You are on page 1of 9

Scientia Horticulturae 189 (2015) 150158

Contents lists available at ScienceDirect

Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Conditioning garlic seed cloves at low temperature modies plant


growth, sugar, fructan content, and sucrose sucrose fructosyl
transferase (1-SST) expression
T. Guevara-Figueroa a , L. Lpez-Hernndez a , M.G. Lopez e , M.D. Dufoo Hurtado a ,
M.E. Vzquez-Barrios a , L. Guevara-Olvera b , R.G. Guevara Gonzlez c ,
D.M. Rivera-Pastrana a , H. Torres-Robles d , E.M. Mercado-Silva a,
a
Programa de Posgrado en Alimentos del Centro de la Repblica (PROPAC), Facultad de Qumica, Universidad Autnoma de Quertaro, Facultad de
Qumica, Departamento de Investigacin y Posgrado en Alimentos, Centro Universitario Cerro de las Campanas, 76010 Quertaro, Qro., Mexico
b
Departamento de Ingeniera Bioqumica, Instituto Tecnolgico de Celaya, Av. Tecnolgico y A. Garca Cubas S/N Col. FOVISSSTE, Celaya Guanajuato CP
38010, Mexico
c
Laboratorio de Biotecnologa, Cuerpo Acadmico Ingeniera en Biosistemas, Facultad de Ingeniera, Universidad Autnoma de Quertaro, Cerro de las
Campanas S/N Col. Las Campanas, Santiago de Quertaro CP 76010, Mexico
d
Consejo de Ajo del Estado de Aguascalientes, Mexico
e
Departamento de Biotecnologa y Bioqumica, Centro de Investigacion y de Estudios Avanzados del IPN, Unidad Irapuato, Mexico

a r t i c l e

i n f o

Article history:
Received 25 August 2014
Received in revised form 18 March 2015
Accepted 19 March 2015
Keywords:
Allium sativum
Conditioning
Fructan
Sugar
Gene
1-SST

a b s t r a c t
Bulb size and regularity of shape and absence of defects determine garlic commercial quality, with these
factors being related to variety, environment and cultural practices. Low temperatures and short photoperiods at the beginning of development, followed by high temperatures and long photoperiods at the
end of the crop cycle are important for good bulb formation. Conditioning seed cloves at low temperatures before they are sown ahead harvesting and also decreases the incidence of undifferentiated bulbs;
however, its effects on growth pattern, fructan accumulation and gene expression of enzymes associated
with their synthesis are unknown. During 4 different seasons, sets of two hundred garlic bulbs Coreano
were conditioned at 5 C (5C) for 5 weeks and others sets were stored at room temperature (RT), the
cloves were separated and selected by weight (58 g). Three treated replicates, two hundred cloves per
replicate, were sown in commercial elds at Cosio Aguascalientes Mxico. Representative samples of
plants were analyzed for growth parameters, sugar and fructan contents, and 1-SST expression at different developmental stages. During the conditioning at 5C, the buds of different cloves were used to
1-SST expression. The 5C plants had shortened crop cycle (3242 days), and their growth rate was higher
(0.23 cm d1 ) compared to RT plants (0.013 cm d1 ). The fructan accumulation was also accelerated, and
this process could be associated with leaf senescence, apparently modulated by the high glucose content
in leaves of 5C plants (7.7 mg 100 g1 ) in comparison with the RT plants (1.5 mg 100 g1 ). However, the
5C plants did not reach either the yield or quality features of the RT plants. 1-SST expression was higher in
the garlic buds conditioned at 5C than in those of RT plants; however, with respect to plant development,
the expression was higher in all organs of RT plants. The conditioning of garlic bulbs at 5 C was effective
in shortening the harvest time and in eliminating the incidence of defects, allowing them to be harvested
when the garlic supply is low. The changes in fructan and sugar metabolism inuence growth pattern.
Published by Elsevier B.V.

1. Introduction

Corresponding author. Tel.: +52 442 192 1307x5579; fax: +52 4421921304.
E-mail addresses: lorenzogo@yahoo.com (L. Guevara-Olvera),
ramonggg66@gmail.com (R.G. Guevara Gonzlez), gene torres@hotmail.com
(H. Torres-Robles), mercado501120@gmail.com (E.M. Mercado-Silva).
http://dx.doi.org/10.1016/j.scienta.2015.03.030
0304-4238/Published by Elsevier B.V.

Garlic is the second most important species of the Allium genus.


World production in 2012 reached 24.8106 tons (FAOSTAT, 2012),
with China being the principal producer (80%). Quality factors
demanded by the international marketing of this vegetable include
bulb size, rmness, regularity of shape, number of cloves (812),
and absence of defects such as branching and undifferentiated

T. Guevara-Figueroa et al. / Scientia Horticulturae 189 (2015) 150158

bulbs (Ofcial United States Standards for Grades of Garlic,


1997).
The garlic bulb consists of a basal plate bearing the cloves, which
include the storage leaf and the sprouting leaves that covers a set of
functional dormant leaves that form the bud, or the propagule of a
new plant, which activates its growth under certain environmental
conditions.
The garlic ontogenetic stages include an inductive and morphogenic stage (Ledesma et al., 1997). In the inductive stage, low
temperatures and short photoperiod are important conditions to
promote axillary bud differentiation that will allow clove formation
and bulb growth. In contrast, the morphogenic stage requires high
temperatures and longer photoperiods, conditions that favor photosynthesis, photosynthate transport and the lling of the cloves
(Burba, 2007; Portela, 1996, 2000, 2001; Braz et al., 1997). When
the requirements of garlic ontogenetic stages are not fullled, there
is a high incidence of undifferentiated bulbs and branching. Thus,
fullling these requirements is essential to achieve quality bulbs
that the market demands.
At the end of the crop cycle, bulb formation is a very important stage: weight gain of the plant drastically decreases and the
bulb becomes the greatest sink organ of photoassimilates which are
transformed to fructooligosaccharides (FOS) and high-molecular
weight fructans (HMWF) until plant senescence occurs (Rizzalli
et al., 2002). Varietal characteristics and crop management, as well
as environmental conditions and agricultural practices determine
the quality bulb characteristics for each season (Bertoni et al., 1992).
Takagi and Qu (1995) observed better bulb formation in plants
obtained from seed cloves conditioned at RT and 5 C although
these responses depend on the variety and the range of temperatures used (Volk and Rotindo, 2004). We have previously reported
an early harvest date for garlic cv. Coreano, and an increase of
phenol and anthocyanin contents as a result of cold conditioning of
seed cloves (Dufoo-Hurtado et al., 2013).
The garlic dry matter is mainly composed by fructan (Bekenblia
and Shiomi, 2006) which are polyfructosylsucroses of different
molecular size. These are biosynthesized from sucrose starter unit
and fructose to generate kestose (the smaller fructan) through the
enzyme 1-SST; afterwards more fructose units are added by the
action of 1-FFT enzyme (Baumgartner et al., 2000).
It is known that the environmental temperature directly affects
gene expression in different biochemical pathways like carbohydrate metabolism. For example, a low temperature can change
the sugar accumulation patterns, which could induce the onset of
senescence signals on the leaves (Wingler et al., 2012). However,
there are no data reported yet about these changes during garlic
development, or whether these changes could explain the early
termination of the crop cycle.
It is not well understood how the low temperature conditioning
of the seed cloves affects the plant growth, carbohydrate composition, or fructan accumulation and distribution during plant
development. Therefore, the aim of this work was to study the
effects of conditioning on garlic seed cloves cv Coreano at 5 C
on plant growth, sugars and fructan accumulation, and the 1-SST
expression in different plant organs.

