Note: Descriptions are shown in the official language in which they were submitted.
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ASSAY FOR SEED VIGOUR
FIELD OF THE INVENTION
The present invention relates generally to the field of agriculture.
More specifically, the present invention relates to a method for determining
seed vigour.
BACKGROUND OF THE INVENTION
Seed vigour is an important factor in the economical production of
field and vegetable crops, and in the quality of malting barley. Field crops,
such as canola, may have to be replanted and/or may have lower yield
because of low vigour. The seed of vegetable crops is of high value and low-
vigour in the seed represents a significant extra cost for seed in addition to
production losses. Low-vigour malting barley may loose germination
percentage during shipment and storage and no longer be acceptable for
malting. Thus, farmers, vegetable growers, seed processors, seed merchants,
marketing agents and maltsters need reliable, rapid and economical methods
for determining seed vigour.
Gorecki et al. (Gorecki et al., 1985, Can J Botany 63: 1035-1039)
examined volatile exudates from germinating pea (Pisum savitum) and showed
that as storage time increased, viability decreased. Furthermore, while they
observed no qualitative differences in the volatile exudates produced by
germinating seeds that had been stored for different periods of time, the
quantities of ethanol and acetaldehyde produced by germinating seeds was
somewhat proportional to the age of the seed, increasing as seed age
increased. However, dry seed produced only small quantities of both volatiles.
They also noted that determinations of acetaldehyde and ethanol in the space
over germinating seeds by means of gas chromatography might be a useful
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seed vigour test. Gorecki et al. (Gorecki et al., 1992, Acta Physiologiae
Plantarum 14: 19-27) also analyzed volatile organics produced b~y starchy pea
seeds and fatty cocklebur (Xanthium pennsylvanicum) seeds following water
imbibition, wherein aged seeds showed increasing amounts of ethanol and
acetaldehyde produced, although in cocklebur seeds, acetaldehyde was the
preponderant end product. As will be apparent to one knowledgeable in the art,
imbibition is the movement of water into the seed, which in turn causes seed
swelling and is essential for germination of the seed.
Muskmelon (cantaloupe, Cucumis melo) seeds were aged
artificially for up to 12 d at 45 °C and 100 % relative humidity.
Artificial ageing
reduced their ability to germinate and increased the production of ethanol and
acetaldehyde during imbibition. The authors suggested that ethanol production
in the first hours of imbibition might be used as a method to predict
germination
in muskmelon seed (Pesis and Ng, 1984, J. Expt. Bot. 35: 356-365.).
Zhang et al. (Zhang et al., 1994, Seed Science Research 4: 49-
56) hypothesized that seed-evolved volatiles cause the loss of seed
germinability during storage. It is of note that the seeds were stored at 70%
relative humidity and that the moisture content of the seeds was approximately
6-14%. Various aldehydes were supplied to lettuce, soybean, sunflower carrot
and rice seeds during storage, some of which showed toxicity to seed
germinability. Based on these facts, they suggested that endogenous volatiles,
especially aldehyde, might be an important factor that accelerates seed
deterioration, which often occurs under lower relative humidity and/or
temperatures throughout long-term storage. This in turn suggests that removal
of acetaldehyde and ethanol from seed storage containers would improve seed
vigour, or at least prevent further deterioration, and that it is the amount
of
ethanol or acetaldehyde that a seed is exposed to that determines vigour.
Naturally or artificially aged soybean (Glycine max) seed had
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higher ethanol and acetaldehyde concentrations in seed tissue than did un-
aged seed; however, the difference between aged and un-aged seed varied
greatly with hours of imbibition, and rate of water uptake and temperature
during imbibition (Woodstock and Taylorson, 1981, Plant Physiol. 67: 424-428).
The quantity of certain volatile aldehydes (butanal, hexanal and
pentanal) when released by heat from dry soybeans and expressed as a
percentage of the total volatiles collected were correlated to seed
germination
and seed vigour (Hailsones and Smith, 1989, Seed Sci & Technol. 17: 649-
658).
Unspecified, volatile aldehydes released from germinating
soybean seeds were correlated with field emergence of various seed lots in the
field. The authors suggested that the determination of aldehydes during
germination might be a useful vigour test (Wilson and McDonald, 1986, Seed
Sci. & Technol. 74: 259-268).
Decline in germination and field emergence of bean (Phaseolus
vulgaris) seed previously subjected to accelerated aging at 42 °C and
100
relative humidity was correlated with a decline in ethylene production by the
seed during imbibition (Samimy and Taylor, 1983, J. Amer. Soc. Hort. Sci. 108:
767-769.).
As discussed above, seed vigour is an important factor in the
economical production of crops. The ability to rapidly and reliably determine
seed vigour would significantly reduce costs associated with yield loss,
replanting and return of seeds. There are numerous procedures for determining
seed vigour in the art, however, more accurate and rapid procedures are
desirable.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, there is provided a
method of measuring seed vigour comprising:
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placing a seed under conditions wherein seed metabolism
initiates but the seed is not germinating; and measuring the quantity of at
least
one gas produced by the seed.
According to a second aspect of the invention, there is provided a
device for assaying seed vigour comprising:
a quantity of a detector compound that changes colour when
exposed to ethanol;
an air-tight container including a sealable opening for placing
seeds to be assayed within the interior of the container; and
a regulator for controlling the rate of diffusion of the ethanol from
the interior of the container to the detector compound.
According to a third aspect of the invention, there is provided a kit
for assaying seed vigour comprising:
a quantity of a detector compound that changes colour when
exposed to ethanol;
an air-tight container including a sealable opening for placing
seeds to be assayed within the interior of the container; and
a regulator for controlling the rate of diffusion of the ethanol from
the interior of the container to the detector compound.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 shows chromatograms f.°om headspace gas analysis
of high-vigour (top) and low-vigour (bottom) canola seed. Both chromatograms
are drawn to the same scale. Note that the ethanol peak on the lower
chromatogram has been truncated.
