CHAPTER 16. INTRODUCTION TO VOLUME II: THE ORGANIZATION OF CHAPTERS AND AN EXPLANATION OF ABBREVIATIONS

Volume I of Handbook of Seed Technology for Genebanks dealt with many of the principles of seed testing which need to be understood when monitoring the viability of seed accessions maintained in gene banks. It will have become clear that one of the main problems facing those who have the responsibility for monitoring seed viability in gene banks is that seed dormancy can often interfere with the results of germination tests designed to estimate the percentage viability of accessions. The extent of the problem varies between species and between accessions within species, and the techniques which are most appropriate for minimising dormancy in germination tests also vary. In some species the problems are sufficiently understood so that prescriptions for germination tests have been developed which enable dormancy to be removed completely. In other species sufficient is known to minimise the problem of dormancy so that it is no longer a serious problem. However, there are still many species where existing techniques for dormancy removal are unsatisfactory, and yet others where the information on dormancy is meagre.

This volume provides general approaches, detailed information, guidance and, where available, prescriptions for removing dormancy and germinating the seeds. Since completely satisfactory prescriptions are relatively rare, Chapter 17 deals with general approaches which may help staff in gene banks develop their own techniques for solving dormancy problems.

The subsequent chapters (Chapters 18 to 75) provide information, family by family, on the germination of individual species of crop plants and sometimes their wild relatives. These chapters are essentially for consultation and, since the amount of information is large, considerable use is made of abbreviations. The final chapter (Chapter 76) summarises the germination test recommendations which are available for species outside the 58 families covered by Chapters 18 to 75.

The rest of this chapter is essential to understanding Volume II since it explains the structure and abbreviations used. It also provides guidance on the preparation of solutions commonly used in dormancy-breaking treatments.

THE STRUCTURE OF CHAPTERS 18 TO 75 AND ABBREVIATIONS USED IN THIS VOLUME

Each chapter which deals with a single family begins with a short introduction which includes, where available, the algorithm for devising dormancy-breaking techniques developed by staff at the Wakehurst Place Gene Bank (see Chapter 17). A comment is also provided on seed morphology if this is considered to be of help in devising appropriate treatments to promote germination. Prescriptions for germination test procedures and recommendations for dormancy-breaking treatments from various sources, but primarily the ISTA and AOSA rules, are tabled for species within genera where more detailed information is not provided within the chapter. Most alternating temperature regimes are diurnal cycles of 16h/8h and, to save space, only exceptions to this general rule are noted in these tables. The following abbreviations are used within these tables, and also Chapter 76, to describe the source of information.