2. Materials and methods


2.1. Materials and experimental setup
Garlic seed bulbs (A. sativum, L.) cv. Coreano from four different seasons (20092010 to 20122013), were provided by the
Garlic Producer Association of Aguascalientes Mexico, maintained
by our laboratory, and cultivated at Cosio, Aguascalientes, Mexico
(22 21 N, 102 18 W, 2000 meters above sea level). For each

151

season, before the sowing, one set of 200 bulbs was stored at
room temperature (RT), 19 C and 77% RH average, and another
set was conditioned for ve weeks at 5 C (5C) and 98% RH average. The cloves from each bulb were separated, and selected those
with commercial size and weight (58 g), treated with fungicides,
nematicides, and bactericides to avoid pests and diseases in the
eld. The cloves of each treatment were divided in three replicates
(two hundred cloves per replicate) and sown in different commercial elds at Cosio, Aguascalientes. The cloves were sown in double
furrows at a distance of 7 cm from each other. All cultural practices
used were those commercially managed and recommended by the
Garlic Producer Association of Aguascalientes.
2.2. Indicative parameters of plant and bulb growth of garlic
The number of leaves, plant height, leaf width, bulb and
pseudostem diameter, plant weight, and bulbing index (bulb
diameter/pseudostem diameter) were used to evaluate coldconditioning effect on growth. Five different sampling periods were
conducted in each crop cycle. For each sampling period, ve plants
from each treatment and replicate were taken, transported on ice,
and washed before measuring the growth parameters.
The medium part of the leaves, pseudostem and bulb were separated and frozen in liquid nitrogen and stored at 70 C until their
analysis. Total yield and harvest quality were evaluated at the end of
each season. The harvest time was determined by the leaves senescence and a clear and complete differentiation of the cloves in the
bulb. The plants from a section of 5 m of the furrow were separated and curing in eld for two weeks (whole plants were left
on the eld until the foliage was dry), after which the bulbs were
weighed and sorted by defects and size accordance to commercial
garlic standards. The data were then extrapolated to one hectare.
2.3. Total fructan content
2.3.1. Fructan extraction
A pool from each plant tissue (bulb basal region, pseudo stem
and leaves) was made from ve plants, frozen with liquid nitrogen and freeze-dried before extraction; 0.1 g of freeze-dried sample
powder (0.5 mm particle size) of each organ was mixed in 80 C
water for 15 min then cooled. 50 mL of the mix was ltered (Whatman #. 1) to obtain the fructan raw extract. Total fructan content
was determined using the Fructan HK Assay test kit (Megazyme
International Ireland, Ltd., Wicklow, Ireland), following method
999.03 from the AOAC (McCleary et al., 2000) and the instructions
of the manufacturer.
2.4. Simple carbohydrates, fructooligosaccharides and fructan
prole determination
The carbohydrates were extracted from 100 mg of freeze-dried
tissue placed in 8 mL of water at 80 C, stirred for 15 min, then
cooled and ltered (Whatman #. 1) and made up to 10 mL. The
suspension was diluted to 0.5 mg/mL, and then ltered through a
Millipore nylon membrane (0.2 m). The solution was sonicated
and chromatographically analyzed.
2.4.1. High-performance anion-exchange chromatography with
pulsed amperometric detector (HPAECPAD)
A 25 L sample was injected into an ion chromatograph
Dionex ICS-3000 (Sunnyvale, CA, USA) equipped with a CarboPac
PA-100 (4 mm 50 mm) guard column and a CarboPac-PA100
(4 mm 250 mm) column. Fructan separation was achieved using a
gradient of sodium acetate, in 0.15 M NaOH, at a ow of 0.8 mL/min,
as follows: 05 min, 45 mM NaOH; 560 min, 0375 mM sodium
acetate; 6065 min, 500 mM sodium acetate; and 6575 min,

152

T. Guevara-Figueroa et al. / Scientia Horticulturae 189 (2015) 150158

45 mM NaOH, at a column temperature of 25 C. Applied potentials, for detection by the amperometric pulse, were as follows: E1
(400 ms), E2 (20 ms), E3 (20 ms), and E4 (60 ms) of +0.1, 2.0, +0.6,
and 0.1 V, respectively (Martens and Frankenberger, 1990).

data were analyzed by ANOVA and the Tukey test (p < 0.05) for
means comparison, using the statistical software, JMP v.5.9. The
Gompertz equation was used to t the plant height data over
time or DAS. The model includes three constants of physiological
importance:

2.5. 1-SST gene differential expression

y = Aee

The dynamic of differential gene expression was performed


during the 20112012 season analyzing the buds of functional
leaves from ve seed cloves conditioned at 5 C or RT, and from
different pools from ve plant tissues per replicate (bulb basal
region, pseudostem and leaves) at 69, 155 and 194 days after
sowing (DAS) of plant development. Every week conditioned
cloves; and samples of plant development were frozen with
liquid nitrogen, and stored at 70 C until analysis. RNA was
extracted from these samples using the RNeasy Plant Mini Kit
(Qiagen, Germany). cDNA was synthetized from the same RNA
amount for all the samples using the First Strand cDNA Synthesis
Kit (Thermo Scientic, Wilmington, Delaware, USA) and stored
at 20 C until use. Primers were designed for sucrose:sucrose
1-fructosyltransferase and ACTIN (as control) amplication (1-SST,
Accession AAM21931.1: Fwd 5 -TTTTTTTTTCCGACGGCTTC3 , Rev 5 -GGTACCAATGATCGGGAATA-3 ; ACT, Accession
AY821677: 5 -TTGCTGGTGATGATGCTCCAAGGGC-3 ; Rev 5 TGGTACGGCCACTGGCATAGAGGGA-3 ),
using
Vectro
NTI
Advance v.11.5.0 program (Invitrogen, Life Technologies, Carlsbad, California, USA), and synthetized by Industrias Bioselec S.
A. de C. V. (Mexico). Amplication by Reverse Transcriptase-PCR
program was executed as follows: denaturation at 95 C for 5 min,
followed by 35 cycles of 30 s at 95 C, annealing temperature
(60 C) for 45 s, and 1.5 min at 72 C (elongation). The enzyme
used was Titanium Taq Polymerase (Clontech, Mountain View,
California, USA) following the protocol described by the manufacturer. RT-PCR products were observed by 1.2% agarose/EtBr
gel electrophoresis, then, puried by Wizard PCR preps DNA
purication system (Promega, Madison, WI, USA) and sequenced
at Laboratorio Nacional de Biotecnologa Agrcola, Mdica y
Ambiental (LAMBAMA, IPICYT, SLP, Mexico). Expression level
was calculated by densitometry with ImageJ software program
(National Institutes of Health, USA).

where A determines the maximum value of the asymptotic


growth, B is the maximum growth rate, C is the turning point or
the sudden change in growth rate and t is the time or DAS.

2.6. Statistical analysis


Each season, three sets of two hundred cloves (replicates) of
both treatments were sown in three experimental furrows in the
same eld. A randomized blocks design was used with three replicates; ve plants were taken periodically by replicate, and then
analyzed for growth parameters, sugar and fructan contents. The

(BCt)

3. Results
3.1. Plant growth
Between 44 and 56 DAS no signicant differences were observed
on plant height; nevertheless, in the 87103 DAS period, 5C plants
showed greater height (4265 cm) than RT plants (3647 cm).
These differences were more evident at 122 and 137 DAS, 5C
plants were taller (68.778.2 cm), and had greater bulb diameter (36.447.4 mm), greater bulbing index (2.64.4), and a greater
weight gain (64133 g) than RT plants, although RT plants had more
leaves (8.59.8) with a greater width (2136.6 mm).
5C plants completed the crop cycle between 156 and 158 DAS
while RT plants completed it between 191 and 201 DAS, resulting in an earlier harvest of 3242 days in 5C plants. The statistical
comparison at the end of the crop development of both treatments
(157 and 191 DAS for 5C and RT plants respectively) showed different results than those described above (Table 1). After 191201
DAS, RT plants exhibited more and wider leaves, greater height,
greater pseudostem and bulb diameter, and more weight than were
observed in 5C plants. These data indicate that the growth of RT
plants continued after 157 DAS and they reached larger sizes than
did 5C plants by the end of their cycle.
Using the height data for all seasons and the conditioning treatment of the seed cloves into Gompertz model, the constants
values and the t goodness of model was calculated (Table 2).
The model adequately predicted the plant growth, noting that the
plants from 5C cloves had a limit of maximum growth and a turning
point (inection point on growth curve) signicantly lower than
plants from RT cloves. However, the growth rate of plants from 5C
cloves was almost double than shown by plants from RT cloves.
Early harvest represents an economic advantage when the supply of the product in the market is low. For that reason, the
protability of storing seed cloves at low temperatures should
be calculated taking into account the total yield and bulb quality.
The yield per hectare and the undifferentiated and defective bulbs,
as well as the production of each commercial size obtained in each

Table 1
Growth variables values at the end of the crop cycle of Garlic Coreano, obtained from seed cloves stored at room temperature (RT) or conditioning at 5 C (5C) and planted
on different seasons. Data are means standard deviation of 15 determinations.
Season treatment
2009/2010
RT
5C
2010/2011
RT
5C
2011/2012
RT
5C
2012/2013
RT
5C

DAS

Number of leaves

Plant height
(cm)

Leaf width
(mm)

Pseudostem diameter
(mm)

Bulb diameter
(mm)

BI

Weight
(g)

194
157

11.4 1.4a
5.3 0.2b

81.1 7.8a
63.4 7.5b

27.1 5.0a
18.9 3.3b

23.9 4.7a
11.9 3.6b

51.0 7.4a
45.4 6.6b

3.4 1.0b
7.8 2.9a

175.5 51.9a
80.0 29.2b

198
158

12.7 1.0a
9.2 0.8b

81.9 8.6a
68.8 6.1b

28.1 4.1a
21.1 3.6b

16.6 4.5a
7.6 2.3b

65.8 5.9a
53.5 5.1b

4.2 1.2b
7.7 2.8a

153.1 41.2a
96.2 24.1b

201
155

7.7 1.4a
6.6 3.0b

95.9 8.4a
77.7 11.0b

26.9 5.3a
22.2 4.5b

17.4 4.8a
16.0 4.9b

59.6 11.1a
56.9 9.4b

4.1 0.9a
3.8 0.9a

237.1 81.5a
154.3 79.5b

191
156

7.5 1.3a
5.1 1.2b

71.4 5.9a
77.3 6.7a

31.0 4.6a
27.6 3.7b

17.7 4.0a
13.6 3.0b

64.4 10.5a
57.0 5.4b

3.7 0.6b
4.3 0.7a

168.4 62.5a
129.6 37.5b

Different letters between the same column and factor, indicate signicant differences (Tukey P < 0.05). Days after sowing (DAS), bulbing index (BI).

