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a Pickseed West, Inc., 30190 Hwy. 34 SW, Albany, OR 97321
b USDA-ARS National Forage Seed Production Research Center, 3450 S.W. Campus Way, Corvallis, OR 97331
* Corresponding author (barkerr{at}onid.orst.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: AOSA, Association of Official Seed Analysts • GLM, General Linear Models • PSW, Pickseed West, Inc. • SRF, seedling root fluorescence • RCB, randomized complete block • VFL, variety fluorescence level
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Since its discovery as a phenotypic marker, SRF has been used to separate Italian ryegrass from perennial ryegrass (Gentner, 1929). Generally, seedling roots of Italian ryegrass growing on white filter paper secrete an alkaloid (annuloline) that fluoresces under ultraviolet light, but the majority of perennial ryegrass seedlings do not fluoresce. Fluorescent perennial ryegrass populations exist however, as do populations of nonfluorescent Italian ryegrass (Nyquist, 1963; Nitzsche, 1963; Okora et al., 1999).
An accurate test that detects both physical and genetic contamination of Italian ryegrass in perennial ryegrass cultivars is necessary, especially if the perennial ryegrass seed is to be used for permanent turf. The phenotypic differences between the two species in foliage color, leaf width, and growth rate can have significant impact in high quality perennial ryegrass turf. Italian ryegrass often exhibits more rapid vertical growth, less tillering, with coarser and lighter-colored foliage than perennial ryegrass. Contamination of perennial turf stands with annual type ryegrasses would detract from the otherwise aesthetically pleasing appearance of perennial ryegrass with dark-green color and fine turf texture. Other than a physical plant grow-out test for each seed lot, SRF is the only accepted test for detecting the presence of Italian ryegrass in seed lots of perennial ryegrass; however, this test is not exact, and false positives can easily occur due to a lack of complete test standardization, age of seed, and seed analyst subjectivity in scoring results. Additionally, tolerances are not applied to test results from year to year, or field to field.
The SRF trait has introgressed from Italian to perennial ryegrass, and plant breeders can document SRF levels of new ryegrass cultivars (labeled VFL for variety fluorescence level) before they enter a seed certification system (AOSA, 1994, 1998). Breeders began documenting inherent fluorescence levels for new and existing ryegrass cultivars in 1991 (Association of Official Seed Certifying Agencies, 1991, p. 126–127 and 132–133). By winter 1999, fluorescence values of 148 perennial ryegrass cultivars and 9 Italian ryegrasses were documented (USDA, 1999).
This study was initiated to obtain information that would assist ryegrass breeders in documenting SRF levels in developed cultivars, and to provide a basis for proper interpretation of SRF test results for turf marketing purposes. Wide variation caused by environmental influences where seed is grown would reduce dependability of the SRF test, which is so extensively used in the USA. Objectives were (i) to determine if fluorescence expression of a cultivar (population) remains stable through generations of seed increase, and (ii) to determine if the geographical location or year of seed multiplication affects fluorescence expression of ryegrass cultivars.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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4 cm of water in mid-May prior to anthesis. Additional artificial irrigation was not supplied throughout the seed development season. All possible pair cross combinations were made among stock plants in late May and early June 1990 by mutual bagging of inflorescences in 9.5- by 40-cm glycine bags. Supports were assembled to keep bags erect and intact under field conditions until seed maturation. Mature spikes from each pair cross were harvested in early July, and kept separate by maternal parent. Seed from each maternal parent of each cross was hand-threshed and conditioned using hand screens and a commercial laboratory seed blower. Seed was stored in a cool (5°C), dry room until needed for germination.
Seed from several pair crosses were sown on white filter paper during October 1990. Rules from the Association of Official Seed Analysts (AOSA) were followed for germination and SRF tests (AOSA, 1994). Seedling roots were scored for the presence (+) or absence (-) of fluorescence. Only roots that had brilliant fluorescence without lifting from the filter paper and those that did not show evidence of SRF after lifting from the filter paper were used in population development. Selected seedlings were transplanted to 4.0- by 19.5-cm plastic cone pots (conetainers) filled with commercial potting soil mix. Seedlings were cultured in a greenhouse at a daily temperature of 20°C with a natural 10-h daylight period and ambient humidity. Artificial night heating was not supplied, but the temperature was prevented from dropping below 2°C. As progeny seedlings developed into multitillered plants, each was divided into 20 ramets and placed together within a conetainer rack.
When inflorescence emergence initiated in early May 1991, heading dates (Day of Year when one spike of the parental plant was emerged from boot), vegetative leaf vernation (visually scored on expanding vegetative leaves: F = folded, R = rolled, or I = intermediate), presence (+) or absence (-) of floral awns, and relative assessment of glume length (subjectively evaluated relative to the total length of spikelet) were recorded for each progeny. These characteristics have been used to separate Italian and perennial ryegrass (Nittler and Kenny, 1972; Humphries, 1980; Jung et al., 1996). Maternal progenies were discarded if ramets were weak or failed to express reproductive spikes.