AOSA	AOSA (1981). Rules for testing seeds. Journal of Seed Technology, 6, 1-126.
Atwater	Atwater, B.R. (1980). Germination, dormancy and morphology of the seeds of herbaceous ornamental plants. Seed Science and Technology, 8, 523-573.
Ballard	Ballard, L.A.T. (1972). High sensitivity to temperature of the germination responses of seeds of Townsville stylo (Stylosanthes humilis H.B.K.). Proceedings of the International Seed Testing Association, 37, 779-791.
B&B	Ballard, L.A.T. and Buchwald, T. (1971). A viability test for seeds of Townsville stylo using thiourea, Australian Journal of Experimental Agriculture and Animal Husbandry, 11, 207-210.
Butler	Butler, J.E. (1975). Germination of Stylosanthes humilis (Townsville stylo) in short cycles of alternating temperature. Seed Science and Technology, 3, 523-528.
Cameron	Cameron, D.F. (1967). Hardseededness and seed dormancy of Townsville lucerne (Stylosanthes humilis) selections. Australian Journal of Experimental Agriculture and Animal Husbandry, 7, 237-240.
CHML	Chin, H.F., Hor, Y.L. and Mohd Lassim, M.B. (1984). Identification of recalcitrant seeds. Seed Science and Technology, 12, 429-436.
Everson	Everson, L. (1949). Preliminary studies to establish laboratory methods for the germination of weed seed. Proceedings of the Association of Official Seed Analysts, 39, 84-89.
Fornerod	Fornerod, C. (1975). Remarques sur la germination des semences potageres en laboratoire. Revue Horticole Suisse, 48, 6-9.
G&R	Gordon, A.G. and Rowe, D.C.F. (1982). Seed Manual for Ornamental Trees and Shrubs. Forestry Commission Bulletin 59, 132pp., HMSO, London.
Heit	Heit, C.E. (1948). Laboratory germination test results with herb and drug seed. Proceedings of the Association of Official Seed Analysts, 38, 58-62.
Holm	Holm, A. McR. (1973). Laboratory procedures for germinating Townsville stylo seed pods. Journal of the Australian Institute of Agricultural Science, 39, 75-76.
ISTA	ISTA (1985). International rules for seed testing. Seed Science and Technology, in press. (We are most grateful to Dr. S. Cooper for providing draft copies of the new rules prior to publication.)
M&O	Maguire, J.D. and Overland, A. (1959). Laboratory germination of seeds of weedy and native plants. Washington Agricultural Experiment Station Circular 349, 15pp.
McIvor	McIvor, J.G. (1976). Germination characteristics of seven stylosanthes species. Australian Journal of Experimental Agriculture and Animal Husbandry, 16, 723-728.
M&M	Mott, J.J. and McKeon, G.M. (1979). Effect of heat treatments in breaking hardseededness in four species of Stylosanthes. Seed Science and Technology, 7, 15-25.
Oakes	Oakes, A.J. (1984). Scarification and germination of seeds of Leucaena leucocephala (Lam.) De Wit. Tropical Agriculture (Trinidad), 61, 125-127.
O&W	Olvera, E. and West, S.H. (1985). Aspects of germination of leucaena. Tropical Agriculture (Trinidad), 62, 68-72.
Riley	Riley, J.M. (1981). Growing rare fruit from seed. California Rare Fruit Growers Yearbook, 13, 1-47.
R&S	Rogers, B.J. and Stearns, F.W. (1955). Preliminary studies on the germination of weed seeds. Proceedings of the North Central Weed Control Conference, 12, 7.
SGCF	Steinbauer, G.P., Grigsby, B., Correa, L. and Frank, P. (1955). A study of methods for obtaining laboratory germination of certain weed seeds. Proceedings of the Association of Official Seed Analysts, 45, 48-51.

Most chapters then go on to provide a more detailed summary and analysis of seed germination responses to treatments, genus by genus, for the more important species where information is available. This information is restricted mainly to orthodox species, but information on a few recalcitrant species is provided where dormancy is known to be a potential problem. The layout of information within each genus is as follows.

At the beginning of each genus a catalogue is provided of those species for which some information is given. The catalogue includes botanical synonyms and common names. It should be noted that sometimes identical synonyms may be given for species within another genus. Where this occurs and information is provided for the second genus the reader should consider the information provided for both genera. The information on dormancy-breaking techniques and germination test regimes for each genus is divided into seven sections. Note that although the term dormancy is used in the titles below information on factors other than dormancy per se (see definition in Chapter 5, Volume I), particularly hardseededness (Chapter 4, Volume I), is also included.

I. Evidence of dormancy

This section simply provides evidence of whether dormancy can be a problem and attempts to place it in context - often by giving details of how long after harvest 'post-harvest' dormancy typically remains a problem under ambient conditions. Differences in the degree of dormancy between species within the genus are sometimes noted. Other problems may be noted in this section. For example, it is sometimes necessary to draw attention to the classification of seed storage behaviour (see Chapter 1, Volume I) where this has been in some doubt.

II. Germination regimes for non-dormant seeds

In the majority of cases this section provides details of the prescribed germination test conditions for species within the genera given by the ISTA and the AOSA - where these are available. The ISTA and AOSA rules are divided into three parts here.

The first information is the method (or methods) of providing the medium for the germination test. The abbreviations used and their meanings are:

TP test on top of paper, that is, place the seed on filter papers, blotting papers or paper towels in a petri-dish or similar container.

BP test between paper, including rolled paper towels and pleated papers.

S test in (sterilized) sand.

TS test on top of (sterilized) sand.

Often more than one medium is suggested. In this case choose whichever is the more suitable for your laboratory.

