Dormancy in seeds imposed by the seed coat. - Springer Link

Dormancy in seeds imposed by the seed coat. By

Lela V. Barton. With 5 figures.

I. Introduction. The resting condition of many seeds, especially those of different farm and garden crops, is maintained only as long as the seeds are in dry storage. As soon as a suitably moist medium and a favorable temperature are provided germination proceeds almost immediately. In many other seeds, the embryo is in a genuine (endogenous) dormancy condition which must either disappear "spontaneously" or must be broken by the action of certain environmental conditions before germination can take place. In a surprising number of seeds, however, dormancy is imposed by the presence of a "hard" seed coat, a phenomenon which was recognized long ago. In such seeds, germination depends on changes in the seed coat. It should be kept in mind that hard coats are quite frequently either combined with dormant embryos, or that they may enhance the manifestation of a dormancy condition in the embryo. However, these interrelations with embryo dormancy will be treated in other chapters of this volume 1 ; the present review, which is based on selected, representative literature, is limited to a consideration of the causes of hard-coatedness in seeds, and a brief summary of methods to overcome this condition.

11. Impermeable seed coats. 1. Occurrence and variability. Hard-coatedness is usually caused by impermeability of the seed coat or some of its layers to water or to gases, though both mechanical and chemical inhibition of germination mayaiso be factors in dormancy. In some cases, e.g. avocado, Persea gratissima (EGGERS 1942) and tung, Aleurites fordii (SHARPE and MERRILL 1942), germination is promoted by removal of the seed coat but the nature of this effect is unknown. Impermeable coats are characteristic of certain species and even of certain families of plants. The family Leguminosae is the one most commonly known to possess seeds with impermeable coats, but certain members of other families (Gramineae, Malvaceae, Oannaceae, Geraniaceae, Ohenopodiaceae, Oonvallariaceae, Oonvulvulaceae, Solanaceae, and others) also produce such seeds. Within any given species there may be a variation in the percentage of hard seeds produced from year to year. For example, crops of the white sweet clover, 1 "Seed dormaney: General survey of dormaney types, and dormaneyimposed byexternal agents" by L. V. BARTON, pp.699-720 (speeifieally, p. 702); "Temperature and seed dormaney" by P. STOKES, pp. 746-803 (p.774 et seg.); and "Light and seed dormaney" by M. EVENARI, pp. 804-847 (p. 824 et seg.).

A. Lang (ed.), Differentiation and Development © Springer-Verlag Berlin Heidelberg 1965

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Dormancy in seeds imposed by the seed coat.

Melilotus alba Desr., vary from 2% to more than 90% hard seed, depending upon the particular growing season· (BARTON, unpubl.). Furthermore, within a given seed lot there is a wide variability in the degree of impermeability of the individual seeds. For example, when 1000 old seeds of the East Indian lotus, Nelumbo nucitera Gaertn., were placed in water in the laboratory at Boyce Thompson Institute for Plant Research, Inc., Yonkers, New York, they softened and became swollen at the rate of two or three seeds per year for five years (BARTON, unpubl.). Theoretically, then, these seeds would be providing new plants for 300 to 500 years, though actually all of them may become permeable at an earlier date. More than 20 % of seeds of Robinia pseudo-acacia L. remained hard two years after planting in sand (KONDO 1929). Eighty-three of these hard seeds were then placed in water where about 6 % were still hard after 14 years. Thus, the character of impermeable seeds permits plants to be distributed in time instead of space and that may be an important factor in the continuance of the species. Also it has been reported by DEXTER (1955) that hard seed of alfalfa (Medicago sativa L.) provide survival insurance under adverse sowing conditions such as frost or drought. Other studies in which the survival value of impermeable coats is considered include those by lIARRINGTON (1916), JUBY and PHEASANT (1933), OHGA (1923, 1926a) and PORTER (1949); the subject is also considered in the book by BARTON and CROCKER (1948).

2. The restrictive action of impermeable coats. a) Water absorption. This is the most common type, found in the Leguminosae and almost all of the other families with hard-coated species which were listed in the preceding section. It is generally assumed that the most common way by which such seeds become permeable in nature is through attack by soil microorganisms. This is very probable, but direct evidence is scarce. PFEIFFER (1934), working with Symphoricarpos racemosus (a species with dormant embryos), showed that the softening and disintegration of the seed coats is caused by decomposition of wall substances by fungi. If the seeds are kept under conditions favorable for this process (moist storage, optimal temperatures) but are maintained free from fungi the coats remain unchanged. In some species, the seed coats become permeable in prolonged dry storage. This has been reported, e.g., for alfalfa (Medicago sativa) by LUTE (1928) and guayule (Parthenium argentatum) by BENEDICT and ROBINSON (1946); thelatter authors believe that the changes are caused by slow oxidation processes. In other species, the seeds seem to lose impermeability because of purely climatic influences, such as frost, as reported by MIDGLEY (1926) for alfalfa, or winter conditions in general, as described by MARTIN (1922) for Melilotus alba. The seeds of Rhus ovata have been found to become permeable after exposure to brush fires, this effect being caused by rupture of the coat (STONE and JUHREN 1951 1 ). Ingestion of hard-coated seeds by birds (SWANK 1944) or domestic animals (lIARMON and KEIM 1934, P. MÜLLER 1934, BURTON 1948) and passage of these seeds through the alimentary tract of these animals also result in increased germination of the seeds. Artificial measures to break impermeability of seed coats will be summarized later (p. 739 et seq.). 1

For further discussion, see chapter by P.

STOKES,

specifically p. 753.

Impermeable seed coats.