FIGURE 2 shows the relationship between amounts of ethanol
(expressed as gas chromatography peak areas) in head space gas and
bioassay results for 26 canola seed samples (sample set 1 ). Thirteen pairs,
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each containing a high- and a low-vigour sample of one of a number of
varieties were examined. The low-vigour reference biomass was expressed as
a percentage of the high-vigour reference biomass in each pair in order to
correct for genetic variation in growth rate from variety to variety. Genetic
variation would otherwise have exacerbated measured differences in seedling
vigour in the reference bioassay. The shaded region incorporates peak areas
less than 500,000 counts and indicates an ethanol range that may be
associated with high vigour.
FIGURE 3 shows the relationship between amounts of
acetaldehyde (expressed as gas chromatography peak areas) in head space
gas and bioassay results for 26 canola seed samples (sample set 1 ). The
samples were paired as described in Figure 2.
FIGURE 4 shows the relationship between amounts of unknown
E (expressed as gas chromatography peak areas) in head space gas and
bioassay results for 26 canola seed samples (sample set 1 ). The samples
were paired as described in Figure 2.
FIGURE 5 shows the relationship between amounts of ethanol
(expressed as gas chromatography peak areas) in head space gas and
bioassay results for 93 canola seed samples, which include 32 varieties or
hybrids and 19 seed treatment formulations of fungicide and insecticide
(sample set 2). The shaded region incorporates peak areas less than 500,000
counts and indicates an ethanol range that may be associated with high vigour.
FIGURE 6 shows the effects of seed moisture percentage on
amounts of ethanol (expressed as gas chromatography peak areas) in head
space gas of high- and low-vigour canola seed lots. The head space gas was
analysed 24 ~ 2.5 h after water was added to the seed.
FIGURE 7 shows a device for colourimetric determination
of seed vigour. Seed of high and low vigour was added to 250-ml flasks, seeds
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were brought up to 20% moisture and the flasks were sealed and fitted with
ethanol-indicating diffusion tubes. The figure shows the colour development
after 24 hours at room temperature.
FIGURE 8 is a close-up of Figure 7.
FIGURE 9 is a side view of containers of canola seed. These
containers have an integrated colour disc in the lid for colourimetric
determination of vigour.
FIGURE 10 shows a device for colourimetric determination of
canola seed vigour by means of an integrated colour disc in the container lid.
Figure 10 is a top view of the containers shown in Figure 9. High-vigour
(left)
and low vigour (right) canola seed was made up to 20 % moisture and sealed
in the containers for 24 h.
FIGURE 11 shows a device for colourimetric determination of
canola seed vigour by means of an integrated colour disc in the container lid.
High-vigour (left) and low vigour (right) canola seed was made up to 20
moisture and sealed in the containers for 24 h.
FIGURE 12 shows the determination of gaseous ethanol
standards in the concentration range suitable for estimation of seed vigour by
analysis of head space gas. A commercial hand-held gas monitoring
instrument was used for the determinations.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which the invention belongs. Although any methods and
materials similar or equivalent to those described herein can be used in the
practice or testing of the present invention, the preferred methods and
materials are now described. All publications mentioned hereunder are
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incorporated herein by reference.
DEFINITIONS
As used herein, "seed vigour" refers, depending on the context, to
the ability of seed to germinate rapidly and achieve a high percentage of
germination; to produce seedlings that emerge rapidly from the soil and have a
high percentage of emergence; to produce seedlings that grow rapidly and
demonstrate superior tolerance to various stresses including but not limited
to
cold, weeds and insects; and to the ability of seed to withstand storage or
shipment with a minimum loss of the ability of seed and its seedlings to
germinate, emerge from the soil, grow and tolerate stresses. Not all aspects
of
poor vigour may be found in the same seed. For example, low-vigour seed
may have a high germination percentage but produce seedlings that grow
slowly compared to high-vigour seed of the same genotype grown under the
same conditions.
As used herein, "moisture" refers to the amount of water present
in a material expressed as a percentage of the undried weight of the material.
As used herein, "head space" refers to the space over a
substance in a sealed container. As used herein, head space gas refers to the
gases in the head space.
As used herein, "hypocotyl" refers to the section of stem of a
seedling between the cotyledons and the root-shoot junction.
As used herein, "small", "medium" and "large" seed refers to
seeds wherein there are more than 100 seeds per gram (small, s), more than
and up to 100 seeds per gram (medium, m) and 10 or fewer seeds per gram
(large, I).
Described herein is a method of measuring vigour of various
types of seed by measuring volatile gases, for example, ethanol, emitted from
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the seed when exposed to various conditions. These conditions may include,
for example, exposing the seed to sufficient moisture to initiate metabolism
but
less moisture than required for germination, purging the air in a sealed
container with nitrogen or other gases, and including various inhibitors or
stimulators of metabolism in the water added to the seed. For example, ethanol
produced by canola (Brassica napus and Brassiea rapa) or barley (Hordeum
vulgare) seed in an enclosed container stored for 24 hours at room temperature
after the seed has been made up to 20% moisture is measured by gas
chromatography, other gas detection instrumentation or by colour change in an
indicator substance. For illustrative purposes, an example of other gas
detection instrumentation is the Pac III Single Gas Monitor manufactured by
Dr=ger, Sicherheitstechnik GmbH, Luebeck, Germany. As discussed below,
the quantity of ethanol determined by any method of analysis indicates the
vigour of the seed.
Plants and other organisms obtain energy from stored carbohydrate by
either aerobic or anaerobic metabolism. Aerobic metabolism converts
carbohydrate to carbon dioxide and water, whereas anaerobic metabolism
converts carbohydrate to ethanol, other organic compounds and carbon dioxide
by fermentation. The former is more energeticaiiy efficient than the latter.
As
described below, when seed ages or looses vigour for other reasons, there
appears to be a shift to a higher proportion of anaerobic metabolism with
elevated ethanol production compared to more healthy seed of the same
species.
As will be appreciated by one knowledgeable in the art, 20%
moisture is sufficient to initiate metabolism within the seed but is not
sufficient
moisture to induce germination. It is of note that other suitable moisture
levels
and incubation times which induce similar conditions as well as other methods
which induce these conditions are within the scope of the invention and may be
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used within the invention. For example, seed moisture may be 10-50%, 15-40%
or 15-35%. The incubation time may vary from 2 hours to 48 hours or from 18
to 30 hours.
