全 文 ：作物学报 ACTA AGRONOMICA SINICA 2012, 38(3): 522527 http://www.chinacrops.org/zwxb/
ISSN 0496-3490; CODEN TSHPA9 E-mail: xbzw@chinajournal.net.cn

This work was financed by Projects in the National Science & Technology Pillar Program (2009BADB3B02).
* 通讯作者(Corresponding author): MU Chun-Sheng, E-mail: mucs821@nenu.edu.cn, Tel: 0431-85098113
Received(收稿日期): 2011-05-10; Accepted(接受日期): 2011-12-15; Published online(网络出版日期): 2012-01-04.
URL: http://www.cnki.net/kcms/detail/11.1809.S.20120104.1651.013.html
DOI: 10.3724/SP.J.1006.2012.00522
Optimum Harvest Time of Vicia cracca under Stress Condition
WANG Ying1,2, HOU Yu3, LI Xiao-Yu2, SUN Hai-Xia2, LIN Ji-Xiang2, and MU Chun-Sheng1,
1 Key Laboratory of Vegetation Ecology, the Ministry of Education / Institute of Grassland Science, Northeast Normal University, Changchun 130024,
China; 2 Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130012, China; 3 College of Chemistry and
Biology, Beihua University, Jilin 132013, China
Abstract: Timely harvest is critical to achieve maximum seed viability, vigour and yield. Many native grasses display seed shat-
tering and other seed production problems. Shattering during harvest is a major problem in seed production of Vicia. cracca (L.).
The objective of this study was to further determine the optimum harvest time of V. cracca by simultaneously analyzing pod and
seed morphological and physiological characters, as well as seed stress tolerance. Pods were harvested at three-day intervals from
peak anthesis until pod shattering in pod development. The results showed that pod dry weight (DWT) and thousand seed weight
(TSW) reached the maximum, as well as pod moisture content (PMC) reached the minimum from 36 to 42 days after peak anthe-
sis (DAPA). The maximum percentage germination, germination rate and radicle length were at about 36 DAPA under NaCl and
Na2CO3 stresses. Therefore, V. cracca can be harvested at six days before pod shattering without affecting yields and seed quality.
Keywords: Salinity; Alkalinity; Harvest time; Pod; Seed
逆境下广布野豌豆的适宜收获时间
王 颖 1,2 侯 宇 3 李晓宇 2 孙海霞 2 蔺吉祥 2 穆春生 1,*
1 东北师范大学草地科学研究所 / 植被生态科学教育部重点实验室, 吉林长春 130024;2 中国科学院东北地理与农业生态研究所, 吉
林长春 130012;3 北华大学化学与生物学院, 吉林吉林 132013
摘 要: 及时收获对于获得最大的种子活力和种子产量是很重要的, 许多牧草都具有种子脱落及其他种子生产的问
题。裂荚是广布野豌豆种子生产中的一个重要问题。本文的研究目的是通过分析种子和荚果的生理学特征、种子的
抗逆能力来进一步确定广布野豌豆(Vicia cracca)的收获时间。在广布野豌豆荚果发育过程中, 每隔 3 d收获 1次荚果,
到荚果开始出现开裂为止(盛花后第 42 d)。结果显示, 盛花后 36~42 d, 荚果干重、千粒重达到最大及荚果含水量达
到最小, 在此期间 NaCl 和 Na2CO3胁迫下的种子发芽率、发芽速率及胚根长均达到最大值。因此, 广布野豌豆在裂
荚前 6 d收获不影响种子产量和品质。
关键词: 盐; 碱; 收获时间; 荚果; 种子