T. Guevara-Figueroa et al. / Scientia Horticulturae 189 (2015) 150158

153

Table 2
Parameters estimating garlic development by the Gompertz Model using the plant height (cm) and days after sowing (DAS) data.
Season
treatment

r2

RMSE

Parameters

Estimated

All seasons
5C

0.884

8.824

Asymptote

83.841

2.090

79.746

87.937

Growth rate
Inection point
Asymptote

0.023
48.956
96.587

0.002
1.664
2.717

0.020
45.693
91.261

0.026
52.219
101.912

Growth rate
Inection point
Asymptote
Growth rate
Inection point
Asymptote
Growth rate
Inection point
Asymptote
Growth rate
Inection point
Asymptote
Growth rate
Inection point
Asymptote
Growth rate
Inection point
Asymptote
Growth rate
Inection point
Asymptote
Growth rate
Inection point
Asymptote
Growth rate
Inection point

0.013
68.996
65.747
0.0461
24.605
90.908
0.012
58.332
79.282
0.023
50.524
105.326
0.014
82.284
82.580
0.026
40.535
111.051
0.011
79.737
101.081
0.019
66.107
87.291
0.016
61.167

0.0007
68.996
1.334
0.005
2.867
5.540
0.002
6.811
3.567
0.003
5.309
7.131
0.002
6.042
3.601
0.004
3.477
9.032
0.002
9.231
5.557
0.002
3.485
2.951
0.001
2.634

0.012
68.996
63.132
0.036
18.986
80.051
0.009
44.982
72.289
0.018
44.643
93.044
0.011
70.877
75.523
0.018
33.720
93.350
0.008
61.645
90.190
0.015
59.278
81.508
0.014
57.004

0.015
74.224
68.362
0.056
30.224
101.766
0.015
71.682
86.275
0.028
56.405
117.608
0.017
92.690
89.637
0.033
47.350
127.011
0.014
96.049
111.972
0.022
72.937
93.074
0.019
67.329

All Seasons
RT

20092010 5C

0.920

7.38

20092010 RT

0.878

8.93

20102011 5C

0.946

5.95

20102011 RT

0.933

7.73

20112012 5C

0.934

7.67

20112012 RT

0.883

10.36

20122013 5C

0.926

6.77

20122013 RT

0.881

7.56

Error STD

Lower

Upper

Conditioning at 5 C (5C), stored at room temperature (RT), regression coefcient (R2), root mean square error (RMSE).

Table 3
Total yield and harvest quality of garlic bulbs harvested at different seasons from seed cloves stored at room temperature (RT) or conditioning at 5 C (5C) for ve weeks.
Data are expressed as ton/ha. Undifferentiated bulbs not included into total yield.
Season treatment

Total yield ton/ha

Undifferentiated bulbs

Defects

C<6

C6

C7

C8

C9

C10

C11

C12

C > 12

20092010 RT
20102011 RT
20112012 RT
20092010 5C
20102011 5C
20112012 5C

16.3
15.8
20.1
12.3
9.7
10.0

4.6
0.3
3.3
0.0
0.0
0.0

0.8
0.7
1.1
2.4
0.4
0.5

0.3
0.1
1.4
0.4
0.4
0.4

0.5
0.2
0.2
1.1
0.8
0.6

2.8
0.6
0.4
1.8
2.7
1.8

2.5
1.3
0.6
3.1
3.7
2.5

0.6
2.9
3.9
2.5
1.7
1.7

3.7
3.6
3.7
0.9
0.0
1.9

3.2
3.8
4.8
0.1
0.0
0.3

1.0
2.6
2.9
0.0
0.0
0.3

0.9
0.0
1.1
0.0
0.0
0.0

Garlic size; diameter in cm: C6, 4.55; C7, 55.5; C8, 5.56; C9, 66.5; C10, 6.57.0; C11, 77.5; and C12, 7.58.

season were analyzed (Table 3). The overall yield was lower for
5C (9.712.3 ton per ha) than for RT plants (15.820.1 ton per ha).
Additionally, bulbs from RT plants showed higher calibers (C8 and
C9) compared with those from the 5C plants (C7C9). Nevertheless
bulbs from 5C plants did not show undifferentiated bulb production
whereas for RT plants, this production was high (0.34.6 ton per
ha). However, the successive conditioning at 5 C during consecutive seasons decreased more the overall yield (data not show) being
important for growers to maintain RT plants for seed production.
3.1.1. Changes in total fructan content
The conditioning only had a signicant effect on total fructan
content at 53 DAS (Table 4). However, as expected, the fructan
content in the bulbs was signicantly higher than in pseudostems.
The interaction temperature*organ conrmed these results; the
bulbs of both treatments presented higher values than those found
in pseudostems with no signicant differences between the two
treatments. These results conrmed that bulbs have a high capacity for storing these carbohydrates, and that pseudostems function
as temporary storage sites for these compounds, which are then
transported to the bulbs as the growing cycle takes place.

Table 4
Means comparison of fructan content (%DW) of bulb and pseudostem during development of garlic plants generated from seeds cloves conditioning at 5 C for 5
weeks or stored at room temperature (RT). Data from 2011 to 2012 season, each
data set represents the mean of 30 samples.
Factor/DAS
Temperature
5 C
RT
Organ
Bulb
Pseudostem
Temperature*organ
5 C*Bulb
RT*Bulb
5 C*Pseudostem
RT*Pseudostem

53

102

137

157

36.4a
28.9b

31.9a
18.1a

28.2a
26.3a

30.5a
25.0a

37.9
27.4b

35.0
24.9b

40.9
13.6b

45.2
10.4b

48.4

39.0a
36.8a
33.8a
20.9b

35.4a
34.7a
20.7a
29.2a

46.1a
35.6ab
6.4b
20.8ab

56.0a
34.4b
5.03c
15.7bc

54.2a
36.6ab

194

27.9

19.2b

Different letters between the same column and factor, indicate signicant differences (Tukey P < 0.05). Room temperature (RT). Days after sowing (DAS).

154

T. Guevara-Figueroa et al. / Scientia Horticulturae 189 (2015) 150158

Fructan content at harvest time in 5C bulbs (157 DAS) was


higher than in RT bulbs (Table 4), indicating a higher accumulation of these carbohydrates in 5C plants, while in RT plants, this
content had smaller changes. On the other hand, fructan content in
pseudostems of 5C plants decreased four times in the same period,
whereas this same organ in RT plants had smaller changes. This
suggests that fructan transport in 5C plants was faster than in RT
plants because the pseudostem of 5C plants quickly depleted these
compounds, while in RT plants this occurred at a lower rate. This
may explain the higher growth shown by RT plants at the end of
the crop cycle.

3.2. Changes in the simple carbohydrates and


fructooligosaccharides (FOS)
The sugars and FOS of up to 5 fructose units (DP 5) analyzed by
HPAEC-PAD showed changes associated with conditioning treatment of the seed cloves. Leaves of RT plants showed high sucrose
content throughout the crop cycle (Fig. 1A), which decreased at the
last stages of the crop cycle (from 48 to 28 mg 100 g1 ). Fructose
content increased, reaching maximum levels at harvest time (36 mg
100 g1 ), and glucose showed the lowest levels during the whole
crop cycle (0.2 to 3.2 mg 100 g1 ). Leaves of 5C plants also showed

Fig. 1. Sugar (glucose, black circles and solid lines; fructose, white circles with dotted lines; sucrose, black triangles and short dashes) and fructooligosaccharides, FOS
(kestose, light gray bars; neo-kestose, white bars; nistose, gray bars; DP5, black bars) content during crop development of different garlic organs: leaves, pseudostems and
bulbs of RT plants (A, B, and C, respectively), and 5C plants (D, E and F, respectively). All data is presented by means of three samples with standard deviation bars. Data from
2011 to 2012 season.