729

b) Restriction 01 gaseous exchange. The best-known case of a seed in which the coat is permeable to water but pervents germination by interfering with gas exchange is undoubtedly that of Xanthium, the cocklebur. It has been thoroughly studied by CROCKER (1906), SHULL (1911) and THORNTON (1935). The coat is impermeable to oxygen, and of the two seeds of a bur (the compound fruit of the plant), the upper one shows the phenomenon much more strikingly than the lower, the oxygen supply to its embryo being reduced to as little as one sixtieth. Other seeds with coats relatively impermeable to oxygen are those of Orataegus mollis (DAVIS and RosE 1912), Ambrosia trifida (DAVIS 1930), Fraxinus pennsylvanica (SPAETH 1934), potato (STIER 1937) and apple (VISSER 1954). Lettuce (Lactuca sativa) seeds possess a cuticular membrane on the inner side of the endosperm which is relatively impermeable to carbon dioxide (BORTHWICK and ROBBINS 1928); seeds of Oucurbita pepo have two layers which are impermeable to both oxygen and carbon dioxide, the inner coat being less impermeable than the outer but nevertheless more important as the outer one is pierced by the micropyle (R. BROWN 1940). In some of the seeds, such as lettuce and apple, the gas impermeability of the membrane interferes with germination only under particular conditions such as high temperature (VISSER l. C., BORTHWICK and ROBBINS l. c.). The noxious weed, Digitaria sanguinalis (L.) Scop., also has seed coats which do not exclude the water, but impose a restriction on gaseous exchange which causes dormancy (DELOUCHE 1956). Similar effects were noted for seeds of Agropyron smithii Rydb. and Poa pratensis L. VOSE (1956) also has attributed the coat effect of seeds of Phalaris to an inhibition of gaseous exchange, which he described either as a lack of oxygen or an accumulation of carbon dioxide having a narcotic effect on the embryo. An unusual situation is present in Typha latifolia. The seeds of this plant also have coats which restrict the access of oxygen to the embryo; however, as in this species germination is favored by low oxygen tensions, the removal of the coat results in an inhibition of germination (MORINAGA 1926).

3. The factors determining impermeability of seed coats. a) Struetural properties 01 impermeable seed eoats. Earlier work. Many authors have considered the possible cause of seed coat impermeability.

As early as 1876, NOBBE suggested that impermeability was due to a waxy

layer over the seeds. Since that time other workers have found the cuticle thicker in seeds which are slow to germinate. WHITE (1908) attributed the impermeability of small leguminous seeds to the cuticular layer over the palisade cells or the coats, and that of large leguminous seeds to the cuticle and a portion of the palisade cells. It is thought that some seeds become soft when the upper walls of the Malpighian cells become separated from each other (REEs 1911) or when the tips of these cells are planed through (LUTE 1928). Substances in the so-called light line, which is present in the epidermis (Malpighian cells) of the seed coat of many legumes (see Fig. lA) and also in other seeds, were believed to cause impermeability in seeds of Melilotus (COE and MARTIN 1920) and Nelumbo nucifera (OHGA 1926b). SHAW (1929), on the other hand, thought that the "light line" was due to a bulge in the cells acting as a convex lens to focus more light rays there, and that the line disappeared upon release of the tension, as, for example, by removal of one palisade cello

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However, STEIN V. KAMIENSKI-JANCKE (1958), on the basis of microscopic and histochemical studies on the seed coats of Gleditsia triacanthos L., Oeratonia siliqua L. and several other Leguminosae, came to the conclusion that the light line is a narrow band in the secondary layers of the cell wall consisting exclusively, or almost so, of cellulose whereas the rest of the wall contains both cellulose and pectic substances. SHAW (l. c.) attributed the impermeability of the Nelumbo coat to a water-proofing substance in the palisade layer, a substance which could be leached out by fat solvents. The changing of pectin into water-resistant substances as the seeds of Gymnocladus dioica Koch matures renders them impermeable according to RALEIGH (1930). Dormancy in the akenes of Alisma plantago-aquatica L. is due to the limitation of water absorption by the inner layer (or layers) of the coat (CROCKER and DAVIS 1914). The outer coat layer absorbs water which cannot reach the embryo because of this restriction imposed by one or both of the inner coats. Legumes. Seed coat structure. A thorough study of the structure of the seeds of Melilotus alba in relation to their impermeability has been undertaken by HAMLY (1932). A drawing of a radial section through the seed co at of this species, taken from HAMLY'S work, is shown in Fig. 1 A. According to HAMLY, the layers of tissue surrounding the embryo are of the same general character as those in other legumes, many of which have impermeable seed coats. Some endosperm ceHs (k), including an aleurone layer (j), are found just outside of the embryo. Next is a nutrient layer (h), so called because it contains starch when the seed is developing, followed by osteosclerid cells with inwardly directed buttresses (g), and the columnar Malpighian cells (e), with special caps (c). Immediately below the caps is the light line (d), and above the caps is a matrix known as the subcuticular layer (b) covered externaHy by a cuticle (a). CAVAZZA (1950) examined the coat of hard seeds of Gleditsia triacanthos and Hedysarum coronarium anatomicaHy and microchemically using polarized light and the electron microscope. He found that, except for the outermost cells, aH cells were impermeable. Thus impermeability does not depend upon the properties of a special celllayer, but is a peculiarity of the normal constituents of most of the cells of the seed coat. WATSON (1948) did not find any definite relation between the structure of the testa of certain Papilionaceae tribes of the legume family and the impermeability of their seeds, since no single structure which prevents the absorption of water was present in every species. Effects of alcohoI soaking and other treatments on seed co at permeability. Several authors have presented evidence showing that the causes of failure to absorb water are different in the different subfamilies of the Leguminosae. VERSCHAFFELT reported as early as 1912 that alcohol soaking had a beneficial effect on seeds of the Oaesalpinoideae but failed to make seeds of Papilionoideae permeable. Whether the mode of entry of water into alcohol-treated seeds is through interstices in the hilum region as described by VERSCHAFFELT, seeds so treated do begin to swell in that region. BARTON (1947), having established that seeds of Oaesalpinoideae and Papilionoideae respond differently to weathering conditions in field plantings (see below, p. 740), showed that seeds of Oladastris, Oytisus, Melilotus, and Robinia, all members of the Papilionoideae, may be made permeable by shaking with a sharp impact in a glass bottle for twenty minutes, but soaking in alcohol for 72 hours has no effect on these seeds. On the other hand, seeds of Oassia,

Structural properties of impermeable seed coats.

731

Cercidium, Gleditsia, Gymnocladus, and Cercis, members of the Caesalpinoideae, are made permeable by the alcohol soaking but not by the shaking. Seeds belonging to the subfamily Mimosoideae seem to occupy a position between those of the other two subfamilies. tL Within the genus Acacia, h for example, 88 % of c A. constricta seeds took ri up water after alcohol treatment while shaking .. - e had very little effect. More A. aneura seeds were made permeable by shaking than by soaking in alcohol f while 100 % of A. greggii seeds became permeable as a result of either treatment.

