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Published online 19 March 2008
Published in Crop Sci 48:709-715 (2008)
© 2008 Crop Science Society of America
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Optimum Harvest Time of Vicia cracca in Relation to High Seed Quality during Pod Development

Ying Wang, Chunsheng Mu*, Yu Hou and Xiaoyu Li

Institute of Grassland Science, Northeast Normal University; Key Lab. of Vegetation Ecology, Ministry of Education, P.R. China, 130024

* Corresponding author (mucs821{at}nenu.edu.cn).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Shattering during harvest is a major problem in seed production of Vicia cracca (L.). The aim of this study was to evaluate a simple empirical relationship and determine optimum harvest time by simultaneously analyzing pod and seed morphological and physiological properties, as well as seed quality. Pods were harvested at three-day intervals from peak anthesis until shattering. Pod and seed sizes from 36 to 42 d after peak anthesis (DAPA) were significantly smaller than at other sampling dates after reaching maximum size. Pods and seeds lost their green color and turned light brown and black, respectively from 36 to 42 DAPA. Seed dry weight (DWT) reached maximum values, and seed moisture content (SMC) reached the minimum and suitable values, respectively from 36 to 42 DAPA. Seeds reached maximum percent germination and germination rate at about 36 DAPA. Thus, V. cracca can be harvested six days before shattering (shattering begins at 42 DAPA) without affecting yield.

Abbreviations: AAT, accelerated aging test • CT, cold test • DAPA, days after peak anthesis • DWT, dry weight • EC, electrical conductivity • GDD, growing degree days • PHS, percentage of hard seed • SA, seed age • SGT, standard germination test • SMC, seed moisture content


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
THE PERENNIAL LEGUME plant Vicia cracca (L.) is one of the most common fodder plants in northern China. It is a wild high-quality forage widely grown mostly for hay, green forage, silage, and grain. However, V. cracca seed production is a major problem because of pod shatter at plant maturity. Pods are fragile at harvest time and seed loss can negatively impact yield. Harvesting too early may result in low yield and poor seed quality, whereas harvesting too late may result in shattering and reduced seed yield (Oplinger et al., 1989). Water management (Garcia-Diaz and Steiner, 2000a; Garcia-Diaz and Steiner, 2000b), modulation of flowering time (Chandler et al., 2005), and interspecific hybridization (Gesch et al., 2005) have been used to reduce seed loss caused by shattering. Unfortunately, many of these methods are difficult to apply in production, while in others seed loss is not effectively prevented.

Because of these difficulties, research has focused on determining optimum harvest time to avoid seed loss (Garcia-Diaz and Steiner, 2000b; Child, 2003; Lemke et al., 2003). Researchers have focused on capsule color change (Miyajima, 1997; Elias and Copeland, 2001), maturity stage (Lawrence, 1960), seed moisture content (Elias and Copeland, 2001; Wang et al., 2006), and growing degree-days (Berdahl and Frank, 1998; Wang et al., 2006). Formation of a black layer in the placental area of the seed was reported to be a good indicator of physiological maturity in sorghum [Sorghum bicolor (L.) moench] (Eastin et al., 1973). Moisture content represented an accurate indicator of physiological maturity for soybean (Fraser et al., 1982). In addition, the stage of seed development influences seed quality for both wild and cultivated species, so timely harvest would avoid problems of both under- and overripe pods (Elias and Copeland, 2001).

High-quality seed is essential for establishing a good stand and producing high yield in any cultivated crop. However, obtaining quality seed is the major problem in seed harvest. Many standardized tests are used to evaluate seed quality, including thousand seed weight, standard germination tests, accelerated aging tests, cold tests, and electrical conductivity tests (Wellington, 1969). Quality indices are then used to determine the optimum time to harvest the highest quality seeds.