The information after the first colon gives the prescribed temperature regime for the germination test. An alternating temperature regime is denoted by A°/B°C (xh/yh), where A°C is provided for x hours per day and B°C provided for the remaining y hours per day; e.g. 20°/30°C (16h/8h) means germinate in a continuous alternating temperature regime in which the seeds are subjected to 20°C for 16 hours followed by 30°C for 8 hours each day. Often alternative temperature regimes are provided. The alternative regimes are separated by semi-colons.

The final information provided (after the second colon) is the total germination test period in days, but of course seedlings may have to be removed and counted at more frequent intervals. Moreover, it is likely that in many cases gene banks will have to continue germination tests beyond these periods.

The provision of both the ISTA and the AOSA prescriptions for germination test regimes (where these are available for a species) allows the reader to compare and contrast the two sets. In the main these are quite similar, but where they occur the differences of detail should be noted.

In addition to the ISTA and AOSA rules this section also includes other published information, where available, on germination test procedures which are satisfactory for non-dormant seeds.

III. Unsuccessful dormancy-breaking treatments

The third section gives details of treatments that have been applied to seeds in attempts to break dormancy, but which have either failed to increase germination more than marginally or may have even led to a reduction in germination - either by inducing dormancy in the seeds or by damaging the seeds in some way. Although at first sight the reader may consider this information to be of no interest - after all the requirement is to promote germination - it is important to be aware of those treatments which should be avoided. Moreover, the reader will begin to notice that similar treatments may appear in more than one section. These apparent inconsistencies and contradictions, often between different reports, are nevertheless probably indicative of the real situation: a treatment which greatly promotes the germination of seeds of one accession, may fail to promote the germination of seeds of another accession, whilst in a third accession germination may be reduced by the treatment. Hence the inclusion of this information here.

IV. Partly-successful dormancy-breaking treatments

Treatments detailed in this section have promoted the germination of some dormant seeds, but have either failed to promote the germination of all the dormant seeds within a single accession, or within a report they may have promoted full germination in some accessions but not promoted full germination in other accessions.

V. Successful dormancy-breaking treatments

The ISTA and/or the AOSA recommendations for breaking dormancy are provided first of all in this section (where available for a species). The style of layout is different from, and less detailed than, that given for other sources of information. The reasons for this are to highlight the ISTA and AOSA recommendations and to avoid repetition of the treatment details, which are given below.

Pre-chill: Seeds are placed in contact with the moist substratum and kept at a low temperature for an initial period before being moved to the germination test temperature. With the exception of tree seeds, the pre-chill temperature is between 5° and 10°C and the initial treatment period up to 7 days, although in some cases - particularly the more dormant of the grasses - this may be extended to 14, or, rarely, 28 days. Tree seeds are kept at 3°-5°C for between 7 days and a year.

Pre-dry: Before imbibition the dry seeds are heated at a temperature not exceeding 40°C with free air circulation for up to 7 days.

Potassium nitrate: The germination test paper is moistened with a 0.2% solution of potassium nitrate (see below for details of solution preparation).

GA₃: The germination test paper is moistened with 200-1000 ppm of gibberellic acid (see below for details of solution preparation).

Pre-wash: Seeds are soaked and washed in running water at between 20° to 25°C for 2 hours or so to remove substances in seed (or fruit) coats which may inhibit germination.

Test at: An alternative germination test regime is suggested if difficulties are encountered at the prescribed germination test temperatures (given in II. Germination regimes for non-dormant seeds).

The information which follows the ISTA and AOSA recommendations (where available) provides details of those treatments which have been reported to be fully effective in promoting the germination of all, or nearly all, dormant seeds within accessions. Note, however, that these treatments may on occasion be the same as those given in the preceding sections: that is a treatment found to be successful for one seed lot may not have been successful when applied by another worker to a different seed lot.

VI. Comment

This section may point out problems with the ISTA/AOSA prescriptions and recommendations, conflicts between various reports in the literature and attempt to provide a guide to devising appropriate germination test regimes. In a few cases germination test prescriptions may be given; in rather more cases the more suitable techniques will be suggested with alternatives in the event of failure. The symbol A in this section indicates unpublished work by the authors of this report.

