PHYSIOLOGICAL MECHANISMS INVOLVED IN SEED PRIMING

Cees M. Ka~s~en1, Anthony Haigh 1,2, Peter van der Toorn 3 and Rolf Weges '

1 Department of Plant Physiology Agricultural University Wageningen, The Netherlands

2 Faculty of Horticulture University of Western Sydney, Hawkesbury Richmond, N.S.W., Australia

3 Seed Technology Department Nunhems Seeds Haelen, The Netherlands

4 Seed Technology Department Incotec Enkhuizen, The Netherlands

INTRODUCTION

The high degree of mechanisation in modern plant cultivation systems demands fast, uniform and complete germination. However, this agricultural demand is the extreme opposite of the evolutionary adaptations which wild species have made to ensure success in their natural environment. Gradients of dormancy in the annually produced seeds of most wild species guarantee the spread of germination over periods up to many years. This wild background is often still noticeable in crop seeds that, therefore, germi­nate slowly, irregularly and to low percentages.

Seed quality can be improved in many ways. Breeding and selection are the most fundamental approaches, but these methods are expensive and time consuming. Treatments of plants during seed production have only inci­dentally improved seed quality. Therefore, seed priming is to date the most promising method to improve the quality of seeds. The method was described before, but it was brought to general attention by Heydecker et al (1973). Osmotic priming consists of the incubation of seeds for a specific period of time at a certain temperature in an osmoticum of -1.0 to -1.5 MPa usually salt or polyethyleneglycol (PEG) (molecular weight 6000) dissolved in water. Priming is generally followed by redesiccation of the seeds to allow storage and handling.

MECHANISMS OF PRIMING

An understanding of the water relations of germination and cell expan-

Recent Advances in the Development and Germination of Seeds Edited by R.B. Taylorson Plenum Press, New York

269

sian is necessary before mechanisms of seed prImIng can be examined. Germi­nation may be regarded as a specialized growth phenomenon. Expansive growth of plant cells results from cell wall yielding and water uptake. Expansion is initiated with a yielding of the cell wall and continues under the effects of turgor. Wall relaxation reduces the cell water potential by dissipation of turgor and gives rise to water influx, which in turn in­creases cell volume (Crosgrove, 1986). For seeds with structures enclosing the embryo additional processes will be necessary to remove the restraint imposed on embryo cell expansion.

Under optimal conditions of supply the water uptake by seeds is tri­phasic. Phase I, or imbibition, is largely a consequence of matric forces. Phase II is the lag phase of water uptake, the water potential (~) of the seed is in balance with the environment. During this phase major metabolic events prepare the seed for radicle emergence. Only germinating seeds enter phase III, which is concurrent with radicle elongation. The duration of each phase depends on certain inherent properties of the seed and the conditions of water supply. Phase II is crucial for the timing of germina­tion. In some species, such as the garden pea, phase II hardly exists, radicles protrude directly following imbibition. In most other species certain preparations for the renewed water uptake have to proceed during phase I I. In the case of dormant seeds it requires special environmental conditions to remove blockages to germination. In all species phase II is temperature sensitive. Evidently, the control of water relations is essen­tial to the control of germination.

Osmotic priming prevents the start of phase III and thus permits pro­longed reaction times for processes during phase II. The benefits of pri­ming are seen after reimbibition. They can be diverse. (1) In general, priming may cause a shorter lag phase because part of the preparation to phase III needs no repetition. (2) In a population of seeds the start of radicle protrusion is often better synchronized. (3) The rate of the growth process may be increased and, (4) more seeds in a popUlation may germinate. In the latter case priming has been instrumental in the breaking of dorman­cy that requires specific temperature conditions just like those that occur in the field during the seasons with low or high temperatures. (5) A few studies have reported an improvement of seed vigour in low quality seeds due to priming.

The general mechanism of prImIng seems to be that irreversible pro­ducts are formed that are tolerant of desiccation and stimulatory to germi­nation. Attempts to identify the physiological mechanisms involved in seed priming have been mainly restricted to a few species. In this paper we will review recent developments in studies on priming of celery, tomato, and lettuce seeds in our laboratories. The perspectives for future research will be discussed.