Thefunction

01 tbe

hilum. While earlier authors regarded the hilum of legume seeds as having no significance in relation to the impermeability of the testa, HYDE showed a more recently (1954) that, at least in the species studied (Trifolium repens L., b T. pratense L. and Lupinus arboreus L.), it serves c as a hygroscopically actid vated valve which permits the seed to lose water e in dry atmosphere but 1 prevents it from absorbing moisture in humid atmosphere. The hilum of the I species studied (see Fig. 1 B) is an ova.l-shaped scar in which tissue derived from !I the funiculus, mainly a layer of so-called counter- Fig. 1A and B. Seed coat structure of Leguminosae. A, Radial section of seed coat of Melilotus alba: a, cuticle; b, subcuticular layer; C, caps palisades, overlies the epi- of Malpighian cells; d, Ught Une; e, lumen of Malpighian cell; f, intercellular g, osteosclerid cells; h, nu trient layers; i, space betweeu dermis of the testa. In seed coatspace; and endosperm; j, aleurone layer; k, inner layers of endosperm. Camera lucida drawing, x 1400. (From HAMLY 1932.) the long axis of the hilum B, Transmedian seetion of the hilum of Lupinus arboreus: F, tissue there is a groove in the derived from the funiculus; T, seed coat; HF, hilar fissure; a, parenchyma cells; b, couuter-palisades; c, cuticle; d, light Une; e, palisade funicular tissue and a epidermis (= Malpighian cells); f, stellate cells; g, tracheid bar. (From HYDE 1954.) fissure in the epidermis of the testa, forming the hilar fissure ; immediately below lies a band of tissue composed of short, large-pitted tracheids, the tracheid bar. It is, in turn, surrounded bya tissue composed of stellate cells the intercellular spaces of which are continuous with those of the subepidermal layer of osteosclerid cells (see Fig. 1 A, fand g); this results in a

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continuous layer capable of moisture conduction throughout the seed coat. When relative hnmidity is low, the fissure of the hilum is open permitting the seed to dry out; when humidity is high it closes. In this process, the counter-palisades function as the motor, drying and shrinking when humidity falls and thereby drawing the margins of the hilar fissure upwards and apart. When relative humidity rises the counter-palisades swell causing the fissure to c1ose. Opening and c10sing can occur within periods of one minute. Since decrease of moisture content results in an increase of hard-coated seeds in most legumes (see below, p.734), the hilum also plays a part in the development of hardcoatedness. The strophiolar eIert. One particular characteristic of the Papilionoideae is the strophiolar cleft described by HAMLY (1932). He studied the structure of the seed coat of Melilotus alba in detail, and attributed the impermeability to a layer of tightly appressed suberin caps over the cells surrounding the strophiole, a structure formed by small elevations on either side of a narrow longitudinal depression c10se to the hilum on the side opposite the micropyle. He held the view that these strophiolar cells in hard seeds are in astate of "metastable equilibrium" which can be upset by mechanical impact or by heating to produce a softening of the coat through a split, the strophiolar c1eft, along the middle lamella. Confirming the importance of the strophiole in relation to impermeability of seeds of Melilotus alba Desr. and M. officinalis Willd., MARTIN and WATT (1944) studied both impermeable and permeable seeds and found that the strophiole is the natural place of initial water absorption. Seeds naturally permeable, or made permeable naturally by weathering or artificially by mechanical abrasion blackened and began to swell first at the strophiole when they were immersed in a solution of osmic acid. HUTTON and PORTER (1937) reported shaking effective for five species of legumes all of which belong to the Papilionoideae. They examined shaken seeds of Amorpha and Lespedeza and found fissures at the base of the hilum depression through which water entered the seed. In contrast to these findings, BURNS (1959), working with Lupinus angustifolius and using dye solutions, found no uptake through the strophiole but always through the hilum. KUHN (1925) had reported similar observations. The onIy time that BURNS found the strophiole to be important in water uptake was in seeds that had been imbibed and then re-dried, this process causing a fissure to be formed in the strophiole.

Other groups. Seeds of wheat are often resistant to the absorption of water. This has been shown by HINTON (1955) to be due to the slow water movement in the endosperm instead of the failure of the coats to absorb moisture. The wheat pericarp absorbs water readily, but the amount of water which reaches the endosperm and embryo is limited by the resistance of the testa. Thus 60 hours may be required for the general distribution in the endosperm of water absorbed by the pericarp in one hour. . The hard seed problem is found in cotton (Gossypium) and is often a deterrent to the breeding of promising new lines (WALHOOD 1956). The hardcoated character is not usually a problem in the commercial growing of cotton, since it has been largely eliminated through breeding and selection. Occasionally, growing conditions cause a production of an abnormally high percentage of hard seeds. Intact seeds coat of Oitrus may delay for several days the uptake of water necessary for germination (COHEN 1956). The chemicalloosening of seed caps held in place by hemicellulose hastens the germination of sugar beet seed (LACKEY 1948).

733

Hereditary factors in seed coat impermeability.

b) Hereditary factors. There is little doubt that the hard-seeded character is inherited, at least to some degree, though ecological factors prevailing during the growth and especially during the maturation of the seeds, as wen as storage conditions after maturation have their influence. A

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Fig.2A-C. Percentage of hard seeds in Phaseolus vulgaris on successive days of tests. A, selection 1130; B, F. from 368x1l30 crosses; C, F, from 368x1l30 crosses. (From LEBEDEFF 1947.)

The genetics of impermeable seed production has been a subject of interest for many years, though reported experimental results are few. LEBEDEFF (1943) presented data which indicate that the differences between the percentages of hard seeds of 36 selections of common white beans (Phaseolus vulgaris L.) are hereditary. Certain external factors such as time of germination test, season of growth, age, and seed moisture content, affected the number of hard seeds, but the significant correlations between the percentages of hard seeds in the different selections persisted. This study was carried further (LEBEDEFF 1947) and 5 selections of beans which exhibited marked differences in the rate of