As will be appreciated by one knowledgeable in the art, other
suitable means known in the art may also be used to determine the amount of
ethanol or other gases emitted from -the seeds. See for example, US Patent
3,455,654, US Patent 3,223,488, US Patent 3,208,827, or US Patent
2,939,768.
In one embodiment of the invention, there is provided an on-farm
assay device as well as a kit containing the components thereof, as discussed
below. The assay device comprises: a quantity of a detector compound that
changes colour when exposed to ethanol; an air-tight container including a
sealable opening for placing seeds to be assayed within the interior of the
container; and a regulator for controlling the rate of diffusion of the
ethanol from
the interior of the container to the detector compound. It is also of note
that the
device is arranged such that the detector compound may be viewed while it is
exposed to the head space gas from the seed.
Examples of suitable detector compounds are described herein
and are also well known in the art. As will be apparent to one of skill in the
art,
the exact concentration of detector compound used may vary according to the
specific seed and the assay conditions.
In some embodiments, the regulator is a perforated piece of
Teflon with the number and size of holes carefully controlled. As will be
apparent to one of skill in the art, the rate of diffusion of ethanol is
controlled by
the regulator and depends in some embodiments on the size and number of
the holes. The rate of diffusion will determine the rate of colour change of
the
detector compound. This, along with the quantity of seed and size of container
will permit colour change at the desired seed vigour threshold (probably
around
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80%) over a specified time period (approximately 24 h). As will be appreciated
by one of skill in the art, other suitable times and percentages may also be
used.
In some embodiments, an inert support, for example, a glass fiber
disc, is impregnated with the detector compound. As will be apparent to one of
skill in the art, other suitable supports include, but are by no means limited
to
unreactive porous materials (e.g. a porous ceramic disc) or powders (e.g.,
diatomaceous earth).
According to another embodiment of the invention, there is
provided a device for measuring seedling vigour comprising a substantially
airtight container and an ethanol detector as described above. In use, the
moisture content of the seed to be tested is measured and is elevated to 10-
50%, 15-45% or 15-35% as discussed below, if necessary, and the seed is
placed in the air tight container. The ethanol detector is then operably
connected to the airtight container such that ethanol evolved from the seed is
measured by the ethanol detector. Evolved ethanol may be measured from 2
hours up to 48 hours or longer, as discussed above. An example of such a
device is shown in Figures 7 and 8, wherein the substantially airtight
container
is a sealed 250-ml flask and the ethanol detector is an ethanol-indicating
diffusion tube. As will be appreciated by one knowledgeable in the art, other
suitable containers and detectors may also be used. For example, a
colourimetric ethanol detector may be constructed as an integral part of the
container. Also for example, the headspace gas may be analysed directly or
indirectly by gas chromatography or other instrumental procedures. It is of
note that there are 300-500 canola seeds in a gram. One-half to 4 g of seed in
2-ml to 12-ml vials are used in the routine gas chromatography procedure.
Versions of the colourimetric on-farm assay use 5 to 250 g. Similar quantities
may be used for other seeds.
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The invention provides kits for carrying out the methods of the
invention. Accordingly, a variety of kits are provided. The kits of the
invention
comprise one or more containers comprising an air-tight container, a quantity
of
detector compound, at least one regulator and a set of instructions, generally
written instructions although electronic storage media (e.g., magnetic
diskette
or optical disk) containing instructions are also acceptable, relating to the
use of
the kit for the intended purpose. As discussed above, an inert carrier may be
impregnated with detector compound and may be arranged to be mounted onto
the container or connected to the container for communication therebetween.
The kit may include a plurality of carriers impregnated with detector
compound.
In some embodiments, the plurality of carriers may be impregnated with
different detector compounds or mixtures of different compounds and/or may
be impregnated with different levels of the compounds. In addition, a
plurality of
regulators may be provided. In some embodiments, the respective regulators
may be arranged to restrict gas flow to differing extents. In yet other
embodiments, the kit may include a high osmotic strength water solution for
adding moisture to seeds.
It is further of note that in some embodiments, the kit components are
impervious to water vapour and do not give off organic vapours. This is
important in achieving an acceptable shelf life for the kit. The kit will
discolour
slowly by itself unless water vapour and organic vapours can be eliminated. In
these embodiments, the container may be composed of glass and/or Teflon.
The invention will be described by way of examples. However, the
invention is not limited to the examples. In the examples, the seed of a
number
of crops are analysed. However, other suitable seeds, for example, other
grains, oilseeds, vegetables, horticultural crops, fiber crops, speciality
crops,
pharmaceutical and nutriceutical crops and non-food crops may also be
analysed. Examples of seeds suitable for analysis with the invention include
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but are by no means limited to alfalfa, barley, buckwheat, cabbage, canola,
clover, flax, lentils, mustard, sunflower, turnip and wheat.
As can be seen in Figures 2 and 5, a gas chromatography peak area for
ethanol of greater than 500,000 appears to be associated with low vigour in
canola. It is of note that gas chromatography techniques (including head space
gas analysis and solid phase micro extraction) may yield different results
depending on details of the technique. This could be overcome by converting
peak areas into gaseous concentrations.
The most important volatile is ethanol, followed by acetaldehyde and
unknown E.
As discussed in Example Vl, the correlation coefficients found at
two moisture levels show that the accuracy of the vigour test is lower at 35%
compared to 20% moisture. Some insight into what this means may be
obtained by performing some crude calculations on the accuracy, which is
described in Example III. In this set of 26 samples, the vigour of 1 sample
was
incorrectly diagnosed. This calculation is based on a 500,000 threshold and
specifying rejection of seed that has less than 79% of the vigour expected for
high quality seed of the same variety. (A cut off of about 80 % has been
suggested by representatives of the canola industry.)
