Seed Testing Laboratories Research Articles

Effect of accelerated ageing on seed coat anatomy

Seed quality assessment plays a vital role in seed certification process. The persons those who involved in seed production and seed marketing business in India must follow the legal sections of Seed Act 1966, Seed Rule 1968 and Seed Control Order 1983. To meet out the legal sections the quality of seed must be verified before it is marketed and the quality parameter should be indicated/labelled on the seed containers/packets. The process to verify and confirming the quality level of seed samples will take minimum 15 days duration. Hence the machine developed by Artificial Intelligence (AI) to separate the seeds quality wise, will highly be useful for Seed Testing Laboratories (STLs) and seed industries. The seeds which shown well developed Internal structure during various test shows undersized development when they are transplanted. This type of problems can also be solved by the X-ray seed analysis. The only limitation in this type of analysis is time consuming. With this view the experiment was conducted with the following objectives, 1. To determine the effect of accelerated aging in seed and seedling quality of selected crops and 2. To determine the changes in seed coat anatomical structure with accelerated ageing. The present study revealed that there was a great correlation between the seed coat anatomy and seedling growth. With this concept the development of Seed Quality Analyzer with Artificial Intelligence (AI) is possible and it will highly be useful for Seed Testing Laboratories, Seed Industries and Farmers.

Cost-effective and reliable genomic DNA extraction from plant seedlings for high-throughput genotyping in seed industries

Genetic purity of seeds is one of the critical aspects in the seed industry. Molecular seed testing laboratories are utilizing PCR based diagnostic tools for genetic purity analysis. High quality DNA is an essential prerequisite for such analyses. Here, we demonstrate a robust and inexpensive DNA extraction method to isolate genomic DNA from variety of crops. Current method (M2) was compared with four commonly used DNA isolation methods for PCR-based genetic characterization and High Resolution Melt (HRM) based hybridity analysis of cotton, okra, tomato and maize using SSR markers. DNA extracted through current method showed excellent yield and quality as compared to other methods. High quality, PCR ready DNA was isolated within 30–50 min and displayed best results for genetic purity analysis using HRM. In contrast, several genomic DNA samples extracted using other methods were found unsuitable for HRM analysis. Our method can be a perfect choice in seed industry, where thousands of samples are processed every day. Notably, using our method single technician can extract DNA from 96 leaf samples within 30–50 min, at a cost of only $0.11/sample. Overall, current DNA extraction method is a reliable and cost-effective solution for large-scale genotyping experiments in the agricultural industry.

Prediction of Germination Percentage Through Electrical Conductivity in White and Coloured Coated French Bean (Phaseolus vulgaris L.)

Background: Rapid detection of seed germination will be a valuable tool for seed testing laboratories and commercial seed companies. The EC test has been accepted into ISTA rules for beans. The potential of the electrical conductivity test (EC) readings in predicting total and normal germination of nine white coated and nine coloured coated French bean cultivar seeds was examined. Methods: Nine different white coated and nine coloured coated French bean (Phaseolus vulgaris L.) cultivars were obtained from different sources. EC measurements for predicting germination were carried out 2, 4, 6 and 8 h during soaking at 20°C. Germination test was conducted on three replicates of 50 seeds in between wet towel papers for 8 days at 25°C in the dark. Result: Regression models were developed between EC and percentages of normal and abnormal seedlings in the germination test. EC readings at 2, 4, 6 and 8 hours were negatively related to percentage of total (R2=0.717, 0.830 P less than 0.05) and normal germination percentages (R2=0.762, 0.897 P less than 0.05). Results suggested that EC measurements carried out in between 2 and 8 hours soaking can be used for the estimation of seed germination in beans.

Analytical purity of old New Zealand forage seed samples and detection of fungal and insect contaminants including ryegrass endophyte and Argentine stem weevil

ABSTRACT Pasture seed samples that were harvested around 1917 in New Zealand were analysed for purity, Epichloë endophyte and insects. Samples consisted of 13 packets of seed varying from 2.2 to 15.0 g and comprising, Chewings fescue (Festuca rubra L.), cocksfoot (Dactylis glomerata L.), oat (Avena sativa L.), perennial ryegrass (Lolium perenne L.) and red clover (Trifolium pratense L.). These findings show that in the period prior to modern seed testing laboratories only four of the 13 samples (31%) could possibly have met today’s seed certification standards as it relates to seed purity. When compared with published purities and weed content of seed of the era these samples appear typical. Endophyte testing on the four ryegrass seed samples and the ryegrass contaminants found in the Chewings fescue and cocksfoot samples showed sample-to-sample variation in endophyte infection from nil to 91%. One cocksfoot sample, most likely harvested from the Banks Peninsula, contained an Argentine stem weevil. Subsequent checking of entomological collection records also revealed specimens being present in the Wellington region from 1916. Together these occurrences precede the earliest published record for the species in New Zealand by ten years and suggest that it may have been widespread at that time.