Most of wild grasses or cultivated crops have evolved
seed dispersal mechanisms. Immediate seed shedding at
maturity is a major problem in the harvesting and use of
those potential valuable grasses or cultivated crops seeds.
Seeds shedding at harvest time and seed loss can nega-
tively impact yield. Harvesting too early may result in
low yield and poor seed quality, whereas delayed harvest
can also results in lower seed yields due to shattering [1-3].
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 [4]. Some methods, such as water manage-
ment [5-6], and interspecific hybridization [7] have been
used to reduce seed shedding. However, many of these
methods are difficult to apply in practice and seed loss is
not effectively prevented. Therefore, researchers have
been focused on determining optimum harvest time to
avoid seed loss [8-9]. Capsule color change [10], pod and
seed size [11], seed moisture content [4], cell size kinetics [12];
maturity stage [13-14], and growing degree days [15] were
used to determine seed harvest time. Among these indi-
cators, seed moisture content represented an accurate and
common indicator for most species. However, pod mois-
ture content that would indicate the optimum harvest
time has not been documented. In addition, using pod
moisture content eliminates the need of destroying seeds
第 3期 WANG Ying et al.: Optimum Harvest Time of Vicia cracca under Stress Condition 523


when visual indicators to predict optimum harvest time
are difficult to perform.
The use of high-quality seeds is essential for good
stand establishment and yield in any crop. Furthermore,
obtaining qualified seeds is important in seed harvest.
Quality indices in standard germination tests, accelerated
aging tests, and cold tests [16] have been used to deter-
mine the optimum time for harvesting the highest quality
seeds. However, there is limited information in the ef-
fects of salt and alkali stresses on seed quality at different
stages. Soil salinity is a major abiotic stress influencing
plant growth and productivity worldwide, and affects
about 7% of the world’s total land area [17]. Germination
and seedling growth, especially for young seedlings, are
all vulnerable to the environmental stresses and growth
will be affected by both macro- and microclimates [18].
To date, although there is a large number of literature
on optimum harvest time, very little is known about pod
physiological characters and seed tolerance to stresses.
The main objective of the present study was to experi-
mentally determine pod and seed physiological charac-
ters in pod development and the effects of salt and alkali
stresses on seeds germination of V. cracca at different
sampling dates, further confirming the optimum harvest
time of V. cracca and improving its seed quality.
1 Materials and methods
1.1 Plant material and cultivation
Seeds were sown in 0.30 m rows at a plot of 8 m × 10
m in 2003, with four replicates. 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 lo-
cated in a semiarid area with a continental monsoonal
climate, and Mollisols with poor plant nutrient levels.
Annual average temperature is 2.4°C to 2.7°C and the
average annual precipitation is 300 to 500 mm. Because
V. cracc is a biennial, the seeds were harvested in 2005
and 2006. Average daily temperature and monthly pre-
cipitation were measured at the Ecosystem Field Station
in 2005 and 2006.
1.2 Sampling procedure
Flowers were tagged in early August at peak anthesis
with red scutcheons (banner petal fully extended [19]). V.
cracca required three days from flowering to pod forma-
tion, and the initial pod shattering started at about 42
days after peak anthesis (DAPA). Seeds were too small,
and seed moisture content was too high to separate seeds
from the pods without damage at 3 and 6 DAPA. There-
fore, tagged pods were harvested at three days intervals
from the beginning at 9 DAPA through 42 DAPA.
1.3 Determination of physiological characters
Forty pods were randomly selected from each sample
in about 10:00 of each sampling day. The fresh weight
and dry weight (DWT) of pods were measured with an
electronic balance reading to 0.0001 g for 40 randomly
selected samples that had been marked at each sampling
day. Then average pod moisture content (PMC) was cal-
culated.
Moisture content (%) =
100[(fresh weight–dry weight)/fresh weight]
Additional pods were harvested and carefully opened
at each sampling day. The seeds were removed and al-
lowed to air-dry in the laboratory for about one month.
Four replicates of 100 seeds were tested for each harvest.
The thousand seed weight (TSW) was calculated as
100-seed weight × 10. The rest seeds then exposed to
4°C to avoid losing germination ability before germina-
tion. Seeds lots harvested in two years were mixed for
determining seed tolerance to stresses.
1.4 Seed quality determination
Before experiment, seeds were treated with 98%
H2SO4 for 25 minutes to promote germination. Germina-
tion test was conducted with 100-seed samples placed on
moist blotter paper in 90-mm Petri dishes. About 10 mL
distilled water, 50, 100, 150, 200, and 250 mmol L–1
NaCl solutions, and 25, 50, 75 mmol L–1 Na2CO3 solu-
tions were added to each Petri dish, so that about half the
volume of each seed was immersed. Seeds were cultured
at 20°C with a 12-hour day-night in a growth cabinet for
10-day germinating. The experiment was arranged by a
completely randomized design with four treatments and
four replicates. Germination of seeds was defined as the
radicle penetrating the seed coat. The number of seeds
germinated was counted every day to determine percent-
age germination and germination rate. The germination
rate was estimated using a modified Timson index, i.e.
germination velocity= ∑G/t, where G is the percentage
seed germination at one-day intervals and t is the total
germination period [20]. The maximum possible value of
this index is 100. The greater the value, the more rapid
the germination. Ten seedlings were randomly selected
from each sample and the radicle length was measured on
the tenth day.
1.5 Statistical Analysis
Data were analyzed using a one-way Analysis of Vari-
ance (ANOVA) in SPSS statistical software version 12.0
(SPSS Inc, Chicago Illinois, USA). Tests of significant
differences among treatments were analyzed using the
Least Significant Difference (LSD) test. The significance
level was set at P<0.05. Two-way ANOVA were used to
analyze pod DWT, PMC and TSW at the different sam-
pling dates over two sampling years. Two-way ANOVA
analyses were also conducted for percentage germination,
germination rate and radicle length of different salt and
alkaline concentrations at different sampling dates.
2 Results
The average daily temperature was similar in the two
sampling years (16.7°C and 16.8°C, respectively); how-
524 作 物 学 报 第 38卷

