NL1009006C2 - Method for determining the quality of pre-sprouted, sprouting and sprouted seeds and apparatus for analyzing and apparatus for separating pre-sprouted, sprouting and sprouted seeds. - Google Patents

Description

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Reg. No. 156357 PB / PB

METHOD FOR DETERMINING THE QUALITY OF PREFERRED. Germinating and germinated seeds and apparatus for analyzing and apparatus for separating pre-germinated, germinating and germinating seeds

The present invention relates to a method for analyzing and determining the quality of seeds during pre-germination, seeds that have been pre-germinated and germinated seeds by irradiating the seeds with electromagnetic radiation. Due to this irradiation, the chlorophyll formed in the seeds shows direct fluorescence. The present invention relates to a device for analyzing the above-mentioned seeds, at least consisting of a part for irradiating the seeds with electromagnetic radiation and a part for measuring the chlorophyll fluorescence signal. The present invention also relates to a device for separating seeds, at least consisting of a supply part for the seeds, a part for irradiating the seeds with electromagnetic radiation, a part for measuring the chlorophyll fluorescence signal returned from the seeds. , and a separation portion that operates based on the chlorophyll fluorescence signal returned from the seeds. Hereby, seeds 25 are understood to mean a plant product arising after sexual or non-sexual fertilization of the ovule. Chlorophyll is understood to mean all forms of the chlorophyll molecule, such as chlorophyll, proto-chlorophyll, known as leaf green, and so on.

3 0 Measuring the chlorophyll formed from seeds has proven to be a good method for monitoring the pre-germination process of seeds. It has been found that chlorophyll is formed during the pre-germination and germination phase of seeds. As a result, the amount of chlorophyll in the seed increases during pre-germination. The cotyledons present in the seed can color green 1009006 - 2 - during germination in the seed. The cotyledons of protochlorophyll change to chlorophyll under the influence of light. To date, there are no methods of non-destructively monitoring seed germination. The chlorophyll content in the seed is therefore a good measure of the progress of the germination process of seeds.

Seeds can be roughly divided into two categories, i) Seeds with a fairly transparent seed skin for red and far red light, such as seeds of white beans, corn, pepper and cucumber and ii) seeds with a semi-transparent seed skin for red and far red light, such as seeds of cabbage, tomato and brown bean. With the invention, great certainty can be obtained regarding the germination of the seed. Seeds that do not form chlorophyll or only after a long time chlorophyll will form during pre-germination and under the influence of light will not with certainty start to germinate or be of low quality. Quality is defined as germination rate, germination rate, germination uniformity, vapor, percentage of normal seedlings and health.

2 0 Seeds that start to form chlorophyll in the seed after a certain time of pre-sprouting will germinate more evenly and yield fewer abnormal seedlings. With this new method, seeds can be specifically pre-germinated ("priming"). The seeds are measured for the formation of chlorophyll during pre-germination. When a certain value of the chlorophyll content is reached, the seeds are dried back. This value, as measured by the equipment used, for unsprouted pepper seeds is about 6 pA, and for pepper seeds about to sprout about 20 pA. In this way, the seeds are synchronized in germination, so that after drying the seeds and after rewetting the seeds, they germinate very uniformly and with a very high germination percentage. Furthermore, with the described method, seeds can be sorted after being sprouted in, for instance, the fruit (paprika or tomato) or in the field (barley). This is also called presprouting in the literature. These dried but already 1009006 - 3 - sprouted seeds will no longer produce germs or abnormal germinating plants, or in the case of barley, adversely affect the malting process. With the present invention, the metabolism of the seed during germination can be studied. This has, among other things, applications in studying the influence of external factors such as temperature, available amount of water and light on germination.