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softening of hard seeds under certain environmental conditions were intercrossed. Original parent plants and plants from the first and second generation crosses were all grown together in a single environment. Seeds were harvested and the moisture content of all was adjusted to about 6.61 %. Seeds from the two original «soft-shelled" selections remained the same, while those of the three original "hard-shelled" selections were even more resistant to water absorption than the initial lot. Seeds from F 1 crosses were intermediate between the parents or approached the "soft-shelled" parent, while those of F 2 produced hard seeds ranging between the parents. This effect is shown in Fig. 2 for the "hard-shelled" parent, Selection 1130, and for the F 1 and F 2 crosses from the "soft-shelled" Selection 368 with Selection 1130. In Selection 368, not shown in the figure, most of the seeds became soft by the seventeenth day of the test. While it was concluded from these studies that hard-seededness is controlled by genes, the 'author pointed out that the resuIts could be duplicated only under similar environmental conditions. Changing either environment or method of drying the seeds would probably give different results. WHITE and STEVENSON (1948) were able to produce hard or soft seeds of Melilotus alba by selecting and inbreeding the strains. This has also been done for hairy vetch, Vicia villosa Roth. (HÜBNER 1938). ZENARI (1929), on the other hand, tested the seeds of various Leguminosae, Malvaceae, and Oistaceae both genetically and physiologically and concluded as a result of a three-year selection program that the hard-coat character is irregular and cannot be considered hereditary. Rather he related it to the degree of maturity of the seed, with definite influences by climate, soil, shade, and rainfall. Also, JAMES (1949a) found no inheritance of impermeability in seeds of crimson clover (Trifolium incarnatum L.). On the contrary selfed parents with a difference of about 60 per cent in seed coat permeability produced offspring with a difference of less than one per cent in the first generation. As a result of three years of inbreeding he concluded (1949a, p.266), "From the data presented it appears doubtful that impermeability is inherited unless the possible heritable factors are masked by environmental factors to the extent that the different effects cannot be separated."

c) Environmental factors during seed maturation. Humidity and moisture. There is much evidence to indicate that environmental factors alter the number of impermeable seeds produced as weIl as the degree of impermeability. Conditions, especially of temperature and moisture, surrounding the seed during maturation on the plant and during subsequent storage are of importance in this connection. For example, seeds of Robinia pseudoacacia L. were 100% hard when they ripened in arid climates (GASSNER 1938), while those ripening under more moist conditions showed only a moderate amount of hardness. These pods open in the late summer of the year following their formation and hence seeds from 2 successive years are present on the same tree at the same time. Seeds from the current year's growth showed medium percentage of hardness, while 100% of those from the previous year's growth were hard. The low relative humidity prevailing during the maturation of the latter seeds favored the formation of impermeable seeds according to the author. LEBEDEFF (1943) found that drying of seeds of Phaseolus vulgaris L. increased the number of hard seeds. No hard seeds were present in the control lot which contained about 15 % moisture. The percentage of hard seeds increased to almost 90 % as the lots were dried over calcium chloride to a moisture content

Environmental factors and hard-coatedness.

735

of about 6%. Not only did the number of hard seeds increase with each reduction of moisture content, but the coats remained impermeable for a longer time, as indicated by the reduced rates of softening and germination in the seeds with the lower moisture contents. However, LEBEDEFF found that individual selections differed in the development of hard seeds as noted above. Light (photoperiod) . That light may be an important factor in the production of impermeable coats was shown by LONA (1947) who found that seeds of Ohenopodium amaranticolor, which had been permitted to mature under short-day conditions, showed high germinability. However, plants which were brought to flowering by short days but then transferred to long-day conditions produced seeds which gave little or no germination without seed coat treatment, either mechanically or with concentrated sulphuric acid. Examination showed that seeds produced under long-day conditions possessed unusually thick seed coats, which hindered germination, not by restricting water absorption, but by offering resistance to the breaking out of the root. Other factors (harvest time, seed size, flowering sequence, nutrition, etc.). Further proof that the hard seed content depends to some degree upon the climate is furnished by a study of seed germination of lots of alfalfa, M edicago sativa L., collected from several states (DEXTER 1955). In general, plants at low altitudes produced fewer hard seeds than those from higher altitudes. Also, seeds harvested late in the season were more apt to be hard than those from earlier harvests. In contrast to the findings of others, seeds from irrigated fields had about the same hard seed content as those from dry farming lands. However, the assessment of moisture effects on the production of hard seeds as they mature on the plant must await measurements of critical moisture contents of the soil. JAMES (1949b), having demonstrated the failure of inheritance to account for seed impermeability (see p. 734), turned to a study of environmental factors. He tagged flowers of crimson clover and found a highly significant positive linear relation between sequence of flowering and hard-coatedness, the later flowers producing smaller seeds with a larger percentage of impermeable coats. MIDDLETON (1933) also found that 48-72% of small seeds of five sampies of Korean lespedeza (Lespedeza stipulacea Maxim.) were hard as compared with 1 to 3% in large seeds, and 26 to 50% in those of intermediate size. Using halfplants of crimson clover in balanced incomplete blocks, JAMES and BANCROFT (1951) were able to show that a higher level of calcium increases the production of hard seed; a higher level of potassium decreases the percentage of hard seed; and phosphorus is without effect on seed impermeability. Additional statistically designed experiments of this nature would make possible the evaluation of other environmental factors in their relation to hard seed production. KONDo (1929) found that the percentage of hard seeds of Astragalus sinicus L. in Japan depended upon the locality in which they were grown. Also, the early maturing varieties produced most hard seeds, while fewer were produced by the late maturing ones. LOFTUS HILLS (1942) conducted experiments to determine the effect of delayed harvest on the proportion of dormant and hard seeds in Trifolium subterraneum L. in New South Wales. Delaying harvest up to six weeks after the normal maturity of the seeds reduced the percentage of both dormant and hard seeds. He also found that storage at high temperature, especially under conditions of high humidity, reduced the number of dormant seeds. The development of hard-coatedness in seeds of birds-foot trefoil, Lotus corniculatus L., increased with increasing maturity and decreasing moisture

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content (C. S. BROWN 1955). There was no effect of soil moisture, fertility, light intensity, or atmospheric humidity. Mature, hand-harvested seed was 85 to 95 % impermeable.

d) Storage conditions. ( M oisture loss; temperature; etc.) Since large percentages of seeds of agricultural value possess impermeable coats when harvested, it becomes of importance to determine methods of storage which will soften these seeds so that they are not lost in commercial plantings. With this problem in mind, several investigators have studied the effect of different storage conditions on different seeds. GLOYER(1928) found that beans stored in the laboratory in winter gradually dry out and become hard. Drying seeds with 16% moisture to less than 10% caused a rise in hardness from 0 to 50%. ESDORN (1930) claimed that thoroughly ripe seeds of Lupinus luteus L. on the plant show no hardness, but the development of impermeable coats begins with storage. Hardness started to develop in 24 hours at 18° but no hard seeds were formed at 0°-8° up to December after harvest. Furthermore, seeds stored in a cold place over winter remained permeable, but developed hard seeds during the summer, softened to some extent the following winter and then hardened again the next summer. Humidity was also important. Storage at 18° caused hardening in open air or in a desiccator over sulphuric acid, but not in a moist chamber 1 • HELGESON (1932) attributed impermeability in sweet clover seeds to dehydration in the last stages of maturity. Slightly immature seeds were almost all permeable, but became 90% hard in 4 days when desiccated over calcium chloride. Storage of these imml1tture seeds at 7° with 85 % relative humidity prevented the development of impermeability. HÜBNER (1938) found that seeds of some varieties of Vicia pannonica increased in hardness with drying while others did not, and that the size of the seeds had relatively little effect. WYCHERLEY (1960), in contrast to the preceding authors, found that storage under constant damp conditions not only depressed the total viability of seeds of certain tropical legumes (Calopogonium mucunoides, Centrosoma pubescens, Flamingia congesta, Pueraria phaseoloides) , but also increased the fraction of hard seeds. He considers this as evidence that the entry of water into these seeds occurs by a path which is hydrostatically controlled.

