lost some viability after 12 months storage compared with non-stored seed.     

The Effects of Priming and Accelerated Ageing upon the Nucleic Acid Content of Leek Seeds and their Embryos

Using seed priming and accelerated ageing techniques, a singlelot of leek (Allium porrum) seeds was manipulated to producefour lots of seeds with different germination performance. Changesin content of the major nucleic acid species in whole seedsand embryos of two of these lots (primed and unprimed), weredetermined over the early stages of germination. The major effectof priming was an increased level of RNA species in the seedsand embryos, and this difference was maintained during germination.Comparison of nucleic acid levels in the dry seeds of thesetwo lots and two others (aged and aged then primed) indicatedthat there was no correlation with germination performance.Similar comparisons of the nucleic acid levels in the embryosof seeds imbibed for 1 d showed only a limited correlation betweenrRNA levels and germination performance. Analysis of these datasuggests that accelerated ageing has an adverse effect uponendosperm cells, which results in the degradation of their nucleicacids during priming. Furthermore, the viability of these agedseeds also falls during priming. The data also indicate thatratios of rRNA to DNA correlate with germination performanceof the four lots of seeds studied. It is proposed that sucha relationship is indicative of the efficiency of a primingtreatment, and may be useful in comparisons of naturally varyingseed lots. Key words: Leek, seed, germination, priming, nucleic acids     

Physiological and enzymatic activity of pepper seeds (Capsicum annuum) during priming

Pepper ( Capsicum annuum L. cv. Keystone Resistance Giant 3) seeds were monitored during priming to determine if seed treatments which accelerate the rate of germination could be correlated with specific physiological changes within the seeds. Pepper seeds primed with −0.90 and −1.35 MPa NaCl solutions at 23°C for 18 days did not completely equilibrate with the osmotic potential of the priming solution. Seed respiratory rates indicated that priming extends the lag phase of germination following imbibition. Soluble protein levels increased 115% in primed seeds, and the uptake and incorporation of [¹⁴C(U)] labelled amino acids into the acid insoluble fraction increased throughout the priming treatments. Alcohol dehydrogenase (EC 1.1.1.1, anaerobic metabolism), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44, pentose phosphate pathway) activities remained stable throughout the priming treatment, but were higher after 6 days. than the water-imbibed controls. Aldolase (EC 4.2.1.1. glycolysis) and isocitrate lyase (EC 4.1.3.1, glyoxylate cycle) activities increased with imbibition and were 61 and 56% (respectively) higher in primed seeds as compared to the water-imbibed controls after 12 days. Treatment with the −0.90 MPa NaCl solution was more effective than the −1.35 MPa solution in improving the rate of germination, yet there were no significant differences between the protein concentrations or enzyme activities of the two priming treatments. However, the incorporation of labelled amino acids into pepper seeds was significantly higher in the −0.90 MPa priming treatment.     

The effects of re-use of polyethylene glycol priming osmotica upon the development of microbial populations and germination of leeks and carrots

Three batches of leek seeds were osmotically primed successively in the same polyethylene glycol solution in a bubble column at a seed concentration of 100 g/litre for seven days at 15°C. Three batches of carrot seeds were similarly primed in a separate solution for six days at 15°C. The concentration of microorganisms in the solutions increased rapidly during priming of the first seed batch for both seed types, but increases during priming of the second and third batches were small. The seeds were the main source of the microorganisms; priming reduced the numbers of colonies of filamentous fungi and increased those of bacteria and yeasts. The priming treatments improved the percentage germination of the three seed batches of primed carrots and reduced the mean time to germination in both species and the mean time to emergence in compost. Percentage emergence was not affected by priming except in the third batch of primed carrot seed. The presence of large numbers of microorganisms in the priming solutions did not greatly affect seed performance when the same osmoticum was used three times with leeks and twice with carrots. Priming did not affect the number of abnormal seedlings.     