CELERY

A celery "seed", being morphologically a schizocarpic fruit, consists largely of endosperm surrounded by a thin testa and relatively thick peri­carp. The small linear embryo is located at the micropylar end of the endosperm and measures about one third of the length of the endosperm. The embryo grows to at least twice its size before visible germination occurs. The need for embryo growth in the mature seed may be the main reason that germination of celery and other Umbelliferae is slow and irregular. Osmotic priming more than doubles germination rate, increases the uniformity of germination and raises the upper-temperature limit for germination (see e.g. Brocklehurst and Dearman, 1983).

270

-0- water

-/;.- PEG

-e- PEG/water

4 8 12 16 20 24

Time, days

370r---~--~--~--~--~--'

5 330

~ 290

·iii 250

210

B 1-~1

A-/;' ___ /;._/;._

170 L..-. __ ....... __ ....... __ ....... __ "'--__ "'------'

o 4 8 12 16 20 24

Time, days

1400 C

1200

~ 0_

1000

5 800 c:

~ 600

400

200 0 4 8 12 16 20 24

Time, days

-0- water

-/;.- PEG

-e- PEG/water

-0- water

-/;.- PEG

-e- PEG/water

fig. 1. Embryo growth (A), changes in mean embryo cell size (6) and increases in total embryo cell number (C) in celery seeds cv. Monarch during germination in water at 15 ·C in 12 h light, 12 h dark of non-primed (0) or primed seeds (e) and during pri­ming in -1.2 MPa PEG (a). Primed seeds were surface dried before transfer to water. Embryo length was measured in longi­tudinally cut seeds. The number of cells was calculated from counts in a standard surface area of the hypocotyl just below the apical meristem. The mean cell size was calculated from these counts. Arrows indicate moment of germination (from Van der Toorn, 1989).

271

In water 501'6 of celery seeds cv. Monarch germinated after 6.1 day at 15 ·C in light (Van der Toorn, 1989). Prior to germination the embryo length increased from 0.45 to 1.5 mm (Fig lA). In -1.2 MPa PEG embryos grew at a much slower rate than in water, moreover the growth process ceased at an embryo length of 0.95 mm. When, after 17 days in PEG, seeds were rinsed and transferred to water, embryos extended from 0.9 to 1.5 mm in 1.7 day. For non-primed seeds this growth took 2.7 days. Evidently, priming not only permitted embryos to grow to twice their original size, as was also re­ported for carrot seeds (Austin et al. 1969), but it also improved their growth rate during subsequent incubation in water.

To further analyze embryo growth the number and mean size of embryo cells was determined by microscopic observation of longitudinal sections of a standard area of the hypocotyl just below the apical meristem. In wate~ the mean cell size increased during the first 3 days from 190 to 250)lm and stabilized thereafter (Fig 18). The original number of 300 cells in­creased from the second day to reach 1100 cells at the moment of protrusion (Fig lC). Incubation in PEG reduced the cell division rate to about a quarter of that in water. After 16 days the number of cells stabilized at about 800. Directly after transfer to water the cell division rate in­creased to that observed in non-primed seeds. (In the transfer experiments the number of cells after 17 days was smaller than in the continious PEG treatment probably due to a slight decrease in the osmotic potential of the PEG solution).

The size of cells formed during PEG incubation varied. Initially larger cells were formed but in the latter half of the incubation cells formed were of the same size as observed at the start. Directly after transfer to water at day 17 cell size increased dramatically. This reduced the number of cells that were required to reach the critical embryo length of 1.5 mm. Thus, during osmotic priming newly formed cells hardly expanded but preparations were made for very quick cell expansion after transfer to water.

