METHOD AND APPARATUS FOR DETERMINING SEED QUALITY BY FLUORESCENCE

Abstract: The invention relates to a method and apparatus for analysing pre-germinating, germinated, germinating and germinated seeds and an apparatus for sorting such seeds and for determining the stage of (pre-)germination and the quality of the seeds by measuring the quantity of chlorophyll fluorescence. The invention makes it possible to construct a sorter apparatus in which the seeds are selected on the basis of the quantity of chlorophyll. As the intensity of the chlorophyll fluorescence is directly related to the conversion of the chlorophyll into chlorophyll, it will now be possible to sort the seeds as to the stage of germination and quality. It is now possible to construct an analysis apparatus in which the chlorophyll fluorescence is measured of pre-germinating, pre-germinated, germinating and germinated seeds and a distribution is made of these fluorescence signals. Tests with pepper seeds are described.

Description: METHOD AND APPARATUS FOR DETERMING SEED QUALITY BY FLUORESCENCE The invention relates to a method for analysing and determining the quality of seeds during pre-germination, seeds that are pre-germinated and germinated seeds by irradiating the seeds with electromagnetic radiation. The chlorophyll formed in the seeds shows prompt fluorescence as a result of this irradiation. The present invention relates to an apparatus for analysing 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 an apparatus for sorting seeds, at least consisting of a feeder part for the seeds, a part for irradiating the seeds with electromagnetic radiation, a part for measuring the chlorophyll fluorescence signal re-emitted from the seeds, and a separation part which is active on the basis of the chlorophyll fluorescence signal returning from the seeds. By seeds a plant product is meant which is formed after sexual or non-sexual fertilisation of the ovule. By chlorophyll all appearances of the chlorophyll molecule are meant, such as the chlorophyll known as leaf green, protochlorophyll, etc. Measuring the amount of formed chlorophyll of seeds appears to be a good method to monitor the pre-germination process of seeds. It was found that during the pre-germination and germination stages of seeds chlorophyll is formed. As a result the quantity of chlorophyll in the seed increases during the pre-germination. The cotyledons present in the seed can colour green during the germination in the seed. The cotyledons consisting of protochlorophyll will convert into chlorophyll under the influence of light. - 2 - Up until now there have not been methods for monitoring the germination of the seed in a non-destructive manner. The chlorophyll content in the seed therefore is a good measure for the progress of the germination process of seeds. Seeds can roughly be classified into two categories, i) Seeds with a seed envelope which is reasonably transparent to red and far red light, such as seeds of white beans, maize, pepper and cucumber and ii) seeds with a seed envelope which is semi-transparent to red and far red light, such as seeds of cabbages, tomatoes and brown beans. With the invention considerable certainty can be obtained as to the onset of germination of the seed. Seeds that do not form chlorophyll or do not form chlorophyll until after a longer period of time during the pre-germination and under the influence of light will with considerable certainty not germinate or be of inferior quality. Quality is defined as germinative power, speed of germination, uniformity of germination, vigour, percentage of normal seedlings and health. Seeds which after a certain period of pre-germination form chlorophyll in the seed and are sorted on that basis, will germinate more evenly and yield less abnormal seedlings. With this new method seeds can be primed specifically. During the pre-germination the seeds are measured on the formation of chlorophyll. When the chlorophyll content has reached a certain value, the seeds are dried back. Said value is as measured with the known apparatus for non-germinated pepper seeds approximately 6 pA, and for pepper seeds that are about to germinate approximately 20 pA. In this way the seeds are synchronized in germination, so that after drying and re-moistening the seeds they germinate very uniformly and with a very high germination percentage. By using the described method seeds can furthermore be sorted out after having germinated in for instance the fruit (pepper or tomato) or in the field (barley) . In literature this also called pre- sprouting. These dried but already germinated seeds will no longer germinate or yield abnormal seedlings, or in the case of barley be detrimental to the malt process. By using the present invention the metabolism of the - 3 - seed during germination can be studied. Among other things this can be applied in the studying of the influence