A method for determining chlorophyll in seeds is known from an article by Tkachuk and Kuzina, in "Chlorophyll analysis of whole rapeseed kernels by near infrared reflectance", Canadian Journal of Plant Science 62: 875-884, 1982. spectrophotometer a light beam of known wavelength directed at the seed. After 15 reflection, the device determines the fractional absorption of the light beam. Preferably, it is measured in the range 400-2400 nm. The reflection spectrum is now a measure of the chlorophyll content. The chlorophyll content is determined for the purpose of having the lowest possible chlorophyll content in the oil of the pressed seeds. The major drawback of this method is that it is necessary to measure at different wavelengths, preferably 16, in order to obtain a good and reliable reading. This method is too insensitive and too complex to build into an analysis device or sorting device.

It is known in the art that chlorophyll can be measured with chlorophyll fluorescence. R.M. Smillie, S.E. He-therinton, R.N. Orantley, R. Chaplin and N.L. In "Application of chlorophyll fluorescence to 30 postharvest physiology and storage of mango and banana fruit and the chilling tolerance of mango cultivars," Wade used the chlorophyll fluorescence technique, Asean Food Journal (1987), 3 (2), 55-59. to measure the photosynthetic activity of fruit. They examined the changes in photosynthetic thesis activity during ripening and under low temperature measurements by measuring the chlorophyll fluorescence signal of the peel. The rate at which the chlorophyll-1009006-4-fluorescence signal decreased during exposure to low temperatures was used to select cold insensitive varieties. They do not mention the ability to track germinating seeds over time by measuring direct fluorescence in order to monitor seed metabolism. However, there is no question of measuring the chlorophyll content in seeds of plants with the intention of monitoring the germination process and metabolism of the seed. For fruits and leaves, the chlorophyll fluorescence sig-10 changes during measurement, because the chlorophyll is photosynthetically active. This is in contrast to the chlorophyll in a seed. The direct amount of chlorophyll fluorescence is measured here, ie immediately when the seed is irradiated with electromagnetic radiation. The amplitude of the chlorophyll fluorescence signal is now a measure of the amount of chlorophyll. Smillie's measurement method takes a total of an hour to adjust the plant material to darkness and a few seconds to track changes in the chlorophyll fluorescence signal. The measuring method according to the invention can be done in a fraction of a second. The usual measurement method to measure the photosynthesis activity of plant material is with the pulse amplitude modulation (PAM) fluorometer from U. Schreiber, described in "Detection of rapid induction 25 kinetics with a new type of high frequency modulated chlorophyll fluorometer" Photosynthesis Research (1986 9: 261-272. The photosynthesis activity is not directly dependent on the amount of chlorophyll in plant material. The chlorophyll fluorescence signal is directly dependent on the amount of chlorophyll. Therefore, to determine the photosynthesis activity, correction must be made for the amount of chlorophyll. This is done by calculating a quotient so that this quotient is independent of the amount of chlorophyll. In contrast, the method according to the invention described uses the measured amount of chlorophyll fluorescence as a measure of the amount of chlorophyll.

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According to H.K. Lich-tenthaler in "In vivo chlorophyll fluorescence as a tool for stress detection in plants", Application of Chlorophyll Fluorescence in Photosynthesis, H.K. Lichtenthaler (ed.) 5 1988, 129-142, Kluwer Academic Press, Dordrecht, does not measure the amount of chlorophyll. The parameters measured for calculating chlorophyll fluorescence photosynthesis may even increase in signal size as the amount of chlorophyll decreases. This is because the reabsorption of the emitted chlorophyll fluorescence decreases due to the lower concentration of chlorophyll. H.K. Lichtenthaler and C. Buschmann in "Reflectance and chlorophyll fluorescence signatures of leaves" in Proceedings IGARSS'87 Symp. Ann. Arbor. MI (USA) 18-21 May 1987, 15 1201-1206 see that around 690 and 730 nm the chlorophyll fluorescence signal of leaves is not linear with the amount of chlorophyll. Here it was also shown that a lower amount of chlorophyll can give a higher chlorophyll fluorescence signal. From these two articles it appears that the literature indicated that it was not to be expected that there was a relationship between the amount of chlorophyll and the signal size of the chlorophyll fluorescence. Therefore, it is not plausible for an experienced person in the field of chlorophyll fluorescence that chlorophyll fluorescence can be used to determine the increase in the amount of chlorophyll.