For expansion embryos need space and nutrients. Celery embryos grow at the expense of endosperm tissue. Van der Toorn (1989) showed that the hydrolytic activity continues in endosperm cells during priming. Endosperm cell walls probably consist largely of galactomannans. During priming the activity of endo-~mannanase, one of the galactomannan hydrolyzing enzymes, was hardly reduced below that in germinating non-primed seeds. Therefore, progress in endosperm hydrolysis might be another benefit of primed over non-primed seeds that might explain quicker germination of the former seeds.

It is not known yet why embryos need to achieve a length of 1.5 mm before starting protrusion of the testa and pericarp layers opposing the radicle tip. In non-primed seeds after 4 days of incubation in water changes were observed in the 2 to 3 cell layers at the micropylar end of the endosperm which indicated that hydrolysis and breakdown had occurred. We suppose that these endosperm weakening processes occur independently of the general endosperm breakdown required for earlier embryo growth.

TOMATO

Tomato seeds are flattened and ovoid in shape, up to 4 mm in length, with a curved linear embryo embedded in non-starchy endosperm. In such seeds the tissues enclosing the embryo may influence germination by mecha­nically restraining the expansion of the embryo. Recent studies of germina­ting tomato seeds have revealed much about the mechanisms involved in the control of germination and have thus indicated possible mechanisms involved

272

in the priming of tomato seeds.

As with capsicum seeds (Watkins and Cantliffe, 1983), the germination of tomato seeds was associated with a weakening of the resistance offered by the endosperm opposing the radicle tip. In seeds of a gibberellin­deficient mutant in the background of cv. Moneymaker weakening did not occur and germination was prevented (Groot and Karssen, 1987).

A study of the changes in the water relations of germinating tomato seeds cv. UC 82B has provided confirmatory evidence for the role of the endosperm (Haigh and Barlow, 1987). During imbibition of tomato seeds water uptake continued until water potential equilibrium was established between the seed and the imbibitional solution. The embryo was found to be capable of expansive growth prior to its emergence, but the endosperm tissue enclo­sing the embryo restricted further hydration until such weakening occurred so as to permit radicle emergence. Priming of tomato seeds may lead to more rapid germination by modifying these mechanisms.

Tomato seeds mainly benefit by priming in a strong reduction of the germination lag-time, particularly at moderate temperatures around 15 ·C (Haigh, 1988). The rapid germination of primed and redried tomato seeds was found to result from changes to all three major steps involved in germina­tion: imbibition, radicle cell-wall loosening and endosperm weakening. Water uptake by primed seeds was more rapid than that by non-primed seeds and may have resulted from the slightly lower osmotic potential of primed seeds during imbibition or from improved hydraulic conductivity. Some solute accumulation occurred during priming as was suggested by a number of authors (see Bradford, 1986, for references).

During priming cell-wall loosening occurred in the radicle. It was initiated 30h after the beginning of the treatment. As this occurred at the same time as in non-primed seeds in water this process must not be inhi­bited by the low water potential. The embryos from primed tomato seeds were capable of expansion at the earliest time at which they could be excised

0.55

0.50

Z 0.45

ai U 0.40

.2 0.35 0 QJ

~ 0.30 U c :J 0.25

Q.

0.20

0.1;-L 0

26 C 15 C o = 0.96 R = 0.87

50

/ /0 100 150

t 50. hours

Fig. 2. Correlation between the time needed for 50% germination and the puncture force required to break through the layers that enclosed the tip of the radicle of primed tomato seeds cv. Moneymaker. Seeds were primed in -1.2 MPa PEG at 26 ·C during 1 to 15 days and dried before germination occurred in distilled water at 15 ·C (0) or 26 ·C (D). Endosperm resis­tance was determined according to Groot and Karssen (1987). (T. Heimgartner and C.M. Karssen, original data).

273

from the seeds. This indicated that the cell wall yielding which had oc­curred during priming was not reversed by drying. Thus the time normally necessary to initiate cell wall yielding during germination of non-primed seeds had been completely eliminated by priming. However, as the timing of radicle emergence was controlled by endosperm weakening, the changed embryo cell wall properties may have not affected the timing of germination, but rather the rate of expansion subsequent to radicle emergence. The more rapid expansion of embryos from primed seeds was attributed to changes in radicle cell wall extensibility during priming (Haigh, 1988).

