Journal of Integrative Agriculture 2017, 16(0): 60345-7

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RESEARCH ARTICLE

6-benzylaminopurine treatment maintains the quality of chinese

chive (Allium tuberosum Rottler ex Spreng) by enhancing
antioxidant enzyme activity

JIA Li-e1, LIU Sheng1, DUAN Xiao-ming1, ZHANG Chao1, WU Zhan-hui1, LIU Ming-chi1, GUO Shao-gui1,
ZUO Jin-hua1, WANG Li-bin2

1
Vegetable Research Center/Key Laboratory of Fruits and Vegetable Storage and Processing/Key Laboratory of Biology and
Genetic Improvement of Horticultural Crops (North China) of Ministry of Agriculture/Key Laboratory of Urban Agriculture (North)
of Ministry of Agriculture, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, P.R.China
2
College of Food Science and Technology, Yang zhou University, Yangzhou 225127, P.R.China

Abstract
Chinese chive usually develops an off-flavor after a short storage time. To explore effective ways to maintain the postharvest
quality of Chinese chive, the effect of exogenous application of 6-benzylaminopurine (6-BA) on postharvest quality and
antioxidant activity of chive was evaluated, and the mechanism of the physiological responses of chive to 6-BA treatment
was explored. Chives were sprayed for 10 minutes with 100, 300, or 500 mg L–1 6-BA or with alkaline solution as the con�-
trol, then stored at (2±1)°C with a relative humidity (RH) of 80–85%. We found that 300 mg L–1 6-BA significantly delayed
yellowing and chlorophyll degradation, maintained the total phenolic and flavonoid content, and improved the activities of
antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). In conclusion, we
identified exogenous application of 6-BA as an effective method for maintaining postharvest quality of Chinese chive. In
addition, our finding that the activities of antioxidant enzymes increase in response to exogenous 6-BA provides new insights
into the mechanism of cytokinin-based postharvest fresh-keeping.

Keywords: 6-benzylaminopurine (6-BA), Chinese chive, postharvest quality, antioxidant activity