Measurement of air-dried seed moisture can be accurately done in the
laboratory. Once the moisture percentage is known, the appropriate amount of
water can be added to achieve the desired final moisture percentage. Most
farmers have a moisture meter or ready access to moisture determination. In
some embodiments, the user may determine the moisture content, then add
the appropriate amount of water in order to achieve the required moisture
percentage. Alternatively, a water solution of high osmotic strength (such as
a
polyethylene glycol solution) could be used to limit the moisture uptake of
the
seed to a certain percentage.
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While not wishing to be bound to a specific theory, it appears as though
most crops produce high amounts of ethanol when vigour is low. Only some
crops, though, like canola, have low ethanol emissions when vigour is high.
Those that have relatively high ethanol emissions from high-vigour seed may
include the three main crops discussed in the relevant literature, peas, beans
and soybeans. The difference in ethanol between low and high vigour with
these crops may not be great enough to develop a test.
EXAMPLE I -DETERMINATION OF REFERENCE VIGOUR STATUS
A biological assay was used to determine the reference, or "true",
vigour status of various seed lots. The reference vigour status of each sample
of seed was determined by germinating about 100 seeds on a stainless steel
screen suspended about 1 cm above an aerated complete nutrient solution.
Aerosol from the bubbling solution was sufficient to thoroughly wet the screen
and seed, thus providing conditions for germination. Reference vigour
determinations were performed in chambers with the following day and night
conditions: day--16 hours light (55-60 pmol s' m-2), > 90 % RH, 22°C;
night - 8
hours dark, >90% RH, 17 °C. Two variations of the biological reference
assay
were employed. In the first variation, percentage germination and percentage
of germinated seedlings with greater than 7 mm hypocotyl elongation were
measured each day for five days after the start of imbibition. At the end of
the
fifth day the seedlings were harvested and fresh and dry weights of roots and
shoots determined. A vigour rating scale was established based on the range
of observed values for germination, hypocotyl elongation and root and shoot
biomass production. In the second variation, daily measurements of
germination and hypocotyl elongation were not performed, although seedlings
were harvested and weighed at the end of the fifth day as described. Vigour
rating was based on fresh weight of roots and shoots. Although the first
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variation was more elaborate and provided more information concerning
physiology and growth, both variations provided similar vigour assessments.
EXAMPLE II - GAS CHROMATOGRAPHY METHOD OF DETERMINING
VIGOUR
Seed was weighed into vials of various sizes. If required, water
was added to the seed to make it up to desired moisture contents. The vials
were sealed and incubated for 24 h at selected temperatures. Subsamples of
seed were dried previously to constant weight at 60°C in order to
determine the
moisture content of the original seed sample. After 24 hours, volatile
compounds that had accumulated in the headspace gas of the seed were
analysed by gas chromatography. Analytes were pre-concentrated and
separated from water vapour in the headspace gas by means of automated
solid phase micro-extraction. A 30-metre capillary column with a non-polar
immobile phase was used to separate the analytes, which were detected and
quantified by means of a flame ionization detector.
The separated analytes appeared as peaks in the gas
chromatography output. The peaks were identified by two procedures. Firstly,
purified known compounds were introduced into the seed head space gas and
empty vials in order to match the retention time of known compounds with
those of unknown peaks in the chromatograms. Secondly, the gaseous output
from the gas chromatography column was introduced into a mass spectrometer
and information on the masses of eluted compounds and their degradation
products was obtained. Compounds were identified by correlation of the
measured masses with those of known compounds. Mass spectrometric
analysis was performed by Dr. G. Eigendorf, University of British Columbia.
Typical chromatograms obtained from gas chromatographic
analysis of the headspace gas over high- and low-vigour canola seed lots are
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shown in Figure 1. The ethanol peak appearing at approximately 5.5 min was
much larger in most of the low-vigour seed samples compared to the high-
vigour seed samples.
Ethanol, acetaldehyde and Unknown E were found to be highly
correlated with vigour (correlation coefficients > 0.7). Acetaldehyde is an
intermediate found in the conversion of glucose to ethanol. It is of note that
Unknown E may represent another volatile intermediate or endproduct in the
metabolism of glucose, such as pyruvic acid or acetic acid. On the other hand,
Unknown E may be unrelated to the metabolism of glucose and may be any
low-molecular weight, volatile compound generated by plants. Furthermore, it
has been shown that pentane is present in the head space gas and is weakly
correlated (correlation coefficient < 0.5) to vigour. Pentane is an end
product of
fatty acid oxidation and may play a role in diagnosing seed quality as well.
Other compounds found to be weakly correlated with vigour were Unknown B,
Unknown C and Unknown F. Preliminary data indicates that Unknown B may
be propane. It is possible that a vigour rating determined as a combination or
combinations of these peaks may be more reliable than a vigour rating using
ethanol alone. The potential combinations may be obtained by calculated
functions of measured quantities of individual compounds or by analytical
techniques that measure one or more of the compounds as a group and may
not distinguish among the compounds. As will be appreciated by one
knowledgeable in the art, the gases may be measured using means and/or
indicator compounds known in the art. One compound, dimethyl sulphide, was
not significantly correlated to vigour.
EXAMPLE III - EVALUATION OF GAS CHROMATOGRAPHY METHOD OF
DETERMINING VIGOUR
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Samples of 13 pairs of canola seed lots (Brassica napus) were
obtained. Each pair consisted of a sample of a high-vigour and a low-vigour
seed lot. In addition, both members of each pair were the same variety of
canola. The original designations of high and low vigour for these seed lots
were estimates only.
All 26 seed samples were subjected to the reference vigour
bioassay as described above. It was found that seed samples of different
varieties of canola differed somewhat in their vigour, apparently due to
genetic
variation. Since all varieties of canola must have adequate yield before they
can be registered, we are not concerned with measuring genetic variations in
vigour among varieties, but, instead, we wish to identify those seed lots that
have less than optimal vigour within their variety. Thus, in order to compare
the
low-vigour seed lots to the high-vigour lots, the high-vigour seed lots were
assigned a vigour status of 100. Within each pair, the low-vigour lot was
compared to the high-vigour lot with respect to shoot fresh weight, root dry
weight, germination rate and hypocotyl elongation rate. Each parameter for a
low-vigour lot was expressed as a percentage of the parameter value for its
paired high-vigour lot. The mean of the four percentage values was taken as
the vigour rating for each low-vigour seed lot.