During priming the mechanical resistance of the enclosing tissues decreased to the same extent, but at a slower rate, as during incubation in water of non-primed seeds. As a consequence, at the start of imbibition the resistance in primed and dried seeds was much lower than in non-primed seeds (Haigh, 1988). In primed seeds of the cultivar Moneymaker the re­duction of the time to 50% germination was a function of the reduction of the mechanical resistance (Fig. 2).

Fig. 3.

274

60 A

50 i-~ ;f.

i-~T 1 40 f- 1--~ u: 30 £

9l 20 " ~ 10

0 0 2 4 6 B 10 12

Ol.ratlon of Priming. days

60 B

;f. 50

~ ~ 40

~ u: 30 .~

9l 20

I 10

r--0

0 5 10 15 20 25 30

Time, hOl.l'S

Endo-6-mannanase activity in tomato seeds cv. Moneymaker during (A) priming in -1.4 MPa KN0 3 at 25 ·C (A.)and (B) germination in water at 25 ·C for non-primed (e) and 6 day primed and dried seeds 01). Enzyme activity was assayed visco­metrically according to Groot et al. (1988). (D.C. Keulen and A.M. Haigh, original data).

The decrease in the mechanical restraint of the endosperm layers enclosing the radicle tip prior to radicle emergence is under the control of gibberellins produced by the embryo (Groot and Karssen, 1987). The endosperm cell walls were found to be rich in galactomannan and endosperm weakening was associated with the induction of endo-i3-mannanase activity and an increase in the activity of mannohydrolase (Groot et al., 1988).

Endo-i3-mannanase activity was studied in relation to priming. Priming seeds and germinating primed seeds showed uniformily higher activity than observed in germinating non-primed seeds (Fig. 3). The endosperm of tomato seeds was found to consist of two distinct cell types found in separate locations within the seed. At the micropylar end of the seed the endosperm cells had thin walls, whereas those in the rest of the seed had thickened walls. All cells, except those of the root cap, contained protein bodies. During priming protein body breakdown was more extensive in the micropylar region endosperm cells than was observed prior to germination in non-primed seeds (Haigh, 1988). These changes during priming appear to associate with the endosperm weakening and may be part of the enhancement of germination caused by priming. Similar morphological changes were observed in Money­maker seeds (1. Zingen-Sell, personal communication).

However, as in both non-primed and primed seeds the mechanical re­sistance never fully disappeared, an additional step would appear to be necessary for radicle emergence to occur. This second step would immediate­ly precede radicle emergence and would probably resemble a cell separation process similar to abscission rather than be an extensive breakdown of wall material. As it is impossible to predict the exact time of radicle emer­gence in individual seeds, this second step may be very difficult to iden­tify.

These studies have shown that tomato seeds prime because the endosperm does not weaken sufficiently to permit expansion of the radicle. The mecha­nism by which some endosperm weakening was permitted but the final weak­ening for radicle emergence was prevented was not identifiable. A main area for further investigation is what mechanisms are involved in the removal of the last barrier for radicle protrusion.

LETTUCE

Germination of lettuce achenes ("seeds") is often restricted to rather low maximum temperatures of around 20 to 25 ·C. This phenomenon is often termed thermoinhibition or thermodormancy but essentially it is an example of a phenomenon common to most wild species: dormant seeds only germinate at a restricted range of temperatures, dormancy breaking widens the range and dormancy induction narrows it (Karssen, 1982). In some species like lettuce the maximum temperature (Tmax) for germination varies, in other species like muskmelon (Bradford et al., 1988) the minimum temperature.

Priming of lettuce seeds is thus basicly breaking of dormancy by rather low temperatures. In experiments with seeds of cv. Musette, which had an extremely low T50 (the temperature for 50% germination) of 15 ·C (Table 1), the Tmax increased to over 34 ·C when pretreatment occurred at 2 ·C. Unfortunately, such a treatment also caused seed vernalization leading to bolting (Weges, 1987). Therefore, moderate temperatures around 15 ·C are more sui table.