popular culinary herb in Asia. Significant cultivation of this

1. Introduction
Chinese chive (Allium tuberosum Rottler ex Spreng.) is a
Received 25 October, 2016 Accepted 10 January, 2017
JIA Li-e, Tel: +86-13301162561, E-mail: jialie@nercv.org;
Correspondence LIU Sheng, Tel: +86-10-51503050; Fax: +86-
10-51505002; E-mail: liusheng@nercv.org
© 2017, CAAS. All rights reserved. Published by Elsevier Ltd.
doi: 10.1016/S2095-3119(17)61663-0
crop is found in a vast geographic range, from Asia through
the Middle East to Europe, and there is a long history of
chive cultivation for culinary and medicinal use. This leafy
vegetable is a herbaceous and perennial plant that can grow
to approximately 45 cm and is clustered on rhizomes. The
green leaves are smooth, slim, linear, flat, have a distinctive
mild garlic flavor, and are rich in vitamins, fiber, mineral com-
pounds and sulfur compounds that have antibiotic properties
(Imahori et al. 2004). However, chives are highly perishable
and quickly lose freshness after harvest. Leaf senescence
combined with the delicate chive leaf texture exacerbate the
deterioration of quality and significantly shorten postharvest
 *** et al. Journal of Integrative Agriculture 2017, 16(0): 60345-7
shelf life (Imahori et al. 2007).
Some attempts to maintain the postharvest quality of
chive have been made, such as storing at a low temperature
(Ishii and Okubo 1984), storing at 2°C in different types of
unsealed plastic film bags, such as high density polyethylene
(HDPE)+Molecular sift or HDPE+Calcium carbonate (Hou
et al. 2003), storing under CO2-enriched atmospheric con-
ditions (Imahori et al. 2007) and treating with postharvest
ultraviolet C exposure (Zheng et al. 2011). However, these
methods have met with limited success and have limited in
real-world applications.  respectively, was dispersed in 900 mL of distilled water.
Recently, consumers have become more conscious of
the potential adverse effects of synthetic chemical treat-
ments, and researchers have been committed to finding
high-quality and safe postharvest treatment products that
lead to minimal changes in dietetic and sensory properties
during storage (Siddiqui et al. 2011). The exogenous appli-
cation of cytokinins can reduce chlorophyll degradation and
delay senescence (Garcia and Barrett 2002). The cytokinin
compound, 6-benzylaminopurine (6-BA), which is involved
in preventing chlorophyll degradation in rocket (Eruca sativa
mill.) (Koukounaras et al. 2010) and in improving the quality
of Pakchoi (Brassica chinensis L.) (Gao et al. 2015), has
been approved as postharvest bio-pesticide by the United
Stated Environmental Protection Agency (US-EPA). The use
of 6-BA effectively reduces postharvest yellowing in many
green tissues, such as leaves, floral tissues and spears
(Rushing 1990; Tian et al. 1994; Downs et al. 1997; Costa
et al. 2005; An et al. 2006). It is known that leaf senescence
is due to the decrease in reactive oxygen species (ROS) me-
tabolism-related enzyme activities (Borrel et al. 1997; Shen
et al. 1997; Fang and Zheng 2002; Zheng et al. 2011). It
has been found that higher levels of SOD and POD enhance
the elimination of H2O2 and thus diminish the formation of
hydroxyl radicals and prevent chlorophyll damage (Toivonen
and Sweeney 1998) and that 6-BA delays senescence by
enhancing ROS-related enzymatic activities in broccoli
(Brassica oleracea L.) (Costa et al. 2005). Furthermore,
the enhancement of ROS-related enzymatic activities as a
result of 6-BA treatment, if any, could have a beneficial effect
on quality. Hence, in this study we evaluated the effect of
6-BA on both chive postharvest quality and the activities of
antioxidant enzymes.
2. Materials and methods
2.1. Plant material, treatments and storage
Chinese chives (Allium tuberosum Rottler ex Spreng. cv.
791) were grown in an organic vegetable garden in Tong-
zhou, Beijing, China and harvested at commercial maturity.
3