Analysis of volatiles in headspace gas of each seed sample (after
storage at 20 % moisture for 24 hours at room temperature) was determined in
triplicate by gas chromatography. Significant correlations (P < 0.05) between
the reference vigour rating and measurements of ethanol, acetaldehyde and
unknown E in the headspace gas are shown in Figures 2-4. The ethanol found
in headspace gas of each canola seed lot is compared with its reference vigour
value in Table 1.
Because sample set 1 is composed of paired high- and low-vigour
seed lots of the same varieties, it is suitable for estimating the accuracy of
the
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head space gas method of determining vigour. Representatives of the canola
industry have indicated that a vigour test should be capable of identifying
seed
samples that have lost more than about 20 % of their vigour (we will choose 21
for the current accuracy estimate). In addition, results of ethanol analysis
in
this and other sample sets shows that ethanol peak areas up to 500,000 are
consistent with high vigour. Thus, vigour will be incorrectly diagnosed if 1 )
reference vigour is less than 79 % and ethanol peak area is less than 500,000,
or 2) reference vigour is 79 % or greater and ethanol peak area is 500,000 or
greater. Based on these criteria only one of the 26 samples in sample set 1
would be incorrectly diagnosed.
EXAMPLE IV - EVALUATION OF THE GAS
CHROMATOGRAPHY METHOD BY ANALYSIS OF CANOLA SEED OF
DIFFERENT VARIETIES AND PESTICIDE TREATMENTS
Ninety-three samples of canola seed (Brassica napus and
Brassica raps) were collected from farmers, seed laboratories, seed merchants
and other sources. The collection (sample set 2) included 32 varieties or
hybrids of canola that were either untreated or treated with 19 different
formulations of fungicide and insecticide. The set of 93 samples represented
canola genetics and seed treatments in common use in agriculture. About half
of the seed samples were found to be low vigour as determined by the
reference bioassay. The samples, though, are too diverse with respect to
genetics and seed treatments to be organized in high/low vigour pairs as was
done in sample set 1. Duplicate 0.5-g subsamples of seed were made up to 20
moisture in sealed vials and, after 24 ~ 2.5 h at room temperature, were
subjected to head space gas analysis by gas chromatography. Results for
ethanol determinations are shown in Figure 5. Because the data cannot be
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presented in high/low vigour pairs, variation in the horizontal axis includes
genetic variation as well as vigour loss. We cannot distinguish between these
two sources of variation in unpaired samples. Correlations of all measured low
molecular weight components with results of the reference vigour bioassay are
shown in Table 2. The results of sample set 2 show that ethanol, acetaldehyde
and Unknown E are good indicators of seed vigour in a diverse collection of
canola seed samples.
EXAMPLE V - RELATIONSHIP BETWEEN GAS CHROMATOGRAPHY
RESULTS, REFERENCE BIOASSAY RESULTS AND OTHER MEASURES
OF VIGOUR
Seventeen samples of untreated canola seed lots (sample set 3)
were subjected to head space gas analysis, reference bioassay and a series of
tests used in the art as measures of vigour for a variety of crops. Although
there are many tests of vigour used in the art, field biomass, cold stress and
germination tests were selected for study. Recent research (R. Elliott. 2002.
Presentation to the Seed Vigour Research Review, Canola Council of Canada,
Saskatoon.) indicated that they are the most useful of available tests for
estimating canola vigour. It is generally agreed in the art that seedling
biomass
determinations in the field are the most accurate representation of vigour.
However, field measurements are not normally performed because they are
laborious, costly and usually cannot be performed before spring seeding.
Several laboratory-based cold stress tests are used to estimate the ability of
seed lots to tolerate cold soil. The most common test of vigour is the
standard
7-d germination test. Germination tests of shorter duration may more
accurately estimate vigour because they tend to estimate rate of germination
rather than total germination (W. Buckley, unpublished data). Dr. R.H.
Elliott,
Agriculture and Agri-Food Canada, kindly provided data on the analysis of
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sample set 3 by the field biomass, cold stress and germination tests.
Correlations of field biomass, cold stress and germination test results with
the
reference bioassay and ethanol results are shown in Table 3. Both the
reference bioassay results and the ethanol results were strongly correlated
with
the other vigour measurements. The results provide additional evidence that
determination of ethanol in head space gas of moist seed is a reliable method
of determining vigour.
EXAMPLE VI - EFFECT OF SEED MOISTURE PERCENTAGE ON
DETERMINATION OF ETHANOL IN HEAD SPACE GAS
One-gram samples of high- and low-vigour canola seed were
weighed into 10-ml head space gas sampling vials. Water was added to adjust
the seed moisture from 5 to 50 % and low-molecular weight volatile compounds
in the head space gas were determined after 24 ~ 2.5 h incubation at room
temperature. Moisture percentage had a pronounced effect on the evolution of
ethanol from low-vigour canola seed and only a minor effect on the high-vigour
seed (Figure 6). Similar results were obtained for acetaldehyde and Unknown
E. From these results it might appear that a moisture content of about 35
might be ideal for the head space gas analysis. However, variation in
analytical
results increased with higher moisture percentages. To test the significance
of
this effect on the ability to distinguish between high- and low-vigour
samples, a
set of canola seed lots was tested at 20 and 35 % moisture (seed set 4; 40
samples of several varieties either treated or untreated with fungicide;
approximately equal numbers of high- and low-vigour seed lots; note that seed
set 3 was a subset of seed set 4). Correlation coefficients between the
reference vigour test and ethanol, acetaldehyde and Unknown E were -0.78, -
0.72 and -0.73, respectively, for the seed at 20 % moisture, which is
consistent
with the other sample sets studied. However, at 35 % moisture correlation
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coefficients were -0.60, -0.57 and -0.62, respectively. The results indicated
that the ability to resolve high- and low-vigour seed based on ethanol,
acetaldehyde or Unknown E determinations is substantially better at 20
moisture compared to 35 % moisture. Thus, control of the moisture percentage
of the seed is important when using ethanol determinations to estimate seed
vigour. Adding water to seed without controlling the moisture percentage
within
reasonable limits may lead to unreliable determinations. The optimum moisture
percentage may be in the range of 15 to 25 %. Seed germination begins at 35
- 40 % moisture in canola seed.