T. Guevara-Figueroa et al. / Scientia Horticulturae 189 (2015) 150158

a high sucrose content (Fig. 1D), but this increased until reaching
its highest values at 137 DAS (62.7 mg 100 g1 ) and then fell drastically at harvest time (5.8 mg 100 g1 ). Fructose content showed
a moderate increment (4.212.9 mg 100 g1 ) respect of the values observed for RT plants whereas glucose content showed wider
variation (0.37.7 mg 100 g1 ) in a shorter period of time, (53137
DAS). These data indicated a greater capacity for sucrose synthesis
or accumulation in 5C plants leaves up to 137 DAS, but this capacity drastically decreased in association with the bulb formation and
clove differentiation.
The highest glucose content observed in 5C plants leaves (137
DAS), could indicate a physiological change related to the sugar
signaling process associated with senescence of leaves.
On the other hand, the pseudostem of RT plants (Fig. 1B) showed
important differences in sugar content compared with that of 5C
plants (Fig. 1E). RT pseudostems showed a high sucrose content
at 53 and 102 DAS (24.6 and 44.6 mg 100 g1 ), which quickly
decreased at 137 and 157 DAS, and then increased again at the
harvest time, 194 DAS (41.9 mg 100 g1 ). Exhibiting a similar tendency, fructose content showed high values (11 mg 100 g1 ) at 53
DAS, which signicantly decreased at 157 DAS (0.6 mg 100 g1 )
and increased again at 194 DAS (37.8 mg 100 g1 ). Glucose content was very low during the rst 157 DAS and high at harvest time
(36.0 mg 100 g1 ). 5C plants pseudostems showed lower sucrose
content from 53 to 137 DAS (4.55.7 mg 100 g1 ), which also
increased at harvest time or 157 DAS (28.8 mg 100 g1 ), while
fructose and glucose were not detected within the 53137 DAS
period, and only high contents were registered at harvest time, 157
DAS (49.5 mg 100 g1 and 9.8 mg 100 g1 for fructose and glucose,
respectively).
As with the leaves and pseudostems, the RT plants bulbs also
showed important differences in sugars content (Fig. 1C) when
compared with the 5C plants bulbs (Fig. 1F). RT plants bulbs
showed increases in sucrose during the period from 53 to 137 DAS
(7.443.7 mg 100 g1 ), after which these levels reduced drastically
at 157 DAS and increased again at harvest time, 194 DAS (18.2 mg
100 g1 ). Fructose and glucose were not detected in RT plant bulbs.
The sucrose content of 5C plant bulbs was only determined at 53
DAS (11.1 mg 100 g1 ) and no content at 102 and 137 DAS was
recorded. It was determined again at harvest time, 157 DAS reaching its highest value (19.2 mg 100 g1 ). Fructose was determined
at 53 DAS (12.1 mg 100 g1 ) and at harvest time, 157 DAS showing
a lower value (0.6 mg 100 g1 , Fig. 1F). Similarly, to RT plants, 5C
plants did not show glucose content.
In a general way, the presence of these sugars in the three organs
could be related to the fructan synthesis and restructuring process
in those organs, which seems to be modulated by the length of
the crop cycle. This means that RT plants, with a longer crop cycle,
sucrose uctuations also occurred for longer periods; while for 5C
plants, that uctuation took place in a shorter time.
Fructooligosaccharides (FOS) content also showed important
differences between the two treatments. Leaves of RT plants
(Fig. 1A) showed lower levels of kestose (0.43.7 mg 100 g1 ),
neo-kestose (0.96.5 mg 100 g1 ), and nistose (0.32 mg 100 g1 )
whereas in 5C plant leaves, the FOS content was higher (kestose,
4.411.4 mg 100 g1 ; neo-kestose, 48.9 mg 100 g1 ). This data
showed that conditioning at 5 C induced a high temporary accumulation of FOS in leaves of 5C plants; this effect could be due to
an increased induction of 1-SST and 6G-FFT activities, key enzymes
of 1-kestose, and neo-kestose synthesis, respectively.
1-kestose content in RT plant pseudostems (Fig. 1B) decreased
as the crop cycle proceeded (6.73.4 mg 100 g1 ). Neo-kestose
showed values between 7 and 8 mg 100 g1 at 53 and 102 DAS
respectively, and after that, it decreased at 137 DAS and then
increased from six to 11 mg 100 g1 at 157 and 194 DAS. Nistose remained relatively constant while the FOS DP5 (degree of