ever, average daily temperatures from July to October
(during pod development) in 2005 were lower than these
in 2006 (Fig. 1-A). The average monthly precipitation
varied markedly in both sampling years (47.1 mm versus
36.0 mm, respectively), and the average monthly precipi-
tation from July to October was higher in 2005 than in
2006 during pod and seed development (Fig. 1-B).
2.1 Seed and pod physiological characteristics
The TSW differed markedly among the sampling days
(Fig. 2-A) and the maximum values were about 16.5 and
16.8 g from 30 to 42 DAPA in 2005 and 2006, respec-
tively. Pod DWT increased from 9 to 30 DAPA and de-
creased thereafter. PMC decreased significantly after
peak anthesis (Fig. 2-B). The greatest gain in pod DWT
occurred at 30 DAPA, which was 0.0418 and 0.0429 g in
2005 and 2006, respectively. The average PMC reached a
maximum value at 9 DAPA and then decreased until pod
shattering. The average PMC remained steady at ap-
proximately 10.0% from 36 to 42 DAPA.
A two-way ANOVA indicated that seed developing
stage [seed age (SA)] affected TSW, however, year and
their interaction had no significant effect on TSW. Pod
age (PA) and year individually affected pod DWT and
average PMC, and a similarly significant effect resulted
from their interaction (Table 1).
2.2 Seed stress tolerance characteristics
Seed germination differed markedly among the sam-
pling dates under NaCl and Na2CO3 stresses (Fig. 3).
Seeds showed no germination ability and lack of vigor
from 9 to 21 DAPA, with the percentage germination and
germination rate both equal to zero. The percentage ger-
mination increased rapidly from 24 to 42 DAPA and
relatively unchanged from 33 to 42 DAPA at the same
NaCl and Na2CO3 levels (Fig. 3). Percentage germination
remained stable from 33 to 42 DAPA under low salt
concentrations (0 to 150 mmol L–1), and then decreased
significantly (Fig. 3-A). While the percentage germina-
tion showed the continued decrease with the salt levels at
27 and 30 DAPA. The germination ability was lowest at
24 DAPA compared with other harvest dates. Percentage
germination declined markedly under increased NaCl
levels and was zero in treatments from 150 to 250 mmol
L1 NaCl. Percentage germination under Na2CO3 stress
exhibited similar trends to that under NaCl stress (Fig.
3-B). No significant changes were observed under 0 and
25 mmol L–1 Na2CO3 stresses and the mean value was
more than 90% in percentage germination from 33 to 42
DAPA, however, notable decreases under 50 and 75
mmol L–1 Na2CO3 were detected. A decrease in percent-
age germination at 24 and 27 DAPA was most evident,
with decreasing to about zero from 25 to 75 mmol L–1
Na2CO3.
Germination rate differed significantly among the
sampling dates and decreased noticeably under increased