METHODOLOGICAL ASPECTS OF ASSESSING THE COMPETENCE OF LABORATORY PERSONNEL DETERMINING THE QUALITY OF SEED POTATOES

Most of the quality indicators regulated by the standard for seed potato certification are determined by the subjective component, that is, the competence of the personnel. However, the regulatory framework does not establish criteria for assessing competence, which poses uncontrolled risks in the laboratory. The purpose of the study is to develop and investigate the methodology for assessing the competence of laboratory personnel involved in the testing of seed potatoes and determine the extent of application of the methodology. The methods of probability theory and mathematical statistics are used for the tasks of selection and research of statistical criteria that correspond to the law of distribution of the obtained data and provide the necessary reliability for the available sample volumes. The statistics and the method of determining the threshold value, which mathematically describes the decision-making on competence, are provided for the binomial distribution law. The properties of the proposed statistical method for assessing the competence of personnel in terms of finding the maximum differences in the results of the analysis, at which the personnel are recognised as competent, are investigated. The study demonstrates the dependence of the method capacity on the data volume. It is determined that to obtain a confidence probability of 0.95 for quality indicators, the content of which in the average sample according to the technical conditions is minimal, the sample size should be 600-1000 tubers. At the same time, up to 100 tubers are sufficient for indicators whose content is under 5%. The method proposed by the authors showed that the average sample of 250 tubers recommended by the standard is entirely justified in terms of attracting resources during certification but must be adjusted during competence monitoring in a testing laboratory that confirms its competence by the requirements of DSTU ISO IEC 17025:2019. The findings presented can be used by the personnel in the seed testing laboratories as a measure to minimise the risks caused by the impact of personnel incompetence

GERMINABILITY OF VARIETIES OF BEAN (Phaseoulos Vulgaris L.) AND OKRA (Abelmoschus Esculentus L. Moench) UNDER LOW TEMPERATURE STORAGE CONDITION

Bean and okra are common vegetables cultivated in the most regions of Sri Lanka. The seeds of these crops are mainly stored in cold storage as bulk before releasing retail lots to sales outlets. Commercial growers and gardeners buy seeds in bulk or in packages at retail stores. Although certified seeds are frequently tested by the seed testing laboratories, they do not guarantee the maximum field emergence. Certified seed lots of two pole bean varieties, ‘Keppetipola Nil’ (KN) and ‘Bandarawela Green’ (BG) and two okra varities,’MI-5’ and ‘Haritha’ were stored in poly sack and polypropylene bags under controlled temperature (17±1 °C) and relative humidity (RH) (52-55%) over a two-year period. Bulk seeds of bean and okra varieties packed in polypropylene and poly sack bags depicted a high germination percentage throughout the storage period for two years. Seed quality parameters, seed germination, seed moisture, field emergence and vigour index significantly varied with the storage duration and declined in varieties. Significant differences in moisture, field emergence and vigour index were recognized as affected by the packing materials in all varieties except MI-5. Both Poly sack and polypropylene packing materials were found suitable to maintain viability at the minimum seed certification standards for two years of storage period. Poly sack was more appropriate than polypropylene to keep viability for more than 2 years of storage under low temperature and RH condition. These findings would help seed handlers including seed producers and seed sellers to store crop seeds between growing seasons in the tropical environment.

Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids

Potato spindle tuber viroid and other pospiviroids can cause serious diseases in potato and tomato crops. Consequently, pospiviroids are regulated in several countries. Since seed transmission is considered as a pathway for the introduction and spread of pospiviroids, some countries demand for the testing of seed lots of solanaceous crops for the presence of pospiviroids. A real-time RT-PCR test, named PospiSense, was developed for testing pepper (Capsicum annuum) and tomato (Solanum lycopersicum) seeds for seven pospiviroid species known to occur naturally in these crops. The test consists of two multiplex reactions running in parallel, PospiSense 1 and PospiSense 2, that target Citrus exocortis viroid (CEVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd) and tomato planta macho viroid (TPMVd, including the former Mexican papita viroid). Dahlia latent viroid (DLVd) is used as an internal isolation control. Validation of the test showed that for both pepper and tomato seeds the current requirements of a routine screening test are fulfilled, i.e. the ability to detect one infested seed in a sample of c.1000 seeds for each of these seven pospiviroids. Additionally, the PospiSense test performed well in an inter-laboratory comparison, which included two routine seed-testing laboratories, and as such provides a relatively easy alternative to the currently used tests.