The present invention now has for its object to provide a method by which it is possible to sort seeds on the basis of the amount of chlorophyll formed during pre-germination, by quality and germination stage, without destroying the seeds. Characteristic of the invention are the very high sensitivity and the very high speed with which chlorophyll fluorescence can be measured in the interior of the seed. Another object of the invention is to provide a device with which seeds can be analyzed and sorted quickly and accurately for germination stage and quality.

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The method according to the invention is now characterized in that the electromagnetic radiation has such a wavelength that chlorophyll present in the seed exhibits direct fluorescence, which fluorescence is measured.

The method according to the invention can very well be carried out in a device as mentioned in the preamble, characterized in that the electromagnetic radiation has a wavelength such that chlorophyll present in the seed 10 shows direct fluorescence, which fluorescence is measured in the measuring part.

The present invention is based on a fluorescence measurement that is very specific for the chlorophyll present. Other substances that affect the color of the seed but do not fluoresce will not contribute to the fluorescence signal. Small differences in the amount of chlorophyll in the seed can also be detected according to the invention. This is due to the principle that a fluorescence measurement is very sensitive.

According to the invention, it now appears that a difference in the chlorophyll content of individual seeds is directly demonstrable, even if the casings are completely uniformly colored and opaque to the eye. There is no known data in the literature on the non-destructive measurement of the amount of chlorophyll fluorescence in relation to the seed germination stage before the root tip is visible. A suitable method for measuring the chlorophyll content is by irradiating at least a part of the chlorophyll molecules with electromagnetic radiation, preferably with a wavelength between 400 and 700 nm, so that at least a part of the chlorophyll molecules in a electronically excited state. The excited molecules lose their energy mainly in the form of heat dissipation and about 3% in the form of fluorescence emission, which is preferably measured between 600 and 800 nm.

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When, according to the invention, the intensity of the chlorophyll fluorescence of each seed is individually measured, the seeds can be analyzed and sorted for the stage of pre-germination and quality.

The invention is very sensitive, completely non-destructive and very fast. These are the characteristics of the invention which make it possible to make a sorting device with which seeds can be selected on the basis of the amount of chlorophyll fluorescence. Because the amount of fluorescence by chlorophyll has a direct relationship to the conversion of protochlorophyll into chlorophyll in the seed, sorting can now be done at the stage of pre-germination and quality.

The method according to the invention can also be used for scoring seeds for germination during a germination test which is done to determine the quality of seeds. In a germination test it is important to be able to predict at a very early stage whether the seeds will yield a seedling. The current germination tests are mostly subjective, because the appearance of a root tip is scored. This observation depends on the observer and is labor intensive. With the present invention, seeds can be scored for germination based on the chlorophyll content, so that the germination test can be automated and thus carried out objectively. Another advantage of the present invention is that the seeds can be scored at a much earlier stage of germination, so that this saves time in conducting germination tests. It is of course also possible to analyze and select the seeds for chlorophyll formation at a further stage of germination. This is possible when plant parts other than the root emerge from the seed. Examples are the hypocotyl and the cotyledons. These plant parts contain chlorophyll for many seed species. In an analogous manner as described above, the seeds can be sorted and analyzed for the appearance and formation of chlorophyll.

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The present invention is applicable to many types of seeds of horticultural crops, agricultural crops, ornamental crops, forest crops. The invention works in seeds in which the amount of chlorophyll changes during the germination process and in which the seed skin (setni) is transparent for the excitation light for the chlorophyll and the fluorescence of the chlorophyll and for seeds in which the plant parts coming out of the seed contain chlorophyll.

