Handbook Of Seed Technology For Genebanks Volume II

CHAPTER 16. INTRODUCTION TO VOLUME II: THE ORGANIZATION OF CHAPTERS AND AN EXPLANATION OF ABBREVIATIONSshould be noted that sometimes identical synonyms may be given for species within anothergenus. Where this occurs and information is provided for the second genus the reader shouldconsider the information provided for both genera. The information on dormancy-breakingtechniques and germination test regimes for each genus is divided into seven sections. Notethat although the term dormancy is used in the titles below information on factors other thandormancy per se (see definition in Chapter 5, Volume I), particularly hardseededness (Chapter4, Volume I), is also included.I. Evidence of dormancyThis section simply provides evidence of whether dormancy can be a problem and attempts toplace it in context - often by giving details of how long after harvest 'post-harvest' dormancytypically remains a problem under ambient conditions. Differences in the degree of dormancybetween species within the genus are sometimes noted. Other problems may be noted in thissection. For example, it is sometimes necessary to draw attention to the classification of seedstorage behaviour (see Chapter 1, Volume I) where this has been in some doubt.II. Germination regimes for non-dormant seedsIn the majority of cases this section provides details of the prescribed germination testconditions for species within the genera given by the ISTA and the AOSA - where these areavailable. 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 germinationtest. 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 towelsin 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 suitablefor your laboratory.The information after the first colon gives the prescribed temperature regime for thegermination test. An alternating temperature regime is denoted by A /B C (xh/yh), where A Cis 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 whichthe seeds are subjected to 20 C for 16 hours followed by 30 C for 8 hours each day. Oftenalternative temperature regimes are provided. The alternative regimes are separated by semicolons.The final information provided (after the second colon) is the total germination test period indays, but of course seedlings may have to be removed and counted at more frequentintervals. Moreover, it is likely that in many cases gene banks will have to continuegermination 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 twosets. In the main these are quite similar, but where they occur the differences of detail shouldbe noted.In addition to the ISTA and AOSA rules this section also includes other published information,file:///D /52/ch01.htm[11/27/2012 11:26:48 AM]

CHAPTER 16. INTRODUCTION TO VOLUME II: THE ORGANIZATION OF CHAPTERS AND AN EXPLANATION OF ABBREVIATIONSwhere available, on germination test procedures which are satisfactory for non-dormantseeds.III. Unsuccessful dormancy-breaking treatmentsThe third section gives details of treatments that have been applied to seeds in attempts tobreak dormancy, but which have either failed to increase germination more than marginally ormay have even led to a reduction in germination - either by inducing dormancy in the seeds orby damaging the seeds in some way. Although at first sight the reader may consider thisinformation to be of no interest - after all the requirement is to promote germination - it isimportant to be aware of those treatments which should be avoided. Moreover, the reader willbegin to notice that similar treatments may appear in more than one section. These apparentinconsistencies and contradictions, often between different reports, are nevertheless probablyindicative of the real situation: a treatment which greatly promotes the germination of seeds ofone accession, may fail to promote the germination of seeds of another accession, whilst in athird accession germination may be reduced by the treatment. Hence the inclusion of thisinformation here.IV. Partly-successful dormancy-breaking treatmentsTreatments 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 singleaccession, or within a report they may have promoted full germination in some accessions butnot promoted full germination in other accessions.V. Successful dormancy-breaking treatmentsThe ISTA and/or the AOSA recommendations for breaking dormancy are provided first of all inthis section (where available for a species). The style of layout is different from, and lessdetailed than, that given for other sources of information. The reasons for this are to highlightthe ISTA and AOSA recommendations and to avoid repetition of the treatment details, whichare given below.Pre-chill: Seeds are placed in contact with the moist substratum and kept at a low temperaturefor an initial period before being moved to the germination test temperature. With theexception of tree seeds, the pre-chill temperature is between 5 and 10 C and the initialtreatment period up to 7 days, although in some cases - particularly the more dormant of thegrasses - this may be extended to 14, or, rarely, 28 days. Tree seeds are kept at 3 -5 C forbetween 7 days and a year.Pre-dry: Before imbibition the dry seeds are heated at a temperature not exceeding 40 C withfree air circulation for up to 7 days.Potassium nitrate: The germination test paper is moistened with a 0.2% solution of potassiumnitrate (see below for details of solution preparation).GA3: The germination test paper is moistened with 200-1000 ppm of gibberellic acid (seebelow for details of solution preparation).Pre-wash: Seeds are soaked and washed in running water at between 20 to 25 C for 2 hoursor 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 atthe prescribed germination test temperatures (given in II. Germination regimes for nondormant seeds).file:///D /52/ch01.htm[11/27/2012 11:26:48 AM]