111. Mechanical restrietion of germination by the seed coat. In certain seeds, the coats appear to permit water absorption and gaseous exchange, but they offer mechanical resistance to penetration by the germinated seed. This condition may be illustrated by the coat-bound condition of germinating pepper (Capsicum frutescens L.) seeds (BARER 1948). Under the semiarid conditions of southern California, pepper seedlings sometimes emerge from the soil with the cotyledons and plumules still enclosed in the seed coats (Fig. 3). Further drying after emergence prevents the subsequent release of cotyledons and plumules. Coat-binding may be prevented by keeping the air humidity and soilsurface moisture at a high level. Tomato seedlings are also sometimes affected in this manner, as are those of cucumbers, pumpkins, melons, and beans (BARER 1948, E. HILTNER 1933, LOPRIORE 1904). 1 All temperatures in degrees centigrade (0 Cl.

737

Mechanical restrietion by hard coats. Germination inhibitors.

LONA (1947), as noted above (p. 735), found that unusually thick seed coats of Chenopodium amaranticolor offered resistance to the breaking out of the young seedling root. Mechanical resistance of the seed coats to the germination of Fraxinus excelsior L. is overcome in a moist medium during aperiod at low temperature, but not at a temperature as high as 20° (BARTON and CROCKER 1948).

Fig. 3. Pep per seedlings which developed from seeds with various degrees of coat binding; all of same age, taken from same flat in the greenhouse. Plant at left germinated normally. Plants at top and middle right were the result of tardy release of the cotyledons from seed coats. Cotyledons of plants at lower right were not released at all. (From BAKER 1948.)

Other cases in which the seed coat (or other structures, like the endocarp) prevents or delays germination because of its mechanical resistance have been described in Alisma plantaga (CROCKER and DAVIS 1914), Rubus idaeus (R. C. ROSE 1919) and other plants.

IV. Germination inhibitors in the seed coat. EVENARI (1949) in a review article on germination inhibitors, lists 121 known plant-produced germination inhibitors. Of these only 12 are present in fruit or secd coats: Beta saccharifera, Brassica oleracea var. capitata, Cucurbita pepo, Erucaria boveana, Fagopyrum esculentum, Helianthus annuus, Hirschfeldia incana, Matthiola bicornis, Nico(1:ana rustica, Onobrychis cristagalli, Sinapis alba, and Sinapis arvensis. Inhibitors in still other seed coats, however, have been reported in the literature. Some examples of this type of dormancy are given below. Seed coats of certain varieties of cabbage (Brassica spec.) restriet germination, especially of freshly harvested seeds. That this is due to a chemical inhibitor Handbuch d. Pflanzenphysiologie, Bd. XVj2.

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is indicated by the retardation of germination by the water-soluble fraction of alcoholic extracts of the seed coats (Cox et al. 1945). Water soluble substances toxic to germinating seeds have been found by FRÖSCHEL (1939, 1940) and TOLMAN and STOUT (1940) in the pericarp tissue of sugar beet (Beta vulgaris L.). These substances not only retarded germination, but also killed the radicles of the germinated seeds. Washing the beet seed balls before placing them to germinate removed part or all of the toxic effects. Fig.4 shows the effect on germination and on conditions of the sprouts. In a later paper (1941) STOUT and TOLMAN analyzed the nature of the toxic action. It was found to be largely due to ammonia released by enzymatic hydrolysis from the nitrogenous compounds in the aqueous extracts of the seed balls. Experimental data indicated that the toxic action of the released ammonia was not entirely due to the resulting increase in PR' REHM (1953) confirms the formation of ammonia but attributes it to microbial action mainly during germination. He finds, however, that the dry sugarbeet fruits also contain inhibitory substances, both inorganic and organic in nature; the inhibitor COlliplex of sugar-beet fruits thus seems to consist of two differFig. 4A and B. Comparison of the condition of sprouts from sugarent components, a "primary" beet seed balls not washed and those wahsed prior to germination in Petri dishes. A, unwashed seed balls of variety 550, 1 week after and a "secondary" one. they had been placed in a Petri dish on cotton moistened with water. Damage to the sprouts by the substances that arise from SROELOV (1940) reported the seed ball is very apparent. B, seed balls of variety 550 that were inferior germination of seeds washed 22 hours in running water before being placed on moistened cotton in a Petri dish. The healthy appearance of the sprouts is of Sinapis alba, Erucaria apparent even after 2 weeks of growth. (From TOLMAN and STOUT 1940.) Courtesy of the United States Department of Agriculture. boveana, and Matthiola bicornis when near their fruit coats and stated that the valves and beaks of these fruits contain a substance which inhibits the germination of seeds enclosed in them.

Germination inhibitors. Methods for overcoming hard-coatedness.

739

According to KAUFMANN (1943) the mucous covering of the seeds of Oucumis sativus contains a water- and ether-soluble material that strongly hinders germination. An unidentified inhibiting factor has been found in the outer seed coat of Oitrus (COHEN 1956). Aqueous extracts from hulls of dormant crabgrass [Digitaria sanguinalis (L.) Scop.] seeds inhibited the germination of nondormant seeds (DELOUCHE 1956). The inhibitive property was not destroyed by boiling for 30 minutes. Similar extracts prepared from nondormant seeds were not inhibitive. The elimination of an inhibitor in seed coats may be associated with light exposure. For example, the seed coat of Lythrum salicaria contains an inhibitor which is eliminated by exposure to light (PAECH 1953). This inhibitor does not affect the speed or degree of swelling of the seed. In the absence of light, hydrochloric acid treatment or abrasion will bring about germination of Lythrum. An unusual role of the seed coat in chlorophyll deficiency in Oitrus seedlings has been described by TAGER and CAMERON (1957). They state that as many as 100% of seedlings in a seed bed may exhibit albinism. However, when the seed coats were removed and excised embryos were planted, chlorophyll deficiency was evident in less that 1 % of the seedlings. Since slitting the seed coats had no effect on albinism, the permeability of the coats did not seem to be a factor affecting chlorophyll. Chlorophyll deficient seedlings were produced when excised embryos were germinated in contact with seed coats, thus indicating the presence in the seed coat of an inhibitor of chlorophyll formation. The authors refer to the work of others who had inoculated Oitrus seeds with fungi and obtained a strong indication that chlorophyll deficient seedlings were produced only when certain fungi were present. This latter work has not been published as far as the present author is aware, but it brings to light another possible explanation of coat-inhibiting effects.