Continuous monitoring seed testing equipaments using internet of things

The lack of functional, calibrated equipment, along with using inconsistent testing method contribute to the variable, unreliable results produced by seed laboratories. The objective of this article is present to presente the development of an integrated system consisting of hardware, middleware, and application to continuously monitor and record the performance of seed testing equipment from the beginning to the completion of each test in seed testing laboratories. The central element of the proposed system is the CoIoT middleware (Context + IoT), which provides mechanisms for managing IoT devices and context awareness mechanisms to monitor and record the equipment functions, throughout the seed testing processes. The developed system was tested in the Official Seed Analysis Laboratory (OSTL) of the Brazilian Agricultural Research Corporation (Embrapa). The proposed system was evaluated using the Technology Acceptance Model (TAM), which considers aspects of ease of use and usefulness. To validate the results, a scale of 1 (user totally disagree) to 5 (user totally agree) was used. For ease of use the average was of 4.7, and for usefulness, the average was of 4.4. The results achieved are promising, which encourages the continuity of research to further optimize CoIoT.

Development of Loop-Mediated Isothermal Amplification Assays for the Detection of Seedborne Fungal Pathogens Fusarium fujikuroi and Magnaporthe oryzae in Rice Seed.

Bakanae disease (caused by Fusarium fujikuroi) and rice blast (caused by Magnaporthe oryzae) are two of the most important seedborne pathogens of rice. The detection of both pathogens in rice seed is necessary to maintain high quality standards and avoid production losses. Currently, blotter tests are used followed by morphological identification of the developing pathogens to provide an incidence of infection in seed lots. Two loop-mediated isothermal amplification assays were developed with primers designed to target the elongation factor 1-α sequence of F. fujikuroi and the calmodulin sequence of M. oryzae. The specificity, sensitivity, selectivity, repeatability, and reproducibility for each assay was assessed in line with the international validation standard published by the European and Mediterranean Plant Protection Organization (PM7/98). The results showed a limit of detection of 100 to 999 fg of DNA of F. fujikuroi and 10 to 99 pg of M. oryzae DNA. When combined with a commercial DNA extraction kit, the assays were demonstrated to be effective for use in detection of the pathogens in commercial batches of infected rice seed of different cultivars, giving results equivalent to the blotter method, thus demonstrating the reliability of the method for the surveillance of F. fujikuroi and M. oryzae in seed-testing laboratories.

Spectrophotometry of Artemisia tridentata to Quantitatively Determine Subspecies

Ecological restoration is predicated on our abilities to discern plant taxa. Taxonomic identification is a first step in ensuring that plants are appropriately adapted to the site. An example of the need to identify taxonomic differences comes from big sagebrush (Artemisia tridentata). This species is composed of three predominant subspecies occupying distinct environmental niches, but overlap and hybridization are common in ecotones. Restoration of A. tridentata largely occurs using wildland collected seed, but there is uncertainty in the identification of subspecies or mix of subspecies from seed collections. Laboratory techniques that can determine subspecies composition would be desirable to ensure that subspecies match the restoration site environment. In this study, we use spectrophotometry to quantify chemical differences in the water-soluble compound, coumarin. Ultraviolet (UV) absorbance of A. tridentata subsp. vaseyana showed distinct differences among A.t. tridentata and wyomingensis. No UV absorbance differences were detected between A.t. tridentata and wyomingensis. Analyses of samples from > 600 plants growing in two common gardens showed that UV absorbance was unaffected by environment. Moreover, plant tissues (leaves and seed chaff) explained only a small amount of the variance. UV fluorescence of water-eluted plant tissue has been used for many years to indicate A.t. vaseyana; however, interpretation has been subjective. Use of spectrophotometry to acquire UV absorbance provides empirical results that can be used in seed testing laboratories using the seed chaff present with the seed to certify A. tridentata subspecies composition. On the basis of our methods, UV absorbance values < 2.7 would indicate A.t. vaseyana and values > 3.1 would indicate either A.t. tridentata or wyomingensis. UV absorbance values between 2.7 and 3.1 would indicate a mixture of A.t. vaseyana and the other two subspecies.

