Scientia Horticulturae 225 (2017) 498–504

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

System construction of virus-free and rapid-propagation technology of Baodi T

garlic (Allium sativum L.)
Baoli Fana, Rongfeng Hea, Yuntao Shangb, Liwei Xua, Ningning Wanga, Hui Gaoa, Xiaoying Liua,
⁎
Zhenying Wanga,
a
College of Life Science, Tianjin Normal University, Tianjin, 300387, PR China
b
Tianjin Key Laboratory of Water Resources and Environment, Tianjin Normal University, Tianjin, 300387, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Garlic (Allium sativum L.) is cultivated by extensive vegetative propagation, which may lead to germplasm de-
Baodi garlic gradation, the accumulation of various viruses, a low propagation coefficient, and high production costs. Tissue
Tissue culture culture is the most effective method for the rapid propagation and germplasm enhancement of virus-free garlic.
Inflorescence Here, we present a system for producing tissue culture garlic using inflorescences of Baodi garlic ‘Liu Ban Hong’
ELISA
as explants. By optimizing the type and concentration of plant growth regulators, as many as 11 adventitious
buds could be produced from one explant. The propagation coefficient of bulblets in vitro could reach 10.64 per
explant after optimization of the culture approach and induction parameters. Additionally, tissue culture garlic
was shown to achieve a certain virus elimination effect by ELISA. In this study, we present a complete system for
producing virus-free Baodi garlic from explants. This system provides important technical support for elim-
inating viruses, increasing propagation efficiency, maintaining genetic stability, and improving the quality of
garlic.

1. Introduction
Garlic (Allium sativum L.) is a perennial plant with high nutritional
and medicinal value. Garlic products are popular across the world be-
cause of their antibacterial, anticancer, and anti-cardiovascular benefits
(Li et al., 2016; Ried, 2016; Varshney and Budoff, 2016). Garlic is
usually sterile and thus propagated asexually because it does not pro-
duce seeds (Rotem et al., 2011; Shemesh et al., 2013). The lack of
sexual propagation reduces biodiversity and genetic variation, there-
fore resulting in variety degeneration that can negatively influence
garlic yield and quality. Long-term asexual reproduction also leads to
the accumulation of garlic viruses that affects the commodity value of
garlic (Dijk, 1993). At present, the application of plant tissue culture,
directed mutagenesis, genetic engineering, and polyploidy breeding,
largely improves the breeding of garlic (Etoh et al., 2002; Nabulsi et al.,
2001; Park et al., 2002). Tissue culture is the most effective virus-free
and rapid propagation technology and provides an efficient tool for
producing virus-free garlic plants (Gimenez et al., 2016; Taşkın et al.,
2013).
In garlic tissue culture systems, different explants such as stem apex
(Luciani et al., 2006; Ramakrishnan et al., 2013), root tip (Haque et al.,
1997), young leaf (Fereol et al., 2002; Kahane et al., 1992; Kenel et al.,
2010), storage leaf, leaf primordium (Luciani et al., 2006), immature
inflorescence (Xiong et al., 2000), bulb, and aerial bulb were used for
induction and regeneration. Adventitious buds were induced through
the direct pathway from explants. This pathway has the following ad-
vantages: stable genetic characters, low mutation rates, and short re-
generation times. At present, the shoot tip and basal plate are mainly
used as explants for adventitious bud multiplication with the average
propagation coefficient between 3 and 7 (Mohamed-Yasseen and
Costanza, 1996; Wang and Sun, 1996). The virus elimination rates of
plantlets from scape-tip were 77.6%, this suggested that there is a ca-
pacity of virus elimination in the growth of garlic scape-tip and the
development of plant seed embryos (Ma et al., 1994). High production
of adventitious shoots is one of the capabilities of garlic inflorescence,
which made it an ideal explant for rapid propagation of adventitious
shoot. The propagation rate of inflorescence is 19.2 (Zhang and Li,
2007). This result demonstrates that the inflorescence can be used as an
important explant for virus elimination and rapid propagation of garlic.
Although many researchers have obtained more adventitious buds,
successful cultivation of virus-free garlic has rarely been reported thus
far. The induction of high frequency plantlets is only one of the rate-

Abbreviations: ELISA, enzyme linked immunosorbent assay; OYDV, Onion yellow dwarf virus; LYSV, Leek yellow stripe virus; GMV, Garlic mosaic virus
⁎
Corresponding author at: No.393 Bin Shui Xi Road, Xiqing District, Tianjin 300387, PRChina.
E-mail addresses: skywangzy@mail.tjnu.edu.cn, wzycell@163.com (Z. Wang).