V. Methods of overcoming hard-coatedness. Many methods have been used to eliminate the hard coat effect and thus promote germination. Among the best known ones are mechanical abrasion or impact, high and low temperature treatment - the latter frequently combined with moist storage thus essentially replicating natural weathering conditions - and chemical treatment to remove or dissolve the impermeable portions of the coat. Exhaustive literature reviews can be found in the summary by PORTER (1949) and the books by BARTON and CROCKER (1948), CROCKER (1948) and CROCKER and BARTON (1953). (1) Mechanical scarification by means of a file , abrasion with the aid of sand paper, blowing against needle points and impaction by shaking in a bottle have been used in numerous cases, with Leguminosae and other seeds (e.g., D. H. ROSE 1915, HAMLY 1932, PORTER 1935, BARTON 1947 and many others). Large lots of seeds, especially legume, are machine-scarified in spite of the fact that damage to seedlings often results from such treatment. (2) Temperature treatment. Heating has been commonly used as reported by EWART (1908), K. MÜLLER (1912), HARRINGTON (1916), LUTE (1928), HODGSON (1949) and RUGE and LIEDTKE (1951). Some authors have even used boiling water (TODA and ISIKAWA 1951). These methods must obviously be used with caution since prolonged treatment is apt to injure the seeds. Freezing has been found to reduce the number of impermeable seeds in alfalfa (MIDGLEY 1926). After the first freezing, however, subsequent freezing and thawing had little effect. Also the intensity of the freezing was without effect with Handbuch d. Pflanzenphysiologie, Bd. XV/2.

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the range of 0° to -20°. Impermeable seeds of Melilotus alba are made permeable by plunging them in liquid nitrogen, -195.8° (BARToN 1947). Four dips of 30 seconds each into the liquid were more effective than one dip of one or five minutes' duration. One minute was allowed to elapse between the dips. If the seeds were plunged into water at room temperature before and after each dip, they gave 97% germination when placed subsequently on filter paper moistened with water. Seeds dipped four times without being plunged into water gave only 38 % germination and untreated hard seeds failed to germinate. Liquid nitrogen treatment, even when extended to 15 minutes' duration, had no effect on impermeable seeds of Gleditsia triacanthos. BUSSE (1930) reported that freezing air-dry impermeable seeds of sweet clover and alfalfa in liquid air (-190°) made them permeable. Sweet clover seeds were kept in liquid air for 175 days without injury. Cooling to -80° softened some of the impermeable alfalfa seed but had little effect on sweet clover seeds. BUSSE attributed the increased germination after freezing to formation of very tiny cracks in the impermeable membrane. (3) Weathering. The very low freezing temperature required for rendering hard sweet clover seeds capable of moisture absorption explains the ineffectiveness of freezing and thawing in soil plantings under natural weather conditions. Such weather factors effective in opening the coats of sweet clover have been studied by MARTIN (1945). He found that, in natural seeding in the field or in dry storage over winter in unheated open buildings in Iowa, 80 to 100% of the hard seeds softened by the middle of the following April. In spite of the fact that practically all of the opening of the seed coats to the absorption of water occurred in the interval from March 20 to April 20, a previous exposure of two months or more to fluctuations of temperature near the freezing point was required for effective softening. A constant temperature of 10° as weIl as higher fluctuating temperatures were ineffective in making the seed coats permeable. BART ON (1947) tested the response of members of the Papilionoideae and Caesalpinoideae subfamilies of Leguminosae to weathering effects in field plantings. Filed and intact seeds were planted in the field at Yonkers, N. Y. in December and again the following May. There had been periods of cold weather including some snow, previous to the December planting, but the soil was entirely free of frost, though very wet, on the planting date. In the case of Melilotus alba, 41 % seedling production was secured in the spring from impermeable seeds planted in December (Fig. 5). Only 1 % of the impermeable seeds planted in May produced seedlings. Filed seeds planted in May gave 84 % germination. Filed seeds planted in December absorbed water readily and rotted because of the unfavorable germination temperatures, so no seedlings survived. Seeds of Oladrastislutea, which, like Melilotus, is classified in the subfamily Papilionoideae, gave similar results, though smaller germination percentages were obtained. In Cassia artemisoides and Cercis canadensis, members of the Caesalpinoideae subfamily, no softening of the hard-coated seeds resulted from a winter in the field, but a few seedlings were obtained from such plantings of Gleditsia triacanthos, another member of this same subfamily. These results are of interest in connection with other findings indicating differential behavior of Papilionoideae and Caesalpinioideae seeds to "softening" treatments (see p. 730). (4) Pressure; high frequency. The influence of high pressures on seed germination has been determined for some hard-coated-seeds. DAVIES (1928) applied hydraulic pressures up to 2000 atmospheres to seeds of M elilotus alba and M edicago sativa for periods up to 40 minutes. The germination of Medicago seeds was

741

Methods for overcoming hard-coatedness.

increased over 50% when treated seeds were dried and their germination tested after 30 days and 6 months, while the germination of treated M elilotus seeds was increased by 150 to 200%. Increased germination was brought about by the softening of impermeable coats. In these same seeds and in other leguminous seeds high hydrostatic pressures have been used to reduce the percentage of hard seeds, but in many cases there was a corresponding increase in the percentage of dead seeds (RIVERA et al. 1937). In a range of pressures between 1,000 and 30,000 p.'l.i. (pounds per square inch; approximately 70-2000atm.), these authors

Fig.5A-C. Melilalus alba. A, intact seeds planted in November (3 rows); B, filed seeds planted in May (1 row); and C, intact seeds planted in May. Photographed the following Jnly. (From BARTON 1947.)