VII. References

Within each genus the numbers in brackets refer to the references provided in the last section. These are numbered in alphabetical order with the exception of those references added in final revisions of this manuscript. It is envisaged that gene bank staff will consult only a very few of these references, if at all. Most, if not all, of the relevant information has been extracted and summarised in the sections I to VI.

Shorthand used to describe treatments

A shorthand notation has been devised to present the information in as concise a form as possible. A description of the treatment is given before the colon; the information following the colon gives precise treatment details - where available. Some examples follow.

Alternating temperatures: (4); 20°/30°C, 20°/35°C (16h/8h) (8)

Potassium nitrate: pre-applied, 24h, 0.1-1% (7); co-applied, 0.1, 0.2%, at 25°C (6)

Light: (10); dark, continuous (12); red, 15 min/d (3)

These have the following meaning:

Reference 4 reported that alternating temperatures were used but no treatment details were given. Reference 8 applied alternating temperatures of either 20°C for 16 hours per day and 30°C for 8 hours per day or 20°C for 16 hours per day and 35°C for 8 hours per day. Reference 7 treated the seeds to potassium nitrate solutions between 0.1 and 1% - with several intermediate concentrations - for one day before beginning the germination test which was then carried out on a substrate moistened with water - hence pre-applied. Reference 6 moistened the germination test substratum - hence co-applied - with either 0.1% or 0.2% potassium nitrate (but at no intermediate concentrations) and the germination test was at a constant temperature of 25°C. Reference 10 reported that a light treatment was given but no details were reported. Reference 12 carried out the germination test in the dark. Reference 3 exposed the seeds to red light, but only for 15 minutes per day.

Often incomplete treatment details are provided. References 4 and 10 above provide examples of the layout in such cases. Incompleteness is usually because the information was not provided by the paper referred to, but sometimes we have omitted information that appears to us to be mistaken or misleading.

It is possible that mistakes in interpretation or transcription may have been made. We apologise to the authors of any papers cited if this has occurred and would welcome correspondence pointing out any errors or omissions, and particularly welcome further details of successful dormancy-breaking treatments.

In passing it should be noted that certain regimes are referred to very frequently. For example, diurnal alternating temperature regimes of 20°/30°C, where the higher temperature is applied for 8 hours per day combined with co-application of 0.2% potassium nitrate are often mentioned. This is because it is a germination test regime recommended by ISTA/AOSA for a large number of species and a large number of workers have tested the response of seed germination to this regime as a consequence. However, often this regime appears superior by default - since other regimes will not necessarily have been tested. Consequently the reader is reminded that the information reported here is in that sense limited: other, more favourable, germination test regimes and dormancy-breaking treatments may exist which have not yet been the subject of investigation.

Abbreviations

The following abbreviations have been used to provide treatment details.

cm-2	per square centimeter
°C	degrees Celsius
d	day
g	gramme
GA	gibberellins, the subscript denoting the particular gibberellin; GA3 is the most commonly applied gibberellin.
h	hour
j	joule
kc	kilocycles, that is 1000 cycles
l	litre
m	month
m-2	per square metre
M	Molar, that is the molecular weight in grammes dissolved in a litre
min	minute
ml	millilitre, that is 10-3 litre
mol	6.02x1023 photons - see Chapter 6, Volume I
N	normal, that is the number of gramme-equivalents of the substance dissolved in a litre of solution where one gramme equivalent equals the gramme-molecular weight of the substance divided by its hydrogen equivalence.
nm	nanometre, that is 10-9 meteres - see Chapter 6, Volume I
pH	concentration of hydrogen ions given as the negative logarithm of hydrogen ion activity
ppm	parts per million, equivalent to 0.0001% (see below)
R	Roentgen, a unit of ionizing radiation
s	second
s-1	per second
W	watt - see Chapter 6, Volume I
/	between two temperatures or time period indicates an alternating regime (usually alternating temperature)
/	followed by another symbol indicates per, e.g./d = per day
%	percentage concentration, usually in terms of weight per volume (w/v), that is g/100 ml of solution

How to utilise the information provided for each genus

It is not intended that all seven sections of the information on seed germination provided for each genus should necessarily be read in sequence. The following approach is suggested.