of external factors on germination such as temperature, the quantity of water available and light. A method for determining the amount of 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, 1 982. With a spectrophotometer a light beam of known wavelength is pointed onto the seed. After reflection the apparatus determines the fractional absorption of the light beam. Preferably the measurement is carried out in the 400-2400 nm range. The reflection spectrum is now a measure for the chlorophyll content. The content of chlorophyll is determined with the purpose of having a chlorophyll content that is as low as possible in the oil of the pressed seeds. The main disad- vantage of this method lies in the fact that measurements have to be carried out with different wavelengths, preferably 1 6, in order to obtain a good and reliable result. This method is too insensitive and too complicated to be used in an analysis or sorting apparatus. In the art it is known that by using chlorophyll fluorescence chlorophyll can be measured. In "Application of chlorophyll fluorescence to postharvest physiology and storage of mango and banana fruit and the chilling tolerance of mango cultivars", Asian Food Journal ( 1 987), 3(2), 55-59, R.M. Smillie, S.E. Hetherinton, R.N. Grantley, R. Chaplin and N.L. Wade used the chlorophyll fluorescence technique to measure the photosynthesis activity of fruit. They examined the changes in the photosynthesis activity during ripening and under the influence of low temperatures by measuring the chlorophyll fluorescence signal of the skin. The speed with which the chlorophyll fluorescence signal decreased during exposure to low temperatures was used to select varieties that are insensitive to cold. They do not mention the possibility to monitor germinating seeds in the course of time by measuring prompt fluorescence in order to thus monitor - 4 - metabolism of the seed. 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 fruit and leafs the chlorophyll fluorescence signal changes during the measurement, because the chlorophyll is photosynthetically active. This as opposed to the chlorophyll in a seed. Here the prompt quantity of chlorophyll fluorescence is measured, so instantaneous to the irradiation of the seed with electromagnetic radiation. The amplitude of the chlorophyll fluorescence signal is now a measure for the quantity of chlorophyll. The measuring method of Smillie in total requires one hour for adjustment of the plant material to the dark and some seconds for monitoring the changes in the chlorophyll fluorescence signal. The measuring method according to the invention can be carried out in a fraction of a second. The usual method for measuring photosynthesis activity in plant material comprises the use of the pulse amplitude modulation (PAM) fluorometer of U. Schreiber, described in "Detection of rapid induction kinetics with a new type of high frequency modulated chlorophyll fluorometer", Photosynthesis Research ( 1 986) 9:261 -272. The photosynthesis activity does not directly depend on the quantity of chlorophyll in plant material. The chlorophyll fluores- cence signal however directly depends on the quantity of chlorophyll. For determining the photosynthesis activity therefore a correction for the quantity of chlorophyll has to be made. This is done by calculating a quotient, so that said quotient is independent from the quantity of chlorophyll. The method described according to the invention, however, uses the measured quantity of chlorophyll fluorescence as measure for the quantity of chlorophyll. According to H.K. Lichtenthaler in "In vivo chlorophyll fluorescence as a tool for stress detection in plants ", Application of Chlorophyll Fluores- cence in Photosynthesis, H. K. Lichtenthaler (ed.) 1 988, 1 21 -142, Kluwer Academic Press, Dordrecht, the quantity of chlorophyll cannot be measured with photosynthesis measurements. The parameters measured - 5 - for calculating the photosynthesis by using chlorophyll fluorescence can even increase in signal strength when the quantity of chlorophyll decreases. This is the result of re-absorbtion of the emitted chlorophyll fluorescence decreasing because of the lower concentration of chlorophyll. In "Reflectance and chlorophyll fluorescence signatures of leaves" in Proceedings IGARSS'87 Symp. Ann. Arbor. Ml (USA) 1 8-21 May 1 987, 1 201 -1 206, H.K. Lichtenthaler and C. Buschmann show that around 690 and 730 nm the chlorophyll fluorescence signal of leaves is not linear to the quantity of chlorophyll. It was also demonstrated that a lower quantity of chlorophyll could emit a larger chlorophyll fluorescence signal. From these two articles it appears that the literature indicated that it was not to be expected that there was a relation between the quantity of chlorophyll and the chlorophyll fluorescence signal. Therefore it was not likely to an expert in the field of chlorophyll