EXAMPLE VII - ESTIMATING GERMINATION LOSS IN MALTING BARLEY
The results of our work suggest that loss of germination is one of
a series of changes that happen to seed during loss of vigour. Loss of
germination, though, occurs relatively late in the deterioration process,
whereas
the appearance of ethanol in headspace gas occurs relatively early. Thus,
seed ethanol emissions are expected to be a good predictor of germination
loss. In the malting process, barley is germinated, then dried to yield malt.
A
high germination percentage is very important. After barley has been selected
for malting, though, it is usually stored or transported for varying lengths
of
time. By the time it enters the malting process, the barley may have lost
significant germination percentage. It would be advantageous to identify those
barley lots with a predisposition for germination loss as early as possible.
Thirty barley seed lots were tested for ethanol accumulation in
head space gas. Four grams of seed was made up to 20 % moisture and
sealed in 10-ml head space gas vials and incubated for 24 ~ 2.5 h at room
temperature prior to gas chromatography analysis. The seed was also tested
for susceptibility to germination loss by artificial aging at elevated
temperature
and moisture (the germination loss data was kindly provided by Dr. M.
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Izydorezyk, Canadian Grain Commission). Results of the analyses are shown
in Table 4. The data indicate that elevated ethanol in head space gas may be
a suitable predictor of germination loss in barley.
EXAMPLE VIII - SUITABILITY OF THE HEAD SPACE GAS ANALYSIS
METHOD OF DETERMINING SEED VIGOUR IN A VARIETY OF CROPS
The suitability of the head space gas analysis method for determining
vigour in a variety of crops was examined by means of a screening procedure.
The procedure involved the determination of ethanol in head space gas of un-
aged and artificially aged paired samples of seed. Seed samples of a number
of crops were obtained from various sources. Duplicate 4-g subsamples were
weighed into 10-ml head space gas analysis vials. Sufficient water was added
to make the seed up to 20 % moisture (based on an assumed air-dried
moisture content of 3 %) and the vials were sealed. One subsample was
maintained at room temperature while the other was elevated to 40 °C
for 24 h
then cooled to room temperature. Gas chromatography analysis of head space
gas was performed 24-29 h after the addition of water. Single or double
analysis of each sample was performed.
Accelerated, or artificial, aging of seed is a technique commonly used in
the art for the determination of seed vigour. Seed is maintained in a humid
environment, or at an elevated moisture percentage, and at an elevated
temperature for periods of time that may vary from hours to weeks. Usually the
environment is 100 % relative humidity and the temperature is in the range of
30-40 °C. After accelerated aging has been performed, the germination
percentage of aged seed is determined. Seed that shows a higher germination
after accelerated aging is considered to have higher vigour. It can be
appreciated that the current screening procedure is a combination of
accelerated aging and head space gas analysis. It is reasoned that an
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increase in ethanol emissions by seed subjected to accelerated aging indicates
that the seed is likely to undergo similar changes as vigour declines during
normal storage. Thus, seed lots that show a high ratio (greater than 10) of
aged to un-aged ethanol emissions are considered to be good candidates for
vigour determination by head space gas analysis. The percentage error in
diagnosing vigour from head space gas analysis for crops with low ratios may
be unacceptable.
Thirty crops were tested with the screening procedure (Table 5). All
crops examined showed an increase in ethanol emissions, most to relatively
high amounts, following artificial aging. This suggests that elevated ethanol
emission may be a universal phenomenon in seed that has undergone
deterioration or vigour loss. The ratio of ethanol emissions from aged and un-
aged seed varied from 472 in canola to 2 in vetch and barley. High ratios were
associated with lower ethanol emissions from un-aged seed and not higher
than average emissions from aged seed.
The results for barley (Table 5) were not expected. Although five lots of
malting barley, including four varieties, were tested no sample emitted low
ethanol before aging. However, it is apparent from the results in Table 4 that
high quality barley does have low ethanol emissions. There may be several
reasons for the unexpected barley results. 1 ) Conditions have been poor for
the past three years in Western Canada for the production of malting barley.
2)
Barley, especially in the eastern prairies tends to be infected with fusarium,
which reduces quality. 3) Malting barley must be stored under carefully
controlled conditions in order to maintain its quality after harvesting. Two
of the
five samples screened were not properly stored and storage conditions of the
other three are unknown.
As described in the Background to the Invention, there were four
references found in the scientific literature that referred to the possibility
of
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measuring seed vigour by analysis of gasses emitted from imbibing or
germinating seed. Three of these referred to ethanol or acetaldehyde and one
referred to ethylene. There were four crops in these studies: muskmelon, pea,
soybean and snapbean (a type of bean); and one weed: cocklebur. The four
crops are represented in Table 5. Muskmelon yielded a ratio of 109, the fourth
highest value in the survey. However, muskmelon emitted the lowest amount
of ethanol from aged seeds of any of the crops studied. Although this would
not be a limitation for analysis by gas chromatography, analysis by the
colourimetric method or the instrumental method (Pac III) may not be feasible
because of low ethanol. Peas, soybeans and beans were found to have
aged:un-aged ethanol ratios less than 10. Although we cannot discount the
possibility that we were unable to find a single high quality sample of seed
for
these crops, it appears most likely that high-vigour seed of these crops
normally emits relatively high amounts of ethanol. The differences in ethanol
and acetaldehyde emissions between high- and low-vigour seed samples of
these crops probably are not great enough to develop a reliable assay. This
might explain why a vigour assay based on gaseous emissions from seed has
not been developed previously.
Small-seeded crops appear to be more suited for the ethanol vigour
assay than large-seeded crops. Six of the seven highest ratios were from
small-seeded crops (Table 5). The tendency for medium and large-seeded
crops to emit greater amounts of ethanol from un-aged seed may be related to
the physical size of the seed as well as the quantity of carbohydrate reserves
available in the seed. Large seeds have larger carbohydrate reserves per
embryo and tend to have a larger percentage of carbohydrate by weight.