Up to now, changes in pod and seed sizes at different growing degree days (GDD) that would indicate the optimum harvest time have not been documented. The loss of green color from pods and seeds can be used as an indicator of prime harvest in some, but not all species. For example, color loss is not a good indicator of optimum harvest time in Hordeum brevisubulatum because the head color is not affected by the GDD during harvest (Wang et al., 2006). Thus, the changes in pod and seed sizes at different stages of pod development can be used to rapidly predict harvest time for species where the color indicator cannot be used.

Vicia cracca has only one cultivated genotype, commonly known as "Guangbuyewandou". In this study, we used this genotype to determine pod and seed morphological and physiological properties and measured seed germination during pod development. We then combined these indicators to propose a method for identifying optimum harvest time to improve production.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Plant Material and Culture
Vicia cracca (genotype Guangbuyewandou) plants were sown in 0.30 m rows at a plot of 8 m x 10 m in 2003, with four replications. The experiment was conducted at Ecosystem Field Station of the Institute of Grassland Science at Songnen Grassland in China (44°40' N 123°44' E, 167 masl). The field station is in a semiarid area with a continental monsoonal climate, and Mollisols with poor plant nutrient levels. Annual average temperature is 2.4 to 2.7°C and the average annual precipitation is 300 to 500 mm. Because V. cracca is a biennial, the field study was harvested two years after planting in 2005 and 2006.

Average daily temperature and monthly precipitation were measured on the Ecosystem Field Station in 2005 and 2006. GDD index for the period from peak anthesis to pod shatter was defined as:

Formula
where Tmax = maximum daily temperature, Tmin = minimum daily temperature, Tb = base temperature. In this study, GDD was calculated with a base temperature of 5°C.

Sampling Procedure
Flowers were tagged in early August at peak anthesis with red scutcheons (banner petal fully extended: Peterson et al., 1992). V. cracca required 3 d from flowering to pod formation, and initial pod shatter started 42 days after peak anthesis (DAPA). Seeds were too small, and seed moisture content (SMC) was too high to separate seeds from the pods without damage at 3 DAPA and 6 DAPA. Therefore, tagged pods were harvested at 3-d intervals beginning 9 DAPA and continuing until pod shatter.

Determination of Morphological and Physiological Characteristics
Forty pods were randomly selected from each sample and the color of the pods and seeds was recorded (Munsell, 1977) at each sampling date. Length, width, and thickness were measured in selected pods and seeds at each sampling date using a micrometer reading to 0.02 mm. The fresh weight and dry weight of pods and seeds were measured in 40 randomly selected samples from the marked pods at each sampling date with an electronic balance reading to 0.0001 g. Then average pod and seed moisture contents and seed dry weight (DWT) (%) were calculated.

Formula

Additional pods were harvested and carefully opened at each sampling date. The seeds were removed and allowed to air-dry in the laboratory for about one month before evaluating seed germination.

Seed Quality Determination
Electrical conductivity (EC) of seed leachate was tested according to the ISTA Handbook of Vigour Test Methods (Wellington, 1969). Four replications of 100 weighted seeds were placed in four separate beakers with 100 mL deionized water at 20°C room temperature for 24 h and the conductivity of the solution was measured. At the same time the number of seeds which did not increase in volume (visually assessed) and hence did not imbibe water (termed "hard seeds") was recorded and the percentage of hard seeds (PHS) was calculated. Seeds used to study germination were treated with 98% H2SO4 for 25 min to promote germination. Tests were replicated four times. Germinated seeds were counted every day for percentage germination and germination rate determinations. The standard germination test (SGT) (AOSA, 1988) was conducted on 100-seed samples placed on moist blotter paper in a growth cabinet at 20°C and exposed to 12 h light daily for 10 d. The accelerated aging test (AAT) was conducted by aging seeds at 42°C for 72 h according to the ISTA Handbook (Wellington, 1969) using the wire-mesh tray method and then germinating the seeds as described above for 10 d. The cold test (CT) was done according to the improved method of Elias and Copeland (2001). 100-seed samples were exposed to 5°C for 5 d on moist blotter paper and then transferred to 20°C for 8 d for germination.