Ageing and Osmotic Priming in Wheat Seeds: Effects Upon Certain Components of Seed Quality

Deteriorated wheat seed lots were osmotically primed and surface-dried,dried-back, then aged for two more weeks Osmotic priming didnot affect the final germination in any of the aged seed lots,but mean germination time was decreased The response of unagedseed lot to treatments was studied in the following the timecourses of germination, as well as L-[4, 5-²H]leucine and [6-³H]thymidineincorporation (as a measure of protein and DNA synthesis, respectively)into dissected embryos during the early hours of germinationIt was found that the variation in mean germination time andthe rate of synthetic reactions were related The greatest improvementin the components of seed lot quality was achieved after primingand surface drying The beneficial effects of priming were apparenteven after dehydration followed by two further weeks of ageingCumulative correlations between mean germination time and variousbiochemical tests in variously treated aged seed lots were highlysignificant The physiological processes involved in deteriorationand recovery and the potential application of biochemical testsin detecting vigour changes are discussed Triticum durum, seed, ageing, osmotic priming, mean germination time, protein DNA synthesis     

Responses of Vegetable Seeds to Controlled Hydration

Leek, onion and carrot seeds were imbibed in water and in solutionsof polyethylene glycol (PEG) 6000 over the range –0.5to –4.0 MPa osmotic potential, for periods up to 28 dat 15 C. Seeds started to germinate after 7 and 14 d at –0.5MPa and –1.0 MPa PEG, respectively, but in the lattercase, germination did not exceed 5%. No germination occurredin solutions of lower (more negative) osmotic potential. Seedmoisture content increased with osmotic potential in all threespecies and the relationships were unaffected by the durationof imbibition in solutions of –1.0 to –4.0 MPa,though leek seeds had higher moisture contents than the otherspecies for any given osmotic potential. Linear relationships between response to priming (differencebetween mean germination times of primed and untreated seeds)and seed moisture content were obtained for each species, positiveresponses being obtained above 30–35% seed moisture contentwith optima at 46, 44.5 and 44% seed moisture contents in leek,onion and carrot, respectively. Priming had no effect on embryovolume or cell number per embryo in leek and onion. Carrot embryovolume increased by 43% and cell number per embryo doubled inprimed compared with untreated seeds, whereas seeds imbibedin water showed only a slight increase in cell number per embryoat the stage when radicles were beginning to penetrate the seedcoat. Allium cepa L. cv. Rijnsburger Robusta, onion, Allium porrum L. cv. Winterreuzen, leek, Daucus carota L. cv. Nantaise, carrot, germination, priming, polyethylene glycol, seed moisture, cell number, embryo volume     

Pyrimidine nucleotide and nucleic acid synthesis in embryos and megagametophytes of white spruce (Picea glauca) during germination

Pyrimidine nucleotide synthesis was investigated in isolated germinating zygotic embryos and separated megagametophytes of white spruce by following the metabolic fate of ¹⁴C-labelled orotic acid, uridine, and uracil, as well as by measuring the activities of the major enzymes participating in nucleotide synthesis. The rate of nucleic acid synthesis in these tissues was also examined by tracer experiments and autoradiographic studies conducted with labelled thymidine, and by conventional light microscopy. From our results, it emerges that changes in the contribution of the de novo and salvage pathways of pyrimidines play an important role during the initial stages of zygotic embryo germination. Preferential utilization of uridine for nucleic acid synthesis, via the salvage pathway, was observed at the onset of germination, before the restoration of a fully functional de novo pathway. Similar metabolic changes, not observed in the gametophytic tissue, were also documented in somatic embryos previously. These alterations of the overall pyrimidine metabolism may represent a strategy for ensuring the germinating embryos with a large nucleotide pool. Utilization of ¹⁴C-thymidine for nucleic acid synthesis increased in both dissected embryos and megagametophytes during germination. Autoradiographic and light microscopic studies indicated that soon after imbibition, DNA synthesis was preferentially initiated along the embryonic axis, especially in the cortical cells. Apical meristem reactivation was a later event, and the root meristem became activated before the shoot meristem. Taken together, these results indicate that precise changes in nucleotide and nucleic acid metabolism occur during the early phases of embryo germination.     