All chives were quickly packed in sealed foam boxes with
ice bottles inside to remove the field heat, and immediately
transferred at ambient temperature to the laboratory within 1 h.
Upon arrival in the laboratory, chive plants of uniform size
and color with no signs of wilting or yellowing were selected.
High molecular weight 6-BA (purity≥98%) was purchased
from Sinopharm Chemical Reagent Company (Beijing,
China), and NaOH and HCl were obtained from Beijing
Chemical Works. To prepare 1 L of the 100, 300 and
500 mg L–1 6-BA solutions, 100, 300 or 500 mg of 6-BA,
Next, 10 mL 0.1mol L–1 NaOH was added to dissolve the
6-BA. The pH of the solution was adjusted to pH 7.5 with
1 mol L–1 HCl, and the solution was brought up to 1 L with
distilled water. Alkaline solution without 6-BA (pH 7.5) was
used as the control.
Chives were randomly distributed into four groups each
of 3 kg, and each group was divided into three replicates
of 1 kg. Each set of three replicates was given on of four
treatments. The treatments consisted of spraying chives
for 10 min with (1) alkaline solution (control), (2) 100 mg
L–1 6-BA, (3) 300 mg L–1 6-BA, and (4) 500 mg L–1 6-BA.
The treated chives were then allowed to dry for 25 min at
25°C and 50% RH. Subsequently, the treated chives were
enclosed in plastic boxes lined with microporous film bags
(0.015 mm in thickness) and stored in darkness at (2±1)°C
and 85–90% RH. Samples were taken every 6 days ; one
portion of the samples was used fresh for analyses of visual
appearance, and chlorophyll, ascorbic acid (AsA), soluble
protein, total phenolic and flavonoid content, while the re-
maining samples were frozen in liquid nitrogen and stored at
–80°C for further analysis of SOD, CAT and POD activities.
2.2. Visual quality
Chive visual quality was evaluated based on weight loss
and decay during storage.
To determine the weight loss, three sets (1 kg per set) of
chives in each treatment were weighed at different intervals
using an electronic weighing scale with an accuracy of 0.5 g
(UWA-K-015, Xiamen Andong Electronics Co., Ltd., Fujian,
China). Then the weight loss (%) was calculated using the
formula: Weight loss (%) =(IW−FW)/IW×100, where, IW is
initial chive weight and FW is the weight at each time point
(Siddiqui et al. 2014).
The decay index was evaluated by a nine-member panel
according to the criteria of Cantwell and Thangaiah (2012)
with some modifications. Decay was rated on a scale of
1–5, where: 1=none, 2=trace, 3=slight, 4=moderate, and
5=severe, and a score of 3 indicates the minimum com-
mercial acceptability. Decay index was calculated using
4 *** et al. Journal of Integrative Agriculture 2017, 16(0): 60345-7
the following formula, Decay index=(∑D×Q)/(4×G), where,
D is the degree of decay, Q is its respective decay grams
of chives and G is total grams of chives in each treatment.
The decay index was calculated for three experimental
replicates for each treatment.
2.3. Yellowing incidence and Chlorophyll content
Yellowing incidence of chive was evaluated by a nine-mem-
ber trained panel according to the method described by
Cantwell and Thangaiah (2012) with some modifications.
Yellowing incidence was calculated as the percentage of
the total weight (in grams) of the evaluated chives that were
yellow in appearance. Yellowing incidence was calculated
for three experimental replicates for each treatment.
Chlorophyll content was determined according to the N,
N-dimethylformamide method described by Inskeep and
Bloom (1985) with slight modifications. 0.25 g chives were
immersed in 10 mL N, N-dimethylformamide and stored
at 4°C in darkness overnight. Chlorophyll content was
determined by measuring the absorbance of the solvent
at 647 and 664 nm. Each measurement was performed in
triplicate. Chlorophyll content was expressed as milligrams
per kilogram of fresh extract (mg kg–1). Measurement of
chlorophyll content was repeated for three experimental
replicates for each treatment.