Fig. 1 Average daily temperature (A) and monthly precipitation (B) of 2005 and 2006 in Songnen Grassland in China



Fig. 2 1000-seed weight (A), B pod dry weight and moisture content (B) of V. cracca during seed and pod development in 2005 and 2006
TSW: 1000-seed weight; DWT: pod dry weight; MC: moisture content. Error bars indicate the standard error from means of TSW, pod DWT and
PMC at P=0.05.
第 3期 WANG Ying et al.: Optimum Harvest Time of Vicia cracca under Stress Condition 525


NaCl and Na2CO3 levels (Fig. 4). Germination rate in-
creased from 24 to 33 DAPA and leveled off from 33 to
42 DAPA at the same NaCl (Fig. 4-A) and Na2CO3 (Fig.
4-B) levels. Germination rate decreased to zero from 150
to 250 mmol L–1 NaCl at 24 DAPA. While germination
rates were nearly zero at 25 mmol L–1 Na2CO3 and zero
at 50 and 75 mmol L–1 Na2CO3 at 24 and 27 DAPA.
Radicle length differed significantly among the sam-
pling days and decreased markedly under increased NaCl
and Na2CO3 levels (Fig. 5). Radicle length behaved
similarly from 27 to 42 DAPA, except for the control
(Fig. 5-A). The radicle lengths at 27 and 30 DAPA were
shorter than these at 33 to 42 DAPA when incubated in
distill water. The radicle length was shortest among the

Table 1 Analysis of variation of thousand seed weight, pod dry weight, and pod moisture content of V. cracca
Source df Mean square F Sig.
1000-seed weight
Year 1 0.660 3.309 0.073
Seed age 11 370.682 1859.291 0.000***
Year×SA 11 0.349 1.752 0.079
Pod dry weight
Year 1 0.001 18.573 0.000***
Pod age 11 0.004 126.273 0.000***
Year×PA 11 0.000 3.664 0.000***
Pod moisture content
Year 1 271.642 12.030 0.001***
Pod age 11 41428.646 1834.772 0.000***
Year×PA 11 52.364 2.319 0.008**
**Significant at the 0.01 probability level; ***Significant at the 0.001 probability level.



Fig. 3 Percentage germination of V. cracca during seed and pod development under NaCl (A) and Na2CO3 (B) stresses
Error bars indicate the standard error from means of percentage germination at P=0.05.



Fig. 4 Germination rate of V. cracca during seed and pod development under NaCl (A) and Na2CO3 (B) stresses
Error bars indicate the standard error from means of germination rate at P=0.05.
526 作 物 学 报 第 38卷

sampling days and remained steady under 0 and 50 mmol
L–1 NaCl at 24 DAPA. The radicle length at each sam-
pling day decreased along Na2CO3 treatment (Fig. 5-B).
Radicle length increased from 24 to 33 DAPA and le-
veled off from 33 to 42 DAPA at the same Na2CO3 levels.
Radicle length decreased significantly with the Na2CO3
levels from 24 to 42 DAPA.
A two-way ANOVA indicated that both seed deve-
lopment stage [seed age (SA)] and NaCl and Na2CO3
levels affected percentage germination and germination
rate, and their interactions had significant effect on per-
centage germination and germination rate (Table 2). The
ANOVA results showed that SA and NaCl levels, and
their interactions significantly affected radicle length.
The two-way ANOVA indicated that both SA and
Na2CO3 levels significantly affected radicle length,
however, their interactions had no significant effect on
radicle length (Table 2).



Fig. 5 Radicle length of V. cracca during seed and pod development under NaCl (A) and Na2CO3 (B) stresses
Error bars indicate the standard error from means of radicle length at P=0.05.

