Journal of Experimental Botany, Vol. 51, No. 353, pp. 2031-2043,
December 2000
© 2000 Oxford University Press
Original Papers |
The influence of aerated hydration seed treatment on seed longevity as assessed by the viability equations
1 Department of Agriculture, University of Aberdeen, Aberdeen AB24 5UA, UK
2 Department of Reproduction Technology, CPRO-DLO, Wageningen, The Netherlands
3 Royal Botanic Gardens Kew, Wakehurst Place, Ardingley, West Sussex RH17 6TN, UK
Received 11 February 2000; Accepted 4 September 2000
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Abstract |
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Aerated hydration (AH) treatments of cauliflower seeds for 12 h (12AH) and 28 h (28AH) at 20 °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 °C for 12 months and at 20 °C for 4 months. The improved longevity of low vigour seeds was associated with increased Ki (initial seed viability) and a reduced rate of deterioration (1/
) whereas the Ki 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 °C and from 10.4 to 1.2 months at 20 °C. The improved Ki 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 G1 to G2 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 Ki 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 °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.
Key words:
Brassica oleracea var. botrytis, ß-tubulin, controlled deterioration germination, Ki, rate of deterioration (1/), 4C DNA.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Slow, asynchronous and unreliable germination and emergence, within germinable, low vigour seeds, arise due to seed ageing (Matthews, 1980
) and lead to problems for successful vegetable production. Seed invigoration treatments have, therefore, been developed to improve seed performance during germination and emergence. Most of these involve a period of controlled hydration of the seed to a point close to, but before, the emergence of the radicle after which the seeds are dried back to their initial moisture content before sowing (Basu, 1994; Khan, 1992; Matthews and Powell, 1988). Such treatments include priming in which hydration is controlled in an osmoticum such as polyethylene glycol (PEG) or a salt solution (Heydecker and Coolbear, 1977), solid matrix priming in which seeds imbibe in an inert medium held at a known matrix potential (Taylor et al., 1988), humidification where seeds are hydrated at a high relative humidity (Van Pijlen et al., 1996), and aerated hydration in which seeds imbibe in aerated water for a specified time (Thornton and Powell, 1992). These treatments, and others, have improved the rate, uniformity and reliability of germination and emergence in a range of vegetable species (Basu, 1994; Khan, 1992; Matthews and Powell, 1988).
Explanations for the improvements achieved in seed performance/quality have focused on the metabolic repair of previously sustained deterioration, and germination advancement. Physiological evidence of repair was provided by increased germination after controlled deterioration, seen in cauliflower and Brussels sprouts seeds following aerated hydration (Thornton and Powell, 1992). Germination after controlled deterioration indicates the position of the seed lot on the seed viability curve and hence the extent of deterioration in the initial seed sample. Thus, an increase in germination signifies a reversal of previously sustained deterioration. The effect of temperature, the availability of oxygen and the greater improvement in deteriorated low vigour seeds were cited as evidence in support of metabolic repair during aerated hydration treatment (Thornton and Powell, 1992). Studies on DNA synthesis have also indicated repair. DNA repair synthesis in cauliflower during aerated hydration was indicated when replicative synthesis was inhibited using hydroxyurea (Thornton et al., 1993) and in leek the beneficial effects of priming were correlated with DNA repair (Davison and Bray, 1991). Repair type synthesis made up 30% of total DNA synthesis after 1 d osmotic priming of leek (Ashraf and Bray, 1993) and in the rest of the priming period DNA repair type synthesis contributed to 20% of 3H thymidine incorporation. They (Ashraf and Bray, 1993) also noted enhanced levels of repair and replicative synthesis after 1 d germination
A complementary explanation for the improvement after invigoration is germination advancement (Heydecker and Coolbear, 1977) with emphasis being placed on evidence for advancement in RNA, protein and DNA synthesis. Earlier RNA synthesis occurs during or immediately after priming in lettuce (Khan et al., 1978), tomato (Coolbear and Grierson, 1979) and leek (Bray et al., 1989; Clarke and James, 1991) and enhanced protein synthesis has been observed during priming of a number of species (Khan, 1992). There are fewer reports of increased DNA synthesis during priming although 3H thymidine uptake into the DNA of leek seeds has been found to increase (Bray et al., 1989). DNA synthesis also occurred earlier during the germination of primed leek embryos (Clarke and James, 1991) and in tomatoes, primed seeds entered the S phase of cell division in half the time of untreated seeds (Coolbear and Grierson, 1979). More recently, the possibility of measuring the state of DNA in individual nuclei by flow cytometry has led to work focusing on the effect of invigoration treatments on changes in the cell cycle. Increases in 4C DNA have been observed during invigoration treatments of tomato (Bino et al., 1992; Liu et al., 1996; Cheng and Bradford, 1999), pepper (Lanteri et al., 1993, 1994, 1996) and sugar beet (Redfearn and Osborne, 1997), indicative of DNA replication prior to the onset of cell division and a shift from the G1 to the G2 phase of the cell cycle. Comparable increases in 4C DNA were not observed following humidification of tomato (Van Pijlen et al., 1996).