2.4. Determination of ascorbic acid and protein
content
Ascorbic acid (AsA) was determined quantitatively using the
2, 6-dichlorophenol indophenol dye method (AOAC 2000).
To extract AsA, a crushed tissue sample (10 g) was placed
in a volumetric flask, and 50 mL of 3% metaphosphoric acid
was added. Then the extract was centrifuged at 10 000× g
for 20 min at 4°C and titrated with the standard dye to a pink
end point (persisting for approximately 15 s). The ascorbic
acid content was calculated from the titration value, dye
factor, dilution factor and volume of the sample, and was
expressed as milligrams per kilogram of fresh weight (mg
kg–1). Measurement of ascorbic acid content was repeated
for three experimental replicates for each treatment.
To determine protein content, 4 g chives were homoge-
nized in 8 mL of 25 mmol L–1 phosphate buffer solution (PBS,
pH 7.5), then centrifuged at 13 000× g for 20 min at 4°C.
The amount of protein in the supernatant was determined
according to the method of Bradford (1976) with bovine
serum albumin as a standard. The protein content was
expressed as milligrams per kilogram of fresh weight (mg
kg–1 FW). Measurement of protein content was repeated for
three experimental replicates for each treatment.
2.5. Determination of total phenolic and flavonoid
content
To determine the total phenol content, the extraction pro-
cedure for total phenols described by Vinson et al. (1998)
was used with certain modifications. Briefly, 50 mL 1.2
mol L–1 HCl in 50% aqueous methanol was added to 5 g
chives, and the sample was shaken for 2 h at 80°C and
filtered. This procedure removes vitamin C that is originally
present in chive.
Extractions were prepared in triplicate and stored for a
maximum of 24 h at –20°C. The supernatant was used for
the determination of total phenolic content as described by
Waterman and Mole (1994) with some slight modifications.
An appropriate volume (2 mL) of the filtrate or water (for the
blank) was added to 5 mL Folin–Ciocalteu Reagent (1:10) in
a 100 mL Volumetric Flask. After 6 min, 15 mL 20% Na2CO3
(w/v) was added. Subsequently, alkaline solution was added
to a total volume of 100 mL and incubated for 2 h at room
temperature in the dark with shaking. Absorbance readings
were taken at 740 nm and compared against the blank.
The standard curve was made using gallic acid standard
solutions (0–400 mol L–1) following the same procedure
as above. All determinations were carried out in triplicate,
and the result was expressed as milligrams of gallic acid
equivalents (GAE) per kilogram of fresh weight (mg kg–1).
Measurement of total phenolic content was repeated for
three experimental replicates for each treatment.
Total flavonoid content was measured according to Lin
and Tang (2007) with some modifications. 250 mL metha-
nolic extract was mixed with 75 mL 5% (w/w) NaNO2 and
1.25 mL alkaline solution. After 6 min, 150 mL 10% (w/w)
AlCl3 was added, and the mixture was allowed to stand for
6 min. Then 2.5 mL deionized water and 4 mL 4% (w/w)
NaOH were added. The absorbance was measured at
510 nm and the total flavonoid content was expressed as
milligram rutin equivalents per kilogram of fresh extract (mg
kg–1). Measurement of total flavonoid content was repeated
for three experimental replicates for each treatment.
2.6. Determination of SOD, CAT and POD activities
Frozen tissue samples (4 g) were homogenized in 8 mL
25 mmol L–1 Phosphate Buffered Saline (PBS) containing
8% (w/v) polyvinylpolypyrrolidone (PVPP) and 1 mmol L–1
Ethylene Diamine Tetraacetic Acid (EDTA), then centrifuged
at 13 000× g for 30 min at 4°C. For assays of SOD, CAT and
POD activities, the pH of the extraction buffer was 7.8, 7.0
and 6.4, respectively. The supernatant was collected for the
enzyme assays. SOD activity was determined according to
Luries et al. (1997), and the volume of enzyme correspond-
 *** et al. Journal of Integrative Agriculture 2017, 16(0): 60345-7 5

