CHAPTER 17. GENERAL APPROACHES TO PROMOTING SEED GERMINATION

Deciding appropriate germination test regimes for accessions so that germination tests reflect true viability with minimum interference from dormancy (and other factors which limit germination, such as hardseededness) can be one of the more difficult decisions which gene bank staff need to make. In the following chapters we have summarised useful information on the germination response of seeds of various genera to very many test regimes and dormancy-breaking treatments together with advice on suggested test procedures. In many cases, however, the information available is far from complete and accordingly much of the advice is very tentative. In such cases it is hoped that gene bank staff will be able to improve on the suggestions made. In order to help in this endeavour, in this chapter we discuss four different types of approach to determining appropriate germination environments.

ECOLOGICAL GUIDELINES TO DEVELOPING APPROPRIATE GERMINATION ENVIRONMENTS

A consideration of the ecology of a species may sometimes help to provide some guidance as to appropriate germination test conditions. At the simplest level tropical species generally require higher temperatures for germination than temperate species. However, there are other more subtle responses which relate to the strategy of the plant in relation to its natural environment and a consideration of these may help in the development of germination test conditions which minimise dormancy.

Dormancy may prevent seeds from germinating in the wrong place at the wrong time

As mentioned in Chapter 5 (Volume I) the majority of mature seeds show innate (or inherent or primary) dormancy - a condition necessary to prevent germination on the mother plant before the seed is shed. Innate seed dormancy in most species tends to be gradually lost with time (except in some tree species), often at a rate depending on temperature and moisture content. But induced (or secondary) dormancy, in many respects similar to innate dormancy, may subsequently be induced by conditions which signal that the current environment is inappropriate for germination - e.g. high temperatures or anaerobic conditions. Furthermore enforced dormancy may be temporarily imposed by conditions which again appear to play a role in ensuring that the seed only germinates when there is a reasonable probability of the seedling growing to maturity. Dormancy in all its forms (see Chapter 5, Volume I) therefore tends to prevent seeds from germinating in the wrong place at the wrong time.

Field environment

For example, many annual or ephemeral species produce large numbers of small seeds. At any one time it is common for there to be more dormant seeds in the soil waiting for appropriate conditions than there are growing plants. Since small seeds only contain small food reserves, only a small amount of seedling growth can occur before the seedling needs to start photosynthesising. Thus germination more than a few centimetres below the soil surface or beneath dense vegetation could lead to seedling destruction. It would seem that many species have evolved dormancy mechanisms which avoid these problems. The seed needs to be able to respond to environmental factors that signal when it is at or near the soil surface. There seem to be two major environmental factors which many species use as indicators - light and alternating temperatures; and in most cases the seeds respond to both. Light is obviously only present at or near the soil surface; and diurnal temperature alternations have a maximum amplitude (or diurnal range) at the soil surface: the range rapidly diminishes with depth in the soil profile. Furthermore even at the soil surface the amplitude is diminished by the presence of vegetation. Since vegetation contains chlorophyll which absorbs strongly in the red region of the spectrum, the quality of light beneath vegetation may be inhibiting since far-red light penetrates much more and thus the light quality tends to affect the balance of phytochrome in the seeds in favour of the inactive Pr form (see Chapter 6, Volume I).

Accordingly it is very common for small seeds of annuals and ephemerals to be light-sensitive and to germinate in response to white (or red) light and alternating temperatures. In many cases neither of these factors on their own has a major effect and both are necessary for a maximum response. Both these factors also often interact with nitrate ions.

Maternal environment

It may be worth considering the maternal environment in which the seeds develop as a further guide to distinguishing those species likely to require light treatments in order to promote seed germination. The light-filtering properties of the maternal tissues which surround the developing seeds, whilst they remain moist, can affect the sensitivity of seed germination to light in subsequent environments. Where the maternal tissues have a high chlorophyll content (that is are green) the seeds tend to require a light stimulus for germination (in subsequent tests). In contrast those seeds which have developed within maternal tissues where the chlorophyll content declined whilst the seeds remained moist tend to be able to germinate subsequently in light or darkness (that is are light insensitive). This is because the seeds developing within green maternal tissues would have received light filtered by the chlorophyll. This light would have a low red/far red ratio and most of the phytochrome would be in the inactive Pr form. In the latter case, however, the light would not have been filtered to such an extent; the red/far red ratio of the light received by the seeds would have been greater and thus more of the phytochrome would be in the active Pfr form which promotes germination. This Pfr would remain after desiccation and subsequent imbibition and accounts for the insensitivity of these seeds to light.

Large-seeded species of arid environments

As mentioned in Chapter 6 (Volume I), sustained and/or high-intensity light can also inhibit the germination of some seeds. This response is more common amongst somewhat larger seeds of species of arid environments, where it is more important for seeds to germinate below the soil surface where short-term severe water shortage is less likely, and the probability of the shoot not reaching the soil surface before food reserves are reduced is lessened by the larger food reserves of the seed.

In desert regions where rains are infrequent and of limited duration, ephemeral plants have short life cycles and it is important for them to germinate only after a substantial fall of rain (and not after a minor shower which would not provide sufficient water for survival). In such cases seeds of some species appear to have their own 'rain gauge' which depends on the fact that it takes a certain minimal amount of rain to leach germination inhibitors. Thus in such cases germination may be stimulated by washing the seeds in running water.

Probability of plant establishment and survival

Dormancy mechanisms often also tend to ensure that the seeds do not germinate at times when the probability of the survival of the plant to maturity is poor. For example, many seeds in temperate latitudes tend to germinate after the winter, i.e. at the beginning of the main growing season. These seasonal considerations are often independent of seed size and thus many temperate species germinate best after a period of stratification (i.e. cool temperatures applied to moist seeds). Such a response ensures that seeds do not germinate at the beginning of winter. In contrast, seeds of species from Mediterranean climates tend to avoid germination at the beginning of the hot, dry summer and germination at the beginning of the cool moist winter is more common. In such cases it would be less likely to find marked responses to stratification. This would also be generally true of tropical plants, but see the note below.

