Systems and associated methods for testing viability and / or for identifying and differentiating varieties / varieties for genebank conservation of plant genetic resources and their uses

Abstract

The present invention relates to a system for the non-destructive identification and differentiation of the viability and / or variety / variety of plant genetic resources, said system comprising at least the following: a walled space, the temperature and humidity of which are controlled and which includes at least one location suitable for measuring the plant genetic resources to be tested and suitable for placing a tray for supporting the plant genetic resources; a thermal camera, the thermal camera being positioned facing a location suitable for placing one or more trays suitable for storing the plant genetic resources to be tested; a computer suitable for controlling the thermal camera and for storing and, if necessary, processing the records made by the thermal camera; and a unit for drying the plant genetic resources to be tested; an apparatus for cooling and storing the plant genetic resources to be tested; and one or more trays suitable for measuring the plant genetic resources to be tested. The present invention also relates to a method for the non-destructive identification and differentiation of the viability and / or variety / variety of plant genetic resources and uses thereof.

Description

Technical Field

[0001] The present invention relates to a system for the viability of plant genetic resources and / or the identification and discrimination of their varieties / variety groups in the agricultural field, and said system is suitable for non-destructive (non-invasive) inspection of plant genetic resources. The present invention further relates to a method for identifying and discriminating plant genetic resources and the use of a method independent of the method (by conventional breeding methods or biotechnological or various other methods) by which said system and varieties are produced.

Background Art

[0002] For our Earth project and humanity's access to food in terms of function, it is essential to conserve all elements of the flora, including wild plant species and cultivated plants. In addition to conservation, sustainable use is important as provided by the convention adopted by the United Nations in June 1992 (Convention on Biological Diversity). According to the above, sustainable use is defined as "the use of components of biological diversity in a way and at a rate that does not lead to the long-term decline of biological diversity (thus maintaining its potential to meet the needs and aspirations of present and future generations)". The Food and Agriculture Organization of the United Nations (FAO) has repeatedly pointed out the fact that there is a decline in the diversity of plant genetic resources for food and agricultural purposes, and according to data, more than three-quarters of the varieties used in agriculture have disappeared from cultivation (FAO SoWPGR-2, 2010, Second Report on the State of the World's Plant Genetic Resources for Food and Agriculture; <https: / / www.fao.org / 3 / i1500e / i1500e00.pdf)

[0003] ​​In other words, the genetic preservation of wild and cultivated plant species in gene banks plays an important role in ensuring long-term food safety worldwide. The conservation of agricultural seeds in cold storage began in the 1970s. The methods and conditions for gene bank conservation are detailed in the Hungarian Genebank Standards (1994) and the International Genebank Standards (FAO 2013). Currently, seed bank storage temperatures are generally -18°C ± 3°C at 4-6% humidity using moisture-proof packaging, resulting in a life expectancy of over 100 years (for cereals and pulses) (FAO; Genebank Standards: Food and Agriculture Organization of the United Nations, Commissioner for Genetic Resources for Food and Agriculture, 14th Plenary Session, Rome, 2013). Several methods are available for conserving plant genetic resources. For arable crops and vegetables, the most common method is seed bank conservation. In addition, for certain groups of crops (fruits, climbing plants, certain perennial plants), in-stock gene bank conservation is the main method (gene bank plantation). Furthermore, several new methods are available for the conservation of plant genetic resources (in vitro meristem cultivation, cryopreservation, etc.).

[0004] The key to seed bank conservation is storing a sufficient number of seeds. In fact, seed bank conservation consists of several tasks that must be accomplished. In addition to conservation, the basic tasks of all gene banks include documentation, propagation / replica sowing, dissemination, and viability testing using standard methods. The most commonly used viability test by gene banks is based on seed germination, but after germination, germinated seeds are no longer stored as genetic resources, meaning that the amount of seeds stored during these tests is reduced. A sufficient amount of seeds for gene bank conservation is usually 1,500 to 2,000 seeds, but gene banking practices vary for both positive and negative methods. Viability testing is necessary for genebank conservation because non-viable or poorly germinated seeds are not suitable for the conservation of genetic diversity and lead to the deterioration of the genetic lineage of the variety (WALSH, DG F, WALDREN, S., MARTIN, J; Monitoring seed viability of 15 species after storage in the Irish Endangered Plant Genebank, Biology and Environment - Proceedings of The Royal Irish Academy; 2003, 103 (2), 59-67). The inventors note that germination results alone do not necessarily provide a direct indication of viability. At the same time, even though it is generally known that viability does not directly imply germination ability, germination is still considered an important viability test according to genebank protocols (TIHANYI Z., TOMPA K.; Forest breeding and seedling cultivation: practice, University of Forestry and Wood Industry, Sopron, 1985, 209). In addition to germination, there are numerous viability testing methods available, the number of which increases each year as science and technology advances.

[0005] According to the current state of the art, several devices and methods for viability testing are known for testing plant genetic resources, essentially meaning genebank testing methods of seed lots.

[0006] An apparent viability test is associated with the name Zelenchuk, during which seeds that appear intact are compressed with moderate pressure using a finger, needle, or other instrument; if the seeds withstand, they are considered viable. However, the use of this method is based on subjective reasons (ZELENCHUK, TK; Catalogue of viable seeds in low-lying marsh soils of the Lviv Oblast, Byull. Mosk. Ova Ispyt. Prir., Otd. Biol., 1961, 66 (3), 77-92).

[0007] Pruning is primarily used for important forest species that are widely sown to identify seed health. This method is used to separate weak, abnormally growing, and contaminated seeds (SUSZKA, B., MULLER, C., BONNET-MASIMBERT, M., Seeds of deciduous forest trees from collections for sowing, Mezogazda Kiado, Budapest, 2008, 291).

[0008] The ISTA Tree Seed Committee (ISTA (International Seed Testing Association), 2008, International Rules for Seed Testing, Bassersdorf, Switzerland) has developed a method to identify the viability of primarily tree seeds. The method consists of removing the embryos or seed coats from the fruit after soaking and keeping them in conditions suitable for germination and long enough to show signs of non-viability. Viable embryos are those that continue to develop or remain fresh until the end of incubation.

[0009] The measurement of glutamic acid decarboxylase activity (GADA test) to identify viability was developed by Ellis et al. The essence of their method is that under the influence of the enzyme glutamic acid decarboxylase, healthy seeds produce carbon dioxide, the amount of which can be measured. Seed samples that produce more carbon dioxide have higher viability (ELLIS, RH, HONG, TD, ROBERTS, EH; Seed Technology Handbook for Genebanks, Handbooks for Genebanks n. 2, International Plant Genetic Resources Institute, 1985a, 210; ISBN: 978-92-9043-118-3, ISBN: 92-9043-118-0; ELLIS, RH, HONG, TD, ROBERTS, EH; Seed Technology Handbook for Genebanks, International Board for Plant Genetic Resources. 1985b, 456.; ISBN: 978-92-9043-119-0, ISBN: 92-9043-119-9).

[0010] In the case of vital staining, the detection of viability is based on the fact that polymeric dyes (e.g., indigo carmine, fuchsin, Evans blue) cannot penetrate live cells at all due to the impermeability of the limiting membrane. In contrast, dead cells with damaged cell walls are stained because the dye molecules easily penetrate into them. Nyebuljov (1925) was the first to demonstrate viability using indigo carmine and, based on this, to infer viability (NYEBULJOV DN; On the determination of germination ability without a germination test, Jard. Botan. Inst. Essias Semences Leningrad, Ann. Essais Semences 1925, 7, 31-35).

[0011] Lakon (1942) based the above-mentioned Nebuljov's method on the tetrazolium method (LAKON, G.; Topographical Demonstration of the Germination Ability of Grains with Tetrazolium Salts; Ber. Deut. Bot. Ges. 1942, 60, 299-305). Tetrazolium salts are used for viability tests, and the method provides information about the respiratory state. In the presence of oxygen, living cells reduce various solutions, and tetrazolium salts are therefore considered redox indicators. The disadvantages of the tetrazolium method are that it is very difficult or impossible to perform on small seeds, it does not detect dormancy, and it leads to the complete destruction of the test plant unit.

[0012] Ivanov (1950) used a 0.1% acid fuchsin solution to detect viability (IVANOV, VJ, (1950), Identification of the germination ability of seeds by means of fuchsin, Селек. и Семeн. 17. 60-61).

[0013] The Ivanov method was further developed by Effmann (1966) and Specht (1967) by increasing the concentration of the fuchsin solution to 0.5%, thereby reducing the identification time. Further time and labor savings were achieved by batch sampling, known as "continuous analysis" (Effmann, H., Application of the acid fuchsin method to the identification of grass viability, Thaer-Arch. 1966, 10, 205-212; Effmann, H., Specht, G., Identification of grass seed viability by the acid fuchsin method (continuous analysis), Proc. Int. Seed Test. Ass., 1967, 32, 27-47).