After reading the introduction to the family - and Table 17.1 or Table 17.2 if either is referred to - read sections I (Evidence of dormancy) and VI (Comment).

In most cases these sections will provide sufficient information to decide upon a suitable germination test procedure and whether to apply one or more dormancy-breaking treatments - and, if so, the details of these treatments. Reference to sections II (Germination regimes for non-dormant seeds) and V (Successful dormancy-breaking treatments) may help to clarify the details of these treatments and procedures.

The information provided in sections III (Unsuccessful dormancy-breaking treatments) and IV (Partly-successful dormancy-breaking treatments) should help the reader to understand why certain dormancy-breaking treatments and germination test procedures are to be preferred and why others are best avoided. This information, however, will probably be of more use to those attempting to devise and develop improved germination test procedures and dormancy-breaking treatments if the advice presently available (Comment) is found to be inadequate or inappropriate for an accession.

For gene banks handling comparatively few genera Section VII (References) could form the basis of a reference library of articles on seed germination and dormancy. However this is not essential in view of the information summarised in this manual. More useful will be the information generated from the results of germination tests on material maintained within the gene bank.

Commencing an alternating temperature germination test

If seeds are to be tested in an alternating temperature regime it must be decided which of the two temperatures the seeds are exposed to first. There are three main possibilities:

(1) Initially expose the seeds to the first stated temperature of the alternating temperature regime. For example, in the case of the regime 20°/35°C (16h/8h) the seeds would be exposed to 20°C for 16 hours before their first exposure to 35°C.

(2) Expose the seeds to the lower of the two temperatures first. For example, in the above alternating temperature regime 20°C is the lower temperature and the seeds are thus exposed to 20°C for 16 hours before their first exposure to 35°C.

(3) Expose the seeds to the longer duration of the two phases first. In the above example 20°C is applied for the longer duration and thus the seeds are exposed to 20°C for 16 hours before their first exposure to 35°C.

Application of light during part of alternating temperature cycles

When light is applied during part of an alternating temperature cycle it is the usual practice for the light treatment to coincide with the higher temperature phase (as would occur in a natural environment). If the light is applied once a day for a lengthy period, it is also convenient for the duration of any light treatment to be the same as that for the higher temperature phase of the alternating temperature cycle. This is because the heat generated by the lights (even fluorescent tubes) will affect the maintenance of temperature. Thus if the periods of exposure to the higher temperature and to light coincide throughout then the higher temperature of the germination environment can be set taking into account the heat generated by the lights. It is important that the protocol adopted for the provision of light during alternating temperature regimes be explicitly stated and adhered to. For more information on light and seed germination see Chapter 6, Volume I.

MAKING UP SOLUTIONS

Two of the more common dormancy-breaking treatments pre-applied or co-applied to seeds are potassium nitrate and gibberellic acid. The preparation of solutions of these and other compounds will be required in most, if not all, gene banks. Consequently some notes on the preparation of solutions are provided below.

To make up a 0.2% solution of potassium nitrate, 2 g of potassium nitrate is dissolved in one litre of distilled or deionised water. (It is not essential to make up a whole litre of solution. For example, 1 g dissolved in 500 ml would also provide a 0.2% solution.) To make up a 500 ppm solution of GA₃ dissolve 0.5 g GA₃ in one litre of distilled or deionised water. GA₃ generally takes a long time to dissolve in water and considerable stirring; with a glass rod, may be required before all the GA₃ has dissolved. Strong concentrations of GA₃, above 800 ppm, will reduce pH. To avoid this it is generally advised to use a buffer solution of 0.01 M di-sodium hydrogen orthophosphate dihydrate/di-sodium hydrogen orthophosphate monohydrate for GA₃ concentrations of 800 ppm and above. This solution is prepared by dissolving 1.7799 g of di-sodium hydrogen orthophosphate di-hydrate (Na₂HPO₄2H₂O) and 1.3799 of di-sodium hydrogen orthophosphate monohydrate (Na₂HPO₄H₂O) in distilled or deionised water and making up to one litre. The GA₃ is then dissolved in this buffer solution.