ing to 50% inhibition of nitro-blue tetrazolium (NBT) reduction
at 560 nm was considered one enzyme unit. CAT activity
was analyzed based on Cakmak and Marschner (1992)
with certain modifications, and CAT activity was defined
as a change in OD240 of 0.01 per kilogram fresh weight
per second. POD activity was determined by the method
of Wang et al. (2005), and POD activity was defined as a
change in OD470 of 1 per kilogram fresh weight per second.
SOD, CAT and POD activities were expressed as U kg–1 s–1.
2.7. Statistical analysis
All statistical analyses were done on data pooled from three
independent experiments, and the results were expressed
as the mean value and standard error (SE). Data analysis
was performed using SPSS version 19.0 (SPSS Inc., Chica-
go, IL, USA), with each time point analyzed using analysis of
variance (One-way ANOVA), and T-tests were performed to
identify significant differences between means. Differences
at P<0.05 were considered significant.
than in the control. After 42 days of storage, the total chlo-
rophyll content of the 300 mg L–1 treated chives declined
approximately 7.81%, while in the control chlorophyll content
declined 14.99% (P<0.05) (Fig. 2).
3.3. Effect of 6-BA on the AsA and protein content
of chive
Fig. 3-A shows the AsA content of the control and 6-BA-treat-
ed chives. Irrespective of treatment, AsA content declined
gradually with increasing storage time. However, compared
with the control, the loss of AsA in 6-BA-treated chives
occurred more slowly during storage. Control chives lost
39.72% of the initial AsA by 42 days, whereas chives treated
with 300 mg L–1 6-BA lost 22.94% of the initial AsA by 54
days of storage. Thus, the use of 6-BA significantly retarded
the loss of AsA in the present study.
After harvest, the protein content continuously decreased
in all chives during the first 36 days storage, increased at