If pre-incubation occurred in -0.5 MPa PEG instead of water the treat­ment could be extended and consequently resulted a higher Tmax. Moreover, the priming effect was more stable to drying. The reduction of the improved Tmax was reversible by a second incubation at moderate temperatures (Weges,

275

1987). Therefore, this effect of drying differs from the damage that is caused when seeds that have started extension growth are dehydrated. Compa­rative studies of different seed lots of different cultivars indicated a positive correlation between the T50 of non-primed seeds and the optimal temperature for priming.

Table 1. The temperature for 50% germination of lettuce seeds cv. Musette. Seeds were pre-incubated at different conditions in water or PEG without or with drying to a moisture content of 4.1% (From Weges, 1987).

pre-incubation conditions

none water, 20 h 15 ·C water, 40 h 10 ·C water, 5 d 2 ·C PEG -0.5 MPa, 40 h 15 ·C PEG -0.5 MPa, 72 h 10 PEG -0.5 MPa, 17 d 2·C

not dried

15 23.5 26.5 34 25.5 27.5 34

dried

16.0 21.0 25.0 21.5 23.5 28.0

Psychrometric measurements of osmotic potentials (~n) showed that the increase of T50 was not associated with osmotic adjustment of cells (Weges, 1987). Bradford et al. (1988) point to a comparable situation in osmotical­ly stressed seedling roots where osmotic adjustment to restore turgor was very slow compared to the rapid restoration of the growth rate. The authors concluded that maintenance of cell growth despite decrease in turgor impli­cates changes in cell wall properties.

Fig. 4.

276

1.

30

0.8 ~

c...

~0.6 >-

0.4

01 I

0 Incubation period, h

The yield threshold Y of lettuce seeds cy. Capitan at 24 ·C following pre-incubation in water at 15 • (0), 24 • (~, or 30 ·C (D). The arrow indicates the moment of germination (from Weges, 1987).

In an analysis of the water relations of lettuce seeds cv. Capitan, Weges (1987) pre incubated seeds in water at 15, 24 or 30 ·C. At 15 ·C T50 was increased and at 30 ·C it decreased, 24 ·C hardly changed T50. The rise and fall of T50 was negatively correlated to ~50 (the external osmotic potential that enables 50% germination). During pretreatment at 15 ·C the resistance to osmotic stress (tested at 24 ·C) increased, at 30 • it decreased.

As we concluded before, germination is a specialized growth phenomenon. Therefore, inhibition of germination by osmotic stress may be regarded as a steady-state growth rate of zero. According to Lockhart (1965), as developed by Schopfer and Plachy (1985), at zero growth ~ 50 = ~n+Y,where Y is the minimum turgor at which growth occurs (the yield threshold). Since ~n was not influenced by any pre-incubation tempera­ture, the changes in ~50 indicated changes in cell wall extensibility. Y decreased during pre-incubation at 15 ·C but increased at 30 ·C (Fig. 4). Thus it seems that lower temperatures specifically increases T50, i.e. breaks dormancy, because only at those temperatures cell wall extensibility can be decreased. Higher temperatures have an opposite effect.

For seeds with tissues that enclose the radicle, Y will be composed of a component due to the radicle cell walls (Yr) and one due to the enclosing endosperm, testa and pericarp (Ye). Bradford et al. (1988) concluded that priming of lettuce seeds cv. Empire at 20 ·C lowered Yr at 34 ·c, whereas Yewas not effected. Data obtained by Weges (1987) do not permit accurate determinations of the two components of Y, but evidently, pre-incubation at 15 ·C increased the resistance to osmotic stress of both intact seeds and isolated embryos of cv. Musette, suggesting a decrease of both Yr and Yeo It has to be realised that Empire seeds were clearly non-dormant, 100% germination occurred at 34 ·C, whereas Musette seeds were dormant, the T50 was 20 ·C. ComparIsons between cultivars showed that the deeper the dorman­cy the larger the contribution of the enclosing structures to the restraint of radicle protrusion. Therefore, it is most likely that priming increases cell wall extensibility in both seed parts of lettuce. Endosperm cells opposing the radicle were highly vacuolated prior to radicle protusion and showed clearly mobilized storage materials. Cells at the lateral and co­tyledonary end of the endosperm were not changed (Psaras et al., 1981). Enzymatic studies of endosperm loosening have only described changes associated with visible germination (Bewley et al., 1983). Priming related changes are not known yet.