found that the higher the pressure, the greater the rate and final percentage of germination of seeds of Cladrastis lutea. These seeds were somewhat injured by pressures of 45,000 and 60,000 p.s.i. (about 3,000 and 4,100 atm.). Recent work by EGLITlS and JOHNSON (1957) has indicated that the hard seed of alfalfa (Medicago sativa L.) can be controlled with high frequency energy. Sam pies of seed in paper or wood containers were placed directly between two electrodes of a 5 kW high-frequency generator operated at 27 Me with an output of 1 amp. This treatment increased the water-absorption capacity of the seed and permitted normal germination without affecting the growth of the plants. (5) Chemical treatments. Ethyl and methyl alcohols of various concentrations, turpentine, and chloroform were used by REES (1911) in an effort to dissolve out impermeable fats and waxes from the seed coat with only limited success. The use of alcohol in Caesalpinioideae (VERSCHAFFELT 1912, BARTON 1947) has been mentioned in another connection (see p. 730); SHAW (1929) has used it to render Nelumbo seeds permeable. WYCHERLEY (1960) found that immersion Handbuch d. Pflanzenphysiologie, Bd. XVj2.

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LELA V. BARToN: Dormancy in seeds imposed by the seed coat.

into certain liquids, particularly glycerin, promoted germination in hard-coated seeds of tropical legumes (see p. 736); the treatment was particularly effective if combined with warm temperatures. The most widely used chemical method is treatment with concentrated sulturic acid: it was introduced by L. HILTNER (1899), ROSTRUP (1899) and TODARO (1901) and has been used, among many others, by HORN and COLON (1942), LEMMON et al. (1943), LONA (1947), CAVAZZA (1953), MISRO (1954), SCOTT and INK (1957), WILLIAMS and WEBB (1958). Naturally, care must again be taken to avoid injury to the embryos by too extended treatment. BURNS (1959) showed that sulfuric acid treatment of Lupinus angustitolius seeds dissolved the counter-palisades which obstruct the hilar fissure (see p. 731/732) or eroded pits through the testa. It must however be noted that the mode of action of sulfuric acid is not always as clear. Thus, many grasses germinate better after sulfuric acid treatment (L. HILTNER 1910) but the seeds take up water quite readily without this treatment (V. K. TOOLE 1941, WILLIAMS and WHITE l. c.).

Literature. BAKER, K. F.: The coat-bound condition of germinating pepper seeds. Amer. J. Bot. 30, 192-193 (1948). - BARToN, L. V.: Special studies on seed coat impermeability. Contr. Boyce Thompson Inst. 14, 355-362 (1947). - BARToN, L. V., and W. CROCKER: Twenty years of seed research at Boyce Thompson Institute for Plant Research. London: Faber & Faber 1948. - BENEDICT, H. M., and J. ROBINSON: Studies on the germination of guayule seed. Techn. BuH. U. S. Dept. Agric. No 921 (1946). - BORTHWICK, H. A., and W. W. ROBBINS: Lettuce seed and its germination. Hilgardia (Berkeley, Calif.) 3, 275-304 (1922). - BRowN, C. S.: Hard seed in birdsfoot trefoil. Doct. dissert., CorneH Univ., Ithaca, N. Y. 1955. Abstr. in Dis~ert. Abstr. 10, 1961 (1955). - BRowN, R.: An experimental study of the permeability to gases of the seed coat membranes of Oucurbita pepo. Ann. Bot., N. S. 4, 379-395 (1940). - BURNs, R. E.: Effect of acid scarification on lupin seed impermeability. Plant Physiol. 34, 107-108 (1959). - BURToN, G. W.: Recovery and viability of seeds of certain southern grasses and lespedeza passed through the bovine digestive tract. J. agric. Res. 76,95-103 (1948). - BussE, W. F.: Effect of low temperatures on germination of impermeable seeds. Bot. Gaz. 89, 169-179 (1930). CAVAZZA, L.: Recherches sur l'impermeabiliM des graines dures chez les Iegumineuses. Ber. schweiz. bot. Ges. 60, 596-610 (1950). Abstr. in Biol. Abstr. 25,3347, No 37706 (1951).Considerazioni sull'impiego deH'acido sulfurico nei trattamento ai semi duri. Nuovo G. bot. ital., N. S. 60, 750--759 (1953). - COE, H. S., and J. N. MARTIN: Sweet-clover seed. BuH. U. S. Dept. Agric. No 844 (1920). - COHEN, A.: Studies on the viability of citrus seeds and certain properties of their coats. BuH. Res. Counc. IsraeloD, 200-209 (1956). Oox, L. G., H. M. MUNGER and E. A. SMITH: A germination inhibitor in the seed coats of certain varieties of cabbage. Plant Physiol. 20, 289-294 (1945). - CROCKER, W.: Role of seed coat in delayed germination. Bot. Gaz. 42, 265-291 (1906). - Growth of plants. New York: Reinhold 1948. - CROCKER, W., and L. V. BARToN: Physiology of seeds. Waltham, Mass.: Chronica Bot. 1953. - CROCKER, W., and W. E. DAVIS: Delayed germination in seed of Alisma plantago. Bot. Gaz. 58, 285-321 (1914). DAVIES, P. A.: The effect of high pre3sure on the percentages of soft and hard seeds of Medicago sativa and Melilotus alba. Amer. J. Bot. 15,433-436 (1928). - DAVIS, W. E.: Primary dormancy, after-ripening, and the development of secondary dormancy in embryos of Ambrosia trifida. Contr. Boyce Thompson Inst. 2, 285-303 (1930). - DAVIS, W. E., and R. C. RosE: The effect of external conditions upon the after-ripening of the seeds of Orataegus mollis. Bot. Gaz. 04, 49-62 (1912). - DELOUCHE, J. C.: Dormancy in seeds of Agropyron smtthii, Digitaria sanguinalis, and Poa pratensis. Iowa State Coll. J. Sci. 30, 348-349 (1956). - DEXTER, S. T.: Alfalfa seedling emergence from seed lots varying in origin and hard seed content. Agron. J. 47, 357-360 (1955). EGGERS, E. R.: Effect of removal of the seed coat on avocado seed germination. Yearb. Calif. Avocado Ass. 1942, 41-43. - EGLITIS, M., and F. JOHNSON: Control of hard seed of alfalfa with high-frequency energy (Abstr.). Phytopathology 47,9 (1957). - ESDORN, I.: Untersuchungen über die Hartschaligkeit der gelben Lupine. Wiss. Arch. Landw., Abt. A Pflanzenbau 4, 497-549 (1930). - EVENARI, M.: Garmination inhibitors. Bot. Review 15, 153-194 (1949). - EWART, A. J.: On the longevity of seeds. Proc. roy. Soc. Victoria, N. S. 21, 1-203 (1908).

