Plant Physiol. (1974) 53, 875-878

Seed Aging: Chromosome Stability and Extended Viability ofSeeds Stored Fully Imbibed1

Received for publication October 29, 1973 and in revised form February 7, 1974

T. A. VILLIERSDepartment of Biological Sciences, University of Natal, Durban, South Africa

ABSTRACT

Increase in moisture content of seeds of Lactuca sativa L. andFraxitnus americana L. in air-dry storage caused a rapid declinein longevity and an increase in the rate of accumulation ofchromosome aberrations. Storage of seeds fully imbibed butunable to germinate allowed a high germination capacity tobe maintained for long periods, together with a very low in-cidence of chromosome aberrations. Seedlings grown fromdry-stored seeds showed an increase in morphological abnor-malities with length of storage, whereas seedlings from im-bibed-stored seeds appeared normal. It is suggested that in drytissues, enzyme-controlled turnover and repair may be tem-porarily suspended, and that this may be an important factorin the loss of seed viability in storage. The effect of increasingseed longevity by lowering the moisture content of dry-storedseeds is discussed in relation to this hypothesis. The relevanceof the proposal is also discussed in relation to ecological studies.

air-dry. They ascribed this increased longevity to the state ofdormancy of the imbibed seeds, in which a low metabolic ratecould enable the seeds to survive even though the tissues werehydrated. However, as there is no reason to suppose that im-bibed seeds have a lower metabolic rate than air-dry seeds, evenwhen dormant, this does not explain why imbibed seeds shouldsurvive for longer periods than air-dry seeds.

During experiments on seed dormancy in this laboratory,various seed samples which had been maintained fully imbibedfor long periods without being given the dormancy-releasingstimuli of low temperature or illumination were found to havegreatly extended life spans compared with seeds of the sameprovenances stored air-dry. Moreover, the plants grown fromseeds stored fully imbibed showed very few abnormalities ofmorphology or growth even when seeds from the same har-vests, but stored air-dry, were dead. It was therefore decided toinvestigate the rate of accumulation of chromosomal damage inseeds stored fully imbibed compared with seeds stored air-dryat a range of moisture contents.

It is well known that low moisture content is an importantfactor in maintaining the viability of seeds in dry storage (8),and as a general rule, a decrease of 1 % in moisture contentmay approximately double the life span of a seed sample (5).From a consideration of the results of experiments in whichseed moisture content is varied during storage, it would beexpected that seeds stored for a time in a fully imbibed statewould have a very short life indeed. However, it is a commonecological observation that seeds may lie buried in the soil formany years and still remain viable (2), and therefore the be-havior of seeds in the soil appears at first sight to be anomalous.

Seedlings grown from samples of old seeds may show highmutation rates, and the losses in viability of stored seeds havebeen in part ascribed to the accumulation of deleterious muta-tions (8). Genetic damage may accumulate until eventually theembryos are unable to germinate and grow. Abdalla and Rob-erts (1) suggested that the number of chromosome aberrationsseen in the first cell divisions occurring during germination maybe taken as a measure of the damage accumulated during thestorage period.

Toole and Toole (10) showed that when lettuce seeds ofseveral varieties were maintained imbibed in the laboratory,but prevented from germinating, the seeds remained alivelonger than seeds from the same harvest which had been stored

'This work was supported by funds from the Council for Sci-entific and Industrial Research, and the University of Natal Re-search Fund.

MATERIALS AND METHODS"Seeds" (fruits) of Lactuca sativa L. vars. Arctic King and

Grand Rapids were obtained from Suttons Seeds Ltd., Reading,U. K. Batches of these seeds were placed in closed containerswith separate vessels of sulfuric acid, water, or diluted sul-phuric acid giving relative humidities of 0, 25, 50, 75, and100% at 20 C in the container atmospheres (9). After 2 weeks,the moisture contents of the seed batches were determined, andthe seeds were sealed into separate glass bottles. Other largebatches of seeds from the same provenances were imbibed onmoist filter papers in Petri dishes. These Petri dishes, togetherwith the bottles of air-dry seeds, were placed in dark incubatorsmaintained continuously at 30 C (Arctic King), and 25 C(Grand Rapids), in order to prevent germination of the im-bibed batches of seeds.

