Japanese Society of Grassland Science ISSN1744-6961

ORIGINAL ARTICLE
Blackwell Publishing Asia

Effect of storage temperature and duration on the vigor of zoysiagrass (Zoysia japonica Steud.) seed harvested at different maturity stages
Pei Sheng Mao, Xin Guo Wang, Yu Hong Wang and Jian Guo Han
Forage Seed Laboratory, China Agricultural University, Beijing, China

Keywords Seed maturity; seed vigor; seed storage condition; Zoysia japonica. Correspondence Pei Sheng Mao, Forage Seed Laboratory, China Agricultural University, 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, China. Email: maopeisheng@hotmail.com Received 1 October 2007; accepted 4 November 2008. doi: 10.1111/j.1744-697X.2009.00129.x

Abstract
Zoysiagrass (Zoysia japonica Steud.) is one of the most important warm season grasses used as forage and ground cover in eastern Asia. This study was conducted to evaluate the effects of temperature and duration of storage on germination and conductivity in zoysiagrass seeds. The seeds were harvested at the milk, dough and hard stages, and stored at a low (7C) or an ambient (1530C) temperature for 618 months in sealed polyethylene bags. The standard germination rate of seeds decreased signicantly (P < 0.05) as the storage period prolonged from 6 to 18 months. Germination was almost always better in seeds stored at the low temperature than at the ambient temperature and highest in seeds harvested at the hard stage followed by the dough and the milk stage. Following accelerated aging, germination was highest when harvested at the hard stage and stored for 12 months. Bulk conductivity was lower in seeds stored for 6 and 12 months than those stored for 18 months and in seeds harvested at the hard stage than those harvested at the dough and the milk stage. The results indicated that zoysiagrass seed maintained a higher vigor level when harvested at the hard stage and stored at low temperature (7C) for 12 months or less.

Introduction
Zoysiagrass (Zoysia japonica Steud.), a perennial rhizomatous grass (family, Poaceae; tribe, Zoyseae), is indigenous to China, Japan and Korea. There are large areas of native vegetation on the Liaodong Peninsula and Jiaodong Peninsula of eastern China, which are the primary areas for zoysiagrass seed production. Zoysiagrass can be used to establish athleticeld turf and landscaping turf as well as for grazing and soil stabilization, as this grass exhibits excellent trampling and drought tolerance (Yeam et al. 1985; Akiyoshi et al. 1998; Li et al. 2002; Wang 2003). Also, high-quality zoysiagrass turf could be established in the transition zone which requires low maintenance (Patton et al. 2004; Zuk & Fry 2005). Zoysiagrass performs well, without stagnancy and disease infection in the hot and humid summer climate of Beijing, while some cool season turfgrass such as Poa pratensis and Lolium perenne were found to have more difculty to survive. In the hot and humid summer climate of the transition zone, zoysiagrass is best for establishment of golf course and landscape turf.
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Historically, seeded zoysiagrass had limited use because of poor turf quality, germination rate and seed production (Xu et al. 2005; Patton et al. 2006). Chinese common, harvested from native stands in China, has been the most commonly seeded variety for many years. However, average zoysiagrass seed yields have been less than 370.5 kg ha1 (Ma et al. 2004), as the seed maturity is not uniform and there is a strong tendency for vegetative propagation. Seed viability decreased rapidly with increasing storage time when the dried seed was stored at temperatures of 1530C (Qian et al. 2000). The germination percentage of zoysiagrass seed dropped rapidly when seeds were stored for 1 year in southern China, particularly in areas with high summer temperatures and high humidity. The environmental conditions in which seeds are stored are important factors in determining how long seed can be stored while maintaining an acceptable level of germinability (Marshall & Lewis 2004). Qian et al. (2000) found that the germination percentage of zoysiagrass seed decreased from 96.5% to 40.5% after 5 years in a seed storeroom with temperatures of 1530C.
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Comparing zoysiagrass seed soaked in 20% NaOH solution before storing with seed treated after storage, Chen (2003) found that seeds soaked in NaOH before storage were more susceptible to deterioration during the 4 years of storage. Storage in sealed polyethylene bags at a low temperature (5C) could slow the seed deterioration. However, the effect of storage condition on the vigor of zoysiagrass seeds harvested at different stages of maturity remains unclear. Generally, it is difcult to determine the stages of seed ripeness in grass species. Pegler (1976) suggested that endosperm consistency was a reliable indicator of seed maturity. Six endosperm stages were used in order of progression; watery, milky, creamy, cream cheese (soft dough), cheesy (rm dough) and hard. Simon et al. (1997) summarized the optimal harvest time for grass species according to the endosperm consistency, days after peak anthesis, moisture content and morphological ripeness, however, zoysiagrass was not included in his work. Ma and Han (2005) studied the reproduction characteristics and production techniques of zoysiagrass seed in Jiaozhou, Shandong Province from 20012003. Standard seed germination rate and germination after accelerated aging increased with advancing seed development stages, reaching the highest level when zoysiagrass seed was harvested at the hard stage. Therefore, proper harvest timing appears to be the most critical factor in maintaining high viability of zoysiagrass seed during storage. Accelerated aging (AA) is a method which exposes seeds for short periods to environmental conditions (high temperature and high relative air humidity) causing rapid seed deterioration (Delouche & Baskin 1973; Hampton & TeKrony 1995). This method enables evaluation of seed vigor within a relatively short experimental period and therefore has been widely used in testing various species. The objective of this research was to investigate the effect of storage temperature and duration on the viability of zoysiagrass seed harvested in the milk, dough and hard stage, in terms of standard germination, germination after accelerated aging and bulk conductivity. The resulting data will provide information on optimum harvest time and storage conditions to maintain high quality zoysiagrass seed.