fluorescence that by using chlorophyll fluorescence the increase of the quantity of chlorophyll could be determined. Thus an object of the present invention is to provide a method which enables one to sort seeds as to quality and germination stage on the basis of the quantity of chlorophyll formed during the pre-germination, without destroying the seeds. Characteristic for the invention are the high sensitivity and the high speed with which the chlorophyll fluorescence in the inside of the seed can be measured. A further object of the invention is to provide an apparatus with which the seeds can quickly and accurately be analysed and sorted as to quality and germination stage. The method according to the invention is characterized in that the electromagnetic radiation has such a wavelength that the chlorophyll present in the seed shows prompt fluorescence, which fluorescence is measured. The method according to the invention can very well be performed in an - 6 - apparatus as mentioned in the preamble, which is characterized in that the electromagnetic radiation has such a wavelength that the chlorophyll present in the seed shows prompt fluorescence, which fluorescence is measured in the measuring part. The present invention is based on a fluorescence measurement which is very specific to the chlorophyll present. Other substances which influence the colour of the seed but do not fluoresce, will not contribute to the fluorescence signal. Also according to the invention small differences in the quantity of chlorophyll in the seed can be shown, because of the principle that a fluorescence measuring is very sensitive. According to the invention it now appears that a difference in chlorophyll content of individual seeds can be promptly shown, even when the en- velopes at first sight are entirely uniformly coloured and not transparent. In literature no data are known on non-destructive measuring the quantity of chlorophyll fluorescence in relation to the stage of germination of seeds before the root tip is visible. A suitable method for measuring the chlorophyll content comprises irradiating at least a part of the chlorophyll molecules with electromagnetic radiation, preferably with a wavelength between 400 and 700 nm, as a result of which at least a part of the chlorophyll molecules are electronically excited. The excited molecules mainly loose their energy by heat dissipation and for about 3% by emission of fluorescence which preferably is measured between 600 and 800 nm. When according to the invention the intensity of the chlorophyll fluorescence of each seed separately is measured, the seeds can be analysed and sorted as to the stage of pre-germination and quality. The invention is very sensitive, entirely non-destructive and very quick. These are the characteristics of the invention which make it possible to manufacture a sorting apparatus with which seeds can be selected on the - 7 - basis of the quantity of chlorophyll fluorescence. Because the quantity of fluorescence by chlorophyll has a direct relation to the conversion of protochlorophyll into chlorophyll in the seed, sorting as to the stage of pre- germination and quality is now possible. The method according to the invention can also be used for scoring seeds as to the start of germination during a germination test which is carried out to assess the quality of the seeds. In a germination test it is important to predict at a very early stage already whether the seeds will yield a seed- ling. The present germination tests are mostly subjective, because the seeds are scored visually on the emergence of a root tip. This observation depends on the observer and is labour-intensive. By using the present invention seeds can be scored on germination on the basis of the chlorophyll content, so that the germination test can be automated and therefore be objectively carried out. Another advantage of the present invention is that the seeds can be scored in a much earlier stage of germination, which means a gaining of time in carrying out germination tests. It is of course also possible to analyse and select the seeds on the formation of chlorophyll in a further stage of germination, namely when other plant parts than the root emerge from the seed. Examples thereof are the hypocotyl and the cotyledons. For many kinds of seeds these plant parts contain chlorophyll. In an analogous manner as described above the seeds can be sorted and analysed on the appearance and formation of chlorophyll. The present invention can be applied on many kinds of seeds of horticultural crops, agricultural crops, ornamental plants and forestry crops. The invention works with seeds in which the quantity of chlorophyll changes during the germination process and in which the seed envelope is (semi)transparent to the excitation light for the chlorophyll and the fluorescence of chlorophyll and with seeds in which the plant parts that