155

polymerization = 5) increased from 0.1 to 3.2 mg 100 g1 . For


5C plant pseudostems (Fig. 1E), values of 1-kestose were high
(7.28.8 mg 100 g1 ) at 53 and 102 DAS and decreased to 3 and
2.9 mg 100 g1 at 137 and 157 DAS; while the neo-kestose content
increased from 3.1 to 9.9 mg 100 g1 at 53 and 137 DAS and then
decreased at 157 DAS (1.7 mg 100 g1 ). Nistose values increased
from 1.5 to 3.5 mg 100 g1 at 53 and 102 DAS, and decreased
after that period until harvest time. DP5 increased from 0.5 at 53
DAS to 5.7 mg 100 g1 at 137 DAS and then decreased at harvest
time. The data show that in this organ there are synthesis and
FOS transformation processes, in which the production of complex
FOS proceeded from simpler compounds like 1-kestose and neokestose. It was also possible to observe that these processes were
faster in samples from 5C plants.
FOS content in bulbs of RT plants (Fig. 1C) increased throughout
the crop cycle; it was observed that the highest values occurred at
the end of plant development, while 5C plant bulbs showed this
same tendency, but with lower content (Fig. 1F).
3.3. Changes in high-molecular weight fructans (HMWF) prole
during development
Peaks and areas detected by the HPAEC-PAD equipment whose
retention times ranged between 21.5 min (DP5 peak) to 48.1 min
were registered. All areas were added for each organ, sampling
time and conditioning treatment. These data directly indicate the
changes in the fructan content whose polymerization degree is
higher than DP5 (Fig. 2).
Leaves of RT and 5C plants showed capacity for storing fructan of high-molecular weight (>DP5) in the rst stages of the crop
cycle; however, 5C plants displayed a higher accumulation capacity than RT plants at 102 DAS, (27 and 9.6 U min1 for 5C and RT,
respectively), which decreased at the subsequent sampling dates.
This could indicate possible changes in metabolism and transport
of these carbohydrates from leaves to pseudostems and bulbs.
Pseudostems also showed differences between the two conditioning treatments. 5C plants pseudostems showed the lowest
values at harvest time (4.3 U min1 ) compared with the same organ
in RT plants (24.6 U min1 ). These data suggest that the capacity for
storing these carbohydrates remains for a longer period in RT plants
pseudostems than in 5C plant pseudostems, but that this capacity is
eventually depleted, which probably is related to bulb development
and clove differentiation at the nal stages of the crop cycle.
RT plant bulbs showed a steady increase in the accumulation of high-molecular weight fructans from 53 DAS to the end
of crop cycle (194 DAS), when the highest value was recorded
(40.3 U min1 ). 5C plant bulbs also showed an increase in the fructan accumulation, from 53 until 137 DAS, when a maximum value
was recorded (42.5 U min1 ), but afterwards, decreased at harvest
time (26.2 U min1 ).
The nal HMWF decrease in the bulbs of 5C plants could be due
to having fewer and more narrow leaves, and the smaller diameter
of the pseudostems, as well as to differentiation process. Therefore these factors could, decrease the photosynthesis capacity and
fructan biosynthesis, resulting in a lower availability for fructan
transport from pseudostems to bulbs.
3.4. Changes in the 1-SST gene expression during the
conditioning of seed cloves and harvest time
Sequencing PCR product and alignment on blastx
database (NCBI) showed homology with sucrose:sucrose 1fructosyltransferase [Allium sativum, Accession AAM21931].
Genetic expression analysis showed differences in the 1-SST
expression level between treatments. During all weeks of

156

T. Guevara-Figueroa et al. / Scientia Horticulturae 189 (2015) 150158

Fig. 2. Changes in high molecular weight fructan content during crop cycle expressed as sum of areas under chromatograms HPAEC-PAD. RT plants (A) and 5C plants (B).
Bars represent means of three samples with standard deviation bars. Data from 2011 to 2012 season.

Fig. 3. 1-SST gene expression level in garlic sprouts during conditioning at 5 C or room temperature (A), and in different organs during crop development (B). Room
temperature (RT). Conditioning at 5 C (5C). Bulb (b). Pseudostem (Ps). Leaves (L). Days after sowing (DAS). ACTIN was used as positive control to normalize the expression
levels. Data from 2011 to 2012 season.

conditioning at 5 C, the expression levels were higher in the


5C buds and reached their maximum during the last two weeks of
conditioning (Fig. 3A). A different expression pattern was observed
at 155 DAS in leaves, pseudostems and bulbs of RT plants, which
showed a higher 1-SST expression level than the 5C plants in
all analyzed organs. Similarly, at the harvest time of RT plants
(194 DAS) expression levels of this gene were higher in all organs
than those observed for 5C plants at their harvest time (155 DAS,
Fig. 3B). These data could indicate that the ability of 5C plants to
synthetize fructan is induced since the low-temperature conditioning, reaching its highest level at the end of the treatment before
sowing. Nevertheless, this capacity was markedly diminished at
harvest time. In contrast, RT plant organs (leaves, pseudostems
and bulbs), maintained a higher expression level during the crop
cycle, which may indicate that the activity of fructan synthesis in
these plants was retained until harvest. This seems to correlate
positively with fructan content discussed in the previous section.

4. Discussion
The early harvest time of Coreano garlic resulting from conditioning the seed cloves at 5 C coincides with the accelerated
bulb formation period previously reported by Del Pozo et al. (1997)
and Braz et al. (1997) in garlic varieties from Chile and Brazil.
Other studies (Rahim and Fordham, 1988, 2001) had observed an
advancement of 15 days in harvest time when they conditioned
seed cloves for 30 days at 5 C; their scanning electron microscopy
observations indicated that clove primordia appeared two weeks
before in 5C plants compared with that of the control plants. Additionally, they observed that bulb development and differentiation
were completed at 87 and 117 DAS in both treatments, respectively.
Nevertheless, they also indicated that the bulbs of 5C plants had
fewer but larger cells; these data conrm our results of the early
harvest, and seem to explain the low yield and bulb sizes observed.
Khokhar et al. (2007) working with onions, also observed a

T. Guevara-Figueroa et al. / Scientia Horticulturae 189 (2015) 150158

shortening of the harvest time, with fewer leaves, lower bulb fresh
weight, and lower yields. These authors also observed high induction of ower stalk bolting in 5C plants and they hypothesized
that the metabolism of plants conditioned at low temperature was
strongly inuenced by this structure, which produced plants with
fewer leaves. In this work, the ower stalk incidence was over
90% higher in 5C plants than in RT plants, which could modify the
metabolism, accelerate carbohydrate transport, and the senescence
of leaves and pseudostems. In contrast, Ade-Ademilua et al. (2009)
conditioned seed cloves at 8 C for 15 days, and after 70 DAS in
open eld observed a higher number of leaves, greater leaf area,
larger size, higher fresh weight and dry weight when compared
with plants with no conditioning. However, they did not report the
treatment effects on yield and bulb quality at the end of the crop
cycle. Thus, our results only match partially with their results.
The data indicate that the conditioning at 5 C induced a fast
emergence and growth of garlic 5C plants during the early stages of
the crop cycle; however, this was reversed during the intermediate
and nal stages by the RT plants.
The Gompertz model conrmed that the conditioning of seed
cloves at 5 C for ve weeks induced a faster plant growth (high
growth rate), but did not reach the sizes achieved by the RT plants,
although these constants did not explain why 5C plants had a
smaller size and lower yields. Reis et al. (2014) used different
mathematical models (logistic, von Bertalanfy, and Gompertz) to
describe similarities and differences between garlic varieties in the
dry matter accumulation during growth. They indicated a good t
of the experimental data with the logistic model; however, other
models also showed high values of R2 .
The ndings of Rahim and Fordham (1988) with respect to
the early formation of clove primordia in 5C plants allowed us to
hypothesize that early clove differentiation induced early leaf and
pseudostem senescence that ended the crop cycle.
The bulbing index gives an indication of the physiological and
metabolic changes during plant development, which could indicate
the stage at which the photosynthates are directed toward bulbs,
and that the plant is ready to generate cloves as a propagule. Fig. 4
shows the change of this index during different seasons for RT and
5C plants and shows at least three stages. The rst stage (4050
DAS) corresponds to initial plant development when nutrients are
transferred from the seed clove to the new plant, the initial

Fig. 4. Bulbing index, BI (bulb diameter/pseudo stem diameter) for all seasons
(20092013). Solid circles represent means for room temperature (RT) conditioning
plants. White circles, means for 5 C (5C) conditioning plants. Solid line, Gompertz
regression for RT plants; and dotted line, Gompertz regression for 5C plants. Solid
arrows represent the different growth stages of RT plants, and white arrows represent the different growth stages for 5C plants.