DNA and histone synthesis are uncoupled during germination of maize embryos

The rate of synthesis of DNA and histones was studied in germinating maize embryos as a function of the length of the germination period. To that end excised embryos from seeds germinated for different periods of time were pulse labelled either with [¹⁴C]protein hydrolysate or with [³H]TdR. Specific activities were determined for the total cellular proteins and the total histone fraction obtained by acid-extraction of the cellular homogenate and BioRex70 ion exchange chromatography. The results show that the early germination period is characterized by a lack of coupling between the histone synthesis and that of the nuclear DNA. The early histone synthesis peak might be necessitated by the reprogramming of the embryo genome that takes place during germination.     

Effect of Matric-Priming Duration and Priming Water Potential on Germination of Four Grasses

In this study, a matric-potential control system was used todetermine the effect of matric-priming duration and primingwater potential on the germination response of Bouteloua curtipendula(Michx.) Torr., Cenchrus ciliaris L., Eragrostis lehmannianaNees, and Panicum coloratum L. Seeds were primed at water potentialsof –1·5 to –7·7 MPa for up to 14 d.Optimum germination generally occurred in treatments primedat high water potential for the shortest period. Germinationof seeds primed at lower water potential and for longer periodsexhibited a negative germination response relative to the control.Seeds were not redried after the priming treatment. Seed-wateruptake measurements suggest that a reduction in the lag timeof imbibition accounted for at least some germination-rate enhancementin the positive-priming treatments Key words: Germination, matric-priming, imbibition     

Cell division versus cell elongation: the control of radicle elongation during thermoinhibition of Tagetes minuta achenes

Endogenous embryo factors, which act mainly in the radicle, prevent germination in Tagetes minuta at high temperatures. These factors act to prevent cell elongation, which is critical for radicle protrusion under optimal conditions. Once the radicle has emerged both cell elongation and cell division are required for post-germination growth. Germination can be induced at high temperatures by fusicoccin, which rapidly stimulates cell elongation. In addition, priming seeds at 25 °C on polyethylene glycol (PEG) 6000 and mannitol could also induce germination on water at 36 °C, indicating that priming prevents radicle protrusion at a point subsequent to the point of control in thermoinhibited achenes. Flow cytometry studies revealed that DNA synthesis occurs during thermoinhibition and the inhibition of DNA synthesis during this process inhibits subsequent germination on water under optimal conditions, suggesting a protective role for DNA synthesis in thermoinhibited achenes of T. minuta.     

DNA synthesis and mRNA accumulation during germination of embryos of the orchidSpathoglottis plicata

Summary Germination of embryos of the orchid,Spathoglottis plicata Blume involves the formation of a protocorm in which DNA synthesis and cell divisions are confined to the proximal end whereas cells at the distal end undergo enlargement. Although³H-thymidine was incorporated into the distal cells of the embryo during the early period of germination, DNA synthesis was not followed by mitosis and cytokinesis. Poly(A)-RNA detected by in situ hybridization of sections to³H-poly-(U) was present uniformly in all cells of the embryo of the dry seed. However, coincident with the formation of the apical meristem, there was an increase in the density of auto-radiographic silver grains in the cells of the embryo, with a concentration of silver grains in the proximal part. The results indicate that regulatory events in the embryo prior to seed maturity determine the fate of its proximal and distal parts during germination.Dedicated to the memory of Professor John G. Torrey     

Free radical generation in Pinus sylvestris and Larix decidua seeds primed with polyethylene glycol or potassium salt solutions.