Twenty-nine clonal maternal progenies were assembled into three synthetic populations based on previously defined foliage phenotype, similar anthesis date, and fluorescence expression (Table 1). Populations 1 and 2 phenotypically resembled perennial ryegrasses, and Population 3 was a mixed phenotype of Italian and perennial ryegrass. Populations 1 and 3 had 10 maternal progenies each, and Population 2 had nine maternal progenies. A fourth population was constructed using six clonal progenies from Gulf and other diploid accessions of unknown Italian ryegrass (i.e., variety not stated classification). These were obtained from Agri-Seed Testing and Tangent Seed Lab International, two private seed testing laboratories. The six clonal progeny parents used in Population 4 had the Italian ryegrass phenotype, but five were nonfluorescent variants (Table 1). Thus, populations were assembled to represent low, medium, and high fluorescence levels based on parental SRF expression, and to represent both perennial and annual growth habit characteristics.
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To avoid possible winter kill in central Oregon, parental clones of the four populations to be established at Madras, were maintained at the Pickseed West, Inc. (PSW) research farm, Albany, OR, during autumn 1991. Starting February 1992, ramets of these parents were transplanted to larger conetainers (7 by 25 cm) and kept at a daylight exposure of 9 h accomplished by covering plants with 6-mm black plastic covers at 1630 h PST daily. The plants were uncovered at 0730 h each morning. Populations were transplanted to field nurseries at Madras on 10 Apr. 1992. Plots at Madras were only fertilized at transplanting with a broadcast application of granular formulation of ammonium sulfate (21-0-0-24, N-P-K-S) at 45 kg N ha-1 and for each season of the test. From the time of transplanting until 1 wk before seed harvest, plots at Madras were irrigated weekly to maintain plant turgor and overall vigor for the test duration. Supplemental irrigation was not necessary at the other sites during the years of seed increase.
The first spring flush of weeds was controlled at the western Oregon locations by interrow applications of Paraquat (1:1-dimethyl-4, 4'-bipyridinium dichloride) herbicide at a rate of 0.24 L ha-1. Treatment was applied with a knapsack sprayer. Subsequent weed control was done by hand hoeing. Weed control at the Madras site was done by hand hoeing throughout the entire test, after a preplant broadcast application of glyphosate {N-(phosphonomethyl) glycine} herbicide at the rate of 4.09 L ha-1.
Seed was harvested with a handheld serrated knife in late June to early July at all three locations by cutting each parental line when seed began to shatter (natural disarticulation). As seed was harvested, it was dried under ambient field conditions in a covered shed. Seed was threshed, conditioned, and weighed for each ramet, and then bulked by maternal line to create half-sib families.
Syn 2 and Syn 3 Generation Increases
Five replicates of 100 random seeds for each maternal family were counted from the Syn 1 or Syn 2 increase of each population using a commercial lighted counter board. Selection for caryopsis size was avoided. Since it was necessary to retain at least 100 viable seeds of each family line for a final fluorescence test, any family which did not produce total seed 
600 in 1992 was discarded from future increase (Table 1).
Direct seeded rows were established in a RCB experimental design with five replications at the western Oregon locations. Rows consisted of progeny seeds for each half-sib family of all populations. Rows were 1 m long and spaced 61 cm apart. Seed was planted 8 mm deep by hand. Syn 1 seeds were sown on 18 October 1992 at Corvallis, and during the first week of November 1992 at Aurora. Syn 2 seed produced was sown at Corvallis on 20 Oct. 1993 and at Aurora on 28 Oct. 1993 to produce the Syn 3 generation. Identical numbers of family lines for each population were sown at both western locations. Plots were planted using carbon banding (Extension Services of Oregon State University, Washington State University, and the University of Idaho, 1995). Diuron {3-(3, 4 dichlorophenyl)-1, 1-dimethylurea} was applied preemergence to control weeds. Spring weeds were controlled as noted above. Plots at Corvallis and Aurora were sown into areas used as cereal borders the previous generation, and new cereal borders were established. Plot rows were carefully examined throughout the growth period for Italian ryegrass that might appear as a weed; none were found. Border crops were scouted for Italian ryegrass weed contamination and removed by hand prior to anthesis in the populations.