3. Results Control 100 mg L–1 6-BA

300 mg L–1 6-BA 500 mg L–1 6-BA
3.1. Effect of 6-BA on the visual quality of chive A 55
50
There was a marked loss of weight during the storage period
for each treatment (Fig. 1-A). During storage, the control 40
Weight loss (%)

**
showed excessive weight loss, with an average rate of
**
1.98% per day, whereas chives treated with 100, 300 and 30 **

500 mg L–1 6-BA lost weight at a rate of 1.46, 0.98 and 1.23% **
20
per day, respectively. We concluded that the weight loss in **
**

6-BA treated chives was significantly lower than that of the *

10 **
**
control after 42 days of storage (P<0.05). *
**
*
*
Fresh chives were green, with a typical characteristic 0
odor, but the external appearance of the control chive de- 24 30 36 42 48 54
Storage time (d)
teriorated rapidly between 42 and 48 days (Fig. 1-B). All
1.0
treatments could delay the development of decay and did B
0.8
not differ significantly from each other. The decay index *
*

of 300 mg L–1 6-BA-treated chives was significantly lower **

0.6
than that of the control (P<0.05), although it increased
Decay index

during storage.
**
0.4 **
**

3.2. Effect of 6-BA on yellowing incidence and chlo-

*
rophyll content of chive
*
0.2 *

Yellowing was observed in the control after 42 days of stor- 0.0

age, and there was a significant increase in yellowing inci- 30 36 42 48 54 60
Storage time (d)
dence between the beginning (14.63%) and end (29.23%)
of storage (Fig. 2-A).
Fig. 1 Effect of 6-benzylaminopurine (6-BA) on weight loss (A)
The 6-BA treatments clearly delayed chlorophyll degra-
and the decay index (B) of chives during 54 days of storage at
dation during storage (Fig. 2-B). The 6-BA treated samples 2°C and 80–85% relative humidity (RH). Values are presented
showed chlorophyll degradation, but the process was slower as the means±SE. *, P<0.05; **, P<0.01.
6 *** et al. Journal of Integrative Agriculture 2017, 16(0): 60345-7

A Control 300 mg L–1 6-BA

30
Control A 45
300 mg L–1 6-BA

Ascorbic acid content (mg 100 g–1)

40
Yellowing incidence (%)

25

30
20

20
15

10
10
30 36 42 48 54
Storage time (d) 0
B 0 6 12 18 24 30 36 42 48 54
65 Storage time (d)
Chlorophyll content (mg 100 g –1)

B 46
60 44

Protein content (mg g–1)

* **
**
55 40

50
Control
300 mg L–1 6-BA
45
40
0 6 12 18 24 30 36 42 48 54
Storage time (d)
Fig. 2 Effect of 6-benzylaminopurine (6-BA) on yellowing
incidence (A) and chlorophyll content (B) of chives during 54
days of storage at 2°C and 80–85% relative humidity (RH).
Values are presented as the means±SE. *, P<0.05; **, P<0.01.
42 days and then declined again (Fig. 3-B), The protein
content of the treatment group was higher than that of the
control after 42 days of storage, but, this difference was not
significant. Hence, the 6-BA treatments could delay protein
degradation in chive.
3.4. Effect of 6-BA on the phenol and flavonoid con-
tent of chive
The phenolic compound content fluctuated in all chives
(Fig. 4). Phenolic compound content decreased in the con-
trol and 6-BA-treated samples, but a difference in content
was only apparent after 42 days of storage. At this point
in time, the total phenol content of the control declined
sharply, while that of the 6-BA treatments increased to a
value that was not significantly different from the initial
content (Fig. 4-A). Similar to phenolic content, the flavonoid
content of 6-BA-treated chives decreased significantly after
24 days of storage and, after a longer period of storage,
increased again to a value not significantly different from
the initial content (Fig. 4-B). However, unlike phenolic
content, the change in flavonoid content of both the control
36
32
28
24
0 6 12 18 24 30 36 42 48 54
Storage time (d)
Fig. 3 Effect of 6-benzylaminopurine (6-BA) on ascorbic acid
(A) and protein (B) content of chives during 54 days of storage
at 2°C and 80–85% relative humidity. Values are presented
as the means±SE.
and 6-BA-treated chives was similar.
3.5. Effect of 6-BA on antioxidant enzyme activities
of chive
It is known that SOD detoxifies superoxide ion to H2O2 ,
which is finally converted to H2O and O2 by CAT and POD
(Martino et al. 2006; Carocho and Ferreira 2013). The ac-
tivities of defense-related enzymes (SOD and CAT) in both
the 6-BA-treated and control chives increased gradually
during the first 36 days and then showed a sharp decrease
during the last 18 days of storage (Fig. 5-A and B). The
POD activity in all chives increased during the first 42 days
of storage, and there was a slight drop over the last 12
days (Fig. 5-A). However, in 6-BA-treated samples, all de-
fense-related enzyme activities were higher than the control
samples during the storage period (Fig. 5), and the differ-
ence in SOD activities was significant (Fig. 5-A, P<0.05).
These results indicate that 6-BA treatment stimulates SOD,
CAT and POD activities to different extents.
 *** et al. Journal of Integrative Agriculture 2017, 16(0): 60345-7 7