In tropical rain forests the temperature and water supply is always adequate for growth and so seeds of such species often show little dormancy. Seeds of many of the woody perennial species are also recalcitrant. Such seeds do not have to survive long periods in the dry conditions, and fresh supplies of short-lived seeds are produced regularly by the mature plants. Many seeds of this type show little dormancy, and many are killed by cool temperatures (e.g. less than 10° to 15°C) which they would never normally experience. However, even in tropical forests some species are not recalcitrant, and some may show light responses to ensure that they only germinate when a gap arises in the forest canopy and/or the soil is disturbed after clearance, thus ensuring that early growth is not inhibited by low light intensity.

There are, of course, exceptions to these general principles. In particular it should be noted that laboratory treatments which emulate environments which the seeds would never experience in their natural habitat may sometimes promote germination. For example, low temperatures applied to moist seeds of some species of tropical and sub-tropical ecotypes can promote germination. Nevertheless the few examples above may be sufficient to indicate that a consideration of the ecology of the species may give useful clues as to the type of laboratory germination techniques which may be most appropriate.

Further reading

Cresswell, E.G. and Grime, J.P. (1981). Induction of a light requirement during seed development and its ecological significance. Nature, 291, 583-585.

Grime, J.P., Mason, G., Curtis, A.V., Rodman, J., Band, S.R., Mowforth, M.A.G., Neal, A.M. and Shaw, S. (1981). A comparative study of germination characteristics in a local flora. Journal of Ecology, 69, 1017-1059.

THE ANATOMICAL APPROACH TO DEVELOPING APPROPRIATE GERMINATION ENVIRONMENTS

This approach has been developed largely by B.R. Atwater from long experience of attempting to germinate seeds of flower and ornamental species. Atwater's observations suggest that dormancy and germination requirements in these species are closely related to the maturity of the embryo and the structure and permeability of the seed coat coverings in the mature seed.

Atwater has described eight basic forms of seed structure. These categories have been described in Chapter 3 (Volume I). Within each of these categories it is suggested that the seeds have similar dormancy and germination patterns, but it should be emphasised that the mechanisms suggested are hypotheses rather than established facts.

I. Seeds with dominant endosperm (ENDOSPERMIC SEEDS) and immature dependent embryos (that is embryos not ready to germinate)

A. BASAL RUDIMENTARY EMBRYOS

The rudimentary embryos are the principal cause of delayed germination in this group. Inhibitors found in the endosperm may also contribute to the delay. Treatment consists of neutralization of the inhibitors with leaching, or with extra oxygen at low temperature followed by higher temperatures favourable to rapid embryo growth. Gibberellic acid increases the rate of embryo development if added to the substrate.

B. AXILLARY LINEAR EMBRYOS

Linear embryos are similar to the above. An additional block is added by the seed coats which may limit oxygen entry, but seed coat permeability may be increased when the seeds are exposed to light. Gibberellic acid added to the substrate increases the rate of embryo development.

C. AXILLARY MINIATURE EMBRYOS

Miniature seeds having minimal embryos are protected by thin seed coats showing a light requirement for oxygen permeability. Germination is prompt. Gibberellic acid is an aid, but not a substitute for light.

D. PERIPHERAL LINEAR EMBRYOS

Peripheral embryos which surround the food storage organ rather than being contained within it have multiple seed coverings which contain inhibitors, and which form barriers to both oxygen and water. Treatment is primarily leaching of the inhibitor but cold or light treatments may be required to increase permeability. Removal of the seed covering structures is also effective.

Summaries of seed anatomy, blocks to germination, successful germination test regimes and families exhibiting these characteristics are provided in Table 17.1 for endospermic seeds.

TABLE 17.1 ENDOSPERMIC SEEDS: Anatomy and Germination

		A. BASAL RUDIMENTARY EMBRYO	B. AXILLARY LINEAR EMBRYO	C. AXILLARY MINIATURE EMBRYO	D. PERIPHERAL LINEAR EMBRYO
SEED ANATOMY	embryo	small, show little differentiation,	linear in a central axillary	linear-spatulate in a central	curved in a peripheral position
		basal location	Position	axillary position	surrounding the perisperm or endosperm
	cotyledons	obscure and limited to a few cells	minimal: thin, narrow and shorter than the stalk	not expanded and equal to the stalk	thin, narrow and equal to the stalk
	endosperm	occupies most of seed and surrounds embryo	occupies half or more of seed and surrounds the central embryo	occupies half or less of the seed and surrounds the central embryo	centrally placed within the curved envelope
	seed coat	permeable and fibrous or reticulated	thin, reticulous or fibrous: may be semi-permeable	thin and fragile	thin testa, leathery outer coat and often parts of the calyx
	seed size	medium, 2-4 mm long	medium-large, 3-10 mm long	small, 1 mm long or less	medium-large, 2-6 mm diameter
	example	Anemone coranaria	Cyclamen persicum	Nemesia strumosa	Portulaca grandiflora
	embryo	must develop before germination, hence delay	must develop before germination, hence delay
BLOCKS TO GERMINATION	endosperm	inhibitors to germination may be present
	seed coat	permeable, no block to germination	may be semi-permeable	permeable if light provided	may be impermeable and contain inhibitors
	temperature	temperate species 15°C; others 20°C, 20°/30°C	15° to 20°C; 10°/30°C or 20°/30°C	10°, 15° or 20°C; 20°/30°C, 10°/20°C or 5°/30°C	10°, 15° or 20°C (rarely 3°-6°C); 20°/30°C
	test duration	14-28d; in extreme cases 100-155d	14-28d; in extreme cases 40-60d	10-28d; in extreme cases 40-60d	5-28d; in extreme cases 65-75d
	KNO3	*0.2%	*0.2%	*0.2%	0.2%
SUCCESSFUL GERMINATION TEST	GA3	*100-400 ppm	*400-800 ppm	120-400 ppm
	pre-chill	2w at 3°-5°C, or (rarely) test throughout at 5°-8°C	8w at 5°C	2-4w at 5°C
REGIMES (*most widely applied treatments)	light	probably insensitive	*insensitive or sensitive (promotory, may aid imbibition)	*sensitive (promotory); supply red light for up to 70h either continuously or 12h per day	*may be insensitive or sensitive (promotory)
	seed coats	partial removal of pericarp	may be necessary to remove endosperm over radicle		*remove (calyx and) outer coats; pierce, chip (clip)
	pre-soak	hot water; 0.5h in 1N KOH or other alkaline solutions			pre-wash in water
PLANT FAMILIES WITH SPECIES EXHIBITING THESE CHARACTERISTICS		ARALIACEAE, FUMARIACEAE, PAPAVERACEAE, RANUNCULACEAE	ERICACEAE, GENTIANACEAE, PRIMULACEAE, RESEDACEAE, SOLANACEAE, TROPAEOLACEAE, UMBELLIFERAE	BEGONIACEAE, CAMPANULACEAE, CRASSULACEAE, HYPERICACEAE, LOBELIACEAE, SAXIFRAGACEAE, SCROPHULARIACEAE, SOLANACEAE	AIZOACEAE, AMARANTHACEAE, CACTACEAE, CAPPARIDACEAE, CARYOPHYLLACEAE, CHENOPODIACEAE, MESEMBRYANTHACEAE, NYCTAGINACEAE, POLYGONACEAE, PORTULACACEAE