Electron paramagnetic resonance (EPR) spectra of Pinus sylvestris and Larix decidua seeds show that priming with PEG+200 mg kg(-1) gibberelic acid (GA(3)) results in appreciably higher free radical contents than in unprimed control samples. Only relatively minor changes in the free radical levels were observed in seeds primed with K(+) salts. However, both priming treatments have been reported previously to result in faster germination rates compared to controls without changing the germination percentage. In measurements on individual seeds of L. decidua, there were no significant differences between the mean free radical levels in viable and non-viable seeds within each treatment group. Thus, the elevation in free radical levels in the PEG+GA(3) treatments appear to be a direct consequence of the priming treatment and do not correspond to the initiation of germination.     

The phiX174-type primosome promotes replisome assembly at the site of recombination in bacteriophage Mu transposition.

Initiation of Escherichia coli DNA synthesis primed by homologous recombination is believed to require the phiX174-type primosome, a mobile priming apparatus assembled without the initiator protein DnaA. We show that this primosome plays an essential role in bacteriophage Mu DNA replication by transposition. Upon promoting transfer of Mu ends to target DNA, the Mu transpososome undergoes transition to a pre-replisome that permits initiation of DNA synthesis only in the presence of primosome assembly proteins PriA, DnaT, DnaB and DnaC. These assembly proteins promote the engagement of primase and DNA polymerase III holoenzyme, initiating semi-discontinuous replication preferentially at the Mu left end. The results indicate that these proteins play a crucial role in promoting replisome assembly on a recombination intermediate.     

Developmental Aspects of Ethylene Biosynthesis during Somatic Embryogenesis in Tissue 4Medicago sativa

Rates of ethylene production were determined in highly embryogenicand virtually non-embryogenic tissue cultures of Medicago sativassp. falcata during a 10-d induction period on medium containing2, 4, -dichlorophenoxyacetic acid and kinetin, and during thefirst 10 d of somatic embryo formation on growth regulator-freemedium. In cultures of both genotypes, ethylene production increasedrapidly, peaked after 10 d and then declined again rapidly.This fall in ethylene production rate also occurred when thecultures were kept on the growth regulator-containing medium,but it was more pronounced in cultures which were transferredto growth regulator-free medium, i.e. under conditions favouringsomatic embryo formation. There were only minor differencesin the rates of ethylene production between the two genotypes,mainly after transfer to growth regulator-free medium. Cobaltand nickel ions strongly inhibited ethylene biosynthesis throughoutthe culture period under investigation. They did not, however,affect embryo induction, but exerted strongly inhibitory effectson somatic embryo formation. Amino-oxyacetic acid and aminoethoxyvinylglycinehad no effect on ethylene production during the inductive period.It is concluded from these experiments that the high rates ofethylene production during embryo induction are not essentialfor subsequent embryo differentiation. Key words: auxin, ethylene, Medicago sativa, somatic embryogenesis, tissue culture     

Germination of Rye Embryos Following Hydration-Dehydration Treatments: Enhancement of Protein and RNA Synthesis and Earlier Induction of DNA Replication

On germination of low viability embryos of rye, var. LovaszPatonai, the rate of protein synthesis increases during theearly hours of imbibition and high rates of DNA replicationcommence around the 9th hour. If embryos are imbibed for 3 or6 h then dehydrated back to their original weight, their rateof protein synthesis when next imbibed closely corresponds tothat of embryos germinated for a period equal to that of thehydration pre-treatment plus the duration of the second imbibition.Pre-treatment also enhances subsequent RNA synthesis and embryoshydrated for 9 h then dehydrated start major DNA synthesis atonce as water is again supplied. Many changes occurring duringthese periods of hydration pre-treatment must therefore be stableto subsequent dehydration. Damage occurs to areas that are firstactive in protein and RNA synthesis if pre-treatments extendbeyond 9 h and subsequent germination of the embryo is thenimpaired. The implications of these results are discussed inrelation to the effects of hydration pre-treatments upon enhancedgermination and the stability to dehydration of the productsof early RNA and protein synthesis.     