Germination rate = {Sigma} (Gi/Di), Gi: number of normal seedlings germinated in the ith day; Di: number of days until the ith reading (Maguire, 1962).

Statistical Analysis
All data were subjected to analysis by SPSS statistical software (version 12.0, SPSS Inc, Chicago, IL) and analysis of variation (ANOVA). Two-way ANOVA were also used to analyze seed DWT, SMC, and seed quality (EC, SGT, AAT, and CT) at the different sampling dates over two sampling years. Differences in different sampling dates were analyzed using the Least Significant Difference (LSD) test. The significance level was set at P ≤ 0.05. Correlation analyses were done between seed EC and PHS.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The average daily temperature was similar in the two sampling years (16.7°C and 16.8°C, respectively); however, average daily temperatures from July to October (during pod development) in 2005 were lower than the same period in 2006 (Fig. 1a ). The average monthly precipitation differed markedly in both years (47.1 mm versus 36.0 mm, respectively), and the average monthly precipitation from July to October was higher in 2005 than in 2006 during pod development (Fig. 1b).


Figure 1
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  		Figure 1. a) Average daily temperature by month (°C) and b) monthly precipitation (mm) of 2005 and 2006 in Songnen Grassland in China.

 
Pod and Seed Morphological Characteristics
Gradual changes in pod and seed colors were observed with progressive development until pod shatter (Table 1 ). At the very early degree-days of pod growth, the pods were light green from 9 to 15 DAPA, turning green from 18 to 27 DAPA. With further development, the pods turned brownish green, and finally turned light brown from 36 to 42 DAPA, and the accumulated GDDs during these periods were from 599 to 689 in 2005 and 568 to 644 in 2006, respectively (Table 1).


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  		Table 1. Pod and seed colors (color designations from Munsell 1977 in parentheses) at different stages and growing degree days (GDD) of pod development of V. cracca averaged over two years.

 
The pods were fragile during this final period. At the early degree-days of seed development, the seeds were transparent from 9 to 15 DAPA, becoming green from 18 to 27 DAPA. With further development, the seeds turned greenish black and then black and became hard from 36 to 42 DAPA.

Pod length, width, and thickness varied significantly by sampling date (Fig. 2a ). Pod length increased slightly from 9 to 27 DAPA and then decreased slightly from 27 to 42 DAPA. The pod width decreased gradually following peak anthesis, and was significantly shorter from 36 to 42 DAPA than at other sampling dates. The thickness increased gradually from 9 to 30 DAPA and then decreased from 30 to 42 DAPA. Pod size changed significantly throughout pod development, as did the seed length, width, and thickness (Fig. 2b). The length, width, and thickness of seeds increased markedly from 9 to 33 DAPA, but the values were unchanged thereafter. Pod and seed sizes from 36 to 42 DAPA were significantly less than other sampling dates after reaching the maximum sizes (Fig. 2a and 2b), and seed size was unchanged from 36 to 42 DAPA after the pods and seeds lost their green color.


Figure 2
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  		Figure 2. a) Pod and b) seed length (L), width (W), and thickness (T) of V. cracca during its development in 2005 and 2006. Pod and seed L, W, and T curves represent the average of two years. Error bars indicate the standard deviation from means of pod and seed L, W, and T at P = 0.05.