CHAPTER 16. INTRODUCTION TO VOLUME II: THE ORGANIZATION OF CHAPTERS AND AN EXPLANATION OF ABBREVIATIONSThe information which follows the ISTA and AOSA recommendations (where available)provides details of those treatments which have been reported to be fully effective in promotingthe germination of all, or nearly all, dormant seeds within accessions. Note, however, thatthese treatments may on occasion be the same as those given in the preceding sections: thatis a treatment found to be successful for one seed lot may not have been successful whenapplied by another worker to a different seed lot.VI. CommentThis 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 devisingappropriate germination test regimes. In a few cases germination test prescriptions may begiven; in rather more cases the more suitable techniques will be suggested with alternatives inthe event of failure. The symbol A in this section indicates unpublished work by the authors ofthis report.VII. ReferencesWithin 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 infinal revisions of this manuscript. It is envisaged that gene bank staff will consult only a veryfew of these references, if at all. Most, if not all, of the relevant information has been extractedand summarised in the sections I to VI.Shorthand used to describe treatmentsA shorthand notation has been devised to present the information in as concise a form aspossible. A description of the treatment is given before the colon; the information following thecolon 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 weregiven. Reference 8 applied alternating temperatures of either 20 C for 16 hours per day and30 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% - withseveral intermediate concentrations - for one day before beginning the germination test whichwas then carried out on a substrate moistened with water - hence pre-applied. Reference 6moistened 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 aconstant temperature of 25 C. Reference 10 reported that a light treatment was given but nodetails were reported. Reference 12 carried out the germination test in the dark. Reference 3exposed the seeds to red light, but only for 15 minutes per day.Often incomplete treatment details are provided. References 4 and 10 above provideexamples of the layout in such cases. Incompleteness is usually because the information wasnot provided by the paper referred to, but sometimes we have omitted information thatappears to us to be mistaken or misleading.It is possible that mistakes in interpretation or transcription may have been made. Weapologise to the authors of any papers cited if this has occurred and would welcomefile:///D /52/ch01.htm[11/27/2012 11:26:48 AM]

CHAPTER 16. INTRODUCTION TO VOLUME II: THE ORGANIZATION OF CHAPTERS AND AN EXPLANATION OF ABBREVIATIONScorrespondence pointing out any errors or omissions, and particularly welcome further detailsof 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 appliedfor 8 hours per day combined with co-application of 0.2% potassium nitrate are oftenmentioned. This is because it is a germination test regime recommended by ISTA/AOSA for alarge number of species and a large number of workers have tested the response of seedgermination to this regime as a consequence. However, often this regime appears superior bydefault - since other regimes will not necessarily have been tested. Consequently the reader isreminded 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 yetbeen the subject of investigation.AbbreviationsThe following abbreviations have been used to provide treatment details.cm - per square centimeter2 Cdegrees CelsiusddayggrammeGA gibberellins, the subscript denoting the particular gibberellin; GA3 is the most commonly appliedgibberellin.hhourjjoulekckilocycles, that is 1000 cyclesllitremmonthm-2 per square metreMMolar, that is the molecular weight in grammes dissolved in a litremin minutemlmillilitre, that is 10 -3 litremol 6.02x10 23 photons - see Chapter 6, Volume INnormal, that is the number of gramme-equivalents of the substance dissolved in a litre of solutionwhere one gramme equivalent equals the gramme-molecular weight of the substance divided by itshydrogen equivalence.nm nanometre, that is 10 -9 meteres - see Chapter 6, Volume IpHconcentration of hydrogen ions given as the negative logarithm of hydrogen ion activityppm parts per million, equivalent to 0.0001% (see below)RRoentgen, a unit of ionizing radiationsseconds -1 per secondWwatt - see Chapter 6, Volume I/between two temperatures or time period indicates an alternating regime (usually alternatingtemperature)/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 solutionHow to utilise the information provided for each genusfile:///D /52/ch01.htm[11/27/2012 11:26:48 AM]

CHAPTER 16. INTRODUCTION TO VOLUME II: THE ORGANIZATION OF CHAPTERS AND AN EXPLANATION OF ABBREVIATIONSIt is not intended that all seven sections of the information on seed germination provided foreach 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 suitablegermination 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 regimesfor 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-breakingtreatments) and IV (Partly-successful dormancy-breaking treatments) shouldhelp the reader to understand why certain dormancy-breaking treatments and germination testprocedures 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 germinationtest 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 formthe basis of a reference library of articles on seed germination and dormancy. However this isnot essential in view of the information summarised in this manual. More useful will be theinformation generated from the results of germination tests on material maintained within thegene bank.Commencing an alternating temperature germination testIf seeds are to be tested in an alternating temperature regime it must be decided which of thetwo temperatures the seeds are exposed to first. There are three main possibilities:(1) Initially expose the seeds to the first stated temperature of the alternatingtemperature regime. For example, in the case of the regime 20 /35 C (16h/8h) theseeds 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, inthe above alternating temperature regime 20 C is the lower temperature and theseeds 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 aboveexample 20 C is applied for the longer duration and thus the seeds are exposed to20 C for 16 hours before their first exposure to 35 C.Where a regime is described as, for example, 30 /10 C (16h/8h) it will be seen that it is notpossible to satisfy all three possible rules. We suggest that it is logical for the first statedtemperature to be the initial temperature to which the seeds are exposed and that, therefore,the first rule should take priority over all others. It is important that the protocol to describealternating temperature regimes be explicitly stated and consistently applied in gene banks.Application of light during part of alternating temperature cyclesWhen light is applied during part of an alternating temperature cycle it is the usual practice forthe light treatment to coincide with the higher temperature phase (as would occur in a naturalenvironment). If the light is applied once a day for a lengthy period, it is also convenient for theduration of any light treatment to be the same as that for the higher temperature phase of thealternating temperature cycle. This is because the heat generated by the lights (evenfile:///D /52/ch01.htm[11/27/2012 11:26:48 AM]