[0014] Gaff and Okong'o-Ogala (1971) used solutions of different concentrations of Evans blue to determine viability (DF, OKONGO-OGALA, O.; The use of impermeant content to test cell viability, Journal of Experimental Botany; 1971, 22, 756-758).

[0015] X-ray examination has been recommended for detecting viability and insect damage in woody plants that are difficult or take a long time to germinate (e.g., Carpinus betulus, Fraxinus spp.) (SZABO LASZLO GY. (ed.); Fundamentals of Seed Biology, Akademiai Kiado, Budapest, 1980, 391). The method is rapid and does not damage the seeds. The test is carried out by placing the seeds on a graduated plate on a film. The exposure is brief, usually lasting a few seconds, and the duration is determined by the woody species, the thickness of the seed, and the sensitivity of the film. Tests using contrast agents are also common. The embryonic tissue of dead seeds absorbs contrast agents that are impervious to X-rays (SMITH, RD, DICKIE, JB, LININGTON, SH, PRITCHARD, HW, PROBERT, RJ, EDS.; Seed Conservation: Turning Science into Practice, Chapter 24. Kew, UK, Royal Botanic Gardens, 2003; SUSZKA, B., MULLER, C., BONNET-MASIMBERT, M., From Collection to Sowing of Deciduous Forest Trees, Mezogazda Kiado, Budapest, 2008, 291).

[0016] WO 08150798 A1 describes an automated, contamination-free method and associated system for sampling and testing seeds. The system consists of a seed loading station, an imaging station that collects image data about the seeds, and a seed orientation station that independently positions and holds each seed in a desired orientation. The method according to WO 08150798 A1 is not considered non-destructive because tissue samples are taken from each seed.

[0017] EP 3097398 A1 describes a seed tissue sampling method suitable for genotyping. It is stated that this method preserves the viability of the seeds, despite the tissue samples taken from the seeds. The tissue samples are taken by manual or semi-automatic drilling of the test seeds.

[0018] An automated system for collecting tissue samples from seeds is described in WO 18236874 A1. Seed tissue sampling is performed so as to preserve the seeds' viability for germination, but the testing method is not truly non-destructive due to the tissue sampling. According to WO 18236874 A1, genotyping is a method for identifying whether genetic variants are present in the tested seeds. There is no mention of a thermal camera method for distinguishing between variants, and the test in WO 18236874 A1 is clearly based on examining the seed's DNA.

[0019] The viability of dormant seeds is examined in patent application US 2020267890 AA using infrared (IR) contrast imaging. Spectral illumination is used to heat the seeds, resulting in contrast IR images that visualize the metabolism of the seed's embryo. The presence of CO2 from plant seed metabolism infers whether the seed is viable (i.e., alive) or not (the CO2 band provides a characteristic signature in the IR spectrum).

[0020] WO 18015495 A1 deals with the prediction of germination of maize seeds by NMR. The method steps are: a) measurement of NMR parameters for maize seeds, b) prediction of germination profile based on NMR measurements, applying a mathematical model.

[0021] WO 2020009978 A1 describes that seed viability is traditionally measured in laboratories using manual labor. WO 2020009978 A1 describes an automated seed planting and evaluation system. The automated system eliminates human error. According to the description, seeds are planted in all cases, which means that the method is not called non-destructive.

[0022] Patent application IN 202231013203 uses an artificial intelligence (AI) based computing system to identify seed quality and germination patterns.

[0023] Seed imaging and related methods are covered by WO 19178238 A1. An imaging and analysis unit (referenced 14 in the specification) collects image data of the test seeds and uses optimized image analysis algorithms to identify seed characteristics (color, size, shape, texture, internal composition, mass, volume, moisture content, and chemical composition). The specification states that by combining two or more imaging diagnostic methods, a more accurate prediction of seed quality can be obtained. In WO 19178238 A1, seed viability is investigated in terms of seed surface contamination, disease, and composition.

[0024] Flowchart 2B of WO 2019173606 A1 shows a block diagram of a method for assessing seed viability, which uses spectral images to infer whether seeds have poor sowing potential. The specification of WO 2019173606 A1 discloses that the spectral imaging device includes a multi- and hyperspectral imager. The spectral images and seed viability are stored in a database and used for spectral code search and / or artificial intelligence-based pattern recognition algorithms.

[0025] WO 2016084452 A1 describes a method for sorting seeds of coniferous plants. The method includes the steps of: a) irradiating seeds with near-infrared light, measuring the spectrum of near-infrared light reflected from each seed, and obtaining reflectance data at each wavelength; b) obtaining reflectance data at two specific wavelengths in the range of 750 to 2200 nm; c) calculating a ratio of retrievable reflectance for each seed; and d) comparing the ratio calculated in step c) with a threshold value for the ratio of reflectance at the two specific wavelengths to identify whether the seed has a viable embryo (wherein the threshold value is used to test whether the seed has a viable embryo). The specification does not refer to measurement parameters or describe sample preparation, and non-cereal / agricultural crop seeds are tested.

[0026] Example 1 of WO 09128998 A1 describes a test relating to seed viability, which is destructive, i.e., the germination of the seeds actually occurs.

[0027] The invention described in WO 2012048897 A1 relates to a method for classifying seeds based on IR spectroscopy. Paragraph

[0085] of WO 2012048897 A1 states that in a specific example of the invention, IR spectroscopy, particularly near-IR spectroscopy, is used to identify free space within seeds, but the specification is silent on the differentiation of varieties / variety groups and on testing seed viability.

[0028] WO 13012860 A1 describes a method and system for modeling seed structure, identifying the structural morphology of seeds / seedlings through spectral analysis. Seeds to be tested are placed one by one in sample containers in a tray, as shown in FIG. 1 . The tray is placed in the analyzer by a robotic arm. The seeds are incubated before measurement; one way to do this is to immerse the seeds in water before measurement. Furthermore, according to the description in WO 13012860 A1, the primary focus is on seed classification, rather than non-destructive viability testing.

[0029] Patent application US 2015135585 AA describes a method and apparatus for non-destructive testing of seeds. US 2015135585 AA lists the following in relation to non-destructive measurements: "NIR, IR, NMR, X-ray, hyperspectral, UV and RGB imaging, although not exclusively." In this case, sampling that does not affect viability is also required, and thus the method is not considered 100% non-destructive.

[0030] The method described in WO 10000266 A1 is also used to classify seeds according to quality. The grading method comprises the following steps: a) obtaining an optical spectrum of the seeds in one or more spectral regions, and b) identifying the quality category of the seeds by comparing the data obtained in step a) with control spectra. The method is particularly suitable for classifying seeds of pine trees of the genus Abies.

[0031] WO 199742489 A1 discloses a method for identifying the maturity and quality of seeds by electromagnetic radiation. Upon irradiation (the radiation source is an LED or laser), chlorophyll in the seeds generates a detectable fluorescent signal. The invention also relates to a device for preserving seeds.

[0032] WO 2001089288 A1 relates to seed classification. The classification is based on spectral data recorded from the seeds. The obtained spectral data is compared with reference spectral data and used to estimate viability. Prior to measurement, the seeds are pretreated with water and partially dried. During the measurement, the seeds are irradiated with light in the wavelength range of 180-2500 nm, i.e., an irradiation method is used here to classify the seeds.

[0033] Patent EP 1188041 B1 relates to a method for determining the germination ability of seeds, in particular cereals. In step 1, the seed grains are irradiated with light and at the end of the irradiation method, at least one characteristic value of the light emitted by the seeds is measured. In step 2, at least one characteristic value of the light emitted by the seeds without an external light stimulus is measured; from this characteristic value, the germination ability is inferred. This prior art technique tests viability, but under the influence of an external stimulus (irradiation).

[0034] At the 4th Ecotoxicology Conference (2014), a physical method for measuring the differentiation of genetically modified maize from non-modified maize was presented (Jozsef Berke, Hajnalka Banati, Borbala Baktay, Otto Szalkovszki, Rita Szabo, Eszter Takacs, Bela Darvas, Ferenc Gyulai; Possibility of differentiation by thermal imaging of seeds from the progeny of the maize variety MON 810 BT; IV. Ecotoxicology Conference; November 2014). During the presentation, it was concluded that with further development of the method, differentiation of groups within the main classes is expected in the case of maize.

[0035] Kranner et al. examined seeds using a thermal camera to estimate viability. They found that infrared thermography can detect biophysical and biochemical changes associated with hydration and germination. The cooling phenomenon observed in the early phase of the thermal profile is explained as the result of the dissolution of low-molecular-weight carbohydrates. According to Kranner et al., the rate of production of these "cooling" components varies over time as a function of seed viability (KRANNER, I., KASTBERGER, G., HARTBAUER, M., PRITCHARD, H.; Noninvasive Diagnosis of Seed Viability Using Infrared Thermography; Proceedings of the National Academy of Sciences of the United States of America; 2010, 107. 3912 7. 10.1073 / pnas.0914197107).