English

Black oat crop occupies the largest area during winter. It is important for animal production, crop rotation and dry matter production of approximately 3.8 million hectares in Rio Grande do Sul State. To ensure sowing, seeds are produced which must have high quality standards and must be evaluated by seed testing laboratories. Given this, the present study evaluated the physical and physiological quality of oat seeds analyzed by the Seed Analysis Laboratory of UNIJUI from 2006 to 2014. 2,910 samples were evaluated; 2,229 were evaluated with seed production process; 357 were evaluated with seed analysis of own use and 324 were evaluated with tetrazolium analysis. The samples obtained through seed production process were analyzed in terms of their physical and physiological aspects, while the own seed and tetrazolium test used were evaluated only in terms of their physiological attributes, following the methodology described in the Seed Analysis Rules. The data were analyzed through descriptive statistics for each variable studied per year, and the averages, maximum and minimum standard deviation and coefficient of variation were identified. The data were also evaluated based on their dispersion, and compared to weather occurrences and&nbsp; national standards, in order to estimate the percentages of samples approved. Seeds produced according to the National Seed system had high levels of physical and physiological quality from 2006 to 2010. However, between 2011 and 2014, 14.0 and 14.5% of the samples were above the standard levels for seeds of other cultivated species and harmful prohibited species respectively. The own used seeds showed greater variability and dispersion, with 18.1 and 31.7% samples below the standards for germination in the years 2006 to 2010 and 2011 to 2014, respectively, while the samples analyzed through the tetrazolium test showed approved levels of 19.4 and 12.5%, respectively. The major physiological qualities were obtained in 2008, 2010 and 2011 and the lowest in 2009, 2012 and 2014. It is noteworthy that the seed quality is related to years with levels of rainfall and appropriate temperatures for vegetative development, physiological maturity and harvest. &nbsp; Key words: Avena strigosa (Schreb.), purity, germination, tetrazolium.

Can the Tetrazolium Test be Used as an Alternative to the Germination Test in Determining Seed Viability of Grass Species?

Currently, the standard germination test (SGT) is the official test for evaluating seed viability in seed testing laboratories around the world. The SGT takes 3 to 4 wk to complete for most grasses, including the dormancy breaking treatment period. The tetrazolium test (TZT), however, offers quick viability determination within 24 to 48 h, even for dormant seeds. This study was conducted to compare SGT and TZT results for tall fescue (TF), annual and perennial ryegrass (ARG and PRG), orchardgrass (OG), bentgrass (BG), Kentucky bluegrass (KBG), and fine fescues (FF). A total of 3432 samples representing the seven species were evaluated for viability by SGT and TZT during 2009 to 2013 at the Oregon State University Seed Laboratory (OSUSL). According to the germination tolerance table of the Association of Official Seed Analysts (AOSA), both SGT and TZT results were within tolerance in 94.2% of TF samples, 95.2% of ARG samples, 90.4% of PRG samples, 93.7% of OG samples, 90.8% of FF samples, 83.9% of BG samples, and 42.4% of KBG samples. Dormancy was the main reason for higher viability by TZT results than by SGT in the same sample, causing the two test results to be out‐of‐tolerance. Based on these results, it can be concluded that the TZT can be used as an alternative viability test to the SGT for TF, ARG, PRG, OG, FF, and possibly for BG. However, the KBG has higher levels of dormancy, which brings the SGT below the TZT results, especially for freshly harvested seeds.