Table 1 Symbols for the combinations of zoysiagrass seed maturity and storage temperature Symbols MA ML DA DL HA HL Seed maturity Milk stage (0%) Dough stage (77%) Hard stage (85%) Storage temperature Ambient temperature Low temperature Ambient temperature Low temperature Ambient temperature Low temperature

Standard germination rate before seed storage. Ambient temperature, 1530C; low temperature, 7C.

The seeds were tested for germination capacity and conductivity after 6, 12 and 18 months of storage. The symbols for the different seed harvest stage/seed storage temperature combinations and corresponding initial germination rate of each seed maturity level prior to storage are presented in Table 1.

Sample pretreatment
Prior to testing, seed samples were soaked in a 70% alcohol (ethanol analytical reagent) solution and the sinking seeds were selected as the working sample. The working seeds were immersed in a 10% NaOH solution, followed by shaking for 15 min in a water bath at 25C. Seeds were then rinsed for 6 h with tap water, then air-dried in preparation for the experiment.

Standard germination
Standard laboratory germination tests (as per ISTA 2003 protocol) were conducted in an incubator (LRH-250-G, Guangdong Medical Instruments Factory, Guangdong, China) set at an alternating 8-h light period at 35C and a 16-h dark period at 20C. Four replications of 100 seeds each were utilized. The seeds were placed on top of three layers of lter paper (11-cm diameter qualitative lter paper produced by Hangzhou Xinhua Paper Industry, Hangzhou, China) in Petri dishes and a 0.2% KNO3 solution was used to initially saturate the lter paper. Distilled water was used for subsequent moistening. Normal seedlings were counted after 10 and 28 days of incubation, with the percentage of normal seedlings determined as described in ISTA rules (2003).

Materials and methods

Seed material
In June 2002, zoysiagrass seeds were collected at different maturity stages from native stands near Jiaozhou, Shandong Province. Milk, dough and hard stage seeds were harvested 10, 31 and 42 days after peak anthesis, respectively (40%, 28% and 24% moisture content). After air drying, equal amounts of seed of each maturity stage were placed in sealed polyethylene bags and stored in a refrigerator (MPR-311D, Sanyo Medicool, Tokyo, Japan) at a low temperature (7C) or in a storeroom with ambient temperatures ranging 1530C.
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Accelerated aging
Distilled water (50 1 mL) was placed in each plastic accelerated aging (AA) box (12 cm length 12 cm width 5 cm depth) and a dry plastic screen tray (diameter, 11 cm; depth, 4.5 cm; mesh size, 0.5 mm 0.5 mm) was inserted, being certain not to splash water onto the screen. At least 400 seeds of each sample were placed on the surface of the screen tray. Seeds
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were spread out in a single layer to ensure uniform moisture uptake from the humid environment. After securing the lids of each AA box, they were placed in the aging chamber (DNP-9162, Shanghai Jinghong Laboratory, Shanghai, China) set at a constant temperature of 41 0.3C for 72 h 15 min. Germination tests were conducted for each sample of manipulated seeds (as per ISTA 2003 protocol) within 1 h after the completion of the AA treatment.