TABLE 17.2 NON-ENDOSPERMIC SEEDS: Anatomy and Germination

		A. AXILE FOLIAR EMBRYOS WITH HARD SEED COATS	B. AXILE FOLIAR EMBRYOS WITH THIN, MUCILAGINOUS, SEED COATS	C. AXILE FOLIAR EMBRYOS WITH WOODY SEED COATS AND INNER SEMI-PERMEABLE LAYER	D. AXILE FOLIAR EMBRYOS WITH FIBROUS SEED COAT AND SEPARATE INNER SEMI-PERMEABLE MEMBRANOUS LAYER
SEED ANATOMY	embryo	spatulate, bent or folded and fills seed cavity	spatulate or bent and fills most of seed cavity	fills most of seed cavity	spatulate and fills most of seed cavity
	cotyledons	large, thickened and dominant over the stalk	large, thickened and dominant over the stalk	expanded and dominant	large and dominant embryo
	endosperm	reduced to a thin layer or lacking	thin layer around embryo or lacking	surrounds axillary embryo forming a lining of the seedcoat or none	thin layer(s) lining the membranous testa
	seed coat	hard	thin, may exude mucilage when wet, or contain a mucilaginous layer within	woody, usually achenes with fused or separate membranous testa
	seed size	medium-large; 2-15 mm long	small-medium; 1-6 mm long	medium-large; 2-10 mm long	wedge-shaped achenes; 1-10 mm long
	example	Ipomoea purpurea	Iberis amara	Verbena x hybrida	Dimorphotheca sinuata
	embryo				inhibitors present in cotyledons
BLOCKS TO GERMINATION	endosperm	possibility of inhibitors in residual endosperm			impermeable to oxygen, gibberellins and inhibitors
	seed coat	impermeable when desiccated; imbibition through specialized valves or after scarification	becomes impermeable to oxygen and other gases as it absorbs water	permeable to moisture, but impermeable to some gases and inhibitors may be present within the seed coat	inhibitors may be present
	temperature	20°C; 20°/30°C	15°C, 20°C; 15°/25°C or 10°/30°C	15°, 20°, 25°C; 20°/30°C, 25°/35°C, 15°/25°C or 15°/30°C	15°,20°C; 15°/25°C or 20°/30°C
	test duration	14-28d; in extreme cases 40-50d	10-28d; in extreme cases 56-90d	9-28d; in extreme cases 40-60d (but excised embryos 2-4d only)	5-14d; in extreme cases 21-40d
	KNO3		*0.2%	*0.2%	0.2%
	GA3		*100-400 ppm	5 ppm	5-10 ppm
	pre-chill				60d at 2°-5°C with high oxygen conc.
SUCCESSFUL GERMINATION TEST	light	insensitive	*insensitive or sensitive (promotory)	*insensitive or sensitive (promotory)	insensitive or sensitive (promotory)
REGIMES (*most widely applied treatments)	seed coat	*file; chip (clip); excise embryo	excise embryo	*excise embryo; pierce, remove over radicle	*excise embryo from endosperm and seed coat; chip (clip) radicle end
	scarification	*mechanical; conc H2SO4 for 15 min to 4 h; absolute ethyl alcohol for 20-72 h; percussion (shake)
	pre-soak	water at 50°-90°C for 30 sec-24h		400 ppm GA3 or warm water for 24h	*in water or 5% chlorox or leach (even with excised embryos) away inhibitors with water
	after-ripen	dry at 95°C for 6 mins; dry over CaCI2		2-3m dry storage
PLANT FAMILIES WITH SPECIES EXHIBITING THESE CHARACTERISTICS		ANACARDIACEAE, CONVOLVULACEAE, GERANIACEAE, LEGUMINOSAE, MALVACEAE, RHAMNACEAE, SAPINDACEAE	BALSAMINACEAE, CRUCIFERAE, LABIATAE, LINACEAE, PLANTAGINACEAE, VIOLACEAE	ACANTHACEAE, APOCYNACEAE, BALSAMINACEAE, BORAGINACEAE, CISTACEAE, DIPSACEAE, EUPHORBIACEAE, HYDROPHYLLACEAE, LABIATAE, LIMNANTHACEAE, LOASACEAE, ONAGRACEAE, PASSIFLORACEAE, PLUMBAGINACEAE, POLEMONIACEAE, ROSACEAE, VALERIANACEAE, VERBENACEAE, ZYGOPHYLLACEAE	COMPOSITAE (only)

II. Seeds with only residual or no endosperm (NON-ENDOSPERMIC SEEDS) and mature independent embryos (that is embryos ready to germinate)

A. HARD SEED COAT

Hard (impermeable) seed coats are present which limit the entry of water for imbibition. Treatment for allowing water entry is necessary and may be mechanical scarification, acid treatment, percussion, high temperature treatment or soaking. (See Chapter 7, Volume I, for more information on treatments to overcome hardseededness.)