[0036] In connection with this publication, the "cooling effect" observed during the evaluation of the measurement results was, in our opinion, caused by steam condensing on the surface of the seeds, and therefore the measurement method cannot be considered reliable as the measurements were not carried out under constant environmental conditions.

[0037] Men et al. conducted a viability test on peas using thermal imaging (MEN, S., YAN, L., LIU, J., QIAN, H., LUO, Q.; A taxonomic method for assessing seed viability using infrared thermography; 2017, Apr Basel 12; 17(4), 845. doi: 10.3390 / s17040845. PMID: 28417907 Free PMC article). Seed samples were individually inspected for temperature and viewing distance every 5 minutes at 24°C for 5 days of germination. In this case, germination was also performed, making the method time-consuming.

[0038] Fernandez et al. studied peas and lichens under controlled conditions (FERNANDEZ-MARIN, B., BUCHNER, O., KASTBERGER, G., PIOMBINO, F., GARCIA-PLAZAOLA, J.I., KRANNER, I.; Non-invasive diagnosis of seed and lichen viability by infrared thermography under controlled environmental conditions; Plant Methods; 2019 Dec 5;15:147). Measurements were performed in an incubator with adjustable humidity and gas composition (suitable for lichens). Over a range of temperatures, the "thermal fingerprints" of peas were observed during water immersion at relative humidity levels of 30% and 60% for 96 hours. Then, outside the measurement chamber, the developing seeds were germinated on moist filter paper under an infrared camera. Infrared images were recorded for 4 days for seeds at a rate of 1 frame per minute and for 170 minutes for lichens at a rate of 1 frame per second. Humidity was continuously monitored throughout the measurements. However, measurements were performed over a long period, essentially recording the germination (water uptake) process for 96 hours in the case of seeds. Thus, on the one hand, the tested seeds were not used for further analysis, and on the other hand, these seeds were considered lost from the genebank's point of view.

[0039] ElMasry et al. provide an overview of research into the use of thermal imaging systems on seeds, including estimating seed viability, detecting fungal and insect damage, assessing seed damage and contamination, and identifying and classifying seed viability. The paper emphasizes that measurements using thermal imaging systems are non-contact and therefore do not damage the seeds tested, unlike destructive laboratory tests (which are time-consuming and labor-intensive). The authors investigated the biochemical and biophysical properties of pea and lettuce seeds, and found that seed water uptake and the rate of decomposition of their nutrient reserves allowed them to identify differences between healthy and senescent seeds. Healthy and senescent seeds were tested for 24, 48, 72, 96, 120, 114, and 168 hours. The methods mentioned in this publication are only briefly described and do not include a full detailed method of measurement (ELMASRY, G., ELGAMAL, R., MANDOUR, N., GOU, P., AL-REJAIE, S., BELIN, E., ROUSSEAU, D.; Emerging thermal imaging techniques for seed quality assessment: principles and applications; Food Res Int. 2020 May; 131, 109025. doi: 10.1016 / j.foodres.2020.109025. Epub 2020 Jan 22. PMID: 32247450 Review).

[0040] In their paper, Liu et al. state that seed viability testing can be greatly facilitated by noninvasive, high-resolution infrared thermography (LIU, L., WANG, Z., LI, J., ZHANG, X., WANG, R.; Noninvasive Analysis of Seed Vitality by Infrared Thermography, Plants (Basel); 2020 Jun 19; 9(6):768. doi: 10.3390 / plants9060768. PMID: 32575514 Free PMC article). Siberian elm seeds were collected from 30-year-old trees, dried at room temperature for 4 days, and stored in nylon bags at -20°C until testing. The following varieties were also tested: Chinese prickly pear, white acacia, soybean, rice, corn, and tomato. The seeds were stored at 4°C for approximately one year. The Siberian elm was tested after 0, 24, 48, 72, 96, and 120 hours. For the experiment, the seeds were artificially aged and then germinated, during which they were photographed with a thermal camera, although the method is not called non-destructive.

[0041] investigated the "infrared thermal fingerprint" of onion seeds (THAKUR, M., SHARMA, P., ANAND, A., PANDITA, VK., BHATIA, A., PUSHKAR, S.; Raffinose and hexose sugar composition during germination correlates with the infrared thermal fingerprint of onion (Allium cepa L.); Front Plant Sci. 2020 Oct 6; 11:579037). Measurements were taken at 8, 12, 16, 20, 24, 28, 32, 36, and 52 hours, during which time nearly constant environmental conditions were maintained (making the method very expensive). Summary of the Invention [Problem to be solved by the invention]

[0042] According to the current state of the art, there is no known method for identifying seed viability and providing more accurate information about seed viability than prior art methods without externally damaging or stimulating the seeds (which would reduce the amount of seeds used for this purpose during genebank studies), and which is also suitable for quality assurance testing of commercially available seeds. Furthermore, For genebank conservation There are no known systems and methods for seed testing that are suitable for testing viability as well as for non-destructively identifying and differentiating varieties / varietals.

[0043] Because the amount of seeds stored in genebanks is limited, it is necessary to use non-invasive, non-destructive viability tests, so that even after such tests, the tested seeds can be stored in the genebank and the same seeds can be used for repeated testing. Therefore, methods using physical effects (incision, compression, etc.) and chemicals (paints) or radiation must be avoided, because after such methods are applied, the tested seeds are no longer suitable for genebank storage. Non-invasive and non-destructive tests that require long periods of time must also be avoided, because such tests must be performed at genebank storage temperatures (typically -20°C), but not at temperatures higher than the temperature at which long-term, irreversible life processes begin in the tested seeds (continuing genebank conservation after such tests, and returning seeds to frozen seed storage after drying, is not permitted). In addition to viability tests, a key challenge in genebank implementation is the identification and differentiation of varieties and cultivars, for which the prior art offers solutions, including destructive and morphological methods in the open field. [Means for solving the problem]

[0044] Therefore, the object of the present invention is to For genebank conservationThe present invention provides a system and related methods for identifying viability, which system does not suffer from the above-mentioned problems of the state of the art and is also suitable for identifying and differentiating varieties and cultivars. Our goal is to provide a novel method and system for its implementation, which provides a more accurate picture of the viability of seeds stored in genebanks and other seeds than is available in the state of the art, where the seeds are not damaged during testing and are still suitable for genebank conservation and further testing after testing.

[0045] We have recognized that measuring seeds stored in genebanks (e.g., at -20°C) using a thermal camera testing system allows for highly accurate and non-destructive identification of seed viability, thereby ensuring the continued suitability of the tested seeds for further genebank conservation. We have also found that this system and method is suitable for identifying and differentiating varieties / varietals, as well as for identifying the viability of plant genetic resources, regardless of the method by which the varieties are developed (traditional breeding methods or novel biotechnological methods). We have further found that the method and system according to the present invention are suitable for testing not only seeds but also other plant genetic resources.

[0046] Therefore, the present invention provides a method for producing a plant genetic resource, preferably a seed, according to the present invention. For genebank conservation The system and associated method for testing viability and / or for identifying and separating varieties / varieties is adapted to test plant genetic resources, preferably seeds and / or varieties / varieties to be identified and separated. For genebank conservation The viability of plant genetic resources, preferably seeds, can be non-destructively identified and reliable information provided about them, and is based on the unexpected realization that the tested plant genetic resources, preferably seeds, retain their suitability for further genebank conservation, further tests, including further viability tests.The system for testing the viability of plant genetic resources, preferably seeds, and / or for identifying and differentiating varieties / varieties, and the related method according to the present invention, is based on the photographing of the tested plant genetic resources, preferably seeds, by a thermal camera during a short heating period, and the measurement parameters are selected in such a way that the measurements performed are reliable.