Fruits of Fraxinus americana L. were purchased from Vil-morin-Andrieux of Paris. Samples were brought to a range ofmoisture contents as described for the lettuce seeds. They werethen sealed into bottles, and stored in the dark at 22 C. A largesample of fruits was imbibed by mixing with moist sand andstored at 22 C in the dark in order to prevent germination ofthe imbibed fruits (unpublished results).At the times stated in the results, seeds from these treatments

were tested for germination under 40-w fluorescent daylightlamps at 22 C. Petri dishes of seeds which had been maintainedcontinuously imbibed were simply brought out from the darkincubators, while samples of seeds from air-dry store wereimbibed on moist filter papers in Petri dishes at the beginningof each germination test. Germination was scored as the emer-gence and geotropic curvature of the radicle.Chromosomes in 1-mm radicle tips of lettuce were ex-

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Plant Physiol.. Vol. 53, 1974

amined by the aceto-carmine squash method (7) after fixationovernight in acetic acid-alcohol, 1:3. Aberrant nuclear divisionswere scored as percentages of 400 late anaphase figures ob-served for each sample of seeds tested from a treatment. Thisinvolved the examination of 40 to 50 root-tip squashes in mosttreatments.To observe the growth of the seedlings following the various

seed storage treatments, batches were allowed to continuegrowth in the Petri dishes after the germination tests. Thesamples grown on to maturity were planted in pots after thehypocotyls had become fully extended, and grown in 16-hr-longdays until flowering and seed set.

RESULTS

Table I shows the moisture contents attained by lettuce andF. americana seeds maintained at a range of atmospheric rela-tive humidities for 2 weeks. The seeds were then stored at thesemoisture contents, and also in a fully imbibed state in contactwith liquid water, as described above. Germination tests carriedout at intervals during the storage period showed that as theseed moisture content was increased, the period of viability ofthe various seed samples became greatly shortened (Figs. 1and 2).The very rapid loss of viability of the Arctic King lettuce

seeds was most probably due to the high temperature of 30 Cmaintained throughout the storage period, but which was nec-essary in order to prevent germination of the fully imbibedseeds. The experiments have been repeated with the lettucevariety Grand Rapids, which remain dormant at 25 C in thedark even though imbibed, and exactly similar results havebeen obtained. It should also be noted that the experimentsusing F. americana were maintained continuously at 22 C.

Figures 1 and 2 show that seeds of both lettuce and F.americana maintained full viability for an indefinite period oftime provided they were maintained in contact with water dur-ing storage. When plants were grown from seedlings producedin the germination tests, an increasing number of abnormalitieswas observed with increase in time of dry storage, includingstunted growth, distorted cotyledons with necrotic patches oftissue, subdivided first leaves, swollen roots and necrosis of theradicle meristems. On the other hand, lettuce plants grownfrom seeds stored for more than 12 months continuously im-bibed seemed to be normal in all respects and grew vigorously(Fig. 3). The plants produced from imbibed-stored seeds flow-ered and set seeds, which in turn germinated fully (98%) andproduced a second generation crop of mature plants of greatuniformity and vigor, without any observable mutations.Chromosome Studies. Lettuce seeds were imbibed under

fluorescent lighting and germinating seeds with radicles of in-creasing length were fixed in acetic alcohol. Using the acetic

Table 1. Moistutre Conztents of Batches of Seeds of F. americantaanid L. sativa after Storage for 2 Weeks at a Rantge of

Atmospheeric Relative Hiumidities

RelativHumidityofater Content of SeedsRelative Hfumidity of

Store at 20 CF. americana L. sativa var. Arctic King

. ~ ~~~~.C ,

0 4.7 3.625 5.4 5.1

50 6.8 7.075 9.5 9.7100 18.6 13.5

a bU0

40 -

20-

9.5186

I I_- Iu 2 4 6 8 10 12

months of storage

FIG. 1. Germination of seeds of F. americana from large batchesstored air-dry (-0-) at 22 C with seed moisture contents of 5.4,9.5, and 18.6%, and also fully imbibed (-O-) mixed with moistsand and kept in darkness to prevent germination during storage.Germination tests were conducted at 22 C under fluorescent light-ing. "Germination" was scored as emergence and geotropic curva-ture of the radicle.

100 imbibed

80.

c 60-0

4040ON

months of storage

FIG. 2. Germination of seeds of lettuce, var. Arctic King, fromlarge batches of seeds stored air-dry (---) at 30 C with seedmoisture contents of 5.1, 7.0, 9.7, and 13.5%, and also fully imbibed(---) in darkness on moist filter papers in Petri dishes. Germina-tion tests were conducted at 22 C under fluorescent lighting.

squash technique, the total number of mitotic figures in radiclemeristems was recorded for various stages of germination, frompre-emergence at 20 hr to 10 mm in length at 48 hr. Cell di-visions were not seen until the radicles were about 2 mm long,and increased to a maximum at about 3.5 mm (Fig. 4).