B. THIN SEED COAT WITH MUCILAGINOUS LAYER

Mucilaginous seed coats limit oxygen availability to the embryo after imbibition. Gibberellic acid is the most effective additive but light and potassium nitrate are also helpful, particularly when treatment with the two agents is combined.

C. WOODY SEED COAT WITH INNER SEMI-PERMEABLE LAYER

Woody-textured and membranous multi-layered seed coverings cause blocks to germination which are most difficult to overcome. They readily admit water for imbibition but contain strong inhibitors which do not leach readily. Some of these are probably located in the thin residual endosperm surrounding the embryo or within the cotyledons of the embryo and are blocked from leaching by the semi-permeable membranous testa incorporated in the coverings. Excised embryos germinate promptly.

D. FIBROUS SEED COAT WITH SEPARATE SEMI-PERMEABLE MEMBRANOUS COAT

The Compositae family forms a specialised group which is similar to the preceding section. Embryos contain inhibitors in their cotyledons. Testa and thin endosperm form a separate membranous semi-permeable coat. An outer fibrous coat offers various dispersal forms. Extracted embryos grow promptly if leaching is complete.

Summaries of seed anatomy, blocks to germination, successful germination test regimes and families exhibiting these characteristics are provided in Table 17.2 for non-endospermic seeds.

Atwater has developed her ideas and experience almost to the stage of providing species prescriptions. Much of this information is summarised in subsequent chapters, but the problems inherent in species prescriptions are discussed below. Note also that certain plant families have species in more than one category of seed anatomy (e.g. Solanaceae, see Tables 17.1 and 17.2). If Tables 17.1 and 17.2 are used to develop germination test environments and dormancy-breaking treatments, it should be remembered that combinations of different dormancy-breaking agents are more likely to be successful than treatment with a single agent alone.

Further reading

Atwater, B.R. (1978). Dealing with stop-go germination in flower seeds. Acta Horticulturae, 83, 175-179.

Atwater, B.R. (1980). Germination, dormancy and morphology of the seeds of herbaceous ornamental plants. Seed Science and Technology, 8, 523-573.

Mullet, J.H. (1981). Germinating those problem seeds. Australian Horticulture, December 1981, 61-67.

PRESCRIPTIONS FOR GERMINATING ALL ACCESSIONS OF A GIVEN SPECIES

This approach to determining germination test environments is exemplified by the Rules of the International Seed Testing Association (ISTA) and the Association of Seed Analysts of North America (AOSA). Commercial seed testing has evolved over the past 130 years to provide a certain degree of assurance to farmers, growers and foresters that any seeds of agricultural, horticultural or arboreal crops that they may purchase are capable of germinating under normal field conditions, emerging above the soil surface and developing into plants which yield an adequate crop. Thus, by assessing the quality of seed lots before they are sown, commercial seed testing minimises the risk to the farmer, grower or forester of sowing seed lots which do not have the ability to produce an abundant crop. Since this assessment will be done by only one of many laboratories and since there is also considerable international trade in seed of many species, assessments by different seed testing laboratories must give similar results.

Compatibility between seed testing stations

To achieve compatibility of germination test results between different laboratories ISTA and AOSA co-ordinate and produce agreements between seed testing laboratories on the procedures to be applied for each species. We have discussed in Chapters 4 and 5 (Volume I) how the proportion of seeds which will germinate in a germination test can be affected by the test environment. Thus if seed testing laboratories used very different germination test conditions the results obtained in different laboratories could differ substantially. To aid compatibility ISTA and AOSA seek agreement between laboratories as to the most appropriate environment for testing the germination of a given species. In theory this implies agreement upon a single germination test environment. In practice alternative environments are often specified for a given species. This is either because of disagreement between laboratories as to the single most appropriate environment, or because of the need to provide alternative procedures for certain seed lots, or because of practical difficulties in providing certain environments (for example, alternating temperatures) in some laboratories.

The rules of the ISTA and AOSA list the prescribed germination test conditions for each species. These prescriptions specify the substrata, temperatures and test duration which seed testing laboratories must adhere to, Additional directions are provided for special treatments which may be required for certain species together with recommendations for breaking dormancy. These prescriptions and directions are summarised in the chapters which follow.

Now, these rules and directions are intended to give the most regular, rapid and complete germination for the majority of samples of seed of a particular species. To that end we presume the rules are successful, but how closely does the above aim coincide with the problems facing those testing seeds within gene banks?

Prescriptions are not available for all species

First, ISTA and AOSA tend to only provide rules for those species in which certian individual seed testing stations have knowledge of the germination behaviour of a large number of seed samples. Thus the species listed in the ISTA and AOSA rules are those in which there is considerable trade in the sees; the seed-propagated crops. In contrast gene banks will often need to germinate seeds of species which are not yet in substantial trade in the developed world (e.g. the tropical pasture species), true seeds of vegetatively propagated crops (e.g. potato, Solanum tuberosum), and seeds of species which do not have crop status such as wild and weedy relatives of modern crop species (e.g. teosinte, Euchlaena mexicana). Thus the first drawback for gene banks of the ISTA/AOSA rules is that for many species on which gene banks require advice on germination test conditions no information is provided.

Prescriptions developed for modern cultivars

Secondly, within those species for which ISTA and AOSA prescribe test conditions the range of seed lots which seed testing stations test for germination, and upon the results of which ISTA precriptions are based, are generally limited to relatively non-dormant, high quality, modern, genetically homogeneous cultivars. That is in most cases the cultivars with which the rules have been developed are likely to have been subjected to considerable selection pressures. In contrast gene banks will be testing seeds of primitive cultivars and genetically heterogeneous landraces which may also show considerable dormancy and/or be of poor quality. Thus the second drawback for gene banks of the ISTA/AOSA rules is that the prescribed conditions for a particula species will not necessarily be suitable for those acessions within gene banks which are dissimilar to modern cultivars - or of poor quality.