[0047] In other words, the essential advantage of the solution according to the invention compared to state-of-the-art systems and associated methods for seed viability and / or variety / variety differentiation is that the system and method for identifying and selecting plant genetic resources, preferably seed viability and / or its variety / variety differentiation according to the invention provides accurate results in terms of both viability and variety / variety identification and differentiation of plant genetic resources, preferably seeds, regardless of plant species and seed form, without requiring lengthy sample preparation and germination (which should be cost-effective), and furthermore the tested plant genetic resources, preferably seeds, maintain their suitability for further conservation in genebanks. [Brief explanation of the drawings]

[0048] [Figure 1] Shown is a graph of average intensity versus time recorded during a thermal camera test on sunflower seeds (FIR means far infrared, i.e. in the far infrared range), the graph clearly shows three distinct warming states (transient state; steady state; saturated state). [Figure 2] Average intensity curves of thermal camera measurements as a function of time for filled and empty sunflower seeds are shown; the curves for the two types of seeds are clearly separated from each other, thus easily identifying the viability of sunflower seeds of unknown viability. [Figure 3] 1 shows a graph of average intensity versus time recorded based on thermal camera measurements of kidney bean seeds. [Figure 4] 1 shows a graph of average intensity versus time recorded based on thermal camera measurements of corn seeds. [Figure 5] 1 shows a graph of average intensity versus time recorded based on thermal camera measurements of einkorn seeds. [Figure 6] 1 shows a graph of average intensity versus time for non-germinated "waste" and germinated seeds of kidney beans considered "waste" as recorded from thermal camera measurements. [Figure 7]1 shows a graph of average intensity versus time for hybrid and genebank maize seeds measured with a thermal camera to separate varieties. DETAILED DESCRIPTION OF THE INVENTION

[0049] The present invention relates to the development of plant genetic resources. For genebank conservation A system for non-destructive identification and differentiation of viability and / or varieties / variety groups, said system comprising at least: a walled space (i) in which the temperature and humidity of the space are controlled; ii) the space includes one or more locations suitable for placing trays for supporting the plant genetic material suitable for measuring the plant genetic material to be tested; a thermal camera, the thermal camera being positioned facing a location suitable for placing one or more trays suitable for storing the plant genetic resources to be measured; a computer suitable for controlling the thermal camera and for storing and, if necessary, processing the recordings made by the thermal camera; Unit for drying test plant genetic resources; Equipment for cooling and preserving test plant genetic resources; One or more trays suitable for measuring the test plant genetic resources The compound comprises:

[0050] The present invention relates to the development of plant genetic resources. For genebank conservation The above system for non-destructive identification and differentiation of viability and / or varieties / varieties, wherein said plant genetic resources are selected from the group comprising seeds, other plant parts, and most preferably the plant genetic resources are seeds.

[0051] According to a preferred embodiment, plant genetic resources For genebank conservation The drying unit of the system for non-destructive identification and differentiation of viability and / or variety / variety groups is a drying chamber or other unit suitable for drying that contains silica gel.

[0052] According to another preferred embodiment, the plant genetic resources For genebank conservation The thermal camera of the system for non-destructive identification and differentiation of viability and / or varieties / cultivars is positioned within the enclosed measurement space together with positions for storing one or more trays of plant genetic resources to be measured, and / or the thermal camera is mounted on a support stand.

[0053] According to another preferred embodiment, the plant genetic resources according to the invention For genebank conservation The thermal camera of the system for non-destructive identification and differentiation of viability and / or varieties / cultivars has a spectral sensitivity of 7-14 μm.

[0054] According to a preferred embodiment of the system for non-destructive identification and differentiation of viability and / or varieties / varieties of plant genetic resources according to the invention, the one or more trays suitable for measuring the plant genetic resources have a segmented design.

[0055] Plant genetic resources according to the present invention For genebank conservation According to a preferred embodiment of the system for non-destructive identification and differentiation of viability and / or varieties / varieties, the material of the one or more trays suitable for measuring plant genetic resources is paper, a suitable plastic or any natural or artificial insulating material, preferably cardboard.

[0056] The present invention relates to the development of plant genetic resources. For genebank conservation The present invention also relates to a method for the non-destructive identification and differentiation of viability and / or varieties / variety groups, said method comprising the steps of: a) drying the test plant genetic material; b) cooling the plant genetic resource after drying according to step a) to a temperature below 0°C; c) setting the temperature and humidity in a measuring space separated by a wall; d) placing the plant germplasm to be measured cooled according to step b) under a thermal camera and measuring the plant germplasm, wherein the measurement lasts for less than 2 hours; e) software processing of the thermal camera recordings of the nominated plant genetic resources; f) drawing conclusions about the viability and / or variety / variety group of the measured plant genetic resources based on the results of the software processing obtained in step e); The designated plant genetic resources to be measured are placed on one or more trays suitable for storing plant genetic resources before step a), b) or c).

[0057] The present invention relates to the development of plant genetic resources. For genebank conservation It also relates to a method for the non-destructive identification and differentiation of viability and / or varieties / varieties, most preferably wherein the plant genetic resource is seeds.

[0058] According to a preferred embodiment of the invention, in step a) the temperature used in the step of drying the plant genetic resource is between 10 and 25°C.

[0059] According to another preferred embodiment of the invention, in step a), the humidity is reduced during the drying of the plant genetic resource, so that the final relative humidity is between 10 and 15%.

[0060] According to a further preferred embodiment of the invention, in step b) the plant genetic material is cooled for at least 24 hours.

[0061] According to a further preferred embodiment of the invention, in step b), the plant genetic resource to be measured is cooled to a temperature of 0 to -18°C ± 3°C.

[0062] According to a further preferred embodiment of the invention, for the cooling according to step b), the plant genetic material is cooled in a sealed container and / or on a tray suitable for measurement.

[0063] According to a more preferred embodiment of the invention, the measurement environment in step c) is a temperature of 16°C ± 4°C and a relative humidity of 70 to 90%.

[0064] According to a preferred embodiment of the invention, in step d), the measurement time of the cooled plant germplasm is less than 1 hour, preferably 30 minutes, most preferably 15 minutes.

[0065] According to a further preferred embodiment of the invention, the method is carried out in the system described above.

[0066] The present invention relates to the development of plant genetic resources. For genebank conservation of plant genetic resources for non-destructive identification and differentiation of viability and / or varieties / varieties For genebank conservation It also relates to the use of the above system for the non-destructive identification and differentiation of viability and / or varieties / varieties.

[0067] The present invention relates to the development of plant genetic resources. For genebank conservation of plant genetic resources for non-destructive identification and differentiation of viability and / or varieties / varieties For genebank conservation The present invention also relates to the use of the above system for the non-destructive identification and differentiation of viability and / or varieties / varieties, wherein most preferably the plant genetic resource is seeds.

[0068] The essence of the present invention is the development of plant genetic resources as detailed below. For genebank conservation The objective of the present invention is to non-destructively identify and separate the viability and / or variety / variety groups of cooled plant genetic resources using a system and associated methods including a thermal camera for non-destructively identifying and separating the viability and / or variety / variety groups of cooled plant genetic resources so that the tested plant genetic resources remain suitable for further genebank conservation.

[0069] Within the scope of this specification, when a numerical value is indicated, it is understood that the last digit of the indicated numerical value indicates the precision of the indicated numerical value according to the rounding rule, for example, 3.0 is understood to mean a range of 2.95 to 3.05.

[0070] For the purposes of this specification, plant genetic resources are defined as all genetic material of plant origin containing functional units of heredity, including reproductive and asexual propagation, regardless of the source of the plant genetic resources (conventional propagation techniques or novel biotechnological methods). Genetic material is defined as the entire genome of the plant present in the plant in the form of DNA. For the purposes of this specification, plant genetic resources are selected from the list of seeds and other parts of plants, preferably seeds. Seeds are the fruit part of flowering plants grown from fertilized sowing, the organ that protects and nourishes the germ. Its components are usually the seed coat or fruit wall, the vegetative tissue (containing or lacking starch, oil) and the germ.

[0071] Viability is generally understood as the property of a plant to withstand adverse conditions without significant damage. Testing the viability of plant genetic resources means whether the tested plant genetic resources are viable, i.e. whether their life processes are active.

[0072] For example, there are numerous methods for testing seed viability. The most common method is germination. This test allows for the elimination of non-viable or poorly germinating seeds (which are unsuitable for preserving genetic diversity). At the same time, as detailed in the state-of-the-art statement, germination results do not always directly infer viability due to seed dormancy and other factors. According to genebank protocols, germination is considered the most widespread viability test, even though we know that viability is clearly not considered equivalent to germination ability.

[0073] The essential conditions for germination of plant genetic resources are the correct temperature, humidity, light and oxygen tension. If any of these conditions are not met, germination will not occur or will not occur properly. Furthermore, of course, many other factors influence the induction and progression of germination, but the four elements mentioned above are the most crucial.

[0074] The first essential requirement for the initiation of germination is the amount of water required (SZABO LASZLO GY. (ed.); Fundamentals of Seed Biology; 1980, Akademiai Kiado, Budapest, 391). However, achieving an optimal water supply for the germination medium is difficult. The standards of ISTA (International Seed Testing Association) and AOSA (Association of Official Seed Analysts) provide extensive guidance on germination methods.

[0075] The identification and differentiation of cultivars or varieties means the organization into a group of individual plant genetic resources belonging to the same taxonomic category and their separation from each other. The cultivated plant unit is the variety (cultivar = cv.). From the point of view of the grower (farmer, gardener, forester), a variety means a cultivated individual or group of plants belonging to the same species, which share one or more essential characteristics but differ from other species in at least one characteristic (Gyulai 1999). So far, the differentiation of cultivars and varieties is only possible morphologically, by international descriptive testing methods (which require the entire growing season and the whole plant) and / or DNA testing methods. Both morphological and DNA testing methods involve the loss of plant genetic resources.

[0076] The system and associated method according to the present invention is non-destructive, since not only do they not take tissue samples from the plant germplasm, but external stimuli do not affect the plant germplasm during the measurement, allowing the tested plant germplasm to be used for further measurements and not lost from a genetic perspective.