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FRÖSCHEL, P.: Onderzoekingen over de physiologie van de kieming. I. Remstoffen. [In Vlamish.] Natuurw. Tijdschr. 21,93-116 (1939). - Untersuchungen zur Physiologie der Keimung. I. Hemp.stoffe. Bio!. Jaarb. ("Dodonaea", Ghent) 7, 73-116 (1940). GASSNER, G.: Uber die Hartschaligkeit von Robiniensamen und eine Methode zu ihrer Beseitigung. Angew. Bot. 20, 293-303 (1938). - GLOYER, W. 0.: Hardshell of beans: its production and prevention under storage conditions. Proc. Ass. off. Seed Anal. N. Amer. 20, 52-55 (1928). HAMLY, D. H.: Softening of the seeds of Melilotus alba. Bot. Gaz. 93, 345-375 (1932).HARMON, G. W., and F. D. KEIM: The percentage and viability of weed seeds recovered in the feces of farm animals and their longevity when buried in manure. J. Amer. Soc. Agron. 26, 762-767 (1934). - HARRINGTON, G. T.: Agricultural value of impermeable seeds. J. agric. Res. 6, 761-796 (1916). - HELGESON, E. A.: Impermeability in mature and immature sweet clover seeds as affected by conditions of storage. Trans. Wisconsin Acad. Sei., Arts and Lett. 27, 193-206 (1932). - HILTNER, E.: Die Hartschaligkeit des Saatguts. In SORAUER'S Handbuch der Pflanzenkrankheiten, 6th edn., vol.l, pt. 1, pp. 443-445. Berlin: Parey 1933. - HILTNER, L.: Über ein neues Beizverfahren für Rübenknäuel und die Vorteile desselben gegenüber den bisherigen Beizmethoden. Österr.-ung. Z. Zuckerind. 28, 18-99 (1899). - Die Keimungsverhältnisse der Leguminosensamen und ihre Beeinflussung durch Organismenwirkung. Arb. biol. Abt. Landw.-Forst. KsI. Gesundh.-Amt 3, 1-102 (1902). - Die Prüfung des Saatgutes auf Frische und Gesundheit. J.ber. Vergg. angew. Bot. 8, 219-238 (1910). - HINTON, J. J. C.: Resistanc of the testy to entry of water into the wheat kerneI. Cereal Chem. 32, 296-306 (1955). - HODGSON, H. J.: Effect of heat and acid scarification on germination of seed of Balua grass, Paspalum notatum Fluegge. Agron. J. 41, 531-533 (1949). - HORN, C. L., and J. E. NATAL COLON: Acid scarification of the seed of two Cuban fiber plants. J. Amer. Soc. Agron. 34, 1137-1138 (1942). HÜBNER,R.: Untersuchungen über die Hartschaligkeit der Zottel wicke und ihre Behebung auf züchterischem Wege. Landw. Jb. 85,421-789 (1938). - HUTTON, M. E.-J., and RH. PORTER: Seed impermeabilityh and viability of native and introduced species of LegumiMsae. Iowa State Coll. J. Sei. 12, 5-24 (1937). - HYDE, E. O. C.: The function of the hilum in some Papilionaceae in relation to the ripening of the seed and the permeability of the testa. Ann. Bot., N. S. 18, 241-256 (1954). JAMES, E.: The effect of inbreeding on crimson clover seed-coat permeability. Agron. J. 41, 261-266 (1949a). - Some factors affecting the production of hard seed in crimson clover. Proc. Ass. S. Agric. Workers 1949, 52-53 (1949b). - JAMES, E., and T. A. BANCROFT: The use of half-plants in a balanced incomplete block in investigating the effect of calcium, phosphorus, and potassium, at two levels each, on the production of hard seed in crimson clover, Trifolium incarnatum. Agron. J. 43, 96-98 (1951). - JUBY, D. V., and J. H. PHEASANT: On intermittent germination as illustrated by Helianthemum guttatum. J. EcoI. 21, 442-451 (1933). KAUFMANN, E.: Beiträge zur Keimungsphysiologie von Cucumis sativus im Zusammenhang mit dem Wuchsstoffproblem. Plan~a (Berl.) 33, 516-545 (1943). Abstr. in Chem. Abstr. 38, 5881 (1944). - KONDO, M.: Über die harten Samen von Astragalus sinicus L. und Robinia pseudacacia L. Ber. O'Hara Inst. lndw. Forsch. 4, 289-293 (1929). - KUHN, 0.: Die Hartschaligkeit bei Lupinus angistufolius. Kühn-Arch. 9, 332-381 (1925). LACKEY, C. F.: Chemicalloosening of seed caps in relation to germination of sugar be.et seed. Proc. Amer. Soc. Sugar Beet Technol. 5, 66-69 (1948). - LEBEDEFF, GA: Heredity and environment in the production of hard seeds in common beans (Phaseolus vulgaris). Puerto Rico Agric. Exp. Stat. Res. Bull. No 4 (1943). - Studies on the inheritance of hard seeds in beans. J. agric. Res. 74, 205-215 (1947). - LEMMON, P. E., R L. BROWN and W. E. CHAPIN: Sulfuric acid treatment of Beach pea, Lathyrus maritimus, and Silvery pea, L.littoralis, to increase germination, seedling establishment, and field stands. J. Amer. Soc. Agron. 35, 177-191 (1943). - LOFTUS HILLS, K.: Dormancy and hardseededness in T. subterraneum. I. The effect of time of harvest and of certain seed storage conditions. J. Austral. Counc. scient. and ind. Res. 15, 275-284 (1942). - LONA, F.: L'influenza della condizioni ambientali, durante l'embriogenesi, sulle caratteristiche deI seme e deHa pianta ehe ne deriva. In: Lavori di botanica, volume pubbl. in occasione deI 70° genetliaco deI Prof. G. GOLA (padova 1947), pp. 313-352. 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STOUT: TODe effect on germinating sugar-beet seed of water-soluble substances in the seed ball. J. agric. Res. 81, 817-830 (1940). - TOOLE, V. K.: Factors affecting the germination of various dropseed grasses (Sporobolus sp.). J. agric. Res. 82,691-714 (1941). VEBSCHAFFELT, E.: Le traitement chimique des graines a. imbibition tardive. Rec. Trav. bot. neerl. 9, 401-435 (1912). - VISSER, T.: After-ripening and germination of apple

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