Table 2 Treatment means and ANOVA results for germination rate (%) of zoysiagrass seed Storage duration (months) Seed maturity and storage temperature MA ML DA DL HA HL Mean 6 50a 73b 76bc 82d 82d 81cd 74C 12 46a 62c 52b 77d 78d 88e 67B 18 9a 33b 27b 55c 69d 79e 45A

Bulk conductivity
Bulk conductivity tests were performed using a DDS-II conductivity meter (Tianjin Second Analysis Instrument Factory, Tianjin, China). Two replications of 50 seeds of each sample were soaked in 100 mL of de-ionized water at 20C for 24 h, after which the conductivity of the water of each sample was measured (ISTA 2003).

See Table 1 for symbols. Values followed by different lowercase letters within the same column are significantly different at P < 0.05. Values followed by different uppercase letters in the bottom row are significantly different at P < 0.05.

Statistical analyses
anova was performed using SPSS for Windows ver. 11.0 (SPSS, Chicago, IL, USA), by taking the germination rate and bulk conductivity measurement as dependent variables. Duncans multiple range test (P = 0.05) was used to compare treatment means of germination and conductivity.

those stored at ambient storeroom temperatures (1530C) across the seed maturity stages and seed storage durations.

Effect of storage treatments on germination after AA zoysiagrass seeds harvested at different maturity stages
Germination rate of AA treated zoysiagrass seeds varied signicantly (P < 0.05) with the storage durations, with 12 months showing the highest rate, followed by 18 and 6 months (Table 3). Within each storage duration, the germination rate of AA treated seeds was signicantly (P < 0.05) higher at the low storage temperature (ML, DL and HL) than at the ambient temperature (MA, DA and HA) for seeds stored for 12 and 18 months, but was not signicantly different (P > 0.05)

Results and discussion

Effect of storage treatments on the germination of zoysiagrass seeds harvested at different maturity stages
Germination rates of zoysiagrass seed decreased signicantly (P < 0.05) as the storage period prolonged from 6 to 18 months, with moderate declines in seeds harvested at the hard stage (Table 2). Within each storage duration, germination was better in seeds stored at the low temperature (7C) than at the ambient temperature (1530C), except the hard seeds (HA and HL) stored for 6 months. Germination was almost always highest in seeds harvested at the hard stage, followed by the seeds harvested at the dough and the milk stage. High seed quality is paramount in establishing uniform stands of a desired plant density. Qian et al. (2000) found that the germination rate, germination index and vigor index were strongly correlated (P < 0.01) with the storage duration. They showed that the germination rate of zoysiagrass decreased 56% after 5 years when the seeds were stored at temperatures of 1530C. Chen (2003) suggested that seed germination rate, germination index and vigor index could be maintained at the highest level when the seeds were stored in sealed bags for 4 years under a low temperature (5C). In the present study, zoysiagrass seeds stored at low temperature (7C) almost consistently showed higher germination rates than
Grassland Science 55 (2009) 15 2009 The Authors Journal compilation 2009 Japanese Society of Grassland Science

Table 3 Treatment means and ANOVA results for germination rate after accelerated aging (%) of zoysiagrass seed Seed maturity and storage temperature MA ML DA DL HA HL Mean Storage duration (months) 6 2a 4a 11b 10b 23c 25c 12A 12 29a 38b 30a 57c 59c 77d 48C 18 3a 10b 18c 38d 51e 65f 31B

See Table 1 for symbols. Values followed by different lowercase letters within the same column are significantly different at P < 0.05. Values followed by different uppercase letters in the bottom row are significantly different at P < 0.05.