[0077] Plant genetic resources according to the present invention For genebank conservationThe system for non-destructive identification and differentiation of viability and / or varieties / varieties comprises at least the following: a space limited by walls, the temperature and humidity of which are controlled, and the space suitable for measuring the plant genetic resources to be measured comprises at least one location suitable for placing trays for supporting the plant genetic resources; a thermal camera arranged facing said one or more trays suitable for storing the plant genetic resources to be measured; a computer suitable for controlling the thermal camera and for storing and, if necessary, processing the records made by the thermal camera; and a unit for drying the plant genetic resources to be tested; an apparatus for cooling and storing the plant genetic resources to be tested; and one or more trays suitable for measuring the plant genetic resources to be tested.

[0078] Within the framework of the present invention, a space limited by walls is understood to mean a measurement chamber that serves as a measurement location. Walls are necessary so that the temperature and humidity do not change during the measurement due to ambient air movements to an extent that would affect the measurement. It is also preferred that all sides of the space limited by walls are bounded by walls with the necessary doors and windows. However, systems in which not only all sides of the space limited by walls, but also some sides, are formed by walls suitable for excluding undesired air movements, also fall within the scope of protection of the present invention.

[0079] The temperature and humidity of the space separated by the wall must be controllable. To perform the measurement successfully, it is necessary to set the temperature appropriately (so that it falls within a specific temperature range), which depends on the plant genetic material being tested (e.g., 16°C for seeds with a diameter of 3 mm or more). In the present invention, a cooling-heating device (e.g., an air conditioner suitable for cooling and heating) is used to regulate the temperature. It is preferable to perform the measurement in a space separated by a good insulating wall (measurement chamber), which makes it easy to maintain the temperature required for the measurement. The temperature required for the measurement can be set not only in spaces separated by artificial insulating walls, but also in caves and cavities found in nature, for example.

[0080] In addition to the temperature of the space bounded by walls, its humidity must also be controllable, i.e., a humidifying device is required. A humidifying device guarantees a constant high humidity in the space bounded by walls. As mentioned above, in order to avoid inappropriate measurement results due to moisture precipitation on the surface of the plant genetic resources, it is important to maintain a constant measurement environment, one of which is maintaining constant humidity. It is important to distinguish between the low relative humidity (10-15%) applied during drying, as described later in this specification, and the high relative humidity (70-90%) used during measurement. On the other hand, the humidity required for measurement can be achieved artificially, for example, by using a humidifying device, or natural conditions where this condition exists must be found (e.g., humid air in caves, mine tunnels, etc.).

[0081] As will be apparent to those skilled in the art, the controllability of both temperature and humidity includes the measurability of these parameters, since control is achieved only by knowing the measured values. Of course, it is possible for the temperature and / or humidity to be exactly right for a particular measurement, and there is no need to alter them with cooling-heating or humidifying devices.

[0082] Plant genetic resources For genebank conservation A component of the system for identifying and differentiating viability and / or varieties / varieties is a location suitable for receiving one or more trays for supporting plant genetic material suitable for measuring the test plant genetic material. This location is created under the field of view of the thermal camera and one or more trays are placed therein for measurement.

[0083] Plant genetic resources For genebank conservationA component of the system for identifying and differentiating viability and / or varieties / varieties is a thermal camera that is particularly suitable for carrying out this easy measurement (see below for details of suitable thermal cameras). The thermal camera is placed in a space limited by walls, facing one or more trays suitable for storing the plant genetic resources to be measured. The thermal camera allows to examine physiological processes of the plant genetic resources, from which their viability can be inferred, and to identify and differentiate varieties / varieties based on the recordings of the thermal camera.

[0084] In the present invention, the thermal camera measurements are controlled by a computer, which is suitable for storing and, if necessary, processing the records (images and video film) made during the measurements. The digital images of the thermal camera measurements are processed using software (e.g., IRPlayer software version 4.0 (Hexium Kft., Budapest, Hungary) is suitable for this purpose), which is able to handle the unique file format of the thermal camera. The software modules used are: image data reading; file merging; data conversion and data saving. Furthermore, the Lumi program, specially created for thermal camera measurements (e.g., Lumi IDSF version 5.42 (SFD Informatika Kft., Keszthely, Hungary)), is used for batch measurement of the intensity of digital images based on fixed and individually defined linear intensities. Additionally, the computer extracts data from a data collector suitable for measuring temperature and humidity using software (ComSoft Basic software (Testo SE & Co. KGaA, Lenzkirch, Germany)).

[0085] The drying unit is used for the storage of plant genetic resources. For genebank conservationIt is part of a system for the identification and differentiation of viability and / or varieties / varieties. Drying plant germplasm before measurement is important for several reasons. Firstly, dried plant germplasm is less likely to form condensation on its surface (which would falsify the measurement results). On the other hand, seeds with low moisture content are stored cooled to -20°C (in this case, freezing of water within the seed and such an increase in volume does not damage the cells).

[0086] The device for cooling and storing plant genetic resources is a part of the system according to the invention, which device is a freezer or cold storage. The function of the device for cooling and storing plant genetic resources is twofold: on the one hand, it is used to cool the plant genetic resources after drying, and also to store the plant genetic resources in a genebank.

[0087] Plant genetic resources For genebank conservation A component of the system for identifying viability and / or varieties is one or more trays suitable for measuring germplasm, which are placed in a space divided by walls, under the field of view of a thermal camera, in a designated position suitable for carrying out thermal camera measurements. The one or more trays serve as the location of the plant germplasm to be measured during the measurement, i.e., the seeds to be measured are placed on them during the measurement. As a result, the trays on which the plant germplasm to be measured (e.g., seeds) are placed and the thermal camera are positioned relative to each other, so that the thermal camera can take images of the seeds resting on the one or more trays. In this way, the thermal camera is positioned so as to face the one or more trays suitable for storing the plant germplasm to be measured.

[0088] For genebank conservation The system for non-destructive identification and differentiation of plant genetic resources for viability and / or variety / variety groups is used to test seeds and other plant parts, preferably seeds. These are the types of plant genetic resources most commonly deposited in gene banks, and therefore these types of viability testing are most frequently requested. By other plant parts, we mean fruits, fruit parts, and other plant reproductive organs.

[0089] Plant genetic resources For genebank conservation The drying unit of the system for non-destructive identification and differentiation of viability and / or varieties / varieties is a drying chamber or other drying unit containing silica gel. Drying with silica gel is carried out gently at low temperatures for long periods of time without causing irreversible changes in the plant germplasm. Drying is necessary for the reasons mentioned above. In our experience, the test plant germplasm is most effectively dried in a drying chamber or, in rare cases, in a drying unit that uses a desiccant (e.g., silica gel).

[0090] Plant genetic resources according to the present invention For genebank conservation The thermal camera of the system for non-destructive identification and differentiation of viability and / or variety / variety groups is placed within an enclosed measurement chamber together with a location for placing one or more trays containing the plant genetic resources to be measured, and / or the thermal camera is mounted on a support stand.

[0091] The humidity and temperature of the enclosed measurement space are essentially the same as those of the moistly walled space and are continuously measured and recorded during the measurement (e.g., suitable equipment for this: Testo 174 H data logger (Testo SE & Co. KGaA, Lenzkirch, Germany)).

[0092] It is also possible to imagine a specific embodiment in which the thermal camera is placed outside the enclosed measurement space; in this case, when selecting the material of the measurement area, a material that does not affect IR signal transmission must be selected. However, it is preferable that the thermal camera, or at least the target of the thermal camera, is placed inside the enclosed measurement space. In practice, an enclosed measurement space means a Plexiglas house with openable windows, and the thermal camera placed in the enclosed measurement room is protected from external influences (e.g., air environment, currents, researcher movements) (the purpose is to minimize factors that may disturb the measurement). The thermal camera required for the measurement is placed on a horizontal and vibration-damped table (thereby minimizing influences that may disturb the measurement).

[0093] A more precise measurement setup is possible by placing the thermal camera on an adjustable stand. Because the sizes of various plant genetic resources vary over a wide range of dimensions (on the order of mm or cm), all of them can be monitored by the thermal camera of the system of the present invention regardless of their size, and no other recording devices are required for the test.

[0094] In fact, the thermal camera of the system according to the invention detects electromagnetic waves in the infrared range. The thermal camera of the system according to the invention preferably has a spectral sensitivity (i.e., working range) of 7-14 μm, the thermal camera has a measuring range of -30 to 1000°C, operates over a wide humidity range (10-95%) and is designed to perform automatic measurement correction (outdoor temperature, distance, relative humidity). Further parameters of the thermal camera are preferably: Sensor Type: Uncooled FPA Microbolometer Zoom: x1, x2 (Digital zoom processing software included) Number of pixels: 640 x 480 Built-in imaging device: Thermal camera (16 bits / pixel) Output: LAN, external display terminal NETD(300K, 50Hz)30mK Power supply: External adapter (230V AC, 50Hz) Includes.