 
Visual indicators such as color have been used to indicate optimal harvest time (Miyajima, 1997; Elias and Copeland, 2001). Physiological measurements, such as seed dry weight, moisture content, or seed quality to predict optimum harvest time are often time consuming and difficult to perform. Methods for determining optimum harvest time in V. cracca based on morphological indicators would minimize these constraints. In addition, using pod and seed size measurements eliminates the need to destroy the pods as when measuring physiological characters and seed germination. Changes in pod and seed size, and accompanying changes in color, could be dependable and rapid indicators providing a simple way for seed producers to judge optimal harvest time. Some researchers indicated that using growing degree days to determine harvest time may not be the best method because GDDs varied between years (Lemke et al., 2003, Wang et al., 2006). In this study, the differences of accumulated GDDs were 31 GDD at 36 DAPA and 45 GDD at 42 DAPA between the two years, which indicated three day intervals between 2005 and 2006. Therefore, GDD from peak anthesis to pod shatter was also not an appropriate index in this genotype of V. cracca because of variability between years.

Seed Physiological Characteristics
Seed DWT increased and SMC decreased significantly over time following peak anthesis (Fig. 3 ). The greatest gain in seed DWT occurred from 36 to 42 DAPA, and the highest DWT values were about 92.6% and 92.1% in 2005 and 2006, respectively. The average SMC reached a maximum value at 15 DAPA and then decreased until pod shatter (Fig. 3). The average SMC remained steady at approximately 10.0% from 36 to 42 DAPA. A two-way ANOVA indicated that both seed development stage [seed age (SA)] and year individually affected seed DWT and average SMC, and a similarly significant effect resulted from their interaction (Table 2 ). These results were consistent with former studies (Hill et al., 2005; Wang et al., 2006).


Figure 3
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  		Figure 3. Seed dry weight (DWT) and seed moisture content (SMC) of V. cracca during seed development in 2005 and 2006. Error bars indicate the standard error from means of DWT and SMC at P = 0.05.

 

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  		Table 2. Analysis of variance of seed dry weight (DWT) and seed moisture content (SMC) of V. cracca.

 
The variation in DWT may be due to translocation of assimilates from the source (e.g., leaves) to the sink (i.e., seeds), which also caused a significant decrease in SMC level following peak anthesis. SMC increased at first and then decreased until full maturity (Fig. 3). One former study (Fraser et al., 1982) reported that SMC represented a more accurate indicator of physiological maturity than seed DWT. On the contrary, we found when seeds reached maximum DWT the moisture contents was near 10% and the plants were suited to direct harvest, threshing, or storage from 36 to 42 DAPA. Seed DWT represented an accurate indicator of optimum harvest time as SMC did in this research. The reason may be that seed DWT was not expressed as a percentage (%), but was expressed as g seed–1 by Fraser et al. (1982). Despite differences in temperature between 2005 and 2006, differences in DWT were not significant between the two years. Average daily temperatures during pod development in 2005 were lower than the same period in 2006; however, the average monthly precipitation from July to October was higher in 2005 than in 2006 during pod development.

Seed Quality Characteristics
Both the EC and PHS were similar in V. cracca in both sampling years. Seed EC decreased rapidly following the peak anthesis, and there was no significant change from 27 to 42 DAPA in 2005 and from 24 to 42 DAPA in 2006 (Fig. 4 ). Seed EC was below 20 µs cm–1 g–1 from 27 to 42 DAPA in both sampling years. The PHS remained 0 from 9 to 15 DAPA, and then increased rapidly from 18 to 27 DAPA; once the maximum value was reached there was little change thereafter. A strong negative correlation (r05 = –0.686** and r06 = –0.684**) was found between seed EC and PHS during seed development. EC reportedly has an inverse relationship to seed germination (Wellington, 1969; Perry, 1981). However, EC values determined in this investigation were less than 20 µs cm–1 g–1, because of the hardness of the seed of this species (genotype Guangbuyewandou) from 27 to 42 DAPA. Optimum harvest time was the last six days before shattering according to DWT and SMC. Thus, EC was not a good indicator of optimum harvest time in this species for seed with high PHS.


Figure 4
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  		Figure 4. Seed electrical conductivity (EC) and percentage of hard seeds (PHS) of V. cracca during seed development in 2005 and 2006. Error bars indicate the standard error from means of EC and PHS at P = 0.05.