For the Madras location, if any maternal family did not produce at least 25 progeny seeds from the 1992 bulk harvest, it was dropped from succeeding increase generations. Thus, future increases at Madras did not quite parallel the family constitution of the Aurora and Corvallis locations because of differential survival and recombination of parental stocks in 1992 at Madras relative to the other two locations (Table 1). Syn 1 seeds were germinated, evaluated for fluorescence expression, transplanted to conetainers, and kept at PSW until transplanted to the field on 20 Apr. 1993. Syn 2 progeny were transplanted 13 to 14 Apr. 1994. Cereal barriers were not possible for the second and third increase generations at Madras; consequently, plots were sown in fallow areas and only distance isolation was used. In these seasons, distance between populations was never <76 m.
Seed was harvested by maternal half-sib family at each location during July. Seed was dried under ambient conditions, threshed, conditioned, weighed for each maternal family row, and then bulked by maternal origin among replications for each generation of open-pollinated increase.
Seed Testing
Seed from both 1992 and 1993 harvests were kept in cool (5°C), dry storage from the time of conditioning until the beginning of seed testing. Germination and SRF tests were conducted during winter 1994–1995 at the Oregon State University seed testing laboratory. Seed produced in 1994 was given a 7-d pre-chill treatment (stratification) at 5°C, as specified by AOSA rules, to allow breakage of latent dormancy (AOSA, 1994). Dormancy was not a consideration for the older harvested seed. Seed tests were conducted for each population–generation–location combination in 16.5- by 24.5-cm acrylic germination boxes. Germination substrate was Whatman (Clifton, NJ) white filter paper moistened by 2.1 g L-1 potassium nitrate solution.
Fluorescence tests were conducted with equal amounts of progeny represented from each half-sib family within a population to provide 100 seeds from a population in each of four replications of the test. The SRF test then closely approximated the 400-seed standard test conducted on commercial seed lots. For example, Population 4 was constituted of six half-sib families; thus, sixteen progeny seeds from each family for a given generation at a location were tested in four replications; (16 x 6 x 4) = 384 seeds tested for one population–generation–location. Progeny seed of each family were hand sown and arranged randomly within a germination box. Germination boxes were arranged in the germinator in a completely randomized design. All boxes were confined to the same shelf in the same germinator for the duration of the experiment. Germinator lighting (photosynthetically active radiation = 89 µmol m-2 s-1), temperature (alternating 15 and 25°C with light during the 8-h 25°C period), and relative humidity (95%) followed standard protocols (AOSA, 1994, 1998). Each seed test was conducted at two different times and data were pooled for the two runs. Seedling root fluorescence determinations were made in a darkroom, using a near ultra-violet two bulb lamp to visualize fluorescence at 7 and 14 d after germination (AOSA, 1994). Fluorescent root streaks, detected after all seedlings were lifted from the filter paper at 14 d, were included in test fluorescence values.
Statistical Analyses
Data were analyzed using the General Linear Models (GLM) procedure (SAS Institute, 1994) to conduct an analysis of variance. Mean separations were by t-tests with the LSMEANS option of GLM. The statistical model considered populations, generations, and locations as fixed effects. Results presented pertain to gross population changes by location and generation of seed multiplication.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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It is not clear why location of seed increase had such a large impact on SRF level. Generation response was similar at Aurora and Madras with little or no change in SRF, but SRF always increased with increasing generation at Corvallis. Populations would not be expected to change in SRF unless there was a selection pressure for SRF either directly or indirectly; or there was contamination from an outside source either from seed or pollen. Foreign pollen may have been a source of contamination because of high concentrations of commercial seed production fields of Italian ryegrass near Corvallis in the southern Willamette Valley (Young and Griffith, 1996). Even though populations at Corvallis were isolated by cereal rye, possible contamination sources may have been higher than suspected. It is unknown how far ryegrass pollen may move, nor how long it stays viable. Had Italian ryegrass pollen contamination been a factor for SRF increase, however, it seems unlikely that each population would have responded similarly because anthesis date of the populations was somewhat different relative to each other (Table 6). Italian ryegrass as a crop in neighboring fields, or as volunteers in land adjacent to the test plots, would have flowered 7 to 10 d earlier than perennial ryegrass. Populations 1 and 2 phenotypically resembled true breeding perennial ryegrass. Perennial and Italian ryegrass overlap in anthesis time during the day (Rampton, 1966), but may differ greatly in date of anthesis.
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Population 4 was phenotypically and physiologically most similar to volunteer Italian ryegrass. It would have been the only population likely to flower coincidentally with commercial Italian ryegrass seed production fields. Mean anthesis date of families within Population 4 at Corvallis was 8 and 9 d earlier than mean anthesis date of families in Populations 1 and 2 in 1992, 6 and 8 d earlier in 1993, and 4 and 7 d earlier in 1994, respectively (Table 6). Anthesis date differences, the presence of a cereal crop physical barrier, and absence of significant other Italian ryegrass characteristics in subsequent generations or grow-out tests suggested that pollen contamination, from either Italian ryegrass commercial fields or from the other populations themselves, did not cause increased SRF at Corvallis.