Relative priorities of the aims of seed testing

Thirdly, for some crops seed testing stations may operate under considerable pressure at harvest time. For example, in temperate countries autumn sown cereal seeds are sown only five or six weeks after harvest. Thus seed testers may need to complete germination tests in as short a time as possible. Hence seed testing stations' aim of providing conditions which give rapid germination. Now in at lest one group of species - the temperate cereals - although the conditions prescribed by the rules may give rapid germination this is only achieved in our experience by using supra-optimal temperatures at the cost of the parallel aims of achieving regular and complete germination which are better achieved at much lower temperatures. In contrast to seed testing stations, gene banks are not under any pressure to give priority to rapid germination over achieving regular and complete germination. Thus the third drawback for gene banks of the ISTA/AOSA rules is that the aims of rapid and complete germination may not always be compatible. Note that this criticism does not apply to the rules for all crops. In the case of tree species, for example, considerable periods are allowed for dormancy-breaking and germination test treatments by ISTA and AOSA rules, and the requirement for complete germination takes precedence over the requirement for rapid germination.

Field planting value

The purpose of commercial testing is to provide information on the comparative field planting value of different seed lots. In general the ISTA and AOSA prescriptions make no attempt to enable those viable seeds which will not germinate under field conditions to germinate in the laboratory test. If, for example, dormancy is a problem in laboratory tests, but not in field sowings (for example, due to differences in temperature between the two regimes) then directions to break dormancy must be provided in the rules - otherwise the field planting value may be underestimated. However, where dormancy is a problem in both laboratory tests and field sowings then to obtain an indication of field planting value the rules may not necessarily provide dormancy-breaking directions - since the removal of dormancy in laboratory tests may result in an overestimate of field planting value.

Again whether or not this objective of ISTA or AOSA prescriptions and recommendations makes them unsuitable for viability testing in gene banks depends on the species. For example, in tree species directions for dormancy removal are suggested to enable viability to be estimated in laboratory tests and these directions must also be followed in field sowings if laboratory results are to provide a reasonable estimate of field planting value. In contrast in some species where hardseededness prevents germination no recommendation is made to render the seed coat permeable. Instead the proportion of hard seeds is reported. Thus the fourth drawback of ISTA/AOSA rules is that no attempt may be made to overcome a wide range of physiological phenomena which may prevent the germination of viable seeds under both laboratory and field conditions. Of these phenomena the most important are hardseededness and strong dormancy. In contrast to seed testing stations, gene banks aim to provide conditions which will enable all viable seeds to germinate both in laboratory tests and in subsequent field or glasshouse sowings.

Other sources of prescriptions

Numerous groups of workers have attempted to provide prescriptions for germination tests of species for which no ISTA/AOSA rules are available or which overcome certain of the deficiencies of the ISTA and AOSA prescriptions for species where rules are available. Particular emphasis has been placed on overcoming dormancy since this can be an important problem for plant breeders, and also for seed testers if considerable dormancy is experienced when the seeds are tested but which is likely to be lost during storage before sowing.

However, much of this information is also of limited use in gene banks. This is because the majority of such information relates to the application of single dormancy breaking agents alone. In Chapter 5 (Volume I) it was explained that if this approach is used then the treatment concentration (if a chemical agent) or duration (if another form of treatment) required to break dormancy in the most dormant seeds would be likely to damage less dormant seeds, particularly if these are also of poor quality.

The aim of developing a species prescription for use in gene banks

We believe that the ultimate desirable aim of a prescriptive test for a given species is to be able to provide within one test environment or procedure those conditions which enable all viable seeds of that species - whether strongly dormant, moderately dormant, weakly dormant or non-dormant, and of high or low quality - to be able to germinate. This means that any stimulus or combination of stimuli used to promote the germination of dormant seeds must not be damaging to other non-dormant seeds in the sample or to intolerant genotypes.

Developing a prescription for a species

Whether this aim can in fact always be achieved is a matter for debate, but two considerations are important in attempting to achieve it. The first is to ensure that the germination test temperature is suitable for poor quality seeds; within this range attempts can then be made to determine the most suitable temperature regime which will minimise the expression of dormancy. In certain species this first step may result in an adequate single germination test environment without any need for further treatment. In temperate cereals this appears to be the case, where a single low constant temperature is appropriate for both purposes. However, in other species simple solutions of this type may not be available and so it is necessary to take a second step and provide further stimuli to break dormancy which do not impair the germination of less dormant seeds. In our opinion this is often best attempted by combining more than one additional stimulus with all stimuli being applied at relatively low dose-levels. This is an attempt to utilise the positive interactions observed between many dormancy-breaking agents (see Chapter 5, Volume I). The treatments developed for Oryza spp. and Vitis spp. provide examples of this type of procedure (these and other prescriptions are described in the appropriate chapters of this volume).

Gene banks considering developing their own germination test prescriptions may be discouraged by the wide range of different treatments - and the even wider range of different treatment combinations - which may be of potential benefit in promoting the germination of viable seeds. Consequently it is worthwhile considering which factors should receive priority in any investigation. We believe that the three most important factors to consider first are to ensure that the seeds have imbibed moisture without damage (see Chapter 7, Volume I) and then to attempt to determine the most suitable temperature (probably alternating temperature, see Chapter 5, Volume I) and light (see Chapter 6, Volume I) regimes for germination.

Provided a prescription is already available for a species which is suitable for application in a gene bank then this would be the simplest approach for gene banks to use. However, even where a prescription is well-tried, gene banks will still need to be alert to the possibility that some accessions may be discovered for which the prescription does not appear to work. Furthermore, for many years to come, there will continue to be a large number of species for which there are no adequate prescriptions. Accordingly alternative approaches to providing suitable germination test environments are also considered in this handbook.

Use of tetrazolium test to determine efficacy of a prescription

Although a gene bank can never be absolutely certain about the efficacy of any approach to germinating seeds, do not forget that the efficacy of a species prescription on a particular accession can be tested by comparing the results of the germination test with that of the topographical tetrazolium test (see Chapter 11, Volume I) on a sub-sample of the seed lot and by also applying a tetrazolium test to the seeds remaining ungerminated at the end of the test. In this way one can determine the proportion (if any) of viable seeds which are either killed by the germination test procedure or remain dormant at the end of the germination test procedure.