CARACTERIZAÇÃO MORFOLÓGICA DE FRUTOS, SEMENTES, PLÂNTULAS E MUDAS DE PAU-BRASIL (Caesalpinia echinataLAMARCK)1

RESUMOEstudos relacionados à morfologia vegetal com espécies florestais nativas têm sido ampliados nos últimos anos, em face da sua importância sob os aspectos taxonômicos ou por serem de grande utilidade na identificação de frutos, sementes, plântulas e mudas, em laboratórios de análises de sementes, em viveiros de produção de mudas ou em estudos que avaliem a regeneração natural em ecossistemas florestais. Porém, diante da grande diversidade de espécies, ainda não é possível encontrar informações suficientes para subsidiar tais estudos e nem coleções em herbários para um grande número de espécies nativas. Assim, este trabalho foi realizado com o objetivo de caracterizar morfologicamente frutos, sementes, plântulas e mudas de pau-brasil (Caesalpinia echinata Lam.) – Fabaceae, Subfamília Caesalpinioideae. Foram realizadas, inicialmente, avaliações das características morfométricas e morfológicas externas e internas dos frutos e sementes. Em seguida, foram realizadas descrições do desenvolvimento inicial de plântulas e mudas. Considerando-se as análises descritivas da espécie, as informações de frutos, sementes, plântulas e mudas podem ser empregadas com segurança em trabalhos de identificação das espécies, tanto em trabalhos de laboratórios e viveiros quanto em estudos de campo sobre a ecologia dessa espécie. Especial atenção pode ser dada ao fruto legume seco, ao embrião, à germinação epígea fanerocotiledonar e à presença de acúleos nas fases jovens (plântulas e mudas), que são importantes para a identificação da espécie. Os caracteres que são comuns a outras espécies de Fabaceae, da Subfamília Caesalpinioideae, corroboram o caráter taxonômico dos aspectos morfológicos.

From knowledge-based research towards accurate and rapid testing of seed quality in winter rape

Winter rape has a life cycle which necessitates rapid testing of seed quality for the seed production sector, namely between July after harvest of the previous crop and August when the next crop establishment begins. As this crop is the fourth most important crop in France, and the most important oilseed crop, the French seed testing station GEVES has managed several research programmes to study seed quality in this species. These collaborative programmes have not only associated seed science with seed technology but also informatics and electronic engineering to provide seed testing tools that are already used by the seed sector, such as conductivity, or innovative tools, such as germination time courses monitored by computer imaging. Forthcoming tools have also been looked at in order to control seed storage potential, which is often used nowadays to anticipate the next season. Biochemical markers have been studied, including seed-specific biotinylated proteins or enzymes involved in free radical (reactive oxygen species) scavenging, such as catalase. Both are well correlated with seed ageing and have been tested to provide an ELISA-like assay for seed testing laboratories. Germination monitoring by computer imaging is now used by research and breeding institutes to phenotype varying seed material, including genotype or mutant collections, primed or ageing seed lots, etc. The next step in seed quality testing in oilseed rape is to characterize variation in germination and seed vigour in order to stabilize optimal yield. This aim constitutes a new international project involving high-throughput phenotyping and genotyping of a large collection of genetically diverse rapeseed genotypes (double haploid populations or diverse cultivars).

A model of a successful utilization of a high genetic potential of maize yield

The principle of a system, defined as a ZP system, implying corresponding relationship among research, seed production and seed marketing, is that each segment within the system has its tasks and responsibilities, as well as, a clear interest. This system was established at the Maize Research Institute, Zemun Polje, almost half a century ago. The crucial characteristic is that this system encompasses obtained results of scientific accomplishments (patent - a released hybrid), optimal utilisation of the environmental conditions, facilities for seed drying, processing and packing, staff and transport capacities. The ZP system provides the economic interest of all participants in studies and the maize seed production. The fundamental base of the quality seed production within the ZP system is a multidisciplinary programme on maize breeding, as well as, 535 released hybrids with standard and specific traits. According to regulations in foreign countries, approximately 100 ZP maize hybrids have been released abroad. Agroecological conditions in Serbia are favorable for the development of the best genotypes and the production of basic and certified maize seed. There 10 processing plants that apply recent technologies in the maize seed processing procedure. Several generations of experts have been trained and gained experience within the maize seed production. Three seed testing laboratories have been accredited by the International Seed Testing Association. According to regulations in Serbia, monitoring of seed production under field conditions, and further on, during the processing practice is done only by designate authorities. This study presents one of successful systems of the seed production organization applicable in countries with similar conditions.

Seed identification using a computerised database

Seed testing laboratories worldwide analyse samples for quarantine assessments to prevent the entry of prohibited and restricted seeds. Current practices of identifying seeds by comparing an unknown seed with samples of known seeds or photographs of seeds are time consuming, costly and inefficient. A Seed Identification Key using a computerised database has been developed to identify prohibited and restricted seeds. There are currently 78 prohibited and 47 restricted seeds in the database. Lucid software was used to develop the Key because of its versatility in handling both text and image data. A total of 21 externally visible seed characters were identified as most suitable for development of the Key. Explanatory images and notes are attached to the character states to assist the user in correct selection of the state. The Key may be helpful to quarantine officers as well as seed analysts working in seed testing laboratories. It may also be used as an educational tool by agricultural scientists, students and others interested in seeds.