Germinating lettuce seeds with radicles 3 mm long were se-lected from the germination tests conducted at regular intervals

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SEED AGING AND CHROMOSOME STABILITY

FIG. 3. Lettuce seedlings grown from germination tests described in Fig. 2. Both samples had been stored for 12 months at 30 C in the darkLeft: seed sample stored fully imbibed on filter paper kept continuously moist: right; seed sample stored continuously air-dry, with a moisturecontent of 5.4%. t

2001

.)

0

E

1601

1201

80[

401

0 *2 4 6

length -of radicle in mm.

8

FIG. 4. Total numbers of mitotic figures in radicle tips of in-creasing length during germination of lettuce, var. Arctic King.Germination occurred on moist filter paper under fluorescent lightsat 22 C, and 1-mm radicle tips were excised and examined by theaceto-carmine squash technique. Seeds had been recently purchasedin sealed containers from commercial suppliers, and not subjectedto any laboratory storage treatment.

on samples of seeds stored air-dry at the range of moisture con-

tents described above, and also from the samples stored im-bibed. Using the acetic squash method, the percentage of aber-rant nuclear divisions was scored for each seed sample.

Figure 5 shows that with an increase in moisture content ofseeds in air-dry storage, the percentage of chromosome aber-rations increased sharply with increasing time of storage. Thetypes of aberrations seen were mainly chromosome bridges,and deletions represented by duplicated chromosome frag-ments. In many of the germination tests carried out on seedsstored air-dry under suboptimal conditions, a high percentageof radicle emergence was frequently found, but in many cases

few of the seedlings were able to continue growth. Such sam-ples always showed a high percentage of chromosome aberra-tions. However, germination tests of the seeds stored fullyimbibed showed in all cases not only very high germinationcounts and rapid establishment of vigorously growing plants,but also a very low incidence of chromosome damage.

For the demonstration of recessive mutations, it is necessaryto continue the observations to the second generation of plantsfollowing an experiment. Seeds harvested from the plantsgrown from imbibed-storage experiments, as described above,showed less than 1 % chromosome aberrations when germi-nated within 1 month of harvesting. Most of the samplesshowed no aberrations whatever, and this was presumed to bebecause of the more careful handling during harvesting and

50

9.7

7'040

0'.0

5-1

02206 8 10 1

0E0

months of storage

FIG. 5. Percentage aberrant nuclear divisions in radicle tips oflettuce, var. Arctic King, in samples withdrawn at- intervals fromlarge batches stored air-dry (-0-) with moisture contents of 13.5,9.7, 7.0, 5.1, and 3.6%, and also fully imbibed (-O-) on moist filterpapers. The distal 1-mm of each radicle at the 3-mm stage of germi-nation (Fig. 4) was excised and examined by the aceto-carminesquash technique. Aberrations were scored as percentages of a totalof 400 late anaphase figures observed, involving from 40 to 50seedlings from each sample at each germination test.

Plant Physiol. Vol. 53, 1974 877

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Plant Physiol. Vol. 53, 1974

storage in laboratory experimentation compared with large-scale commercial seed production and distribution, which mayoften damage crop seeds.

It appears that while dry storage allowed the accumulationof nuclear damage, at rates more or less rapid according tostorage conditions, imbibed storage in contact with water al-lowed the seeds to maintain a high germination capacity and ahigh degree of genetic stability.

DISCUSSION

A large amount of empirical data has accumulated on opti-mum storage conditions for seeds. However, the basic causeof the loss of viability of seeds in dry storage has not yet beendiscovered. Increase in the moisture content of seeds in air-drystorage would be expected to increase their metabolic rates, andtaken together with the deleterious effects of warm tempera-tures and the presence of oxygen (8), it has been assumed thatdry seeds are able to survive because they possess a very lowmetabolic rate.A new approach to the problem of the loss of seed viability

becomes possible if one considers the seed in its normal en-vironment. Several workers have remarked that the ability ofseeds to remain alive in the soil for very long periods seems tobe contrary to the information resulting from seed storage ex-periments.

Although the ability of seeds to remain alive in an air-drystate is a biological advantage as part of the dispersal mecha-nism of the majority of seeds, it is probable that in a naturalenvironment the majority of seeds are air-dry for only a shorttime. After dispersal from the parent plant, the seeds fall intothe surface litter on the soil, where they may become rehy-drated. If such seeds do not germinate immediately, but remaindormant, they are able to survive for long periods of time,either continuously or intermittently imbibed, and thereforewith a high water content.