Another marker of cell cycle activation in germinating seeds is the accumulation of the cytoskeletal protein ß-tubulin (de Castro et al., 1995, 1998). The accumulation of ß-tubulin is mainly related to the formation of cortical microtubules in preparation for cell elongation (Jing et al., 1999). During germination of tomato, cabbage and cucumber, the accumulation of ß-tubulin precedes DNA replication (de Castro et al., 1995; Gornik et al., 1997; Jing et al., 1999). Hydration of tomato seeds at a range of moisture contents has shown that ß-tubulin accumulation can occur at around 29% seed mc, whereas DNA replication requires a minimum mc of around 34% (Van Pijlen et al., unpublished results).
Despite the immediate improvements in seed performance following invigoration treatments, there have been contrasting reports of seed storage potential following treatment. In carrot and tomato both improved and reduced storage potential have been associated with different storage conditions (Alvarado and Bradford, 1988; Argerich et al., 1989; Dearman et al, 1987a, b) and the type of hydration treatment (Alvarado and Bradford, 1988; Dearman et al., 1987a; Mitra and Basu, 1979; Savino et al., 1979; van Pijlen et al., 1996). In leek (Maude et al., 1994) and wheat (Nath et al., 1991) longer hydration treatments reduced seed storage potential and Penaloza and Eira indicated an interaction between the initial seed quality of tomatoes and the length of treatment in determining response to storage (Penaloza and Eira, 1993). Thus after longer hydration treatments (up to 48 h) some lots retained improvements in germination during subsequent storage whilst in others, germination declined (Penaloza and Eira, 1993). In pepper, reduced storage potential after priming was associated with increased 4C DNA (Saracco et al., 1995) and in tomato (Liu et al., 1996; Van Pijlen et al., 1996) it has been suggested that seed storage potential will be reduced if a large number of cells in the radicle tip have entered the G2 phase of the cell cycle during the priming treatment.
Seed longevity during storage is described by the viability equations (Roberts and Ellis, 1989; Ellis, 1991). These specify that under the same storage conditions, seed lots of the same species will deteriorate at the same rate and that differences in storage potential arise only as the result of differences in the initial seed viability or Ki. This suggests that where improved or reduced longevity is reported after the invigoration treatment, this would be the consequence of the effect of treatment on Ki. Any effect of invigoration on the rate of deterioration would be contrary to the predictions of the viability equations.
The aim of this paper was to examine the effects of the aerated hydration invigoration treatment on the longevity of cauliflower seeds, stored at different moisture contents and temperatures, and in particular on two components of the viability equation, Ki and the rate of deterioration (1/). The responses to storage will be considered in relation to evidence for metabolic repair during treatment, as assessed by controlled deterioration, and the advancement of germination as assessed by immunodetection of ß-tubulin and flow cytometric determination of nuclear DNA content.
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Materials and methods |
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Seed material
Samples of cauliflower (Brassica oleracea L. var. botrytis) seeds cv. Alpha 7 Jubro were obtained from Nickersons Seeds, UK in both 1996 and 1997. Periods of controlled ageing were used to produce subsamples from those seeds that retained high germination, but had low vigour (Thornton and Powell, 1992
), i.e. they had slower rates of germination and would be expected to have reduced field emergence and storage potential (Matthews, 1980). Low vigour seeds can be identified using standard vigour tests (ISTA, 1995). For the storage experiments, seeds having high germination but low vigour (as assessed by the controlled deterioration vigour test; ISTA, 1995) were produced from the original high vigour samples by subjecting them to controlled ageing (Thornton and Powell, 1992) at 20% seed moisture content (mc) and 45 °C for either 32 h (1996 seeds) or 18 h (1997 seeds). For the flow cytometry and ß-tubulin analysis, low vigour seeds of a further sample of cauliflower (1997 seeds) were aged at 20% mc and 45 °C for 36 h. The use of different periods of controlled ageing was necessary to allow for the different initial seed qualities of the samples of commercial seeds obtained and therefore to achieve comparable levels of seed quality on each occasion. Aged seeds were allowed to dry back to approximately their initial mc (determined by weighing) in open Petri dishes in the laboratory at room temperature (20±1 °C). This was achieved within 12–15 h after which all seeds were sealed in foil packets at 5 °C before further use.