LONGEVITY AND REPAIR

In the range of conditions involved in commercial seed storage and longterm storage for the conservation of genetic resources the logarithm of any measure of longevity is a negative linear function of the logarithm of percentage moisture content (Roberts and Ellis, 1989). In lettuce seeds it extends from 2.6 to 15% moisture content. Above the critical moisture content, which varies between species, the trend is reversed and, providing oxygen is present, seed longevity increases with increase in moisture content until full hydration is reached. Therefore, storage at very low moisture contents may be as good as in fully hydrated state.

The benefits of hydrated storage were also noticed with aged seeds. Partial hydration of aged lettuce seeds by .exposure to moist air (up to 34% moisture content) or to an osmotic priming solution (up to 44% moisture content) reduced the proportion of seeds which would otherwise germinate abnormally, increased subsequent root lengths and reduced the frequency of visible chromosome damage at first mitoses (Rao et al., 1987). Liou (1987) reached similar results by priming aged cabbage seeds in -1.5 MPa PEG.

277

Thus, in addition to the benifits discussed so far, priming also gives repair processes an opportunity to restore storage damage.

Priming is not always beneficial to seeds. Recently it has been re­ported that primed lettuce seeds show reduced storability at moisture contents between 5.0 and 16% and temperatures between 22D and 55 DC. (Weges, 1987). Bradford et ale (1988) found that primed tomato seeds re­tained their high quality for years when stored below 10 DC, but longevity was reduced by storage at high temperatures even at low moisture contents. In contrast, Thanos et ale (1989) reported that priming of pepper seeds in 0.4 M mannitol at 25 DC improved the resistance of seeds to storage at 25 DC at 96% moisture. Further study is required to explain these conflicting results. Better knowledge of the processes involved in ageing and repair will greatly benefit our understanding of priming effects.

CONCLUSIONS

The significant benefits arIsIng from seed prImIng have been readily explored and are being applied in the cultivation systems of many crops. However, the study of the physiological mechanisms of priming is left with many open questions, particularly at the molecular level. Nevertheless, some general conclusions are starting to appear.

It is evident that inCUbation in osmotic solutions only prevents the processes directly involved in radicle protusion. All other reactions seem to proceed albeit often at a reduced rate as is shown for instance, for respiration in lettuce (Weges, 1987), cell division in celery (Van der Toorn, 1989), and protein and DNA synthesis in leek seeds (Bray et al., 1989). The reduced rates ask for the prolonged priming times that are standard in most protocols.

As a result of priming, seeds may contain increased levels of certain products. When the product is an enzyme, reactions in primed seeds start quicker after imbibition and at a much higher rate (Fig. 3).

It is an open question whether priming stimulates "more of the sam~1 or induces the synthesis of speci fic proteins, I.e. controls at the level of gene expression. Some evidence appears in favour of the latter conclu­sion (Bray et al., 1989). Future research has to show whether the increased molecular activity is indeed essential for the effect of priming.

Comparison of the three species that were described in this paper show both similarities and differences. In all three species priming prepared a quick expansion of radicle cells. In celery embryos new cells also were formed. In all species the embryo expansion depended to a certain extent on changes in the endosperm. The endosperm of celery had to be hydrolyzed and its products had to be absorbed by the embryo to permit growth and to make space. Endosperm breakdown was not prevented by priming conditions. Radicle protusion in celery seeds depended both on a critical length of the embryo (1.5 mm in cv. Monarch) and on hydrolytic changes in the few cell layers of endosperm at the micropylar end. Also in tomato and lettuce seeds radicle protrusion was preceeded by specific hydrolytic changes that only occurred in the endosperm cells that oppose the radicle tip. In tomato seeds endosperm weakening could be measured. The process also occurred during priming at a slightly reduced rate. In lettuce, endosperm resistance seems to differ between dormant and non-dormant seeds.