emerge from the seed contain chlorophyll. - 8 - It is preferred to carry out a chlorophyll measuring in an apparatus as shown in figure 1 . This is a simple embodiment of the apparatus. The light of a LED with a maximum emission at 650 nm is filtered by a narrow band filter at 656 nm. The beam splitter reflects about 50% of the LED light in the direction of the lens, which concentrates the light onto the seed. In this way at least a part of the chlorophyll molecules is electronically excited. At least a part of the excited chlorophyll molecules falls to the ground state while emitting fluorescence. At least a part of the chlorophyll fluorescence is captured with the same lens. The filter ensures that predominantly 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% at a suitable frequency. In this way the fluorescence is also modulated with the same frequency. The alternating current of the photodiode is converted in the lock-in amplifier into a signal that is propor- tional to the intensity of the fluorescence. Said detector can be built in in a sorting apparatus. After measuring the chlorophyll fluorescence for instance a valve can subsequently be operated with an electronic circuit such as a micro processor, which valve takes out the seeds from the main stream that have a higher or lower signal than a pre-determined value. The sorting from the main stream with a valve can take place with any known principle, such as an air stream, liquid pulse or mechanical valve. It is pointed out that sorting can be performed on seeds which are in the air, but also for seeds that are contained in a liquid. Sorting in the liquid can for instance take place in the germination liquid, in which the seeds are sorted on germination in a continuous process. Analysing seeds on the distribution of the quantity of chlorophyll in a sample of a seed batch can be performed with the same preferred apparatus for sorting seeds, but now no sorting has to take place. The chlorophyll fluorescence signals of the seeds are for instance stored in the memory of a micro processor or on an optical or magnetic disc. The measured chlorophyll fluorescence signals can for instance be plotted in - 9 - the form of a table or in the form of a diagram or histogram, so that the - distribution of the chlorophyll fluorescence signals becomes visible. Analysis of the pre-germinated or germinating seeds can also be carried out with an electronic camera. With the image of the camera the chlorophyll fluorescence images of several seeds are measured at the same time. After that a survey can be given of the distribution of the chlorophyll fluorescence signal of all measured seeds, for instance in the form of histogram. Furthermore the germination of the seeds in the course of time can be monitored with this preferred apparatus. This can for instance be carried out by scoring seeds at regular intervals that have a value of the chlorophyll fluorescence signal that is above a certain threshold value. The person skilled in the art will see that in the above-mentioned preferred sorting and analysing apparatus, the LED can be replaced by a lamp with filters or by a laser. Furthermore it is also possible to use a photo multiplier or an electronic camera for the photodiode. For example the camera can be used to make chlorophyll fluorescence images of a number of seeds, in order to make the distribution of the chlorophyll therewith, or chlorophyll fluorescence images of the individual seeds. The invention can also be used in a seed sorter apparatus. It can be built in in all kinds of sorter apparatus. The invention can particularly be used in the known colour sorter apparatus. The light source can be replaced by the electromagnetic radiation source (for instance a laser) and the colour measurer can be replaced by the photo diode. It is also possible to measure seeds in the germination liquid during pre-germination. The invention will now be elucidated on the basis of a number of examples. Examples - 10 - ln the following examples the chlorophyll fluorescence signal of the inside of the individual seeds was measured by using of the method according to the invention. Tests were carried out with pepper seeds (Capsicum an- nuum). Example 1 By using the present invention the chlorophyll fluorescence signals of 200 pepper seeds (Capsicum annuum) were measured individually and on both sides of the seeds at different points in time during germination. The germination tests were carried out on filter paper moistened with a solution of water with 0.2% KNO 3 in plastic trays in a germination cabinet at an alternating temperature of 20°C-30°C, at 20°C in the dark ( 1 6 hours) and at 30°C in the light (8 hours). After 1 36 hours the seeds were visually inspected on the emergence of a root tip. In figure 2 the results of the measurings of the germinated and non-germinated seeds are plotted. In panel A the averages of 20 