157

development of the rst leaves occurs, with smaller increases in


bulb diameter. The second stage did not show pronounced changes
in the bulbing index, indicating that growth is carried out simultaneously by the bulb and pseudostem, and the length of this stage
depended on the conditioning of seed cloves (50100 days at
5C, or 50170 days for RT plants). The third stage shows a faster
increase in the bulb diameter, from 100 days to harvest time in 5C
plants, and from 170 days to harvest time for RT plants.
Rizzalli et al. (2002), and Portela and Cavagnaro (2005) proposed a four-stage growth cycle for different garlic varieties whose
seeds were stored at RT. Patterns of dry matter accumulation
were similar to the plant growth curves indicated in this work; the
rst three phases described by those authors seem to correspond
to the stages outlined in this paper. This growth model seems to
explain the behavior shown by RT plants. However, this assumption does not t the pattern shown by the 5C plants because neither
photoperiod nor thermoperiod seem to control the crop cycle in the
same way. Therefore, we argue that there must be some additional
factor or an undetermined mechanism that generates an advance
response in the cycle, which could alter the thermo or photoperiod
responses.
The early harvest time, smaller bulb size, and lower crop yields
of 5C plants appear to be explained by the early plant senescence
and the physiological changes of carbohydrate synthesis, and their
accumulation in the bulbs, although its mechanism is not clear.
Pourtau et al. (2006) induced leaf senescence in Arabidopsis by accumulation of glucose and fructose. Our study showed that the leaves
of the 5C plants at 137 DAS had a higher glucose, fructose and
sucrose content that in RT plants, which showed no increases of
glucose until 194 DAS. This suggests that the glucose increment
at 137 DAS may induce the early leaf senescence. Argello et al.
(2006) indicated low levels of glucose and fructose in pre-harvest
garlic Paraguayo Rosado for RT plants, which seems to support
this hypothesis. In addition, the low glucose content in the leaves of
RT plants may indicate a delayed senescence and therefore greater
functional life, which explains the higher plant growth rate and
crop yield for RT plants.
Van den Ende and El-Esawe (2014), and Wingler et al. (2009,
2012), showed that the leaf senescence induction by glucose was
associated with a lower nitrogen supply. In this respect, the garlic fertilization program provides 80% of total nitrogen units at the
initial stages of crop development, and in the nal stages the nitrogen supply is eliminated, which seems to support the proposed
hypothesis.
The presence of 1-kestose, neo-kestose, nistose, and DP5
reported in this work, agrees with that reported by Stahl et al.
(1997) in onion samples using the same analytical technique, while
Baumgartner et al. (2000) reported the presence of kestose and
neo-kestose in garlic bulbs, using separation on column techniques
(SEC) and gas chromatography. The results of FOS content and
HMWF as well as the expression of 1-SST gene conrmed the temporary role of storage in leaves and pseudostems, also suggesting
an active replacement of these carbohydrates and their transport
to the bulbs.
Our working group has reported that the conditioning of seed
cloves at 5 C for 5 weeks induced the expression of genes related to
phenolic and anthocyanin metabolism that were associated with an
increased synthesis of these compounds during bulb development
(Dufoo-Hurtado et al., 2013). In this report, it is noted that the same
treatment induced the expression of 1-SST, which catalyzed the
rst step of fructan metabolism in different organs. This explained
the changes observed in the fructan content during plant development. However, the data shown indicate that the gene expression
and accumulation pattern of these carbohydrates is closely related
to the crop cycle. Del Viso et al. (2009) also indicated an overexpression of 6-SFT in Bromus pictus plants that enhanced fructan

158

T. Guevara-Figueroa et al. / Scientia Horticulturae 189 (2015) 150158

accumulation in the leaves bases. Also, gene expression analysis


in seedlings of garlic grown in controlled environment at 4 C for
27 days was reported by Son et al. (2012), indicating that 15 genes
were up-regulated and 4 down-regulated as a result of exposure to
low temperatures.
Our data show that the conditioning of seed cloves for 5 weeks
at 5 C changed the growth pattern of plants, modied the 1-SST
gene expression related to fructan metabolism, and modied the
accumulation process of these carbohydrates. In addition, these
changes could be explained by the early leaf senescence, in which
glucose may play an important role.
Acknowledgments
This research was supported by SEP-CONACyT-136252, FOFIUAQ FCQ201203 Projects and by the Garlic Producer Association
of Aguascalientes. M.D.D.-H. and K.G.Z.-G., thank CONACyT-Mexico
for their MSc fellowships (55735/306031 and 231388 respectively).
T.G.-F. and M.D.D.-H., would like to thank CONACyT-Mexico for
their Ph. D. fellowships (290611 and 339652, respectively). We
thank D. Stuffer (Faculty of Language and Literature) for his help
in reviewing the English grammar and style.
References
Ade-Ademilua, O.E., Iwaotan, T.O., Osaji, T.C., 2009. Pre-planting (cold) treatments
of Allium sativum L. cloves improves its growth and yield under open eld and
open shade conditions. J. Plant Sci. 4 (3), 4958.