It has usually been stated that seeds in the soil are able tosurvive because they are dormant (10), implying that they havea low rate of metabolism. However, dormant seeds are ableto carry out many metabolic functions, including food inter-conversions, membrane synthesis, and organelle production(11). It has been concluded that seed dormancy is not a stateof general inactivity, but must be due to some specific meta-bolic block (3). Furthermore, the possession of a low metabolicrate does not explain why seeds in the soil should live longerthan air-dry seeds. Therefore, considered from the point ofview of the seed in nature, it is obvious that air-dry seed storagefor extended periods is totally artificial. It becomes possibleto accept that it is continued air-dry storage itself which is thecause of seed deterioration, and it is pertinent to ask whatprocesses occur in imbibed, dormant seeds which cannot takeplace in air-dry seeds.The existence of macromolecular repair systems in cells

has now been demonstrated. In addition, organelle turnoverhas been shown to occur and is assumed to be necessary toreplace worn-out or damaged cellular components. It is partic-ularly significant when considering genetic stability, that dam-age to DNA can be repaired under the control of enzymes ina wide range of organisms (4). It seems reasonable to expectthat such repair and maintenance systems should function as anormal, and indeed essential cellular activity.

However, in view of the fact that the increase in chromo-some damage in seeds in dry storage does not occur in imbibed-stored seeds, I suggest that in dry tissues turnover and repairactivities may be temporarily in abeyance, leading to the ac-cumulation of damage to macromolecules and organelles.

It remains to be explained why seed longevity is progressivelyincreased by lowering the moisture contents of seeds in air-drystore. If cellular maintenance activities are suspended in air-dry seeds, any change in the physical conditions of storagewhich would tend to improve the stability of macromoleculeswould prolong the viability of the seed as a whole unit, andtherefore lowering the seed moisture content in air-dry storagewould be expected to increase longevity. Even in a water-saturated atmosphere at 20 C, lettuce seeds do not appear toattain more than about 14% water content, and it is probablethat a higher water content would be necessary before macro-molecular repair and membrane synthesis could occur at anormal rate.

It is significant that certain seeds which rely on the preven-tion of water uptake by impermeable coats as a dormancy-imposing mechanism possess morphological adaptations suchas hilar valves, which ensure that the tissue moisture contentbecomes progressively lower with fluctuating atmospheric rela-tive humidity, and is eventually maintained at the lowest levelpossible (6). Seeds such as citrus, which are damaged by dryingto a low moisture content, probably do not become dry in thenormal course of events in nature, but a sufficient number willbe deposited in positions where they can remain hydrated untilgermination. It is only when such seeds require storage thattheir inability to survive in the dry state becomes a problem.Wet storage in a dormant state would be the obvious answerto this difficulty.

If the loss of viability of dry seeds is caused by the inactivityof repair systems, damage to macromolecules and organellescan only be repaired when the seeds are imbibed for germina-tion, by which time the damage might be too extensive forrepair to be effective. In wet-stored seeds, or in seeds in thesoil, such damage might be repaired as it occurs, and notbegin to accumulate unless, or until, the cellular maintenancesystems themselves become faulty. Experiments are in progressto test the suggestions made in this paper.

LITERATURE CITE

1. ABDALLA, F. H. AND E. H. ROBERTS. 1968. Effects of temperature, moisture,and oxygen on the indluction of clhromosome damage in seeds of barley,broad beans, and peas during storage. Ann. Bot. (N.S.) 32: 119-136.

2. BARTON, L. V. 1961. Seed Preservation and Longevity. Leonard Hill, London.3. CIHEN, S. S. C. AND J. E. VARNER. 1970. Respiration and protein synthesis in

clormant and after-ripened seeds of Arena fatua. Plant Physiol. 46: 108-112.4. HAIN-AWALT, P. C. 1972. Repair of genetic material in living cells. Endeavrour 31:

83-87.5. HARRINGTON, J. F. 1973. Problems of seed storage. In: W. Heydecker, ed.,

Seed Ecology. Butterworths, London. pp. 251-263.6. HYDE, E. 0. C. 1954. The function of the hilum in some Papilonaceae in rela-

tion to the ripening of the seecl. Ann. Bot. (N.S.) 18: 241-256.7. PEACOCK, H. A. 1966. Elementary Microtechnique. Arnold, London.8. ROBERTS, E. H. 1972. Viability of Seeds. Chapman and Hall, London.9. SOLOMON, M. E. 1951. Control of humidity with potassium hydroxide. stil-

phuric acid, and other solutions. Bull. Entomol. Res. 42: 543-554.10. TOOLE, V. K. AND E. H. TOOLE. 1953. Seed dormancy in relation to seed

longevity. Proc. Int. Seed Test. Assoc. 18: 325-328.11. V'ILLIERS, T. A. 1971. Cytological studies in dormancy. I. Embryo maturation

during dormancy. New Phytol. 70: 751-760.

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