In the above we have detailed objections to the wholesale and unquestioning adoption of either ISTA or AOSA prescribed germination test procedures as gene bank germination tests. However, this certainly does not mean that such prescriptions should be ignored by gene banks. The ISTA and AOSA rules summarise a wealth and depth of expertise and experience which is unmatched. Thus where available, the prescribed conditions represent a control against which any proposed alternative should be tested; and, despite our earlier comments, certain of these prescriptions will be suitable for gene bank use.

TAXONOMIC ALGORITHMS TO DETERMINE APPROPRIATE GERMINATION TEST ENVIRONMENTS

As mentioned above, germination test prescriptions are available for only a small proportion of seed-producing species. What environment should be chosen to test the germination of seed accessions of species for which no prescription or no acceptable prescription is available? This is exactly the problem which faced staff at the Royal Botanic Gardens Kew, Wakehurst Place Gene Bank, for almost all their accessions, because this gene bank deals entirely with seed collections of species which do not have great commercial significance. The method that they developed to answer the question posed above provides another approach to developing appropriate germination test regimes for seed accessions maintained in gene banks.

Algorithm: the shortest series of tests to define a suitable test environment

This approach acknowledges that the most suitable environment for testing each accession is unknown. Thus, instead of a single prescription, an attempt has been made to develop the shortest series of tests which is likely to give a solution to the problem of defining a suitable test environment. The decision as to which test to try next depends on the results obtained in the previous test(s), and so we call this series of tests an algorithm. For gene banks a truly universal algorithm would be one appropriate to all seed-producing species, irrespective of taxonomy. However, such an approach would be rather unwieldy and require a large number of tests. Certain similarities in the response of seed germination to environment for species within a family can be recognised. Consequently this approach provides a separate algorithm for each family where sufficient knowledge has been accumulated.

Where available, the algorithms developed by the staff at the Wakehurst Place Gene Bank are presented according to family in Chapters 18 to 75. However, it is necessary here to provide some explanation of how the algorithms were developed, their possible faults, and how they should be applied.

The development of the RBG Kew Wakehurst Place algorithms

Storage at the Wakehurst Place Gene Bank began in 1974. Since then all accessions entered into storage have been tested for germination as soon as possible thereafter. In many cases accessions were tested in more than one germination test environment, the reason for this being an inadequate proportion (less than 85%) of seeds germinating in the first test. Thus for many accessions it was possible to compare germination test results over a number of different environments with and without the imposition of various additional dormancy-breaking treatments. For each accession the best germination test regime was noted, and this regime was then used for all subsequent germination tests of that accession. In addition the results of all the different germination tests carried out on each accession were filed according to species to provide a growing information bank on the response of seed germination to different test conditions. Table 17.3 provides an example of the information generated and filed from such tests for a single accession of Veronica verna.

TABLE 17.3 Example of germination test results recorded by staff at the Wakehurst Place Gene Bank used in subsequent analyses to determine algorithms for determining suitable test regimes: germination (%) and classification of non-germinating seeds after testing seeds of a 1977 collection of Veronica verna under four different regimes (100 seeds per test).

Test Date	Germination Test regime	Total Test Duration (days)	Cumulative Germination (%) at end of test	Non-germinated seeds at end of test1
				Fresh	Mouldy	Empty
16/12/77	21°C	42	0	100	0	0
16/12/77	21°/11°C (12h/12h)	157	48	52	0	0
18/4/78	26°C for 28 days (imbibed) then 11°C	48	100	0	0	0
18/4/78	2°C	73	88	12	0	0

¹ expressed as the percentage of the original number of seeds tested

Over the past ten years or so a considerable amount of information on the response of seed germination to various test regimes (similar to that described above for Veronica verna) has been generated by staff at Wakehurst Place. This raw information has subsequently been analysed, principally by Mr. S. Linington, in the following manner. For a particular family all the routine test data for all accessions (each similar in form to that provided in Table 17.3) were examined. It was (arbitrarily) decided that test regimes in which 85%, or more, of the full seeds germinated would be classified as successful. For each accession all the successful regime(s) (if there were any) were listed. For example, two regimes would be listed as successful for the accession shown in Table 17.3: 2°C constant; and 28 days at 26°C followed by 11°C constant.

When this first stage of analysis was completed a pattern of successful regimes within each family was sought. Emphasis was placed on determining those simpler regimes (that is those employing few dormancy-breaking agents) which were successful for the greatest number of accessions. These regimes are those which are now used as the first step in the algorithms which have been developed. More complicated regimes (involving the use of many dormancy-breaking treatments) which are successful for fewer accessions are then provided as subsequent alternatives (later steps) in the algorithm if the first step does not provide a suitable environment for germination testing.

Possible limitations of the algorithms

There are a number of possible faults inherent in this method of analysis. First, by excluding from the analysis all accessions which failed to reach 85% germination under any test regime, the response of accessions which are either very dormant, or very poor quality and vulnerable in certain germination test environments may have been ignored. Secondly, it is based on tests of accessions within the Wakehurst bank which, at least in the past, were biased towards collecting in temperate regions. Not only may this result in an algorithm more suited to temperate collections, but it may also result in algorithms more suited to certain tribes within each family. For example, the algorithm for the Leguminosae was based largely on accessions of the Papilionoideae tribe and little data was available for accessions from either the Caesalpinoideae or Mimosoideae tribes. Finally it should be noted that the analysis was based on data obtained over only a limited range of treatments. Other, untested, treatments may be superior and there is as yet no way of knowing whether this will be the case.