Laboratory germination
Laboratory germination was assessed on four replicates of 25 seeds set to germinate in Petri dishes on germination papers moistened with 3.5 ml water. The Petri dishes were put into trays lined with moist J-cloths, placed in polythene bags to avoid moisture loss and held in a germination room at 20±1 °C. Germination, defined as the protrusion of the radicle, was counted daily and after 10 d normal and abnormal seedlings were assessed according to ISTA rules (ISTA, 1996). The mean germination time (MGT) was calculated (Nichols and Heydecker, 1968) as: MGT=
fx/f where f is the number of seeds newly germinated on day x and x is the number of days from the start of the germination test.
Controlled deterioration test
Seeds were subjected to the controlled deterioration (CD) vigour test at 20% mc and 45 °C for 24 h (ISTA, 1995; Powell, 1995) both before and after an aerated hydration treatment. Seed mc was raised by allowing samples (usually 1 g) of seed of known weight and mc to imbibe from moist germination papers. The weight at the desired mc was calculated as: Desired weight=(100–initial mc/100–desired mc)xinitial seed weight. The attainment of the desired weight was determined by frequent weighing. Once the desired mc was reached, the seeds were sealed in foil packets and held overnight in a refrigerator (<10 °C) to allow the equilibration of moisture throughout the seed. The seed packets were then placed in a water bath at 45 °C for 24 h after which a germination test was set up as described above. In this case, however, germination was only counted after 10 d, with any seed producing a radicle being counted as having germinated.
Determination of seed moisture content
Seed mc was determined by drying four replicates of 25 seeds for 1 h at 130 °C and was expressed as a percentage of the seed fresh weight. In some cases seed mc after ageing or seed treatment was calculated from the seed weight after ageing/treatment compared with the seed weight at its initial mc before ageing/ treatment. Previous work has shown the calculated mc to be within 0.5% of the mc determined by the oven method.
Aerated hydration treatment
Aerated hydration (AH) was carried out as described previously (Thornton and Powell, 1992) in perspex columns (0.5 m high, 5 cm diameter) containing deionized water and aerated by an aquarium pump. The columns were placed in a growth cabinet at 20 °C and water held in the columns overnight to allow equilibration to 20 °C before adding the seeds. Samples of seeds of known weight and mc were added to the columns and given AH treatment for 12 h or 28 h (12AH and 28AH). On completion of the treatment the seeds were carefully removed from the columns, surface-dried with paper towels and weighed. The mc after treatment could therefore be calculated. The seeds were left to dry in open Petri dishes at room temperature (20±1 °C) for 12–18 h until they reached approximately the same mc as before treatment. The actual seed mc after drying was determined by the oven method.
Polyethylene glycol (PEG) priming treatment
PEG priming of seeds was carried out by placing samples of seed (1–2 g) of known weight and mc in Petri dishes holding germination papers to which 3.5 ml PEG 6000 solution having a water potential of -1.0 MPa had been added. The Petri dishes were placed in polythene bags in the dark at 20±1 °C and samples removed after 5 d and 13 d. Treated samples were rinsed well to remove any PEG, surface dried using paper towels and weighed to allow calculation of the mc after treatment. The seeds were then left to dry at room temperature (20±1 °C) for 12–18 h until they reached their approximate mc before treatment. The actual seed mc after drying was determined by the oven method
Seed storage
Three storage experiments were set up using cauliflower seeds.
Experiment 1:
Seeds (1996 seeds) having either high or low vigour were stored both untreated and after two AH treatments (12AH, 28AH) at two seed mcs (initial, 12%) at 10 °C. The initial mc (Table 1) was equivalent to that following drying back after ageing and/or AH treatment and ranged from 4.51–5.17% (0.047–0.055 g H2O g-1 dry weight). The storage mc of 12% (0.136 g H2O g-1 dry weight) was achieved by allowing samples of known weight and mc to imbibe from moist germination papers as in the CD test. Once the samples reached the desired mc they were sealed in a foil packet and held in a refrigerator overnight for moisture equilibration.