For the present study, from 11 to 42% immigration (contamination) from Italian ryegrass to the perennial-type populations would be needed to account for the generation to generation change of the reported magnitude (Falconer and MacKay, 1996). This amount of contamination from pollen load was highly unlikely considering the wide variation in anthesis dates and the duration of anthesis in perennial ryegrass.
Results reported herein are supported by Copeland and Hardin (1970), who used SRF to detect pollen contamination in commercial ryegrass seed production fields in western Oregon. They found little contaminant Italian ryegrass pollen in perennial ryegrass beyond 6 m from the edge of perennial ryegrass fields, even with large pollen loads from an adjacent Italian ryegrass field. Beyond 12 m from the border, there was no evidence of contamination from Italian ryegrass. Griffiths (1951) pointed out that for perennial ryegrass, as the abundance of noncontaminating pollen increases, in large production fields, fertilization by contaminating sources is reduced. He showed that border rows of plants were highly effective in reducing contamination among perennial ryegrass accessions, and suggested individual ryegrass plants were fertilized primarily by pollen from neighboring plants in their immediate vicinity.
Barker et al. (1997) and R.E. Barker (1997, personal communication) examined SRF levels for three cultivars increased 4 yr from fields of the same cultivars located in both north and south sections of the Willamette Valley. Three laboratory SRF test runs were made on each seed lot. Besides the obvious difference among cultivars for SRF, they found SRF varied within cultivars across years and across test runs; however, differences across seed production environments were not found, indicating that pollen contamination from possible higher Italian ryegrass pollen loads in the south Valley did not generally increase SRF. Seed sampled by Barker et al. (1997)(and Barker et al., 1997, unpublished data) came from perennial cropped fields of established cultivars. Thus, each cultivars' frequency of SRF expression was expected to have been stable across years of certified seed production.
Selection and genetic drift could also have affected the distribution of alleles for SRF in the small population sizes used in this study. Selective advantage between genotypes among varying environments is possible depending on differential tillering capacity, differences in plant height, or anthesis duration among half-sib families. No appreciable differences for these traits were observed, nor were there differences for seed production among maternal clonal lines. A separate experiment using clonal genotypes established at each location would be required to measure environmental conditions on seasonal family variations in growth and seed production.
The Corvallis and Aurora sites had soils with similar pH and general physical properties. The Madras site had drier soils with higher pH and substantially different physical structure than the Willamette Valley soils. Populations at the Madras site were irrigated throughout the duration of the test, while the other two locations relied only on precipitation. Soil temperatures were not monitored during the study. Future research on SRF should include more thorough classification of soil properties, soil water potentials, and initial soil fertility exchange capacity and soluble anion levels.
Ryegrass SRF has long been used as a determinant trait for cultivar integrity in seed certification schemes in the USA. In commercial applications, it is still assumed by many that variation in SRF test results for individual cultivars is entirely genetic in origin. Tolerances across generations or years are not applied to SRF test results. Results reported herein clearly indicate that the environment where ryegrass plants are grown can affect the expression of SRF, but the cause is yet to be determined. Actual contamination from Italian ryegrass may be misrepresented by variable SRF test results of certain seed production lots (1995 and 2000, personal communication with ryegrass seed growers). If causes of SRF variability are not better understood, the test should not be used as a trait to describe cultivars, unless the results of the laboratory test are reinforced with evidence of off-type appearance in seed production field inspections. Linehan and Mercer (1933) suggested that fluorescence testing should be used only for making approximate determinations in classifying ryegrass seeds. Rampton (1938) stated that fluorescence tests cannot be used as infallible guides to classification of questionable lots of Oregon grown domestic ryegrass seed. Our results confirm the conclusions drawn by these early researchers, yet because many cultivars now have an established VFL, the SRF test is considered an infallible method to separate Italian from perennial ryegrass. The VFL is established from at least three seed lots and at least two generations, with one generation being breeder seed. Thus, only early generation seed is often used to establish VFL, and based on results reported herein, the value may change in later generations.
As long as the USA Federal Seed Act allows plant breeders the right to describe a cultivar's inherent fluorescence value, the SRF test will continue to be used as a descriptive cultivar trait and seed marketing tool. Results of research herein make it clear that plant breeders should utilize wide geographic seed lot sampling for applications to document fluorescence levels of new ryegrass cultivars. Because the VFL for a cultivar is used as a baseline for ryegrass seed marketing purposes, the plant breeder must be certain an accurate estimate of mean VFL be reported. Further, some measure of the true variance of VFL should be required to accurately describe a given cultivar.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Received for publication June 25, 2001.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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