Success of the algorithms may vary between families

Despite these reservations, the algorithm approach to determining germination test environment works well at Wakehurst and is likely to be useful in other gene banks. Tests at Wakehurst on previously untested accessions using the algorithms for the Amaranthaceae, Gramineae, Leguminosae and Solanaceae families (provided in Chapters 20, 39, 43 and 67 respectively) have shown that 100%, 50%, 90% and 75% of accessions within each family reached 85% germination or greater. In view of the importance of the Gramineae in food production the limited success of the algorithm for this family is disappointing. One group of species within the Gramineae whose seed is particularly difficult to germinate is the tropical pasture species. Unfortunately tests on five seed lots of tropical pasture species at Reading (Table 17.4) confirm that the present algorithm for the Gramineae (Chapter 39) is not particularly helpful for determining germination test environment in these species.

Demonstration of the use of an algorithm

Nevertheless, Table 17.4 can be used to explain the method of applying the algorithm. Table 17.4 should be studied together with the Gramineae algorithm provided in Chapter 39. In Table 17.4 every feasible combination of dormancy-breaking agents within the algorithm was applied to the seeds, amounting to some 24 separate test environments. Note, however, that only a few of these tests would have been applied if the algorithm had been followed. The pathway through the tests which would have been followed if the Gramineae algorithm had been applied is shown by the boxed values in Table 17.4, the sequence being from the top of the table to the bottom.

Table 17.4 Normal germination (expressed as percentage of full seeds tested) after 28 days in germination test [lower figures in square brackets after 56 days] for five tropical grass seed lots. If the Gramineae algorithm (Chapter 39) had been followed in the manner intended only the tests in square boxes would have been investigated (see text).

Treatment

SPECIES

Brachiaria decumbens

Brachiaria humidicola

(Gatton Panic) Panicum maximum

(Green Panic) Panicum maximum

(Riversdale Guinea) Panicum maximum

Temperature, °C

Temperature, °C

Temperature, °C

Temperature, °C

Temperature, °C

Control (constant temperature)

20

25

35/20

(15/30,10/30)

20

25

35/20

(15/30,10/30)

20

25

35/20

(15/30,10/30)

20

25

35/20

(15/30,10/30)

20

25

35/20

(15/30,10/30)

[4]

[1]

[0]

[4]

[39]

[25]

[18]

[18]

[2]

[0]

[5]

[3]

[0]

[4]

[39]

[26]

[18]

[18]

[3]

[0]

Alternating temperature

[19]

(10,10)

[21]

(74,73)

[46]

(90,92)

[18]

(13,7)

[31]

(57,69)

[22]

([10],[15])

[57]

([75],[77])

[49]

([92],[93])

[18]

([15],[8])

[44]

([58],[70])

10-3 M KNO3

3

3

[28]

(13,17)

0

1

[18]

(66,-)

61

31

[59]

(93,82)

14

14

[13]

(14,14)

3

0

[40]

(64,79)

[4]

[3]

[30]

([13],[19])

[1]

[2]

[64]

([67],-)

[61]

[31]

[64]

([97],[90])

[16]

[14]

[13]

([17],[14])

[6]

[0]

[51]

([64],[80])

Dehusk

15

14

65

0

2

[14]

11

8

7

14

15

[18]

1

2

35

[17]

[15]

[66]

[0]

[2]

[45]

[12]

[10]

[12]

[14]

[15]

[19]

[10]

[2]

[41]

Dehusk + KNO3

14

15

[63]

4

3

12

17

11

[9]

14

21

13

18

3

[61]

[15]

[16]

[63]

[4]

[4]

[40]

[19]

[13]

[26]

[14]

[22]

[14]

[19]

[3]

[69]

8 week pre-chill

0

0

0

0

0

[3]

33

21

16

0

0

[2]

35

54

59

[0]

[0]

[0]

[0]

[0]

[4]

[34]

[21]

[18]

[0]

[0]

[2]

[35]

[54]

[62]

Dehusk + pre-chill

1

7

38

0

0

5

11

27

25

0

2

2

35

31

46

[2]

[7]

[38]

[1]

[0]

[12]

[11]

[27]

[26]

[0]

[3]

[4]

[38]

[31]

[49]

KNO3 + pre-chill

1

1

1

1

0

4

8

20

[25]

2

2

2

49

62

59

[1]

[1]

[1]

[1]

[0]

[5]

[8]

[20]

[25]

[2]

[2]

[2]

[49]

[62]

[62]

Dehusk + KNO3 + pre-chill

0

9

[35]

0

0

6

24

18

30

4

2

7

31

45

[62]

[0]

[10]

[35]

[2]

[0]

[9]

[24]

[18]

[30]

[5]

[3]

[9]

[33]

[45]

[62]

Viability (tetrazolium test)

[94]

[75]

[97]

[25]

[76]

Three details of the work reported in Table 17.4 differ from the Gramineae algorithm provided by the Wakehurst Place Gene Bank. First the germination test temperatures are not identical, but are within 1°C - except for the upper limit of the alternating temperature regime where the difference is 2°C. These minor differences are unimportant and result from differences in the standard temperatures available at Reading and Wakehurst. Secondly the comment in the algorithm to test at constant temperatures either below 20°C or above 25°C depending upon which gave the higher test result was not followed, since previous experience with dormant seed lots of these species indicated that alternating temperatures were essential to promote germination. Thirdly, following on from the last point, seeds were also tested for germination at the two additional alternating temperature regimes of 15°/30°C and 10°/30°C.

To enable the reader to understand fully the use of the algorithm, an explanation is provided below of the sequence of tests and decisions that would have been applied for tests on the accession of Brachiaria decumbens. The first stage in the algorithm was to test the seeds at constant temperatures of 20°C and 25°C. These test results were inadequate (less than 85% germination).

Now, according to the algorithm (Chapter 39) the next step would have been to look for trends in the results of these two tests (i.e. was germination greater at the higher or lower temperature?) and test for germination at either a more extreme higher or a more extreme lower constant temperature accordingly. This, however, was not done for the reasons given above and we proceeded to the second step of the algorithm which was to test a fresh sample of the seeds in an alternating temperature regime of 35°/20°C (the nearest regime available to the standard 33°/19°C Wakehurst regime). The germination obtained in this test was substantially greater than at either of the two constant temperatures, but again inadequate as a germination test regime. Consequently further tests were required.