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Twelve replicate blocks, each including 12 treatments (2 vigour levelsx3 seed treatments [UT, 12AH, 28AH]x2 mc), were arranged in an incubator at 10 °C. Each of three shelves in the incubator held four replicate blocks with the treatment packets randomly distributed within each block and from left to right across the shelf. Each replicate block represented a sampling time within the storage period. The blocks were randomized within the incubator and their positions were re-randomized after each sampling. Samples were removed monthly for 1 year. After sampling, the seeds stored at 12% mc were allowed to dry back to approximately their initial mc (determined by weight) in open Petri dishes in the laboratory (20±1 °C), so that any assessments of rate of germination after storage could be directly compared with that of unstored seeds at the initial mc. Germination and CD vigour tests were set up immediately after drying.
Experiment 2:
Seeds (1996 seeds) given the same 12 treat-ments as in experiment 1 were also stored at 20 °C. In this case, however, there were only four sampling times (replicate blocks) which were randomly distributed between two shelves in an incubator and re-randomized at each sampling. Samples were taken monthly for 4 months. Seeds stored at 12% mc were again dried back to their initial mc after storage as described above.
Experiment 3:
High and low vigour cauliflower seeds (1997 seeds) were stored at both their initial and 12% mc following PEG priming for either 5 d or 13 d (5PEG, 13PEG). The seed mc was raised to 12% as described for Experiment 1. The mc of seeds stored at their initial mc (Table 1
) ranged from 5.03–7.01% (0.050–0.067 g H2O g-1 dry weight) for the high vigour seeds and from 5.98–7.19% (0.061–069 g H2O g-1 dry weight) for the low vigour seeds. Packets of seeds for six sampling times (six replicate blocks) for each of 12 treatments (2 vigour levelsx3 treatments [UT, 5PEG, 13PEG]x2 mc) were randomly arranged within an incubator at 10 °C as in the other experiments. Samples were taken monthly from 1–4 months then after 6 and 8 months. Seeds stored at 12% mc were dried back to their initial mc before use as in the other experiments.
The statistical package GLIM version 4.0 was used for comparison of multiple probit regression lines constructed from the germination data for seed storage at 12% mc. Analysis for the statistical significance of the increase in the scaled deviance caused by constraining multiple seed survival curves to have the same slope or a common origin used the F-distribution with a 95% confidence interval (Crawley, 1993).
Preparation of nuclear samples
Radicle tips (1 mm long) were cut from embryo axes dissected from dry seeds or from hydrated seeds following AH or PEG treatments. Hydrated tissue was dried rapidly at room temperature (20±1 °C) for approximately 0.5 h before sealing in Eppendorf tubes and holding at 5 °C before use. Nuclear samples were prepared by chopping replicates of five radicle tips with a sharp razor blade in ice-cold PBS buffer (pH 7.3) consisting of 0.137 M NaCl, 2.68 mM KCl, 8 mM Na2HPO4, and 10 mM DTT (dithiothreitol) (total 1 ml buffer per sample). The suspension was passed through an 88 µm nylon mesh before addition of 20 µl propidium iodide. At least two replicates per sample were prepared for flow cytometric determinations.
Flow cytometry
A Coulter Epics XL system flow cytometer (Coulter Systems, Luton, Bedfordshire) was used and analyses were performed using peak height detection and logarithmic amplification (Jing et al., 1999). The DNA amount is proportional to the fluorescent signal and is expressed as arbitrary C values in which the 1C value comprises the DNA content of the unreplicated haploid chromosome content. For most samples between 5000–10000 nuclei were analysed. Small deviations between samples were observed in the exact peak positions. These variations were due to variation in the ratio of propidium iodide and total DNA in the sample. Data were analysed using Multicycle software (Phoenix Flow Systems Inc, San Diego, California, USA). Debris such as cell clumps or other fluorescent particles were excluded from the peak analysis using an exponential model. The percentages of nuclei in the G1 (2C), s and G2 (4C) phases of the cell cycle were calculated from the areas under the fitted curves.