[0095] The thermal sensitivity of thermal cameras is a minimum of 25 mK, which is considered twice as sensitive as commercially available camera systems. This allows for the detection of small temperature differences, which are essential for visualizing different thermal structural elements and rapid temperature fluctuations within plant germplasm (e.g., seeds). Another advantage of thermal cameras is that the field of view can be adjusted to the size of the target. Furthermore, remote sensing systems can collect information about research targets, i.e., plant germplasm, without causing any physical damage to the target. In this way, the use of thermal cameras can replace traditional viability and germination tests (after which the plant germplasm is no longer used for further testing). During thermal imaging, the physiological parameters of the plant germplasm remain essentially unchanged, and the impact of thermal measurements on the plant germplasm is negligible. Therefore, if the plant germplasm is viable before testing, its viability will be maintained during subsequent testing.

[0096] A calibration is carried out in all cases before the thermal camera measurements and, if necessary, between measurements. A distinction can be made between two types of calibration: on the one hand, the thermal camera sensor can be calibrated, and on the other hand, the measurement area itself can be calibrated. A thermal camera records one image every 1 to 1.3 seconds.

[0097] The plant genetic resources are placed on one or more trays suitable for measuring the plant genetic resources so that they do not roll, slide or come into contact with each other and so that there is a distance between them that is necessary for the measurement. The trays applied therefore preferably have an uneven surface, for example they are wrinkled, pleated or, particularly preferably, torn. This design of the trays makes it possible to measure several plant genetic resources simultaneously.

[0098] Numerous materials were tested during the design of one or more trays suitable for measuring plant germplasm. During the tests, the temperature changes of the plant germplasm were monitored, along with the effect of the plant germplasm-holding medium on their temperature. Furthermore, a certain degree of materiality was required for the trays, and trays made of such materials could be created that were practical and suitable for the placement of plant germplasm. During the tests, seven different types of seed-holding media were tested: clay, glazed earthenware, glass, expanded PVC (Palfoam™), baking plasticine, wood, and paper.

[0099] During thermal camera testing of the plant germplasm-holding media (i.e., trays), we monitored the heating of cooled plant germplasm using the measurement methods used for plant germplasm. The length of the measurements depended on the type of media. Overall, measurements were taken for 5 to 45 minutes for individual plant germplasm and the media containing the plant germplasm placed on them. Images taken with a highly sensitive, proprietary thermal camera were saved as files with the .idsf extension and then converted to .tif format for easy later visual presentation. In the cases of glass, foamed PVC, clay, and glazed ceramic, we measured only the tray material without the plant germplasm. These measurements revealed that these materials are too good thermal conductors, i.e., they are not suitable as tray materials. In the case of baking Plasticine, we found that Plasticine has a high heat capacity, which significantly affects the temperature changes (warming) of the plant germplasm placed on it, thus reducing the reliability of physiological property results derived from temperature changes of the plant germplasm. Furthermore, experimental studies showed that the length of measurement increased in the case of baking plasticine, which is thought to be due to the heat capacity of the medium for storing plant germplasm. During measurements on cedar boards, it was found that, like plastic, the heat capacity of wood is significant and significantly affects the temperature change (warming) of the plant germplasm placed on it and therefore the reliability of the results of physiological characteristics derived from temperature changes in the plant germplasm. An increase in the length of measurement time was also observed in the cedar wood case, which, like the case mentioned above, is also due to the heat capacity of the medium for storing plant germplasm. During measurements on paper, it was found that the heat capacity of the Plasticine and cedar board cases was high, but the heat capacity of the paper case was low, therefore only slightly affecting the temperature change (warming) of the plant germplasm and therefore the reliability of the results of physiological characteristics derived from temperature changes in the plant germplasm. This is also particularly true for cardboard trays. Based on experimental studies, unlike baking plasticine and wood cases, the length of measurement time was short, which proves the use of paper as a medium for storing plant germplasm and its appropriate heat capacity.The compartmentalized design of the paper tray ensures that individual plant germplasm is physically separated from one another, all achieved with the aid of a grid. Based on the thermal camera recordings taken during the measurements, it was clearly observed that the temperature of the paper tray did not change significantly, even after 5 minutes, and maintained a temperature close to that of the test table. During the test measurements, paper-based cardboard proved to be the best among the tray materials tested. It will be obvious to those skilled in the art that thermal camera measurements can be performed on trays made of various suitable materials that behave similarly to paper trays during measurements (i.e., they do not significantly affect seed heating during thermal camera measurements and are suitable for supporting seeds). These are, for example, trays made of plastic or various natural or artificial insulating materials suitable for thermal camera measurements.

[0100] The system for identifying and differentiating the viability and / or variety / variety of plant genetic resources is mobilized and the system is installed in various laboratories (mobile shipping containers or laboratories of institutions that handle plant resources (e.g., gene banks), or converted vehicles) as described herein.

[0101] The present invention relates to the development of plant genetic resources. For genebank conservation It also relates to methods for identifying and differentiating viability and / or breeds / breed groups.

[0102] Plant genetic resources For genebank conservation The method for identifying and differentiating viability and / or breeds / breed groups comprises the following steps: a) Drying the tested plant germplasm: During this step, the tested plant germplasm of any species is dried at 10-25°C, depending on the variety, using a drying chamber or other suitable drying unit containing silica gel to reduce the humidity to 10-15% relative humidity. A lower relative humidity will damage the plant germplasm. Depending on its type, the plant germplasm will have an average moisture content of 3-7% after drying.

[0103] b) After drying according to step a), cooling the plant germplasm to a temperature below 0°C; the target cooling temperature depends on the plant germplasm. The pre-dried test plant germplasm must be cooled in a refrigerator for at least 24 hours before measurement. During cooling, preferably to -18°C ± 3°C, the test plant germplasm is cooled in a refrigerator, e.g., a "frost-free" freezer (which prevents water vapor from condensing and freezing on the surface of the test sample), in a closed container and / or on one or more trays made of paper or plastic used for the measurement. Closed containers are sealed glass or triple-layer aluminum-plastic bags with welded closures. During measurement, attention should be paid to the fact that dried plant germplasm is often fragile and thus sensitive to mechanical influences, and therefore must always be handled with care, preferably avoiding shocks and collisions during measurement. When placed in the freezer, the paper trays should only be touched at their edges, thereby reducing the chance that the measurement results will be affected by any external factors. When placing the plant germplasm on the tray, particular care must be taken to ensure that the plant germplasm to be measured and the tray are not affected as much as possible by heat applied by contact with hands / fingers.

[0104] c) Setting the temperature and humidity in the walled space for measuring the viability of the plant genetic resources and / or for identifying and differentiating varieties / varieties: the measuring temperature is preferably 16°C ± 4°C and is set using a cooling-heating device. For viability and / or for identifying and differentiating varieties / varieties, a relative humidity of 70-90% is required, which is continuously monitored by suitable measuring and data collection devices.

[0105] d) placing the batch of plant germplasm to be measured cooled according to step b) under a thermal camera and measuring it with the thermal camera (where the measurement lasts for no more than 2 hours): carefully placing the cooled plant germplasm on one or more trays made of paper or plastic with divided plates, whereby the samples are measured in the visible and thermal ranges. The plant germplasm is placed on one or more divided paper trays so that the plant germplasm is separated by an appropriate distance and does not interfere with each other during heating. Gloves should be worn when placing the trays under the thermal camera to prevent the warmth of fingers from affecting the heating of the plant germplasm throughout the measurement. The plant germplasm must not be touched before the measurement; if one of the samples to be measured needs to be adjusted, this should be done with an appropriate tool, for example, tweezers. The duration of the measurement is variable and can be up to one hour, preferably 30 minutes, and most preferably 15 minutes. The reason for reducing the measurement time to 15 minutes is that the heating of the dried and cooled plant germplasm is most intense in the first 5 minutes. During this 15-minute period, the plant germplasm under test shows the most pronounced and characteristic changes, after which the warming slows down.Three distinct phases were observed during the warming of the plant germplasm, which are characteristic of all the species tested: a transient phase, a stationary phase, and a saturation phase (see, for example, Figure 1).

[0106] The transition phase is the initial phase of core heating, at which the core heating process begins. These parts can have different lengths for each measurement batch (here, measurement batch means plant germplasm belonging to the same species / genebank batch), possibly due to the transition process (e.g., speed of tray placement, initial temperature for each batch). The transition phase lasts on average 4-10 seconds.

[0107] The length of the uniform warming phase will vary for individual plant germplasm species and possibly cultivars. The length of the warming-up period varies, lasting on average 150-300 seconds.

[0108] At the saturation phase, heating begins for measurements. The data (slope, intercept, standard deviation) of the line fit to the saturation state are representative for each plant germplasm lot.