It is preferable to perform a fluorescence measurement in a device, as shown in Figure 1. This is a simple form that the device can have. The light from an LED lamp with a maximum emission at 650 nm is filtered through a narrow-band filter at 656 nm. The beam splitter mirrors approximately 50% of the LED light in the direction of the lens, which concentrates the light onto the seed. As a result, at least a part of the chlorophyl molecules get into an excited electron state. At least a portion of the deposited chlorophyll molecules fall back to the ground state under fluorescence emission. At least part of the chlorophyll fluorescence is captured with the same lens. The filter ensures that mainly fluorescence around 730 nm is detected by the photodiode. The lock-in amplifier modulates the LED light with a modulation depth of 100% and a duty cycle of 50% and a suitable frequency. This also modulates the fluorescence with the same frequency. The AC voltage of the photodiode is converted by the lock-in amplifier into a signal proportional to the intensity of the fluorescence. This detector can be built into a sorting device. After measuring the chlorophyll fluorescence, a valve can then be controlled, for example, with an electronic circuit such as a microprocessor, which extracts the seeds from the main current with a higher or lower signal than a predetermined value. Sorting from the main flow with a valve can be done with any known principle, such as an air flow, liquid pulse or mechanical valve. It is pointed out that the sorting can be done on seeds that are in the air, but also on seeds that are in a liquid. Sorting in the liquid can for instance take place in the germination liquid, wherein the seeds are sorted for germination in a continuous process.

Analyzing seeds for the distribution of the amount of chlorophyll in a sample from a seed lot can be done with the same preferred device for sorting seeds, but now there is no need to sort. For example, the chlorophyll fluorescence signals from the seeds are stored in the memory of a microprocessor or on an optical or magnetic disk. The measured chlorophyll fluorescence signals can be plotted, for example, in tabular form, in the form of a graph or a histogram, so that the distribution of the chlorophyll fluorescence signals becomes visible. Analysis of pre-sprouted or germinating seeds can also be performed with an electronic camera. The image from the camera measures the chlorophyll fluorescence image of multiple seeds 20 simultaneously. An overview of the distribution of the chlorophyll fluorescence signal, for example in the form of a histogram, can then be given for all measured seeds. Furthermore, with this preferred equipment, the germination of seeds can be monitored over time. This can be done, for example, by scoring the seeds at fixed times which have a value of the chlorophyll fluorescence signal above a certain threshold value.

For the aforementioned preferred arrangements of the sorter and analyzer, it is clear to an expert in the field that the LED lamp can be replaced by a lamp with filters or a laser. It is also possible to use a photomultiplier or an electronic camera for the photodiode. The camera can be used, for example, to make chlorophyll fluorescence images of a number of seeds, in order to make the distribution of the chlorophyll, or chlorophyll fluorescence images of individual seeds.

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The invention can also be used in a seed sorting device. Installation is possible in all sorting establishments. The invention is in particular applicable in the known color sorting devices. The light source can be replaced by the electromagnetic radiation source (eg a laser) and the colorimeter can be replaced by the photodiode. It is also possible to measure seeds in the germ liquid during pre-sprouting.

. The invention will now be elucidated on the basis of a number of examples.

Examples

In the following examples the chlorophyll fluorescence signal was measured from the interior of individual seeds using the method according to the invention.

15 tests were carried out with pepper seeds (Capsicum annuum).

Example 1

With the present invention, the chlorophyll fluorescence signal of 200 paprika seeds (Capsicum annuum) individually and on both sides of the seed was measured at different times during germination. The germination tests were performed on a solution of water with 0.2% KNO3 wetted filter paper in plastic trays in a germination cabinet at an alternating temperature of 20 ° C-30 ° C, at 20 ° C in the dark (16 hours) and at 30 ° C in the light (8 hours). After 136 hours, the seeds were visually assessed for appearance of a root tip. In Figs. 2, the results of the measurement of the sprouted and non-sprouted seeds are plotted. In panel A, the averages of 20 not 30 sprouted and 180 sprouted seeds are plotted. The increase in the chlorophyll fluorescence signal of the sprouted seeds shows an exponential behavior. The non-germinated seeds, on the other hand, hardly increase in signal size. In panel B, the I 1009006 - 11 signals are plotted individually for 2 germinated and 2 non - germinated seeds. After 96 hours, the 2 seeds were germinated (germ). This new germination seed measurement method allows the germination process with chlorophyll fluorescence to be measured and monitored.