ß-Tubulin analysis
Radicle tips (1 mm) were cut from embryo axes dissected from dry seeds or after AH or PEG treatment. Hydrated tissue was freeze dried and stored at -20 °C before use. Western blotting detection of ß-tubulin was adapted from de Castro et al. (de Castro et al., 1998). Replicates of five radicle tips were ground into a fine powder with a Potter-S grinder and extracted in 60 µl MODIL buffer containing 80 mM TRIS–HCl, 2% w/v SDS, 12.5% w/v glycerol, and 15 mg ml-1 DTT (dithiothreitol). Extraction took place at 95 °C for 15 min before centrifuging at 20000 rpm for 7 min. Protein concentrations of the supernatants were measured following micro-protein assay procedures (BioRad) using BSA as standards. Crude protein samples of 50 µg were loaded on a 8–18% SDS gradient gel (Pharmacia Excel Gel) for electrophoresis separation using a horizontal electrophoresis system (Multiphor II, Pharmacia). Two samples of pure bovine brain tubulin (10, 30 ng) were loaded as reference samples along with a molecular mass marker (RPN 756 Amersham Life Sciences, UK) having protein molecular mass from 14–220 kDa. The average molecular mass of pure tubulin is 55 kDa. The proteins were then transferred to PVDF membrane (Amersham Life Sciences, UK) for 80 min at 4 °C using a Hoefer TE-77 electroblotting unit (Pharmacia). Immunodetection of tubulin followed the method of de Castro et al. with some modifications (de Castro et al., 1995). The immunoblots were washed for 3 h in blocking solution, incubated overnight with 1 µg ml-1 mouse anti-ß-tubulin monoclonal antibody (Boehringer Mannheim clone KMX-1), washed for 0.5 h with TBST Tween 0.5 followed by 0.5 h in blocking solution, and finally incubated for 1 h with the second antibody, anti-mouse-IgG-POD raised against goat. Washings used a minimum of 150 ml solution for 50 cm2 membrane to ensure excess solution and minimize unspecific binding activity of the second antibody. POD chemiluminescence immunodetecting solutions (Boehringer Mannheim) were used for the fluorescence reaction and films were developed according to the manufacturers’ protocol.
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Results |
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Seeds stored at their initial mc at 10 °C or 20 °C retained laboratory germinations above 90% (data not presented). However, there were clear effects of storage and of AH treatment on the germination of seeds stored at 12% mc at these temperatures. These responses of untreated and AH-treated seeds to storage were examined using probit analysis. It was not possible to constrain responses to either a common origin (the same initial viability) or parallel lines (the same rate of viability loss); therefore the free-fitting model was adopted in the analysis.
The initial viability (Ki) of high vigour seeds was significantly reduced by the 28AH treatment imposed before storage at both 10 °C (Fig. 1a; Table 2) and 20 °C (Fig. 1c; Table 2). For the low vigour seeds however, there was no effect on the Ki of seeds stored at 10 °C (Fig. 1b), whilst both the AH treatments imposed before storage at 20 °C gave a significant increase in Ki (Fig. 1d) from 5.85 (80.2% germination, UT; Table 2) to 6.81 (96.5%, 12AH) and 6.65 (95.1%, 28AH ). These increases in Ki reflected increases in the number of normal seedlings produced.
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) or 28 h (
) aerated hydration. The intercept of the y-axis represents the constant Ki from the viability equations (Roberts and Ellis, 1989).
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AH treatment had a clear and contrasting effect on the longevity of high and low vigour seeds at both storage temperatures. In high vigour seeds the rate of deterioration increased after AH treatment (Fig. 1a
, c; Table 2, 1/) with the time to lose one probit of viability decreasing from 28.68 to 5.34 months at 10 °C and from 10.39 to 1.19 months at 20 °C (Table 2). Thus for high vigour seeds there was a 5–9-fold decrease in seed longevity after AH treatment. In contrast, the longevity of low vigour seeds was enhanced after treatment (Fig. 1b, d) with the time to lose one probit of viability increasing by up to 9.3 times at 10 °C (Table 2) and 3.2 times at 20 °C.
These contrasting effects of AH on germination after storage at 12% mc were confirmed by the CD test carried out after treatment and storage, and in this case were also evident during storage at the initial mc. Untreated high vigour seeds retained high CD germination, indicative of little change in the extent of deterioration of the seed, at both storage temperatures and moisture contents (Figs 2a, c; 3a, c) although there was a slight fall in CD germination after 12 months at 12% mc, 10 °C (Fig. 3a). CD germination of high vigour seeds following 12AH also remained high during storage at the initial mc (Fig. 2a, c), although during storage at 12% mc it fell at both storage temperatures (Fig. 3a, c). However 28AH treatment of high vigour seeds resulted in a clear decrease in the CD germination of unstored seeds (Figs 2a, c; 3a, c) which was indicative of an increase in the extent of deterioration in the seeds. There was a further fall in CD germination during subsequent storage except when seeds were held at 12% mc, 20 °C. In the low vigour seeds, untreated seeds showed a steady decline in CD germination during storage at 12% mc (Fig. 3b, d) and although there was considerable variation in CD germination at the initial mc, it tended to decrease (Fig. 2b, d). However, after both 12AH and 28AH of unstored low vigour seeds there was an increase in the CD germination, indicating that the AH treatment resulted in a reduction in the extent of deterioration within the seed and increased vigour. This increased CD germination was maintained during storage at the initial seed mc (Fig. 2b, d) and although it fell slightly at 12% mc (Fig. 3b, d) it remained higher than that of untreated seeds. There was no obvious difference between the effects of 12AH and 28AH treatment on seed response to storage.