[0109] The most characteristic changes in cooled plant germplasm are observed when the temperature difference between the air and the germplasm is greatest, i.e., when one or more trays are removed from the refrigerator; it is therefore essential to carry out this operation as quickly as possible (in the case of a closed measurement space, one or more trays are inserted through a door formed therein). The 15-minute measurement time not only ensures that the measurement does not take too long to preserve the viability of the germplasm, but also ensures that the germplasm can still be stored after re-drying and that the measurement can be repeated. International literature listed in the state-of-the-art section often describes measurement methods lasting more than 24 hours, and the use of thermal camera technology is referred to as a "non-invasive" method. However, in the case of such long measurements, the germination process has already begun (hence, in many cases, germination itself is monitored with a thermal camera), which means that further testing of the same germplasm and its use in genebanks is no longer possible after the seedlings have been produced.

[0110] e) Software processing of thermal camera images of plant genetic resources: The Lumi IDSF 5.42 program is suitable for measuring the intensity of batch digital images based on built-in and arbitrarily defined (so-called custom) functions. In the Lumi IDSF 5.42 program, a selection is made across the surface of the plant genetic resource, and the software measures the average intensity of the pixels belonging to the selected area. This intensity is treated as the average emitted energy. This measurement is a digital value of emitted energy / pixel. IRPlayer 4.0 is software that manages the unique file format of the thermal camera. This software manages the image data captured by the thermal camera during measurement, as well as the image data created by external control software. It has five main modules: scanned image data; file merging; reference point measurement function; built-in palette selection; data conversion; and data backup.

[0111] f) drawing conclusions about the viability and / or variety of the measured plant genetic resources based on the results of the software processing obtained in step e): during the evaluation of the measurement results, an average intensity-time graph is obtained from which a conclusion can be drawn about the viability and / or variety / variety of the measured plant genetic resources.

[0112] In the method of the present invention, before step a), b) or c), the named plant germplasm is placed on at least one tray suitable for storing the plant germplasm to be measured.

[0113] For genebank conservation For the viability test, viable plant germplasm was germinated to verify the thermal camera measurements. Germination was necessary only to validate the results of the thermal camera measurements, i.e., it was not part of the method steps. The goal was to demonstrate the viability of plant germplasm using non-destructive methods. For genebank conservation In each case, control germination of the plant germplasm was carried out according to the criteria applicable to the given species.

[0114] Preferably, the following genetic resources are used as plant genetic resources: For genebank conservation Non-destructive identification and differentiation of the viability and / or variety / variety group can be tested on: seeds, other plant parts (where plant parts are understood to mean fruits, fruit parts, other regenerable plant parts, etc.). Most preferably, the plant genetic resource is a seed.

[0115] The method according to the invention as described above is preferably carried out by For genebank conservation This is carried out using a system for non-destructive identification and differentiation of viability and / or varieties / variety groups.

[0116] Plant genetic resources according to the present invention For genebank conservation The system for non-destructive identification and differentiation of viability and / or varieties / varieties is preferably For genebank conservation Used for non-destructive identification and differentiation of viability and / or varieties / variety groups.

[0117] The present invention relates to the development of plant genetic resources. For genebank conservation of plant genetic resources, including systems for the non-destructive identification and differentiation of viability and / or varieties / varieties; For genebank conservation It also applies to the non-destructive identification and differentiation of viability and / or varieties / varieties, wherein the plant genetic resources are preferably selected from the group consisting of seeds, other plant parts (wherein plant parts are understood as fruits, fruit parts, other regenerable plant parts, etc.), and most preferably seeds. [Example]

[0118] Differentiation of empty and non-empty sunflower achenes - Viability test a) Prior to the measurements, sunflower achenes were dried in a drying chamber at 20°C, starting from a relative humidity of 20-22% and continuously decreasing the relative humidity (reaching and maintaining a relative humidity of 10-15%) to a seed moisture content of 3.9%. b) The sunflower achenes dried according to step a) were cooled in a "frost-free" freezer at -18°C ± 3°C for at least 24 hours on the divided cardboard trays used for the measurements. c) The temperature of the walled space (measurement laboratory) was set to 16°C, and the measured relative humidity was 75-82.2%. d) As soon as possible, the tray containing the sunflower achenes cooled according to step b) was moved from the freezer under the thermal camera and the thermal camera measurement started. The measurement time was 15 minutes. e) The thermal camera images of sunflower achenes were processed and evaluated by software. f) Based on the results of the software processing, conclusions were drawn about the viability of sunflower achenes. To support the reliability of the procedure, sunflower achenes were germinated. A sunflower achene is "full" if it contains seeds, and "empty" if it does not contain seeds, i.e., the achene is empty. Four series of 20-20 sunflower achenes were measured. The sunflower achenes were measured over a 15-minute heating period, with the first 300 seconds of measurement being the most interesting, as this is the period during which the achenes belonging to each group show a strong separation. The far infrared (FIR) curves for full and empty sunflower achenes differ significantly in terms of mean and speed (min, 3 times standard deviation). Empty sunflower achenes are primarily saturated with air, while full achenes primarily contain seeds. During the measurements, the aim was to separate empty and full sunflower achenes based on the thermal camera measurements. The seeds were then germinated as a test. After genebank propagation, empty achenes were sorted for measurements using a gravity-based sorter ("winding"). The slope of the initial transition phase is greater for empty sunflower achenes because the interior of the achenes is filled with air and lacks seeds. Second, the uniform heating phase is invisible for empty seeds, while it is clearly visible for full seeds. Third, the saturation phase is steeper for full seeds (see Figure 2). In Figure 2, the curves showing the average intensity of full and empty seeds clearly separate from each other as a function of time. For ease of interpretation, Figure 2 plots the average intensity as a function of the first 28 seconds of measurement. From these results, a clear inference can be made about the expected viability, since in the case of sunflowers, seeds considered empty usually do not germinate. Unknown achenes can be classified based on the processed data of the thermal camera measurements, and the course of the curve belonging to a given sunflower achene is unique, i.e., it is possible to identify whether a measurement belongs to an empty or full achene. [Example]

[0119] Thermal camera viability testing of kidney beans, corn, and einkorn wheat a) Prior to the measurements, the seeds / grains of common bean, maize, and einkorn were dried in a drying chamber at 20°C, starting from a relative humidity of 20-22% and decreasing continuously (reaching and maintaining a relative humidity of 10-15%). Using this method, a seed moisture content of 6.9% was achieved for einkorn and 6.1% for maize and common bean. For the test measurements, seeds / grains of common bean, maize, and einkorn were selected from 100 seeds / grains per species, with 50 seeds / grains considered waste from a genebank perspective and 50 seeds / grains considered viable (confident from the results of previous control germination tests). b) The beans, corn and einkorn dried according to step a) were stored for 24 hours at -18°C on divided cardboard trays used for the measurements (20 seeds / kernels per tray, a total of 100 seeds / kernels were measured per species) and cooled in a "frost-free" freezer. c) The temperature of the space separated by a wall (measurement room) was set to 14-15°C and the relative humidity to 78.6-88%, and these values ​​were constantly monitored during the measurement. d) The trays containing the kidney bean, corn, and einkorn seeds / kernels cooled according to step b) were removed from the freezer and quickly placed under the thermal camera, and the thermal camera measurement was started. The measurement time for each measurement (tray) was 15 minutes. e) Thermal camera images of kidney bean, corn, and einkorn seeds / kernels were processed using software. f) Based on the results of the software processing, conclusions were drawn regarding the viability of the above-mentioned plant genetic resources. Seeds / grains of kidney bean, corn and einkorn were germinated according to standard for control. As can be seen from the thermal camera measurements of kidney beans (see Figure 3), maize (see Figure 4) and einkorn wheat (see Figure 5) (in our case "discarded" means non-viable seeds / kernels and "normal" means viable seeds / kernels), the curves of mean intensity showed deviations depending on time. After standard germination of kidney bean, corn, and einkorn seeds / grains, the results were compared with those of thermal camera measurements. In the case of kidney bean, there were "rejected" seeds (4 out of 50 samples) that germinated, and "normal" seeds (8 out of 50 samples) that did not germinate or had distorted (dead) sprouts. It is important to note that according to genebank protocols and the Hungarian Standard (MSZ 1992), seeds that produced distorted sprouts are considered non-germinating because they are not predicted to develop into plants. In the cases of einkorn and corn, based on the results of the germination tests, the "rejected" seeds / grains did not actually germinate, and therefore, they are indeed considered discarded. However, among the "normal" seeds / grains, there was a surprisingly high proportion of non-germinating, moldy, and distorted grains. Therefore, in the case of kidney beans, we further investigated whether seeds that germinated but were deemed "waste" based on the measurements could be separated from seeds that did not germinate and were truly "waste" based on the thermal camera test measurements. Figure 6 clearly shows that "waste" seeds that did not germinate based on the thermal camera measurements could be separated from seeds that germinated but were deemed "waste" (i.e., their measured average intensities were different). Furthermore, for all two species, we examined whether seeds that did not germinate but were deemed "normal" were truly "normal" seeds that would germinate based on the thermal camera measurements. The average intensity of the germinated seeds was distinguishable from seeds that did not germinate but were deemed "normal" based on the thermal camera measurements. In particular, in the second uniform heating step, the average intensity curves were distinguishable from each other. [Example]

