Determining the Extent of Frost Damage in Maize Seed Using the Tetrazolium Test

Frost damage in seed corn causes a significant decrease in seed quality. The tetrazolium (TZ) test has been used to identify types of physiological damage in several plant species, including frost damage in corn. Quantifying the amount of seed damaged by frost and the consequent loss of seed vigor are important for making management decisions. The current tetrazolium test procedures provide seed viability information but do not estimate seed vigor. In our project, the tetrazolium staining procedure was modified to allow separation of viable seeds into vigor categories. Vigor categories were defined based on Association of Official Seed Analysts (AOSA) recommendations, previous research, and current observations. The results from the tetrazolium test were compared with standard germination and saturated cold tests during the first 6 months of storage. The staining procedure aided in visual identification of frost damage. Although a strong correlation was found between seed viability as determined by the tetrazolium test, the standard germination test, and the saturated cold test, sorting seeds into vigor categories in the tetrazolium test did not improve the correlation between the tetrazolium test and the saturated cold test. Disciplines Agricultural Science | Agronomy and Crop Sciences | Plant Breeding and Genetics Comments This is a manuscript of an article published as DeVries, Mindy, and A. Susana Goggi. "Determining the extent of frost damage in maize seed using the tetrazolium test. " Crop Management 5, no. 1 (2006). doi: 10.1094/ CM-2006-0414-01-RS. Posted wtih permission. This article is available at Iowa State University Digital Repository: https://lib.dr.iastate.edu/agron_pubs/493 Determining the Extent of Frost Damage in Maize Seed Using the Tetrazolium Test Mindy DeVries, Department of Agronomy, Iowa State University, Ames, IA A. Susana Goggi, Department of Agronomy, Iowa State University, Ames, IA Abstract Frost damage in seed corn causes a significant decrease in seed quality. The tetrazolium (TZ) test has been used to identify types of physiological damage in several plant species, including frost damage in corn. Quantifying the amount of seed damaged by frost and the consequent loss of seed vigor are important for making management decisions. The current tetrazolium test procedures provide seed viability information but do not estimate seed vigor. In our project, the tetrazolium staining procedure was modified to allow separation of viable seeds into vigor categories. Vigor categories were defined based on Association of Official Seed Analysts (AOSA) recommendations, previous research, and current observations. The results from the tetrazolium test were compared with standard germination and saturated cold tests during the first 6 months of storage. The staining procedure aided in visual identification of frost damage. Although a strong correlation was found between seed viability as determined by theFrost damage in seed corn causes a significant decrease in seed quality. The tetrazolium (TZ) test has been used to identify types of physiological damage in several plant species, including frost damage in corn. Quantifying the amount of seed damaged by frost and the consequent loss of seed vigor are important for making management decisions. The current tetrazolium test procedures provide seed viability information but do not estimate seed vigor. In our project, the tetrazolium staining procedure was modified to allow separation of viable seeds into vigor categories. Vigor categories were defined based on Association of Official Seed Analysts (AOSA) recommendations, previous research, and current observations. The results from the tetrazolium test were compared with standard germination and saturated cold tests during the first 6 months of storage. The staining procedure aided in visual identification of frost damage. Although a strong correlation was found between seed viability as determined by the tetrazolium test, the standard germination test, and the saturated cold test, sorting seeds into vigor categories in the tetrazolium test did not improve the correlation between the tetrazolium test and the saturated cold test. Introduction Frost damage is costly to seed producers for two reasons. First, the seed decreases in value because of physiological damage and second, the extent of damage can only be assessed after seed harvest, drying, conditioning, and storage of damaged seed. The delay in assessing seed deterioration is caused by the initially slow decline in seed vigor and viability. The tetrazolium test is traditionally used for determining viability, although its use for seed vigor determination is outlined in the Tetrazolium Testing Handbook [1] and Seed Vigor Testing Handbook [3]. The tetrazolium test is often considered the most accurate test for early determination of frost damage in seed corn. This test requires trained analysts and more