Table 2 Analysis of variation of percentage germination (PG), germination rate (GR), and radicle length (RL) of V. cracca
NaCl Na2CO3 Independent
variable Seed age Concentration SA  C Seed age Concentration SA  C
PG (%) 0.000*** 0.000*** 0.001*** 0.000*** 0.000*** 0.000***
GR (No. d–1) 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000***
RL (cm) 0.000*** 0.000*** 0.000*** 0.013* 0.000*** 0.102
*Significant at the 0.05 probability level; *** Significant at the 0.001 probability level.

3 Discussion
Timely harvest is crucial to maximum seed yield, vi-
ability and vigour. Delayed harvest can result in lower
seed yields due to shattering [2]. Therefore, determining
accurately the time of seed maturity for V. cracca is able
to harvest a good quality seed with a higher yield. It may
also be useful to select shattering resistance in future
breeding programs.
Pod physiological characters differed among the sam-
pling days, but exhibited the similar trends. Pod DWT
difference was not significant from 33 to 42 DAPA,
however, PMC was near 10% and plants were suited to
indirectly harvest from 36 to 42 DAPA. Physiological
characters, such as seed DWT, SMC, or seed quality have
been used to predict optimum harvest time. While these
methods need to destroy seeds and were difficult to per-
form in measuring physiological characters and seed
quality. Changes in seed and pod sizes, and accompany-
ing changes in color, could indicate the optimum harvest
time [11]. However, the visual indicators could not be used
to judge optimal harvest time for all the species [21] ex-
cept for V. cracca. In this study, PMC could be a de-
pendable and rapid indicator providing a relatively sim-
ple way to indicate the optimum harvest time because
pod was fragile and could be broken by hand when PMC
was nearly 10%. Here, we offer one method for other
plants in practice when visual indicators can not be used
to indicate the optimum harvest time. The pod DWT and
PMC were markedly different between 2005 and 2006.
Average daily temperature was lower in 2005 than in
2006, while the average monthly precipitation was higher
in 2005 than in 2006 during pod development.
Prediction of optimum harvest time is often regarded as
a useful approach to balance the seed loss from shedding
and getting a good seed quality as high as possible [22].
The seed vigour increased with the sampling days and
the results was consistent with the former study [23]. Ap-
parently, the seeds were more vulnerable from 24 to 30
DAPA because the seeds were immature, so sensitive to
the stress and easy to be affected by stress conditions.
Elias and Copeland [4] reported that seeds of all canola
(Brassica napus L.) cultivars were sensitive to high and
low temperatures. This may be explained by the physio-
第 3期 WANG Ying et al.: Optimum Harvest Time of Vicia cracca under Stress Condition 527


logical changes (e.g. hormonal mechanism) that occur
after physiological maturity, which can promote germi-
nation [20]. Both germination capacity and seed vigor as
indicated by the germination under salt and alkalinity
stresses support the premise that seeds develop germina-
tion capacity ahead of vigor [24]. However, the seeds were
more tolerant from 33 to 42 DAPA, at that time perhaps
the seeds were mature. Namely, seeds reached maximum
germination and were relatively unaffected by stress
conditions, such as high and low temperatures [11] and salt
and alkalinity stresses. In this study, the radicle length at
27 DAPA was higher under low stress (50 mmol L1
NaCl) than under control. It has been reported that low
salinity stress will improve seedling growth [25]. Fur-
thermore, cold treatment of immature seeds could im-
prove percentage germination and germination rate [11]. In
the study we concluded that the suitable harvest time can
be determined by simultaneously analyzing pod and seed
morphological and physiological characters, as well as
seed tolerance.
4 Conclusion
V. cracca should have the maximum TSW and mini-
mum average PMC (10%) from 36 to 42 DAPA, at that
time the seeds are suitable for direct harvest, then
threshing and storaging without further drying, and the
seed stress tolerance is the best.

Acknowledgment: We thank the Northeast Normal Uni-
versity for using their facilities.
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