Because carbohydrate may be in relatively short supply in small seed, healthy
small seed may have a greater tendency to avoid fermentation than healthy
larger seed, thereby conserving the energy supply. Fermentation, which
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produces ethanol from carbohydrate, is energetically inefficient, and normally
occurs when oxygen is limiting. Oxygen may be limiting in the sealed vials of
the ethanol assay. More importantly, though, the interior of a large seed may
have limited oxygen because of the distance required for diffusion of oxygen
from the surface. Thus, un-aged large seeds might be expected to emit more
ethanol than un-aged small seeds.
EXAMPLE IX - COLOURIMETRIC METHOD OF DETERMINING VIGOUR
USING COMMERCIAL DIFFUSION TUBES
Canola seed was weighed into containers of various sizes. Water
was added to the seed to make it up to 20 % moisture. The containers were
sealed and an ethanol indicator tube (Drager Diffusion Tube, Ethanol 1000/a-D,
Drager Sicherheitstechnik GmbH, Germany) was inserted into a tight-fitting
hole in the vial lid. The vials were incubated for 24 h at room temperature.
Sub-samples of seed were dried previously to constant weight at 60°C
in order
to determine the moisture content of the original seed sample. After 24 hours,
the extent of colour change in the tube indicated the quantity of ethanol
emitted
by the seed and, therefore, the vigour of the seed. The quantity of ethanol
given off over 24 hours by low-vigour canola seed was sufficient to cause a
colour change in the commercial ethanol indicator tube. Thus, the feasibility
of
a simple, economical on-farm test based on colour change was demonstrated.
An example of the difference in colour development 24 hours after moistening
high- and low-vigour seed is shown in Figures 7 & 8.
EXAMPLE X - COLOURIMETRIC METHOD OF DETERMINING VIGOUR
USING AN INTEGRATED COLOUR INDICATOR
The quantity of ethanol and related metabolites given off over 24
hours by low-vigour canola seed at 20 % moisture caused a colour change in a
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colour indicating disc that was an integral part of the seed container. The
colour disc consisted of 80 microliters of a colour developing reagent
absorbed
into a 10-mm diameter glass fiber disc. The reagent consisted of 10 ml
sulphuric acid and 1.2 g potassium dichromate per 100 ml of water solution.
The glass fiber disc with reagent was dried over a desiccant prior to assembly
of the seed container. The container was constructed so that the colour disc
was exposed to gasses emitted from the seed while the disc was visible
through a clear plastic window. An example in which the containers are fl-
ounce hospital-type specimen cups containing 60 g of seed made up to 20
°l°
moisture 24 h prior to recording the colour development is shown in Figures 9
and 10.
EXAMPLE XI - COLOURIMETRIC METHOD OF DETERMINING VIGOUR
USING AN INTEGRATED COLOUR INDICATOR
Five grams of canola seed was added to 10-ml glass vials. The
seed was made up to 20 % moisture and the vials sealed with crimp tops that
included an integrated colour disc. The colour disc was the same as described
in Example X. Figure 11 shows the colour development obtained with high-
and low-vigour seed 24 hours after adding water to the seed.
EXAMPLE XII - AN INSTRUMENTAL METHOD OF ESTIMATING SEED
VIGOUR BY DETERMINING VOLATILES EMITTED BY MOIST SEED.
Commercially available instrumentation other than gas
chromatography may be suitable for measurement of seed vigour. The
instrumentation may have the advantages of lower cost and require less
training than gas chromatography. Figure 12 shows the determination of
gaseous ethanol at concentrations suitable for the analysis of head space gas
of moist seed using a hand held gas monitoring instrument (model Pac III;
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Dreiger).
EXAMPLE XIII - SUMMARY
A method for the determination of vigour in seed has been
described, and details have been provided for canola seed as well as other
seeds. The method is novel because no method currently exists for the
determination of vigour by headspace gas analysis. The method appears to be
reliable, based on the testing of more than 100 canola seed lots. The method
may be suitable for a variety of crops (mostly small-seeded) based on
screening tests of seed of 30 crops. One embodiment of the method uses an
existing colour development technology to distinguish between high and low
vigour in a simple, economical procedure suitable for an on-farm test. The
colour change associated with ethanol production was demonstrated in high-
and low-vigour canola seed lots.
While the preferred embodiments of the invention have been
described above, it will be recognized and understood that various
modifications may be made therein, and the appended claims are intended to
cover all such modifications which may fall within the spirit and scope of the
invention.
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Table 1. Ethanol, acetaldehyde and unknown E (gas chromatography peak
areas) in head space gas of paired high- and low-vigour seed lots of canola.*
High-vigour Low-vigour
lots lots
ReferenceHead space ReferenceHead
gasses space
gasses
Seed bioassay EtOH AA E bioassay EtOH AA
lot %
pair
100 20,389 5,399 0 103 34,148 9,653
12 100 5,441 1,872 0 89 16,691 3,390
8 100 28,940 6,806 0 79 274,664 50,894
6 100 28,391 8,143 0 79 24,240 4,870
7 100 70,172 10,012 0 77 6,267,588243,643 22,
11 100 27,780 8,703 0 74 3,097,329233,091 56,
9 100 22,667 4,427 0 74 1,125,623138,339 7,
13 100 11,757 2,145 0 73 129,176 19,846
2 100 300,722 0 4,591 71 6,184,36989,264 50,
5 100 38,710 9,463 0 67 4,208,946262,515 32,
3 ~ 100 75,696 16,472 0 65 6,426,057333,208 25,
AC Excel100 5,724 2,420 0 57 1,439,61973,715 19,
4 100 16,354 3,113 0 55 5,030,866407,121 78,
*Defini tions:
EtOH
= ethanol:
AA =
acetaldehyde:
E = Unknown
E. Reference
bioassay results of low-vigour lots are expressed as percentages of paired
high-vigour lots. Several varieties are represented; however, each pair
consists of a single variety.