The third step according to the algorithm was to test a fresh sample of the seeds in a medium containing 10^-3 M KNO₃ at, in view of the difference observed between the results at steps 1 and 2, an alternating temperature of 35°/20°C. Again the result of this test at step 3 was an improvement on the result at step 2 but inadequate as a germination test regime since 85% germination was not achieved.

Thus the fourth step was to remove the seed covering structures from a fresh sample of the seeds and test in a medium containing 10^-3 M KNO₃ (since the result at step 3 was greater than the result at step 2) at an alternating temperature of 35°/20°C. Once again the result of the test at this step was an improvement on the previous test result (suggesting that dehusking was a worthwhile treatment), but the percentage of seeds germinating was still unsatisfactory.

Consequently the final step in the algorithm was applied. Namely dehusked imbibed seeds were pre-chilled at 3°-5°C for 8 weeks and then transferred to an alternating temperature regime of 35°/20°C in a medium containing 10^-3 M KNO₃. Unfortunately this test resulted in substantially fewer seeds germinating than the previous test. Thus the conclusion reached by using the algorithm is that dehusked seeds of this accession of Brachiaria decumbens should be tested on a substrate containing 10^-3 M KNO₃ in an alternating temperature regime of 35°/20°C.

Note that the decision making procedure at each step in the algorithm did not result in the algorithm missing more favourable treatment combinations. For example, if the algorithm had been followed dehusked seeds would not have been tested at 35°/20°C without 10^-3 M KNO₃ in the germination medium. In fact this treatment gave a very similar result (that is there was no significant difference) to the treatment with dehusked seeds which did include 10^-3 M KNO₃ in the germination medium. Thus it appears that the use of 10^-3 M KNO₃ in the medium is in fact not necessary, but its use (apparently necessary if the algorithm had been followed) does not result in a significantly lower proportion of seeds germinating. In that sense we can regard the decision-making procedure within the algorithm as being successful, but unfortunately the algorithm itself was not successful since the level of viability indicated by the tetrazolium test was not achieved in these germination tests.

Sensitivity of germination to the temperature regime

In addition to all the possible germination test regimes according to the Gramineae algorithm, Table 17.4 also presents results of tests carried out in two additional alternating temperature environments (15°/30°C and 10°/30°C) and also in combination with 10^-3 M KNO₃ (where sufficient seeds were available). These results suggest that, for some seed lots at least, those gene banks testing tropical grass species would be well advised to include additional alternating temperature regimes as the second step of the algorithm. For three of the seed lots tested here small differences in the alternating temperature regime greatly influenced the proportion of seeds which germinated (Table 17.4). Not only did this result in almost full germination of viable seeds, but it would have avoided the need for several stages of the algorithm in the case of Gatton Panic. (See the section on the genus Panicum, Chapter 39, for advice on an appropriate alternating temperature regime for this species.)

In passing note that the results shown in Tables 17.3 and 17.4 emphasise that seed germination can be particularly sensitive to the temperature regime in which the seeds are tested. It cannot be overemphasised that gene bank staff must make strenuous efforts to get this right first before embarking on investigations as to the efficacy of other dormancy-breaking agents.

Possible use of ungerminated seeds from prior step in algorithm for the current test

In the algorithms provided here additional tests are carried out by drawing a fresh sample of seeds from the accession bulk in storage. In some cases, for example if the supply of seeds is severely limited, it is possible to test the response to additional treatments by applying these to those ungerminated seeds which remain fresh and firm at the end of a previous test. This is particularly suitable for scarification, dehusking or puncturing treatments since it may avoid the need to apply these time-consuming procedures to all seeds tested (see Chapter 7, Volume I). For example, if 70% of seeds tested of a legume accession germinate without scarification it is easier to scarify the remaining proportion of seeds and to return these to the germination environment than to draw a fresh sample of seeds from storage and scarify all of the seeds to be tested.

Duration of germination tests

During each step of the algorithm it is necessary to decide when to terminate the germination test and whether to pursue the next step in the algorithm. Since both aged and dormant seeds may only begin to germinate in a test after a substantial delay (see Chapters 4 and 5, Volume I) it is not possible to provide categorical advice. In Chapter 10, Volume I, an example is provided of how to decide when to terminate a germination test by constructing a germination progress curve. This technique can be used for each step in the algorithm, but each test should not be continued for an excessive period because the next step in the algorithm might result in a more suitable and more rapid procedure. That is if the progress of germination is very slow it might be better to pursue the next step in the algorithm.

A minimum germination test period of 28 days is suggested. A comparison of results after 28 and 56 days in test (Table 17.4), for example, shows substantial germination after 28 days in test for only the Brachiaria humidicola accession tested at 35°/20°C. Consequently it is suggested that the decision to test a further sample of seeds in the next step of the algorithm can generally be made once the 28-day test result is known, but that the current test be continued for a further period dependent upon the shape of the germination progress curve. In tests at a very low temperature (e.g. 6°C), however, it may be better to delay a decision as to whether to continue through the algorithm until the 56-day test result is known.

Use of algorithms at accession receipt

It is suggested that the algorithms could be applied soon after an accession is received if the standard advice on germination test procedures and dormancy-breaking treatments is unsatisfactory or if no advice is available. The number of seeds tested in each of the regimes required by the algorithm need not exceed 100 (and 50 seeds may well be sufficient), but the seeds should be sampled from the accession at random (see Chapter 13, Volume I) and replication in each test is advisable (which will allow the use of the tolerance tables, Chapter 14, Volume I).

Once a suitable test procedure has been developed a further sample of 200 seeds can be withdrawn from the accession and tested by this procedure in order to 'estimate initial accession viability (discussed in more detail in Chapter 15, Volume I). Subsequent monitoring tests of accession viability will also apply the same germination test procedure, but may be either fixed sample size tests (see Chapter 14, Volume I) or sequential tests (see Chapter 15, Volume I). Note that it is envisaged that the algorithms would only be applied once, i.e. when the accession is first received: the subsequent monitoring tests would employ the most appropriate regime already determined.