CHAPTER 16. INTRODUCTION TO VOLUME II: THE ORGANIZATION OF CHAPTERS AND AN EXPLANATION OF ABBREVIATIONSfluorescent tubes) will affect the maintenance of temperature. Thus if the periods of exposureto the higher temperature and to light coincide throughout then the higher temperature of thegermination environment can be set taking into account the heat generated by the lights. It isimportant that the protocol adopted for the provision of light during alternating temperatureregimes be explicitly stated and adhered to. For more information on light and seedgermination see Chapter 6, Volume I.MAKING UP SOLUTIONSTwo of the more common dormancy-breaking treatments pre-applied or co-applied to seedsare potassium nitrate and gibberellic acid. The preparation of solutions of these and othercompounds will be required in most, if not all, gene banks. Consequently some notes on thepreparation of solutions are provided below.To make up a 0.2% solution of potassium nitrate, 2 g of potassium nitrate is dissolved in onelitre of distilled or deionised water. (It is not essential to make up a whole litre of solution. Forexample, 1 g dissolved in 500 ml would also provide a 0.2% solution.) To make up a 500 ppmsolution of GA3 dissolve 0.5 g GA3 in one litre of distilled or deionised water. GA3 generallytakes a long time to dissolve in water and considerable stirring; with a glass rod, may berequired before all the GA3 has dissolved. Strong concentrations of GA3, above 800 ppm, willreduce pH. To avoid this it is generally advised to use a buffer solution of 0.01 M di-sodiumhydrogen orthophosphate dihydrate/di-sodium hydrogen orthophosphate monohydrate for GA3concentrations of 800 ppm and above. This solution is prepared by dissolving 1.7799 g of disodium hydrogen orthophosphate di-hydrate (Na 2HPO 42H 2O) and 1.3799 of di-sodiumhydrogen orthophosphate monohydrate (Na 2HPO 4H2O) in distilled or deionised water andmaking up to one litre. The GA3 is then dissolved in this buffer solution.Solution concentrationsThroughout the report the concentrations of solutions of potential dormancy-breaking agentsare expressed in the style given by the reference. To enable the reader to convert betweenthese different forms of presentation, Figure 16.1 has been provided. In particular thegrammes per litre scale (g/1, or g 1 -1 ) provides sufficient information to enable the reader tomake up the required concentrations of solutions. The only additional information required touse Figure 16.1 is the molecular weight of the potential dormancy-breaking agent. These areusually provided on the labels of chemical containers. Some values are provided here in Table16.1, but more comprehensive information is available from chemical company catalogues andMerck's Index. The latter is particularly recommended. The use of Figure 16.1 is described inthe caption.ADDITIONS AND AMENDMENTSThe chapters which follow are very much a first attempt at pooling practical informationconcerning methods of overcoming seed dormancy and promoting germination. It is intendedthat this report should be referred to on a day to day basis. Readers might like to use thespace after the information on each genus to append their own notes of any additionalinformation they consider useful. Perhaps the most striking point illustrated by the format usedhere is how little is known of satisfactory treatments to break dormancy in seed of so manyspecies. Note that whilst the literature on seed dormancy is considerable, that concerned withregimes capable of promoting full germination for many accessions is extremely limited. Theauthors hope that this realisation will spur readers on to add to this knowledge. We welcomereports of successful dormancy-breaking treatments which can be included in subsequentrevisions or amendments to this handbook.file:///D /52/ch01.htm[11/27/2012 11:26:48 AM]

CHAPTER 16. INTRODUCTION TO VOLUME II: THE ORGANIZATION OF CHAPTERS AND AN EXPLANATION OF ABBREVIATIONSTABLE 16.1. The molecular weights of selected compounds which have been applied toseeds as putative dormancy-breaking agents.Molecular WeightAbscisic acid264Acetaldehyde44Acetamide59Acetic acid60Alanine89Ammonium bisulphide51Ammonium bisulphite99Ammonium chloride53Ammonium nitrate80Ammonium phosphate, dibasic132Ammonium phosphate, monobasic115Ammonium sulphate132Ammonium sulphide68Ammonium sulphite116As

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