5 Example 2

With the present invention, the chlorophyll fluorescence signal of paprika seeds (Capsicum annuum) was individually measured and then divided into 2 classes based on the distribution of the fluorescence. In this example, a laser was used instead of an LED lamp. The low class is for seeds with a chlorophyll fluorescence signal less than 225 pA and the high class for seeds with a signal greater than or equal to 225 pA. The percentage distribution between the 2 classes is shown in Table 15. The germination tests were carried out on a solution of water with 0.2% KNO3 wetted filter paper in plastic containers in a germination cabinet at an alternating temperature of 20 ° C-30 ° C, at 20 ° C in the dark (16 hours) and at 30 ° C in the light (8 hours). The seeds and seedlings were assessed according to the ISTA (1996) International rules for seed testing, Seed Science and Technology 24. In this example, about one third of the seeds had already sprouted. This was visible because the seeds had a brown root tip. Paprika seeds are contained in a fruit which has a high moisture content after ripening of the seeds. Pepper seeds can still be physiologically active due to their moist environment. After fully ripened, pepper seeds can sprout in the fruit. After drying of the seeds, the seeds that have already sprouted in the fruit will no longer give germs or abnormal seedlings. The seeds in the low fluorescence class gave a good turnout of 84%, while the turnout in the high class was 54%. The improvement was 17% compared to the control. In this choice of classes it is therefore evident that the low class is of better quality than the high class.

1009006

Quality of bell pepper seeds without sorting (control) and after sorting in 2 classes by means of chlorophyll fluorescence - 12 -Table 2 chlorophyll fluorescence low high control 5 distribution (%) 54 46 100 normal seedlings (%) 84 54 67 abnormal seedlings (%) 1 4 3 rotten seedlings 12 39 29 germinated seeds 331 1009 006

Claims (6)

Method for determining the quality and stage of pre-germination and germination of pre-germinating, pre-germinated, germinating and germinated seeds by irradiation with electromagnetic radiation, characterized in that the electromagnetic radiation has such a wavelength that chlorophyll present in the seed shows direct fluorescence, which fluorescence is measured. 2. Apparatus for analyzing pre-sprouted, pre-sprouted, sprouting and sprouted seeds, at least consisting of a part for irradiating the seeds with electromagnetic radiation and a part for measuring the signal returned from the seeds, characterized in that the electromagnetic radiation has such a wavelength that chlorophyll present in the seed exhibits direct fluorescence, which fluorescence is measured. 3. Device for separating pre-germinating, pre-germinated, germinating and germinated seeds, at least consisting of a supply part for the seeds, a part 20 for irradiating the seeds with electromagnetic radiation, a part for measuring the signal returned from the seeds and a separation section operating on the basis of the signal returned from the seeds, characterized in that the electromagnetic radiation has such a wavelength that chlorophyll present in the seed exhibits direct fluorescence, which fluorescence is measured in the measuring section. Method according to claim 1, 2 or 3, characterized in that the irradiated electromagnetic radiation has a wavelength between 400 and 700 nm and that fluorescence is measured between 600 and 800 nm. A method according to claims 2 and 3, characterized in that the returned signal from the seeds is measured with a photodiode, photomultiplier or electronic camera. 1009006 - 14 - Method according to claims 2 and 3, characterized in that the irradiated electromagnetic radiation is generated by a laser or LED lamp. 1009006