[0120] Maize variety group separation a) Prior to measurement, corn seeds were dried in a drying chamber at 20°C, starting from a relative humidity of 20-22% and continuously decreasing the relative humidity (reaching and maintaining a relative humidity of 10-15%). Using this method, the moisture content of the corn was determined to be 6.1%. b) The maize seeds dried according to step a) were stored for 24 hours at -18°C ± 3°C on divided cardboard trays used for the measurements (20 kernels per tray, both genebank and hybrid maize varieties were tested) and cooled in a "frost-free" freezer. c) The temperature of the walled space (measurement room) was set to 16°C, and the relative humidity was 70-85%. d) The tray containing the cooled corn seeds according to step b) was taken out of the freezer and placed under the thermal camera as quickly as possible, and the thermal camera measurement was started. The measurement time was 10 minutes (there was no significant change in the subsequent process). e) Thermal camera images of corn seeds were processed using software. f) Based on the results of the software processing, conclusions were drawn regarding the maize seed variety group. The measurements of the genebank corn batches are clearly different from the thermal camera measurements of the hybrid corn, with visible differences in the transitional stages for the varieties. Due to the sensitivity of the thermal camera, it must be calibrated; the alignment of the calibration section is not shown in the figure. The results are compared based on the slope of the fitted curves and trend lines (see Figure 7). The fitted linear trend line shows a clear (positive sign) upward trend in the cases of the hybrid corn batches and the genebank corn batches. However, the mean intensity values ​​and the intersection points of the fitted line (trend line) with the y-axis are significantly different from each other, and based on this, the thermal camera measurements can be used to identify unknown varieties of corn (hybrid or polyfloral). In the case of bulbs and rhizomes, plant parts should be cooled to 1-5°C, and warming can be examined in the same way as for seeds. An essential advantage of the system and method according to the invention is that, compared to current viability and / or cultivar / variety classification methods, thermal camera measurements provide authentic results completely non-destructively. Furthermore, the measurement method according to the invention allows obtaining a wealth of information about plant genetic resources that was previously unavailable. Thanks to the thermal camera of the system according to the invention for testing the viability of plant genetic resources and / or identifying and classifying their cultivars / varieties, it is applicable to all plant genetic resources, regardless of their shape, size, and tissue structure. The measurement time of the thermal camera measurement of the method according to the invention is significantly shorter than that described for the prior art, which is an advantage since it is virtually not necessary to initiate physiological processes necessary for germination. During the measurement, the plant genetic resources are not damaged, can be repeatedly measured, and can still be used from genebank and agricultural perspectives (e.g., propagation).

Claims

1. 1. A system for non-destructive identification and differentiation of viability and / or varieties / varieties of plant genetic resources, said system comprising at least: a walled space, wherein i) the temperature and humidity of the space are controlled; and ii) the space includes one or more locations suitable for measuring test plant genetic resources and for placing trays for supporting the plant genetic resources; a thermal camera positioned facing one or more trays suitable for storing the plant genetic resources to be measured; a computer adapted to control said thermal camera and to store and, if necessary, process the recordings made by said thermal camera; a unit for drying the test plant genetic resources; equipment for cooling and storing the test plant genetic resources; one or more trays suitable for measuring the test plant genetic resources; A system for non-destructive identification and differentiation of viability and / or varieties / varieties of plant genetic resources, comprising:

2. A system for the non-destructive identification and differentiation of the viability and / or variety / variety groups of plant genetic resources, characterized in that the plant genetic resources are selected from the group comprising seeds and other plant parts.

3. 3. A system for non-destructive identification and differentiation of the viability and / or variety / variety groups of plant genetic resources according to claim 2, characterized in that the plant genetic resources are seeds.

4. A system for non-destructive identification and differentiation of the viability and / or varieties / varieties of plant genetic resources according to any one of claims 1 to 3, characterized in that the unit for drying is a drying chamber or other unit suitable for drying containing silica gel.

5. A system for non-destructive identification and differentiation of the viability and / or variety / variety group of plant genetic resources according to any one of claims 1 to 4, characterized in that the thermal camera is arranged in an enclosed measurement space having positions for storing one or more trays for the plant genetic resources to be measured and / or the thermal camera is mounted on a support stand.

6. A system for non-destructive identification and differentiation of viability and / or varieties / variety groups of plant genetic resources according to any one of claims 1 to 5, characterized in that the spectral sensitivity of the thermal camera is between 7 and 14 μm.

7. A system for non-destructive identification and differentiation of the viability and / or varieties / varieties of plant genetic resources according to any one of claims 1 to 6, characterized in that one or more trays suitable for measuring plant genetic resources have a divided design.

8. A system for non-destructive identification and sorting of the viability and / or varieties / variety groups of plant genetic resources according to any one of claims 1 to 7, characterized in that the material of the one or more trays suitable for measuring plant genetic resources is paper, suitable plastic or any type of natural or artificial insulating material, preferably the material of the one or more trays suitable for measuring plant genetic resources is cardboard.

9. 1. A method for the non-destructive identification and differentiation of viability and / or varieties / varieties of plant genetic resources, said method comprising the following steps: a) drying the plant genetic resource to be measured; b) cooling the plant genetic resource after drying according to step a) to a temperature below 0°C; c) setting the temperature and humidity in a measurement space separated by a wall; d) placing the plant germplasm to be measured cooled according to step b) under a thermal camera and measuring the plant germplasm, wherein the measurement lasts for less than 2 hours; e) software processing of thermal camera recordings of the nominated plant genetic resources; f) drawing conclusions about the viability and / or variety / variety group of the measured plant genetic resources based on the results of the software processing obtained in measurement step e); wherein the designated plant genetic resources are placed on one or more trays suitable for storing the plant genetic resources to be measured before step a), b) or c).

10. 10. A method for the non-destructive identification and differentiation of the viability and / or variety / variety groups of plant genetic resources according to claim 9, characterized in that the plant genetic resources are selected from the group comprising seeds, other plant parts, most preferably the plant genetic resources are seeds.

11. 11. A method for the non-destructive identification and differentiation of the viability and / or variety / variety of plant genetic resources according to claim 9 or 10, characterized in that the temperature used in step a) during drying of the plant genetic resources is between 10 and 25°C.

12. 11. A method for the non-destructive identification and differentiation of the viability and / or variety / variety groups of plant genetic resources according to claim 9 or 10, characterized in that in step a) during drying of the plant genetic resources, the humidity is reduced to a final relative humidity of 10-15%.

13. 13. A method for non-destructive identification and differentiation of the viability and / or variety / variety group of plant genetic resources according to any one of claims 9 to 12, characterized in that the cooling of the plant genetic resources in step b) lasts for at least 24 hours.

14. 14. A method for the non-destructive identification and differentiation of the viability and / or variety / variety group of plant genetic resources according to any one of claims 9 to 13, characterized in that during step b) the plant genetic resources to be measured are cooled to a temperature of between 0°C and -18°C ± 3°C.

15. 15. A method for the non-destructive identification and differentiation of the viability and / or varieties / varieties of plant genetic resources according to any of claims 9 to 14, characterized in that for the cooling according to step b), the plant genetic resources are cooled in closed containers and / or on trays suitable for measurement.

16. 16. A method for the non-destructive identification and differentiation of the viability and / or varieties / varieties of plant genetic resources according to any of claims 9 to 15, characterized in that the temperature in step c) is 16°C ± 4°C and the relative humidity is between 70 and 90%.

17. 17. The method for non-destructive identification and differentiation of the viability and / or varieties / varieties of plant genetic resources according to any of claims 9 to 16, characterized in that in step d) the measurement time of the cooled plant genetic resources is less than 1 hour, preferably 30 minutes, most preferably 15 minutes.

18. A method for non-destructive identification and differentiation of viability and / or varieties / variety groups of plant genetic resources according to any one of claims 9 to 16, characterized in that the method is carried out in accordance with the system according to claims 1 to 8.

19. Use of the method for non-destructive identification and differentiation of viability and / or varieties / variety groups of plant genetic resources according to any one of claims 1 to 8 for non-destructive identification and differentiation of viability and / or varieties / variety groups of plant genetic resources.

20. 9. Use of the method for the non-destructive identification and differentiation of the viability and / or varieties / varieties of plant genetic resources according to any of claims 1 to 8 for the non-destructive identification and differentiation of the viability and / or varieties / varieties of plant genetic resources, characterized in that the plant genetic resources are selected from the group consisting of seeds and other plant parts, most preferably the plant genetic resources are seeds.

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