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Table 2. Correlations~ between reference bioassay results (seedling fresh
weight)
and quantities of low-molecular weight volatiles found in head space gas of
moist
canola seed.*
Correlation with seedling
fresh weights
Head space Gasses Correlation coefficient P**
(r)
Ethanol -0.78 < 0.0001
Unknown E -0.73 < 0.0001
Acetaldehyde -0.72 < 0.0001
Unknown C -0.47 < 0.002
Pentane -0.47 < 0.0025
Unknown F -0.36 < 0.022
Unknown B -0.35 < 0.027
Dimethyl sulphide 0.18 < 0.26
*The sample set consisted of 93 canola seed samples and included 32 varieties
or
hybrids that were untreated or treated with 19 formulations of fungicide and
insecticide.
**Probability of incorrectly rejecting the hypothesis that there is no
relationship.
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Table 3. Correlations between reference bioassay, ethanol in head space gas
and
various vigour test results for 17 canola seed lots.
Correlation coefficients (r)
Vigour test Reference bioassay Ethanol
Field fresh weight at 14 d after seedinct0.79 -0.78
Field fresh weight at 21 d after seeding0.80 -0.77
Field fresh weight at 28 d after seeding0.83 0.71
Pre-chill test* 0.77 -0.77
Special cold stress test** 0.84 -0.76
Germination after 4 d on moist blotting0.89 -0.81
paper
Germination after 5 d on moist blotting0.74 -0.79
paper
Germination after 6 d on moist blotting0.68 -0.72
paper
Germination after 7 d on moist blotting0.64 -0.68
paper
(standard germination test)
*Seed was planted in a potting soil, chilled at 5 °C for 7 d, than
warmed to 25 °C
during 16-h light periods and 15 °C during 8-h dark periods for 5 d,
after which the
number of emerged seedlings was counted.
**Seed was planted in potting soil and sand mix (1:1 ), and maintained at 8
°C
during 8-h light periods and 16-h dark periods for 14 d, after which the
number of
emerged seedlings was counted.
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Table 4. Ethanol, acetaldehyde and Unknown E in headspace gas of barley seed
with
and without a predisposition to germination loss.*
Germination Ethanol Acetaldehyde Unknown E
after aging
Lot % Mean SE Mean SE Mean SE
1 22 1431166 104275 12947 1852 3958 204
2 45 424075 230902 2743 1152 1073 457
3 49 123547 103071 333 183 221 215
4 49 1530769 825878 1296 700 1169 566
5 61 15307 4295 220 105 41 41
6 61 12503 10431 181 116 0 0
7 87 14209 11668 125 44 47 47
8 88 16065 8917 303 109 43 43
9 89 16615 16615 9 9 71 71
10 90 118 118 41 41 0 0
11 91 253 253 65 33 0 0
12 93 22860 6336 680 275 30 30
13 94 3944 2055 163 47 32 32
14 95 28420 26712 146 102 37 37
~
i
15 97 0 0 0 . 0 0 0
16 98 7179 470 120 47 0 0
17 98 337929 326282 567 461 597 597
18 98 169 169 38 35 0 0
19 99 133 133 36 36 0 0
20 99 0 0 21 21 0 0
21 99 0 0 27 27 0 0
22 99 2037 1054 65 14 0 0
23 99 0 0 22 22 0 0
24 99 ~ 3450 442 84 38 0 0
25 99 1854 856 148 40 0 0
26 99 0 0 36 36 0 0
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27 99 7352 3383 141 107 95 95
28 100 1701 1701 64 34 18 18
29 100 0 0 15 15 0 0
30 100 29 29 22 22 0 0
*Predisposition to germination loss was demonstrated by artificial aging.
Prior to artificial
aging, all seed lots had 95 % or better germination, except for lot 4 that was
87 %.
Acetaldehyde, ethanol and unknown E in head space gas was determined on un-
aged
samples.
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Table 5. Ethanol emissions from un-aged and artifically aged* seed of various
crops.
No. Ethanol
of (gas
eed otanical lots Mean
size family rop varietiesratio
(s,m,l) tested 0
clZrornatography
peak
ethanol
peak
areas,
means)
area
unaged
aged
(aged:unaged)
s BrassicaceaeCanola 5 507519 4859211472
s BrassicaceaeMustard 2 1785 732664 435
s LeguminoseaeAlfalfa 4 532203 4408895352
m CucurbitaceaeMuskmelon 2 9997 514557 109
s Linaceae Flax 4 269532 470653683
s BrassicaceaeTurnip 1 49080 368697975
s BrassicaceaeRadish 1 7613 533436 70
m PolygonaceaeBuckwheat 1 73706 260229435
s BrassicaceaeCabbage 1 365704 699228219
m LeguminoseaeLentils 2 292793 274209718
s LeguminoseaeClover 1 42224 930759 16
I AsteraceaeSunflower 1 91048 138896615
m Poaceae Wheat (bread)3 585034 423889515
s Apiaceae Carrot 1 411623 36992389
I Leguminoseae, Soybeans 3 577632 21736138
I Poaceae Corn 3 890299 51892146
s Poaceae Ryegrass 1 649565 40244826
m Poaceae Oats 4 858938 41272706
m Poaceae Triticale 1 758970 4113095b
s AsteraceaeLettuce 1 1836862 102274646
s Poaceae Canarygrass 1 1302638 45741134
I LeguminoseaeBeans 4 440974 18847694
m CucurbitaceaeWatermelon 1 327745 13594754
s Liliaceae Onion 1 1008326 38715954
I LeguminoseaePeas 4 1778121 51204604
m Poaceae Rye 3 1906312 56525493
m Poaceae Wheat (durum)2 1589734 39741643
m CucurbitaceaeCucumber 1 1 b89674 41347433
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m Leguminoseae Vetch 1 1289992 2753544 2
m Poaceae Barley 5 2809626 5704220 2
*Seed samples (4 g, except for sunflower, ryegrass and muskmelon, 2 g) were
made up to 20 % moisture in 10-mL head space gas analysis vials. The vials
were
sealed and maintained for 24 h at room temperature (un-aged samples) or at 40
°C (aged samples). During the period of 24-29 h after adding water,
head space
gas in the vials was analyzed.