Control 300 mg L–1 6-BA Control 300 mg L–1 6-BA

A A
0.17 110
Total phenolic content (mg g–1)

SODactivity (U mg–1 FW min–1)

105 **
**
0.16
100 **

95
0.15
90

0.14 85

0.13 0 6 12 18 24 30 36 42 48 54
0 6 12 18 24 30 36 42 48 54 Storage time (d)
Storage time (d) B 0.20
B

CAT activity (U mg–1 FW min–1)

15.0

0.19
Total flavonoid content (mg g–1)

14.5
0.18

14.0
0.17

13.5 0.16

0 6 12 18 24 30 36 42 48 54
13.0 Storage time (d)
0 6 12 18 24 30 36 42 48 54 C 0.300
Storage time (d)
POD activity (U mg–1 FW min–1)

0.275
Fig. 4 Effect of 6-benzylaminopurine (6-BA) on total phenolic
(A) and flavone (B) content of chives during 54 days of storage
at 2°C and 80–85% relative humidity. Values are presented as
0.250
the means±SE.

0.225
4. Discussion

Senescence is a major problem in keeping chives fresh after 0.200

harvesting. Chives deteriorate rapidly and can be preserved
for no more than a week if they are picked and placed in the
air directly. In this study, the application of 300 mg L–1 6-BA
maintained the quality of chives. Moreover, the antioxidant
activity of 6-BA-treated chive was enhanced compared with
the control after 42 days of storage.
Weight loss and decay are two crucial parameters that
are closely related to the commodity value of chives. Most
fruits and vegetables become unsalable as fresh produce
when they lose 3–10% of their weight (Ben-Yehoshua and
Rodov 2003), and it was found that 6% weight loss and a
0.2 decay index may be the limit for marketability of fresh
chives. The weight loss of vegetables is mainly caused by
transpiration dehydration and respiration (Ma et al. 1994).
Dehydration results in the strengthening of hydrolysis, which
eventually causes a decline in disease resistance and stor-
0 6 12 18 24 30 36 42 48 54
Storage time (d)
Fig. 5 Effect of 6-benzylaminopurine (6-BA) on the activities
of reactive oxygen species metabolism-related enzymes in
postharvest chives during 54 days of storage at 2°C and 80–
85% relative humidity. Values are presented as the means±SE.
*
, P<0.05; **, P<0.01.
ability and an increase in decay. In this work all chives were
stored under the same conditions, but the 300 mg L–1 6-BA
treatment could more effectively inhibit the loss of quality
and decay compared with the 500 mg L–1 6-BA treatment.
These findings are in accordance with Gao et al. (2015),
who found that 6-BA treatment could significantly delay the
loss of organoleptic quality, reduce weight loss and inhibit
the respiration intensity of Pakchoi (Brassica chinensis
L.) and that 15 mg L–1 6-BA treatment had a better effect
8 *** et al. Journal of Integrative Agriculture 2017, 16(0): 60345-7
than 30 mg L–1 6-BA treatment. Respiration in postharvest
fruits and vegetables leads to nutrient consumption, which
results in quality loss, dehydration, and decay (Gao et al.
2015). Therefore, during postharvest storage respiratory
consumption should be minimized. 6-BA treatment effec-
tively inhibited the respiratory strength of Pakchoi (Brassica
chinensis L.), while the good performance of respiratory
depression may lie in the antagonistic effect of abscisic acid
(Looney 1979). Gong (2003) also showed that 10 mmol L–1
6-BA treatment of day lily can significantly inhibit respiration
and the production of ethylene, effectively delaying ripening
and senescence and maintaining quality at low temperature
conditions.
We analyzed the influence of 0, 100, 300 and 500 mg
L 6-BA treatment on visual appearance of chive, and our
–1
results showed that 300 mg/L 6-BA treatment can slow
weight loss and the decay process. Compared with a high
concentration (45 mg L–1 6-BA) treatment, low concen-
tration (30 mg L–1 6-BA) treatment significantly delayed
the decrease in endogenous antioxidants of postharvest
Ipomoea aquatic. For example, there was a slower decline
in the levels of ascorbic acid and glutathione, Moreover,
the increase in the production of superoxide anion radicals
(O2–. ) and the levels of H2O2 and malonaldehyde (MDA) in
6-BA-treated samples were reduced, suggesting that 6-BA
treatment could alleviate the senescence of postharvest
Ipomoea aquatic (Lu et al. 2016).
Chlorophyll is one of the most important indicators of plant
physiological activity. Chlorophyll not only affects photo-
synthesis, but also significantly influences the preservation,
production and sale of green leafy vegetables (Ma 2012).
Moreover, the commodity and nutritional value of chives
reduces gradually as chlorophyll degradation occurs with
increasing storage time, which is consistent with the results
from other horticultural products like broccoli (Costa et al.
2005; Xu et al. 2012) and asparagus (Zhao et al. 2011). In
general, chlorophyll degradation is related to the amount of
ethylene release, chlorophyll enzyme decomposition and
chlorophyll magnesium removal (Wu et al. 1991; Tian et al.
1994). In this experiment, chlorophyll content decreased,
resulting in yellowing of the control during storage, while the
6-BA treatment successfully retarded chlorophyll degrada-
tion. The delay in chlorophyll degradation in 6-BA-treated
chives is consistent with the fact that 6-BA promotes the
synthesis and accumulation of chlorophyll by promoting the
biosynthesis of amino acids (Guo et al. 2008; Xu et al. 2012).
In addition to changes in chlorophyll, nutrient content is
an important quality parameter used to estimate the effect
of storage on horticultural crop quality, and AsA and protein
content are related to nutrient content. Another manifesta-
tion of senescence is that the soluble protein content de-
creases rapidly, and the synthesis of net protein decreases,