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Thus, both the germination and CD vigour tests after storage at 12% mc and the CD test after storage at the initial seed mc revealed the improved storage potential of low vigour seeds after AH treatment. In contrast, AH-treated high vigour seeds showed reduced storage potential, particularly after the longer AH treatment. At the storage moisture contents used (approximately 5% and 12% mc), the water within the seed will be in water binding region II as described earlier (Roberts and Ellis, 1989
), therefore the same response to AH treatment was seen for seeds close to the lower and upper moisture content for the orthodox storage response. The CD test completed after different periods of storage also represented a period of storage, in this case in more severe storage conditions of 45 °C and 20% seed moisture content (0.25 g H2O g-1 dry weight) for 24 h. At this moisture content the water within the seed will be in water binding region III. The germination data after the storage treatment plus CD was therefore subjected to probit analysis to determine whether the same effect of AH on seed longevity was seen at this elevated seed moisture content. The data used in this analysis were therefore the germination data following an initial period of storage at 12% mc at either 10 °C or 20 °C, plus storage for 24 h at 20% mc, 45 °C.
The same responses of high and low vigour seeds to storage after AH treatment were evident following this combined 12% and 20% mc treatment (Table 3). Thus high vigour seeds showed reduced Ki after 28AH (Table 2) and their rate of deterioration (1/) increased after 12AH and 28AH at both storage temperatures. On the other hand, AH treatment resulted in increased Ki for the low vigour seeds and the decreased slope of the regression and consequent increased time to lose one probit of viability (, Table 3) indicated slower deterioration.
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Comparisons were also made of the longevity of seeds after a short (5 d) and long (13 d) PEG priming treatment. Storage of both high and low vigour seeds at their initial mc and 10 °C after treatment had little effect on laboratory germination over the 8 month storage period (data not presented), whereas storage at 12% mc resulted in a decline in germination (Table 4a
). For high vigour seeds this fall was small and occurred only after 8 months storage. However, the germination of untreated low vigour seeds fell to 52% after 4 months, whereas that of PEG-primed low vigour seeds remained above 90% over the same time interval. As storage time increased the germination of the untreated low vigour seeds fell further and whilst that of treated seeds also fell, it remained above that of untreated seeds. PEG priming had similar effects on seed vigour (CD germination) during storage (Table 4b) with only a small fall in the CD germination of high vigour seeds after 8 months and the retention of higher CD germinations by PEG primed low vigour seeds indicating improved storage potential after priming. Thus, after 8 months storage seeds previously given 13PEG retained a CD germination of 75% compared with only 2% for untreated seeds.
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Determinations of nuclear DNA levels and of ß-tubulin were made after both AH and PEG priming to examine whether changes in seed vigour following treatment, expressed as either mean germination time (i.e. rate of germination) or controlled deterioration germination, and the contrasting responses to storage could be related to advancement of the cell cycle during treatment. Most nuclei in the radicle tips from dry cauliflower seeds contained 2C DNA, with both high and low vigour seeds containing less than 5% nuclei with 4C DNA (Table 5
). Following AH treatment of high vigour seeds the proportion of nuclei with 4C DNA levels increased to 26% (Table 5) whilst in low vigour seeds there was no increase in the frequency of nuclei including 4C DNA (Table 5). However, the decreased MGT (increased rate of germination) seen in both high and low vigour seeds after treatment (Table 5), and the increase in the CD germination of low vigour seeds from 50% (untreated) to 84% (12AH) and 88% (28AH) indicated a marked increase in vigour.
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A 13PEG treatment of high and low vigour cauliflower (Table 5
) resulted in comparable increases in the frequency of nuclei with 4C DNA along with decreased MGT in all cases. In addition, there was an increase in the CD germination of the low vigour seeds (Table 5). The increase seen in 4C DNA levels was double that observed after the 28AH treatment (Table 5).