Solution concentrations

Throughout the report the concentrations of solutions of potential dormancy-breaking agents are expressed in the style given by the reference. To enable the reader to convert between these different forms of presentation, Figure 16.1 has been provided. In particular the grammes per litre scale (g/1, or g 1^-1) provides sufficient information to enable the reader to make up the required concentrations of solutions. The only additional information required to use Figure 16.1 is the molecular weight of the potential dormancy-breaking agent. These are usually provided on the labels of chemical containers. Some values are provided here in Table 16.1, but more comprehensive information is available from chemical company catalogues and Merck's Index. The latter is particularly recommended. The use of Figure 16.1 is described in the caption.

ADDITIONS AND AMENDMENTS

The chapters which follow are very much a first attempt at pooling practical information concerning methods of overcoming seed dormancy and promoting germination. It is intended that this report should be referred to on a day to day basis. Readers might like to use the space after the information on each genus to append their own notes of any additional information they consider useful. Perhaps the most striking point illustrated by the format used here is how little is known of satisfactory treatments to break dormancy in seed of so many species. Note that whilst the literature on seed dormancy is considerable, that concerned with regimes capable of promoting full germination for many accessions is extremely limited. The authors hope that this realisation will spur readers on to add to this knowledge. We welcome reports of successful dormancy-breaking treatments which can be included in subsequent revisions or amendments to this handbook.

TABLE 16.1. The molecular weights of selected compounds which have been applied to seeds as putative dormancy-breaking agents.

	Molecular Weight
Abscisic acid	264
Acetaldehyde	44
Acetamide	59
Acetic acid	60
Alanine	89
Ammonium bisulphide	51
Ammonium bisulphite	99
Ammonium chloride	53
Ammonium nitrate	80
Ammonium phosphate, dibasic	132
Ammonium phosphate, monobasic	115
Ammonium sulphate	132
Ammonium sulphide	68
Ammonium sulphite	116
Ascorbic acid	176
Boric acid	62
Calcium hypochlorite	143
Calcium nitrate	164
Diethyl dithiocarbanate, sodium	171
Dimercaprol (Dithioglycerol)	124
Dinitrophenol	184
Dithiothreitol	154
Ethrel (Ethephon or CEPA)	144
Gibberellic acid	346
Hydrogen peroxide	34
Hydrogen sulphide	34
Hydroxylamine hydrochloride	70
Indoleacetic acid	186
Ketoglutaric acid	146
Kinetin	215
Mercaptoethanol	78
Methylene blue	374
Napthaleneacetic acid	186
Nitric acid	63
Potassium cyanide	65
Potassium nitrate	101
Potassium nitrite	85
Potassium permanganate	158
Potassium sulphate	174
Potassium thiocyanate	97
Potassium thiosulphate	190
Sodium azide	65
Sodium fluoride	42
Sodium hydroxide	40
Sodium hypochlorite	74
Sodium nitrate	85
Sodium nitrite	69
Sodium sulphide	78
Sodium thiocarbonate	154
Sodium thiocyanate	81
Sodium thiosulphate	158
Sucrose	342
Sulphuric acid	98
Thiourea	76
Uranyl nitrate	394
Urea	60

How to use Figure 16.1

The first three scales of the nomograph differ by factors divisible by ten. To convert between these values place a ruler on the diagram perpendicular to these axes and read off the values. To determine molarity connect up the concentration in grammes per litre with the molecular weight of the compound (see Table 16.1) with the straight edge of a ruler. The point where the ruler crosses the molarity scale gives the value of the molarity of the solution. Alternatively if it were required to make up a solution of a given molarity, connect up this value with the molecular weight of the compound with the straight edge of a ruler and note the point on the grammes per litre scale where the straight edge crosses.

Two examples of the use of the nomograph are shown by broken lines. The uppermost broken line is for potassium nitrate - molecular weight 101. A 0.2% solution is the same as a 2000 ppm solution and is made by dissolving 2 grammes of potassium nitrate in one litre. The resultant solution can also be described as 0.02 M or as 2x10^-2 M. The lower broken dotted line is for GA₃ - molecular weight 346. Thus, for example, a 500 ppm GA₃ solution is 0.0015 M (1.5x10_-3 M) and can be obtained by dissolving 0.5 grammes of GA₃ in one litre of solution.