which leads to the disruption of organism function (Surówka
et al. 2007). The general observation for vegetables is that
the protein level falls with a concomitant increase in free
amino acids during storage because plant senescence
is associated with increased proteolysis and amino acid
interconversion (Imahori et al. 2004). The results of this
work suggest that the regulation of AsA and protein content
might contribute to the beneficial effect of 6-BA treatment
in delaying the vegetable senescence process during
postharvest storage, a response that has been previously
documented by Siddiqui et al. (2011) in fresh-cut broccoli
florets. An et al. (2006) also reported that the decrease
of AsA in spears without 6-BA treatment was significantly
higher than the decrease in 6-BA-treated spears.
The market demand for chives is due not only to their
green-colored appearance and distinctive spicy smell, but
also to their antioxidant functions. The essence of post-
harvest preservation of leafy vegetables is to delay the
process of leaf senescence. Among the many biological
aging theories, the free radical theory of aging has aroused
widespread attention. Free radicals are generated in vivo
as byproducts of normal plant metabolism. If the free rad-
ical scavenging ability of fruits and vegetables decreases
or excessive amounts of radicals are generated, the cell
membranes of fruit and vegetable tissues will be damaged
and the senescence of fruits and vegetables will be accel-
erated (Rao et al. 2011). Thus, the antioxidant capacity
of fruits and vegetables directly affects the quality of fruits
and vegetables. Phenolic compounds, which have a strong
ability to remove free radicals, are important antioxidants
in plants and are also markers of post-harvest senescence
(Zheng et al. 2011; Li et al. 2012). Our results showed that
the phenolic compound and flavonoid content in the 6-BA
treated chives was significantly higher than in the control,
and chives treated with 300 mg L–1 6-BA maintained a higher
antioxidant capacity. This may be due to the fact that a
higher total phenolic content helped to reduce free radical
damage to chive tissues. Numerous studies have shown
that leaf senescence is closely related to ROS metabolism
disorders and decreased ROS scavenging activity (Lu
et al. 2016). Singlet oxygen (‘O2), H2O2, O2–. are the most
important active oxygen free radicals (Elstner 2003). These
substances have highly reactive groups with strong oxidizing
ability. In plants, reactive oxygen free radicals are produced
by a variety of metabolic pathways, such as mitochondrial
aerobic respiration and chloroplast photosynthetic electron
transport (Lin and Hao 2009). An important mechanism of
reactive oxygen free radical-caused plant injury is the direct
or indirect activation of membrane lipid peroxidation, which
results in membrane damage and destruction (Polle 2001).
 *** et al. Journal of Integrative Agriculture 2017, 16(0): 60345-7
MDA is the final product of membrane lipid peroxidation, and
is formed by active oxygen free radical-induced oxidation of
unsaturated fatty acids in vivo (Imahori et al. 2016). MDA
is produced during the postharvest storage of fruits and
vegetables (Chen et al. 2014) and is a marker of cellular
senescence. The reduced MDA content in 6-BA-treated
chives is consistent with the higher phenolic content and
reduced free radical damage of these chives.
It is well known that SOD detoxifies superoxide ion to
H2O2, and H2O2 is converted to H2O and O2 by the actions
of CAT and POD. Thus the activities of these ROS metabo-
lism-related enzymes in plants can ease ROS damage. Our
results showed that 6-BA treatment increased POD, SOD,
and CAT activities to different degrees, and the increased
enzyme activities likely led to increased scavenging of ROS
and delayed senescence of chives during storage. In a
study of the mechanism of fresh daylily senescence, 6-BA
treatment not only increased SOD activity and inhibited the
production of superoxide free radicals and of MDA, but also
delayed the senescence of daylily flowers (Gong 2003). We
found that 6-BA maintained the quality of chive by enhancing
the activities of antioxidant enzymes activities such as SOD,
CAT and POD. Based on our results, we speculate that the
good fresh-keeping effect of 6-BA treatment may be mainly
due to the inhibition of superoxide radical production.
5. Conclusion
In this study we evaluated the effect of 6-BA on posthar-
vest physiology and quality of chives. Based on visual
appearance, we determined that the effective concentration
of 6-BA was 300 mg L–1. Furthermore, we demonstrated
that the application of 6-BA significantly delayed yellowing
and chlorophyll degradation and maintained total phenolic
and flavonoid content, thereby improving the postharvest
quality of chive as reflected by AsA and protein content. In
addition, 6-BA initially enhanced the activities of the chive
antioxidant enzymes, SOD, CAT and POD but suppressed
these activities with increasing storage time. Our results
suggest that the use of 6-BA is an effective method to retain
chive postharvest physiology and quality.
Acknowledgements
This work was financially supported by the Innovation Ca-
pacity Building projects of Beijing Academy of Agricultural
and Forestry Sciences, China (KJCX20140205) and the
Innovation Capacity Building projects of Beijing Academy of
Agricultural and Forestry Sciences, China (KJCX20140408).
We also thank Mr. Bi Yang (the Vice president of Gansu Agri-
cultural University, China) for his careful revision of this article.
9
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