labor than traditional germination tests, but it leads to faster results. Seed damage can be assessed in 24 hours using the tetrazolium test rather than waiting 7 to 8 days for the results of a germination test. Although the tetrazolium test is the first tool for identifying frost damage, germination tests should be used for evaluating subsequent damage in storage [5]. The results obtained with the tetrazolium test must correlate to either the standard germination test or a vigor test such as the cold test to be a valuable tool in seed testing. The use of mechanical detection systems for evaluating tetrazolium -stained seeds has been explored [6, 12]; however, only the color ratio was measured. Scanning tools were used to calculate the ratio of red-stained tissue to white-unstained tissue. This value was termed the tetrazolium staining ratio and was used to determine the viability of the seed. However, the location of staining patterns is as critical as the staining intensity [4]. The precision of these mechanical evaluations is reduced because the location of stained and unstained tissue is not taken into account. Xie and Paulsen [12] increased the accuracy of evaluation by creating a larger algorithm. Their algorithm also accounts for the size of the unstained area based on the assumption that the larger the unstained area, the greater the likelihood that the seed is nonviable. However, they were unable to separate critical locations of unstained areas such as the radicle, plumule, and seminal roots from the entire embryo. This inability to identify staining patterns in critical regions limits the application of mechanical detection for diagnostic evaluations. Therefore, skilled analysts are still necessary for interpreting the tetrazolium test for vigor determination. The traditional staining procedure outlined in the Association of Official Seed Analysts (AOSA) Tetrazolium Testing Handbook [1] states that seeds should be imbibed overnight, bisected longitudinally through the embryo, placed in 0.1% tetrazolium chloride solution, and rinsed with water before evaluation. However, the traditional staining method is inadequate for accurately assessing frost damage. The embryos are stained dark, and staining patterns are difficult to differentiate. The vigor categories of seed stained by the traditional method, especially high and medium vigor, are too similar to separate. We feel that it is important to develop an improved staining method for the tetrazolium test and to clearly define vigor categories for this test specifically relating to frost damage. By improving the identification of frost damage in seed corn, seed producers and brokers have a better tool for making management decisions. The objective of our research was to develop a consistent tetrazolium staining method and evaluation criteria for separating frosted seed into vigor categories. Additionally, we wanted to compare the numbers of seeds in these vigor categories to results from the standard germination and saturated cold tests. Production of Frost-Damaged Seed Corn Hybrids B73xIRF311 and Mo17xIRF311 were produced in isolation in 2003 and 2004 near Ames, IA. These two female lines were chosen to represent different genotypic tolerance to cold temperatures. B73 was developed by the federal-university corn breeding program at Iowa State University and is considered relatively tolerant to cold temperatures. Mo17 was developed by the federal corn breeding program at University of Missouri and is considered relatively sensitive to cold temperatures. IRF311 pollinator is from the Iowa Research Foundation and was selected based on nick with B73 and Mo17. Samples were harvested at three moisture content ranges; 30-35%, 40–45%, and 50–55% moisture content fresh weight (fw) basis. Two field replications were used for this experiment. Ears were harvested with the husks and shank intact. Immediately following harvest, all ears were placed in a cold room at 50°F for a minimum of 4 hours to ensure uniform cooling rates. Control samples were then husked and dried, while remaining ears were frozen in a Conviron growth chamber (Controlled Environment Limited, Winnipeg, Manitoba, Canada). The frost cycle lasted 24 hours, beginning and ending at 50°F and including a minimum of 8 hours below 32°F and 2 hours at the minimum temperature 23°F. After the frost treatment, ears were husked and dried. Samples were dried on the ear in experimental sized forced air driers at a temperature below 90°F. All samples were shelled using an experimental sheller and stored in a cold room (50°F at 50% RH) until the time of testing. Additional seed samples were harvested after the first natural frost event recorded on 2 October 2004. Data from these naturally frosted samples were included in the study as a comparison. However, seeds had reached physiological maturity (as determined by black layer formation) in the field before a natural frost; therefore, all naturally frosted samples were below 35% moisture fw at harvest.