 
Seed germination differed markedly among the sampling dates (Table 3 and 4 ). Seeds showed no germination ability and a lack of vigor from 9 to 21 DAPA, with the percentage germination and germination rate both equal to zero. The percentage of germination and germination rate, determined by SGT, increased rapidly from 24 to 42 DAPA and then leveled off from 33 to 42 DAPA. Percentage germination and germination rate calculated from the AAT increased following peak anthesis, and sampling dates had no effect on germination or rate from 33 to 42 DAPA. The percentage of germination and the germination rate, determined by the CT, increased following peak anthesis and then remained unchanged from 33 to 42 DAPA.


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  		Table 3. Percentage germination (mean ± 1.0 SE) of V. cracca during seed development in 2005 and 2006 by standard germination test (SGT), accelerated aging test (AAT), and cold test (CT).

 

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  		Table 4. Germination rate (mean ± 1.0 SE) of V. cracca during seed development in 2005 and 2006 by standard germination test (SGT), accelerated aging test (AAT), and cold test (CT).

 
The ANOVA results showed that SA and year individually had significant effects on SGT and AAT, and their interactions had a significant effect on SGT (Table 5 ). Neither year nor the interactions between SA and year were significant. The SGT, AAT, and CT were variable during seed development, but remained without significant change from 33 to 42 DAPA (see Table 3 and 4). Apparently, the seeds were more susceptible to stress from 9 to 30 DAPA. For example, high temperature stress affected the AAT, perhaps because seeds were immature, which made them sensitive to the stress. Elias and Copeland (2001) reported that seeds of all Canola (Brassica napus L.) cultivars were sensitive to high and low temperatures. However, in the present study, the percentage of germination and the germination rate in CT were higher than in SGT and AAT from 24 to 30 DAPA. It has been reported that a cold-treatment of seeds could improve percentage germination (Yang et al., 1999) and germination rate (Nordborg and Bergelson, 1999) of wild species. Seeds reached maximum germination and were relatively unaffected by stress conditions, such as high and low temperatures, from 33 to 42 DAPA.


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  		Table 5. Analysis of variance of seed germination of V. cracca as measured by standard germination test (SGT), accelerated aging test (AAT), and cold test (CT).

 
V. cracca reached the maximum seed DWT from 36 to 42 DAPA and the average SMC (10.0%) was suited to direct harvest, threshing, or storage without further drying. Harvesting at full maturity when SMC is near 10.0% is preferred for better threshing and storability (Elias and Copeland, 2001). In this research, the pod moisture content was about 10.0% (data not shown) from 36 to 42 DAPA, and it was easy to separate seeds from pods during threshing. Thus, seeds should be harvested from 36 to 42 DAPA. This was also when the color of the pods became bright brown and seeds turned black. The seed DWT and SMC averaged approximately 90.0% and 10.0%, respectively from 36 to 42 DAPA. Seed EC values remained at 20 µscm–1 g–1 without significant change from 27 to 42 DAPA in both years, making it unsuitable for evaluation of germination potential. Although physiological markers and seed quality were reliable indicators of optimum harvest time, the loss of green color from pods and seeds, and the accompanying change in pod and seed sizes, were more practical and rapid indicators for optimum harvest of this genotype of V. cracca.


   ACKNOWLEDGMENTS
 
We thank the Northeast Normal University for providing use of their facilities. This research was supported by the National TCM Main Project in 11th Five-Year-Period (2006BAD16B06), the Project of Science and Technology Development of Jilin Province (20030554), the National Key Basic Research Program (2007CB106800), and the Program for Changjiang Scholars and Innovative Research Team (PCSIRT) in University (#IRT0519). We are grateful to Hongxiang Zhang and Ping Wang for their technical assistance during the germination test, and also to Guang Yang and Shujing Wang for their insightful comments during manuscript preparation.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
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Received for publication April 14, 2007.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 




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