In general, priming in all three species seems to permit a quick and uniform germination by stimulating cell wall extensibility in the radicle and specific weakening of essential endosperm cell walls.

278

Priming induced repair processes are most probably of a different character than those involved in stimulating fast and synchronous germina­tion.

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Austin, R.D., Longden, P.C., and Hutchinson, J., 1969, Some effects of 'hardening' carrot seed, Annals of Botany, 33:883.

Bradford, K.J., 1986, Manipulation of seed water relations via osmotic priming to improve germination under stress conditions, HortScience, 21:1105.

Bradford, K.J. Argerich, C.A., Dahai, P., Somasco, 0., Tarquis, A., and Welbaum, G.E., 1988, Seed enhancement and seed vigor, in: "Proc. Int. Conf. on Stand Establishment for Horticultural Crops," M.D. Orzolek, ed., Lancaster, Pennsylvani~

Bray, C.M., Davison, P.A., Ashraf, M., and Taylor, R.M., 1989, Biochemical changes during osmopriming of leek seeds, Annals of Botany, 63:185.

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Groot, S.P.C., and Karssen, C.M., 1987, Gibberellins regulate seed germina­tion in tomato by endosperm weakening: a study with gibberellin­deficient tomato seeds, Planta, 171:525.

Groot, S.P.C., Kieliszewska-Rokicka, B., Vermeer, E., and Karssen, C.M., 1988, Gibberellin induced hydrolysis of endosperm cell walls in gibberellin-deficient tomato seeds prior to radicle protusion, Planta, 174:500.

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Heydecker, W., Higgins, J., and Gulliver, R.L., 1973, Accelerated germina­tion by osmotic seed treatment, Nature, 246:42.

Karssen, C.M., 1982, Seasonal patterns of dormancy in weed seeds, in: "The Physiology and Biochemistry of Seed Development, Dormancy ana-Germi­nation", A.A. Khan, ed., Elsevier, Amsterdam.

Liou, T.S., 1987, "Studies on Germination and Vigour of Cabbage Seeds," Ph.D. Dissertation, Agricultural University, Wageningen, The Nether­lands.

Lockhart, J.A., 1965, Analysis of irreversible plant cell elongation, J. Theor.Biol. , 8: 264.

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Rao, N.K., Roberts, E.H., and Ellis, R.Il., 1987, The influence of pre- and poststorage hydration treatments on chromosomal aberrations, seed­ling abnormalities, and viability of lettuce seeds, Annals of Botany, 60:97.

Roberts, E.H., and Ellis, R.H., 1989. Water and seed survival, Annals of Botany, 63:39.

Schopfer, P. and Plachy, C., 1985, Control of seed germination by abscisic acid. III. Effect on embryo growth potential (minimum turgor pressure) and growth coefficient (cell wall extensibility) in Brassica napus L., Plant Physiol. 77:676.

Thanos, C.A., Georghiou, K., and Passam, H.C., 1989, Osmocondi tioning and ageing of pepper seeds during storage, Annals of Botany, 63:65.

Van der Toorn, P., 1989, "Embryo growth in MatureCelery Seeds," Ph.D.

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Dissertation, Agricultural University, Wageningen, The Netherlands. Watkins, J.T., and Cantliffe, D.J., 1983, Mechanical resistance of the seed

coat and endosperm during germination of Capsicum annuum at low temperature, Plant Physiol., 72:146.

Weges, R., 1987, "Physiological Analysis of Methods to Relieve Dormancy of Lettuce Seeds," Ph.D. Dissertation, Agricultural University, Wage­ningen, The Netherlands.

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