Application of Beneficial Microorganisms to Seeds during Drum Priming

Five microbial plant growth promoters or biocontrol agents (Pseudomonas fluorescens CHA0, Pseudomonas sp. AB842, Bacillus subtilis MBI600, Trichoderma harzianum T22 and T. virens G20) were assessed for ability to proliferate on seeds of carrot, parsnip and leek. In small-scale priming systems, both pseudomonads and MBI600 (when applied as cells) at levels between 10⁵ and 10⁶ cfu g^?1 seed were able to colonise all seeds at the end of priming (240 h) despite initial poor recovery after addition of the cells in some cases. Pf CHA0 was a particularly aggressive seed coloniser often comprising the total pseudomonad population at the end of priming. Drying the seed after priming resulted in <1 log₁₀ cfu g^?1 seed loss for the pseudomonads but greater losses for MBI600 on carrot and leek seed. Application of spores of MBI600 resulted in little loss in cfu g^?1 seed following addition of the cells and these levels were maintained throughout the priming period and after drying back. Both T22 and G20 were recovered from carrot and parsnip at the end of priming and in general reflected survival of the inoculum rather than proliferation. T22 and G20 could not be recovered from leek seed following priming. Comparisons were made between proliferation and survival in large-scale drum priming with post-priming application of microorganisms. Pf CHA0 proliferated on carrot and parsnip seed during drum priming and survived the drying back process whereas there was no recovery when applied as a post-priming treatment. In contrast, MBI600 could not be recovered when applied during priming as cells, whereas recovery was good when applied post-priming as spores. T22 spores could be applied in either manner. Post-priming application of metalaxyl and thiabendazole had variable effects on microorganism recovery. The significance of the application of microorganisms to seeds during priming is discussed.     

Maize embryo germination

The cell-cycle progression of germinating embryos of maize (Zea mays L.) was studied from 0 to 72 h after the start of imbibition using DNA flow cytometry on isolated nuclei, and analyses of thymidine kinase activity, histone biosynthesis and levels of proliferating cell nulcear antigen (PCNA). At the start of germination, 75% of the cells were in G1, but this population had decreased to 25% by 72 h. The concomitant increase of cells in S-phase did not occur continuously, but stepwise, indicating that during germination most of the cells enter S-phase as a partially synchronized population. Within the initial 60 h of embryo germination the cells passed through one S-phase; the start and duration of this period of replicative DNA synthesis was further substantiated by the analysis of S-phase-associated events, the biosynthesis of core histones and the enzyme activity of thymidine kinase, which both began to increase at about 12 h after the start of differentiation. Thymidine kinase fluctuated periodically during germination with a transient maximum at 30 h and a second peak at 72 h; histone biosynthesis was not detectable until 12 h after the start of germination. The levels of PCNA protein closely resembled the pattern of thymidine kinase during germination. Together with the cytometric data this allows a clear assignment of cell cycle events to different times of embryo differentiation.Abbreviation PCNA proliferating-cell nuclear antigen Dedicated to Prof. Walter Larcher on the occasion of his 65th birthdayThe authors wish to thank Prof. G. Mikuz (Department of Pathology, University of Innsbruck, Austria) and Prof. G. Stöffler (Department of Microbiology, University of Innsbruck, Austria) for their interest and support. The technical assistance of Mrs. R. Gantschnig is gratefully acknowledged. E.I. Georgieva was recipient of short-term fellowships from the Austrian Academy of Sciences, the Austrian Forschungsgemeinschaft and the Austrian Akademischer Austauschdienst. G. López-Rodas was recipient of a postdoctoral fellowship from the Programa sectorial de Formación de Profesorado y Personal investigador del Ministerio de Educación y Ciencia (Spain). This work was supported in part by Grant SO6011 (to P.L.) from the Austrian Fonds zur Förderung der wissenschaftlichen Forschung and the Dr. Legerlotz-Foundation.