non-germinated and 180 germinated seeds are plotted. The increase of the chlorophyll fluorescence signal of the germinated seeds shows an exponential behaviour. The non-germinated seeds however, hardly increase in size of signal. In panel B for 2 germinated and 2 non-germinated seeds the signals are individually plotted. After 96 hours the 2 seeds had germinated (germ). In this new measuring method for germinating seeds the germination process can be measured and monitored by using chlorophyll fluorescence. Example 2 By using the present invention the chlorophyll fluorescence signals of pepper seeds (Capsicum annuum) were measured individually and subse- quently classified into 2 classes on the basis of the distribution of fluorescence. In this example instead of a LED a laser was used. The low class is for seeds with a chlorophyll fluorescence signal below 225 pA and the high - 1 1 - class for seeds with a signal over or equal to 225 pA. The distribution in terms of percentage in the 2 classes is given in table 2. The germination tests were carried out on filter paper moistened with a solution of water with 0.2% KNO 3 in plastic trays in a germination cabinet at an alternating temperature of 20°C-30°C, at 20°C in the dark ( 1 6 hours) and at 30°C in the light (8 hours) . The seeds and seedlings were evaluated according to ISTA ( 1 996) International rules for seed testing, Seed Science and Technology 24. In this example about a third of the seeds had already been germinated. This was visible because the seeds showed a brown root tip. Pepper seeds are contained in a fruit which after ripening of the seeds has a high moisture content. Pepper seeds can as a result of their moist environment still be physiologically active. After having completely ripened, pepper seeds may germinate in the fruit. After drying the seeds, the seeds that had already germinated in the fruit will no longer germinate or yield abnormal seedlings. The seeds in the class with low fluorescence gave a good initial development of 84%, whereas the initial development of the high class was 54%. Compared to the control this was an improvement of 17%. In this choice of classes it apparently is so that the low class is of a better quality than the high class. - 12 Table 2 Quality of pepper seeds without sorting (control) and after sorting into 2 classes by means of chlorophyll fluorescence chlorophyll fluorescence low high control distribution (%) 54 46 100 normal seedlings (%) 84 54 67 abnormal seedlings (%) 1 4 3 bad seedlings 1 2 39 29 non-germinated seeds 3 3 1

Claims: ■ 1 3- Claims 1 . A method for determining the quality and pre-germination and germination stages 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 the chlorophyll present in the seed shows prompt fluorescence, which fluorescence is measured. 2. An apparatus for analysing pre-germinating, pre-germinated, germinating and germinated seeds, at least consisting of a part for irradiating the seeds with electromagnetic radiation and a part for measuring the signal returning from the seeds, characterized in that the electromagnetic radiation has such a wavelength that the chlorophyll present in the seeds shows prompt fluorescence, which fluorescence is measured. 3. An apparatus for sorting pre-germinating, pre-germinated, germinating and germinated seeds, at least consisting of a feeder part for the seeds, a part for irradiating the seeds with electromagnetic radiation, a part for measuring the signal returning from the seeds, and a separation part which is active on the basis of the signal returning from the seeds, characterized in that the electromagnetic radiation has such a wavelength that the chlorophyll present in the seeds shows prompt fluorescence, which fluorescence is measured in the measuring part. 4. A method according to claim 1 , characterized in that the irradiated electromagnetic radiation has a wavelength between 400 and 700 nm and in that the fluorescence is measured between 600 and 800 nm. -14- 5. An apparatus according to claim 2 or 3, characterized in that the irradiated electromagnetic radiation has a wavelength between 400 and 700 nm and in that the fluorescence is measured between 600 and 800 nm. 6. An apparatus according to claim 2 or 3, characterized in that the returning signal of the seeds is measured with a photodiode, photomul- tiplier or electronic camera. 7. An apparatus according to claim 2 and 3, characterized in that the irradiated electromagnetic radiation is generated by a laser of LED.

Inventors: BINO RAOUL JOHN, JALINK HENDRIK, VAN DER SCHOOR ROB, BINO, RAOUL, JOHN, JALINK, HENDRIK, VAN DER SCHOOR, ROB
Applicants: BINO RAOUL JOHN, CPRO DLO, JALINK HENDRIK, SCHOOR ROB V D, BINO, RAOUL, JOHN, CENTRUM VOOR PLANTENVEREDELINGS- EN REPRODUKTIEONDERZOEK (CPRO-DLO), JALINK, HENDRIK, VAN DER SCHOOR, ROB
Application year: 1999
Main classification: G01N33
IPC codes: A01C1, A01G7, G01N21, G01N33