Argello, J.A., Ledesma, A., Nunez,


S.V., Rodrguez, C.H., Diaz-Goldarb, M.d.C., 2006.
Vermicompost effects on bulbing, dynamics nonstructural carbohydrate content, yield, and quality of Rosado Paraguayo garlic bulbs. HortScience 41 (3),
589592.
Baumgartner, B., Dax, T.G., Praznik, W., Falk, H., 2000. Characterization of the highmolecular weight fructan isolated from garlic (Allium sativum L.). Carbohydr.
Res. 328 (2), 177183.
Bekenblia, N., Shiomi, N., 2006. Hydrolysis kinetic parameters of DP 6, 7, 8, and 912 fructooligosaccharides (FOS) of onion bulb tissues. Effect of temperature and
storage time. J. Agric Food Chem. 54 (7), 25872592.
Bertoni, G., Morand, P., Soubieille, C., Llorens, J.M., 1992. Growth and nitrogen nutrition of garlic (Allium sativum) during bulb development. Sci. Hortic. 50 (2),
187195.
Braz, L.T., Da Silva, E.J., Castellane, P.D., 1997. The effects of pre-planting refrigeration
on bulbs over the development and yield of garlic Chines, Contestado and
Quiteria. Acta Hortic. 433, 499506.
Burba, J.L., 2007. Manejo de Semilla de ajo Frigoinducida. Proyecto Ajo INTA 088.
Ediciones Instituto Nacional de Tecnologa Agropecuaria, Estacin Experimental
La Consulta, Argentina, pp. 13.
Del Pozo, A., Gonzlez, M.I., Barraza, C., 1997. Phenological development of 13 clones
of garlic, (Allium sativum L.): inuence of temperature, photoperiod and cold
storage. Acta Hortic. 433, 389394.
Del Viso, F., Puebla, A.F., Fusari, C.M., Casabuono, A.C., Couto, A.S., Pontis, H.G., Hopp,
H.E., Heinz, R.A., 2009. Molecular characterization of a putative sucrose:fructan
6-fructosyltransferase (6-SFT) of the cold-resistant Patagonian grass Bromus pictus associated with fructan accumulation under low temperatures. Plant Cell
Physiol. 50 (3), 489503.
Dufoo-Hurtado, M.D., Zavala-Gutirrez, K.G., Cao, C.-M., Cisneros-Zevallos,
L., Guevara-Gonzlez, R.G., Torres-Pacheco, I., Vzquez-Barrios, M.E.,

Rivera-Pastrana, D.M., Mercado-Silva, E.M., 2013. Low-temperature conditioning of Seed cloves enhances the expression of phenolic metabolism
related genes and anthocyanin content in Coreano garlic (Allium sativum)
during plant development. J. Agric. Food Chem. 61, 1043910446.
FAOSTAT. 2012. Web page. http://faostat3.fao.org/faostat-gateway/go/to/
download/Q/QC/E (consulted July/2014).
Khokhar, K.M., Hardley, P., Pearson, S., 2007. Effect of cold temperature durations
of onion sets in store on the incidence of bolting, bulbing and seed yield. Sci.
Hortic. 112, 1622.

Ledesma, A., Nnez,


S.B., Argello, J.A., 1997. Bulbing physiology in garlic (Allium
sativum L.) cv. rosado paraguayo II. Characterization of ontogenic stages by
shoot growth dynamics and its relation to bulbing. Acta Hortic. 433, 405416.
Martens, D.A., Frankenberger Jr., W.T., 1990. Determination of saccharides by
high performance anion-exchange chromatography with pulsed amperometric
detection. Chromatographia 29 (2), 712.
McCleary, B.V., Murphy, A., Mugford, D.C., 2000. Measurement of total fructan in
foods by enzymatic/spectrophotometric method: collaborative study. J. AOAC
Int. 83, 356364.
Ofcial United States Standards for Grades of Garlic, 1997. Web page. http://www.
(consulted
ams.usda.gov/AMSv1.0/getle?dDocName=STELPRDC5050266
July/2014).
Portela, J.A., 1996. El ambiente como regulador del desarrollo de las plantas.
Su efecto sobre el ajo (Allium sativum L.). Avances en Horticultura 1 (1),
1940.
Portela, J.A.,2000. Effect of eld temperature on white garlic (Allium sativum L.) vegetative growth. In: Proc. 3rd Intl. Symp. on Edible Alliaceae. The University of
Georgia, Athens, GA, USA, pp. 231234.
Portela, J.A., 2001. Genetic and environmental effects inuencing branching in garlic
(Allium sativum L.). Acta Hortic. 555, 175179.
Portela, J.A., Cavagnaro, J.B., 2005. Growing phases of the white garlic (Allium
sativum) plant in relation to eld temperature and day length. Acta Hortic. 688,
239246.
Pourtau, N., Jennings, R., Pelzer, E., Pallas, J., Wingler, A., 2006. Effect of sugar-induced
senescence on gene expression and implications for the regulation of senescence
in Arabidopsis. Planta 224, 556568.
Rahim, M.A., Fordham, R., 1988. Effect of storage temperature on the initiation and
development of garlic cloves (Alium sativum L.). Sci. Hortic. 37, 2538.
Rahim, M.A., Fordham, R., 2001. Environmental manipulation for controling bulbing
in garlic. Acta Hortic. 555, 181188.
Reis, R.M., Cecon, P.R., Puiatti, M., Finger, F.L., Nascimento, M., Silva, F.F., Carneiro,
A.P.S., Silva, A.R., 2014. Modelos de regresso no linear aplicados a grupos de
acessos de alho. Hortic. Bras. 32, 178183.
Rizzalli, R.H., Villalobos, F.J., Orgaz, F., 2002. Radiation interception, radiation-use
efciency and dry matter partitioning in garlic (Allium sativum L.). Eur. J. Agron.
18, 3343.
Son, J.-H., Park, K.-C., Lee, S.-I., Kim, H.-H., Kim, J.-H., Kim, S.-H., Kim, N.-S., 2012.
Isolation of cold-responsive genes from garlic, Allium sativum. Genes Genomics
34, 93101.
Stahl, B., Linos, A., Karas, M., Hillenkamp, F., Steup, M., 1997. Analysis of fructans from
higher plants by matrix assisted laser desorption/ionization mass spectrometry.
Anal. Biochem. 246, 195204.
Takagi, H., Qu, Y., 1995. Effects of light quality, photoperiod and cold treatment on
in vitro bulbing of garlic soot tip. Acta Hortic. 393, 181188.
Van den Ende, W., El-Esawe, S.K., 2014. Sucrose signaling pathways leading to fructan and anthocyanin accumulation: a dual function in abiotic and biotic stress
responses? Environ. Exp. Bot. 108, 413.
Volk, G.M., Rotindo, K.E., 2004. Low-temperature storage of garlic for spring plant.
HortScience 39 (3), 571573.
Wingler, A., Masclaux-Daubresse, C., Fischer, A.M., 2009. Sugars, senescence,
and ageing in plants and heterotrophic organisms. J. Exp. Bot. 60 (4),
10631066.
Wingler, A., Stangberg, E.J., Saxena, T., Mistry, R., 2012. Interactions between temperature and sugars in the regulation of leaf senescence in the perennial herb
Arabis alpina L. J. Integr. Plant Biol. 54 (8), 595605.

You might also like