Germination status of certified seed samples of paddy, ground nut and cotton collected from different seed testing laboratories of Tamil Nadu

A study was conducted to verify whether the validity period of nine months for the certified seed holds good for paddy, groundnut and cotton produced from various locations and stored under ambient conditions at Coimbatore. The validity period of certified seed samples of 29 paddy varieties (eight varieties each from Madurai and Dharmapuri STLs and 13 from Tirunelveli STL), five groundnut varieties (three varieties from Madurai and two from Dharmapuri STLs) and 11 cotton varieties / hybrids (eight varieties hybrids from Madurai and three from Dharmapuri STLs) were assessed. The results clearly indicated that the validity period of nine months for the certified seeds of paddy and cotton is appropriate. However, the validity period of nine months may be reconsidered for groundnut, since in most of the varieties the germination fell below the minimum standard at 8th month itself. The results also indicated that good quality seed with high initial germination could retain desired levels of germination upto a validity period of nine months. Significant differences due to varieties as well as place of production were noticed for validity period in all the three crops tested. In cotton, irrespective of location of seed collection, the hybrids performed better than varieties.

Precision is an essential component in seed vigour testing

A vigour test assesses, either directly or indirectly, the physiological and physical basis of potential seed lot performance and provides a more sensitive differentiation among seed lots than does the standard germination test. Although seed vigour tests are widely used by the seed industry and are now offered by two-thirds of ISTA seed testing laboratories, vigour testing was only recently (2001 ISTA Congress) approved for inclusion in the ISTA Rules for Seed Testing. The primary reason for this long delay in official recognition has been the lack of uniformity in vigour test results among seed testing laboratories. It is now recognized that greater precision is required for vigour testing than is required for standard germination testing. This recognition has led to improved standardization and uniformity in comparative tests. The objective of this paper is to describe why it has been much more difficult to achieve standardization in vigour testing. It will also outline the procedures, equipment and training needed for seed analysts to achieve and maintain the precision needed in vigour test methods. Finally, it will define the role of seed testing laboratories and the seed industry in the acceptance of seed vigour as a valuable test of physiological seed quality.

A BAYESIAN APPROACH TO ASSESSING LAB PROFICENCY WITH QUALITATIVE PCR ASSAYS USED TO DETECT BIOTECH TRAITS IN CROP SEED

Many seed testing laboratories currently use polymerase chain reaction (PCR) assays to test conventional crop seed for the adventitous presence of biotech trait seed. Seed organizations and companies are spending much time and resources assessing laboratories proficency in running PCR assays. Since many of these assays provide qualitative rather than quantitative results, laboratories must go through a significant effort to obtain adequate assay error estimates. Many sample-processing steps are very similar from assay to assay and therefore error results from different assays may be combined using a Bayesian approach to obtain estimates of assay error rates with increased precision. This paper introduces a relatively simple Bayesian approach that can be used to combine data from a present assay of interest with prior lab data on related assays to obtain updated estimates of assay error rates. If this approach is successfully implemented it can yield as much as a ten-fold reduction in required testing resources.

Inheritance of superoxide dismutase ( Sod-1) in a perennial x annual ryegrass cross and its allelic distribution among cultivars.

Identifying annual ryegrass contamination in perennial ryegrass seed lots has been of major interest in seed-testing laboratories and for seed regulatory agencies in the USA for many years. This study was conducted to characterize a superoxide dismutase locus ( Sod-1) and determine its potential to distinguish cultivated ryegrass species. The inheritance of Sod-1 was evaluated in a three-generation annual x perennial ryegrass mapping population and segregation fitted an expected 1:2:1 ratio for a single locus with two alleles. The molecular form of the Sod-1 locus was determined by H(2)O(2) and KCN inhibitor assays which indicated that the Sod-1, and a second independently segregating Sod-2, locus were both Cu/ZnSod enzymes. The common alleles at the Sod-1 locus were scored in 13 annual and 24 perennial ryegrass cultivars to determine the potential of using this locus for species separation. The Sod-1b allele was homozygous in 98% of perennial ryegrass individuals from 24 cultivars, but those not 100% homozygous for Sod-1b were seed lots with unknown contamination from annual ryegrass. These results indicate that the Sod-1b allele in the homozygous condition is a good indicator of perenniality. All eight annual ryegrass cultivars originating in Europe or Asia had a low frequency of Sod-1b homozygous individuals or none at all. The five cultivars originating in the Western Hemisphere, however, had genotype frequencies for homozygous Sod-1b of up to 56%. The potential of the Sod-1 locus to serve as a test to separate the two growth forms depends on the source of the annual-type contamination.