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between the temperatures after 6 months storage. Germination was almost always highest in seeds harvested at the hard stage, followed by the seeds harvested at the dough and the milk stage. The accelerated aging test has been recognized as a good predictor of seed storability. Artola and Carilllo Castaeda (2005) found that there was a strong relation between storage capacity and vigor. In our study, the timing of the harvest strongly affected the germination rate and vigor of zoysiagrass seed. Standard germination rate and germination rate after AA treatment were the highest in the hard seed, followed by dough seed. Milk seeds had signicant gains in germination from 612 months, but quickly lost the germinability and vigor after 18 months of storage. Although standard seed germination progressively decreased with increasing storage duration, the germination rate after AA treatment reached its highest level when the seed was stored for 12 months. Zoysiagrass seeds harvested at the hard stage and stored for 12 months maintained the highest vigor level. During the development of zoysiagrass seed, the seed weight increases with seed maturity, even as seed moisture content decreases. The seeds initiate the accumulation of reserve substances at the dough stage and complete the accumulation at the hard stage (Ma and Han 2005). Generally, germination capacity increased with seed maturity and the germinability of seeds harvested at all maturity levels could be maintained with storage at the low temperature. Germinability of the seeds tended to decrease with increased storage duration. Although milk seeds had the potential to germinate, the germinability of the seeds diminished greatly after 18 months storage, as the accumulation of reserves was incomplete. The germinability of hard seed was the highest, as the physiological process of reserve transformation and accumulation was complete. It is well documented that seeds harvested at an early stage of development are germinable but generally have poor vigor, indicating that germinability does not mean complete physiological development (Coolbear et al. 1997). In the present study, the germination rate after AA treatment reached the highest after being stored for 12 months, but had the lowest germination when stored for only 6 months. This phenomenon may result from the need for an after-ripening period prior to reaching physiological maturity. Despite the fact that the seed has gained its nal dry weight at the hard stage, the physiological development of a seed may be far from complete at that time. Zoysiagrass seed quality improved when the harvested seeds were stored for 612 months.

Table 4 Treatment means and anova results for conductivity (s cm1 g1) of zoysiagrass seed Seed maturity and storage temperature MA ML DA DL HA HL Mean Storage duration (months) 6 87.6ab 90.2b 85.4a 109.9c 74.1a 79.4a 87.8A 12 146.7c 108.1b 109.9b 91.4b 65.2a 66.3a 98.0A 18 160.3d 134.7bc 146.0c 129.6b 69.5a 68.1a 118.0B

See Table 1 for symbols. Values followed by different lowercase letters within the same column are significantly different at P < 0.05. Values followed by different uppercase letters in the bottom row are significantly different at P < 0.05.

18 months, though this was not the case in seeds harvested at the hard stage (Table 4). Within each storage duration, the effect of storage temperature on the conductivity was non-signicant (P > 0.05) in the hard seed. Milk and dough seeds stored for 12 and 18 months showed decreased conductivity with decreased temperature. The conductivity of seeds harvested at the hard stage trended to be lower than that of seeds harvested at the dough and the milk stage. Bulk conductivity test can demonstrate the degree of the loss of solutes from the seeds, which reects the extent of membrane deterioration resulting from seed aging (Roberts 1973). Higher seed vigor was found to correlate with lower bulk conductivity values, which means that the seed has maintained its membrane integrity. The lower bulk conductivity values in seeds harvested at the hard stage and stored 6 12 months generally coincide with the higher values of standard germination (Table 2).

Conclusion
The viability of zoysiagrass seed, in terms of standard germination, germination after accelerated aging and bulk conductivity, was affected by seed maturity, storage temperature and duration. The seed deterioration process could be minimized with proper harvest timing (hard stage), storage temperature (7C) and duration (12 months).

Effect of storage treatments on bulk conductivity of zoysiagrass seed harvested at different maturity stages
The mean conductivity of seeds stored for 612 months was signicantly (P < 0.05) lower than that of seeds stored for
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Acknowledgment
Special thanks to Mark Majerus, former manager of the USDANRCS Plant Materials Center, Bridger, MT, USA for his comments on this manuscript.
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Grassland Science 55 (2009) 15 2009 The Authors Journal compilation 2009 Japanese Society of Grassland Science