The presence of ß-tubulin in untreated cauliflower seeds (Fig. 4) indicated constitutive levels of ß-tubulin although there was less present in the dry control of low vigour than in high vigour seeds. Accumulation of ß-tubulin was observed during the AH treatments, with faster accumulation in the high vigour seeds compared to the low vigour seeds (Fig. 4). PEG treatments of high vigour cauliflower gave comparable ß-tubulin levels to the control, but there was a large increase in low vigour seeds during the AH treatment.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Both an aerated hydration invigoration treatment and PEG priming improved the storage potential of low vigour cauliflower seeds whilst, in contrast, high vigour seeds showed reduced longevity after AH treatment. The contrasting effects of AH on low and high vigour seeds were associated with changes in the components of the viability equation, namely Ki and 1/
. The decrease in the predicted initial viability or Ki observed in high vigour seeds and increase in Ki of some low vigour seeds indicated that the initial position of the seeds on the viability curve, or the extent of deterioration of the seed, was altered following treatment. The subsequent effects of treatment on the longevity of high and low vigour seeds could therefore be clearly related to the changes in the extent of deterioration within the seed. A similar reduction in Ki and reduced storage potential has been observed in high vigour tomato seeds following priming (Argerich et al., 1989). The changes in longevity in the present work were, however, further confounded by the consistent increase in the rate of deterioration (1/) of high vigour seeds after AH compared with slower deterioration (decreased 1/) in low vigour seeds. These differences in the rates of seed deterioration as a result of AH treatment are contrary to the predictions of the viability equations which suggest that under the same storage conditions, different lots or cultivars of the same species will show the same rates of deterioration (Roberts and Ellis, 1989; Ellis, 1991).
Interestingly, these effects of AH on seed longevity were observed in seeds stored at moisture contents giving two different levels of water binding (Vertucci and Leopold, 1986). Thus AH treatment influenced longevity at the initial seed mc (
6% mc, water binding region II), at 12% mc (region II) and when a storage period at 12% mc (region II) was followed by 20% mc (region III) in the CD test. Seeds show very different levels of physiological activity at these two levels of water binding (Vertucci and Leopold, 1986). In region II low levels of oxygen uptake and RQs of 0.5 are indicative of oxidative reactions that are not the result of mitochondrial electron transport or glycolysis, whereas mitochondrial respiration begins at the transition from region II to region III. Seed moisture content influences deterioration in all three water-binding regions, but in view of the different levels of activity, it was proposed that the mechanism of deterioration differs in the different regions (Vertucci and Leopold, 1986). The influence of changes in the molecular mobility of water at different seed mcs on the kinetics of deteriorative reactions as a result of water occurring in the crystalline, glassy or rubbery state has also been described (Walters, 1998). The range of moisture contents at which seeds were stored in the present work would certainly influence both the nature and the kinetics of deteriorative reactions. Nevertheless, AH influenced the longevity of high and low vigour seeds at all storage moisture contents.
Explanations for differences in the rate of deterioration after AH may lie in the relative roles of metabolic repair and advancement in high and low vigour seeds. In low vigour seeds, the increase in CD germination of AH-treated unstored seeds indicated a reduction in the extent of deterioration within the seeds after AH treatment, as seen previously in Brussels sprouts and cauliflower seeds and put forward as evidence of metabolic repair (Thornton and Powell, 1992). This reversal of deterioration provides an explanation for the increased Ki of low vigour seeds after treatment. In contrast, the fall in the CD germination of unstored high vigour seeds after AH was similar to that previously seen after a 32 h AH treatment of cauliflower seeds (Thornton and Powell, 1995). This was explained as possible ‘over-advancement’ during AH treatment, leading to seeds being more susceptible to drying after treatment and/or the effects of high temperature and mc during the CD test. It was noted recently that if primed seeds are subjected to a mild water or temperature stress before drying, the deleterious effects of priming on longevity are eliminated (Bruggink et al., 1999). Since such treatments also induce desiccation tolerance in germinating seeds (Bruggink and Van der Toorn, 1995), this suggests that it is the period of desiccation of high vigour seeds after AH that is damaging. A deleterious effect on the high vigour seeds would certainly reduce the Ki as seen here, with consequences for potential seed longevity.
The increase in the proportion of 4C DNA and ß-tubulin present in high vigour cauliflower seeds following AH provided clear evidence that germination advancement does indeed occur in high vigour seeds. In contrast, there was no change in the 4C DNA in the low vigour seeds after AH, although the increase in ß-tubulin suggested that germination advancement had begun, but was delayed compared with high vigour seeds. Different responses of seed lots of pepper to priming have also been observed, with aged, and therefore low vigour, seeds showing reduced or no accumulation of 4C DNA compared with unaged high vigour seeds (Lanteri et al., 1994, 1996). These differences in the onset of the G2 phase of the cell cycle in high and low vigour cauliflower seeds may explain their contrasting responses to storage. Previously, rapid deterioration of tomato seeds has been associated with an increase in 4C DNA following a normal priming treatment (Liu et al., 1996; Van Pijlen et al., 1996) as seen here for the high vigour cauliflower. In contrast, priming of fresh tomato seeds (Liu et al., 1996) and humidification treatments of tomato (Van Pijlen et al., 1996) did not allow DNA replication and these seeds were more resistant to deterioration. Cells in the G2 phase of the cell cycle, containing 4C DNA, are more sensitive to factors affecting nuclear division and chromosome morphology such as radiation and free radicals (Sybenga, 1972). Thus the high vigour cauliflower seeds given 28AH and showing increased 4C may be more sensitive to both drying after treatment, and hence their reduced vigour and Ki, and/or a period of storage. In contrast, the absence of, or low 4C DNA in low vigour seeds may result from their capacity to undergo metabolic repair during the initial stages of hydration, as indicated by their increased CD germination. This would lead to a delay in the onset of germination activities and therefore DNA replication. This situation would be analogous to the delay in DNA synthesis seen in aged seeds in the early stages of germination (Osborne, 1983; Thornton et al., 1993) which has been explained by the repair of DNA. In addition, it has been reported that ß-polymerase-mediated DNA repair activity, with the characteristics of an excision-type repair, occurs immediately on imbibition of rye and oats (Elder and Osborne, 1993). Similar repair may occur on hydration of low vigour seeds during invigoration treatments.
In contrast to the AH treatment of low vigour seeds, PEG priming led to a clear increase in both 4C DNA and ß-tubulin after a 13 d treatment. Different responses to priming treatments in terms of both the effects on germination and on the cell cycle have been associated with the osmotic potential of the solution and duration of treatment (Bino et al., 1992; Lanteri et al., 1993; Van Pijlen et al., 1996; Gurusinghe et al., 1999). Lanteri et al., suggested that lower levels of 4C DNA are likely following the activation of repair during longer priming treatments at lower osmotic potentials (Lanteri et al., 1996). However, in this comparison of the effects of AH and PEG priming, the longer PEG treatment resulted in higher 4C DNA than did the shorter AH treatments at a higher osmotic potential. Furthermore, increases in CD germination, which are indicative of a reduction in the extent of deterioration within the seed, occurred following both AH and PEG priming suggesting that metabolic repair had occurred during treatment.
It was not possible to make comparisons between the changes in 4C DNA and ß-tubulin during PEG priming and the subsequent storage potential of high and low vigour seeds as there was little change in the germination and vigour of the high vigour seeds during the storage period. Nevertheless, there was a clear improvement of the storage potential of the low vigour seeds after both 5 d and 13 d PEG priming with the greater improvement after 13 d. However, in contrast with the AH-treated seeds that showed improved storage potential, these PEG primed seeds showed a large increase in both 4C DNA and ß-tubulin. Thus in this case the accumulation of nuclei with 4C DNA did not reduce the storage potential of the seed.
The AH and PEG priming treatments consistently resulted in increases in the rates of germination of both cauliflower seeds although these were not always accompanied by the accumulation of nuclei containing 4C DNA. Increases in germination rate in the absence of evidence of DNA replication have also been observed in pepper (Lanteri et al., 1993; Saracco et al., 1995), leek (Ashraf and Bray, 1993; Clarke and James, 1991), maize (Cruz Garcia et al., 1995), and tomato (Gurusinghe et al., 1999). On the other hand, the proportion of nuclei with 4C DNA was proportional to the increase in mean germination time in both pepper (Lanteri et al., 1996) and tomato (Bino et al., 1996). Furthermore, Gurusinghe et al. commented on the considerable variation that they observed in cell cycle responses to priming in many flower and vegetable species (Gurusinghe et al., 1999). The present work supports the hypothesis that this variation may be linked to initial seed quality and the extent to which metabolic repair is activated before cell cycle activities are initiated. Indeed the increases in germination after controlled deterioration associated with increased rates of germination in the absence of increases in 4C DNA levels would support this view.
This work therefore indicates that aerated hydration and PEG priming treatments can improve seed storage potential. However, the interaction between AH treatment time and initial seed quality emphasizes the importance of these factors in determining the subsequent effect on components of the viability equations and hence the seeds’ response to storage. This interaction may well explain the different responses to storage previously observed following invigoration treatments. Indeed, the importance of length of treatment was recently emphasized by Cheng and Bradford in their study of hydrothermal priming time in tomato (Cheng and Bradford, 1999). They observed that seeds having faster initial germination (i.e. high vigour seeds) required a shorter priming duration to achieve more rapid germination. This study of the interaction between treatment time and seed quality and subsequent effects on storage potential would support their observations.
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4 To whom correspondence should be addressed. Fax: +44 1224 273731. E-mail: a.a.powell{at}abdn.ac.uk
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