http://dx.doi.org/10.1016/j.scienta.2017.07.042
Received 24 March 2017; Received in revised form 18 July 2017; Accepted 25 July 2017
Available online 04 August 2017
0304-4238/ © 2017 Elsevier B.V. All rights reserved.
B. Fan et al. Scientia Horticulturae 225 (2017) 498–504

limiting conditions for virus-free garlic production; other important Table 1

factors are the rooting, transplanting, survival, and absence of viruses in plant growth regulators ratio of media.
the garlic. Hyperhydricity is also a common physiological disorder
Media Murashige and Skoog mineral solution
during plant in vitro culture and can seriously affect the regeneration
and micropropagation of plants (Liu et al., 2017). Many researchers 6-BA (mg L−1) KT (mg L−1) NAA (mg L−1)
have obtained a large number of regenerated plants from callus by in
1 3.0 0 0
vitro culture, but lost a large number of plants in the rooting and
2 2.0 0 0
transplanting process, which reduces the overall reproductive efficiency 3 1.0 0 0
(Matsubara and Chen, 1989; Osaws and Sugawara, 1980). These diffi- 4 3.0 0 0.1
culties make in vitro culture a time-consuming and labor-intensive 5 2.0 0 0.1
process with restricted applications. In an attempt to overcome these 6 1.0 0 0.1
7 0 3.0 0
obstacles, many researchers have successfully improved the survival
8 0 2.0 0
rate by optimizing the transplanting conditions of regenerated garlic 9 0 1.0 0
plants (Jea, 1994; Kwangsoo et al., 2006; Mohamed-Yasseen and 10 0 3.0 0.1
Splittstoesser, 1995; Suh and Park, 1993). However, the effect is not 11 0 2.0 0.1
12 0 1.0 0.1
very good because of acclimation. Xiong et al. obtained bulblets from
induction of plantlets, and found that the transplanting survival rate
could be improved (Xiong et al., 1999). In addition, bulblets have many BA), 1-naphthylacetic acid (NAA) and Kinetin (KT) (Sangon Biotech), as
advantages: they are easy to store and transport, have stronger re- detailed in Table 1. The media were autoclaved at 121 °C for 20 min.
sistance, and do not require hardening. Previous research indicates that The cultures were incubated at a temperature of 25 ± 2 °C under 16 h
bulblets can be grown directly and normally after dormant periods si- daily illumination with white fluorescent light (1500 lx).
milar to those of common garlic (Li et al., 1995).
In this study, we used inflorescence of Baodi garlic “Liu Ban Hong”
2.3.2. Development of adventitious buds and bulblet induction
as explants and improved the media for inducing buds by optimizing
The explants were grown for 6 weeks until the height of ad-
the plant growth regulators type and concentration. We then con-
ventitious buds reached 3–5 cm, and then were transferred into another
structed a complete technical system of the regenerants from explants
medium (MS medium adding 120 g L−1 soft white sugar and 6.8 g L−1
to virus-free plants, combined with the induction of bulblets and im-
agar, pH = 7.5) (Xiong et al., 2000). The plantlets were grown in the
proved cultivation techniques. The system provides an important
artificial climate chamber (Climacell CLC707, MMM) at 25 ± 1 °C
technical basis for garlic virus elimination, rapid propagation, main-
with 16 h light/8 h darkness, and with the 1500 lx illumination in-
tenance of good genetic stability, and improvement of bulb yield and
tensity. The calculation formula of bulblets induction frequency is the
quality.
total number of bulblets/the total number of adventitious buds.
2. Material and methods
2.3.3. Harvesting and storage of bulblets
2.1. Plant material The developing bulblets were grown in the selective media for 6
weeks then taken out and washed. After drying, they were stored at 4 °C
Baodi garlic “Liu Ban Hong” is a famous high-quality variety in for 3 weeks in the dark and then planted in the field directly.
Tianjin. The separate cloves of garlic were planted in Tianjin Normal
University Biotechnology Park at the end of October 2014, the average 2.3.4. Morphological observation of regenerated plants and bulbs
maximum air temperature was 17 °C and the average minimum air Bulblets that passed the dormant period were planted at the end of
temperature was 10 °C. October 2015 in the field with film mulching, the average maximum air
temperature was 15 °C and the average minimum air temperature was
2.2. Reagents 6 °C. Film uncovering, weeding, watering, and soil loosing were per-
formed in May 2016. The garlic bulbs were ripe and harvested in late
Double antibody sandwich (DAS) ELISA test kit for the detection of June 2016. The garlic bulbs were divided into three groups according to
Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), and the standards and then the diameter of bulbs were measured. Ten bulbs
Garlic mosaic virus (GMV) purchased from the Agdia company (USA). were measured in each of the three groups with the mean value cal-
culated from 30 measurements.
2.3. Methods
2.3.5. DAS-ELISA detection
2.3.1. Adventitious bud induction using inflorescence The detection antibody, enzyme labeled antibody, PNPP substrate,
The Baodi garlic “Liu Ban Hong” was planted at the end of October GEB sample extraction buffer, and polyethylene microporous plate
2014 in the field. When the garlic entered its reproductive stage and the were purchased from Agdia. The enzyme conjugate was alkaline
ratio between flower stem length and pseudo stem length was ap- phosphatase labeled antibodies to 3 viruses (OYDV, LYSV and GMV),
proximately 1to1.2, the inflorescences with 1 cm long stem were cut with GEB extraction buffer as the negative control. Virus elimination
and stored for 2 weeks at 4 °C. The inflorescences without stem were effect of common Baodi garlic and the garlic from bulblet planting was
cleaned in running water for 30 min, disinfected in 0.1% HgCl2 for detected by ELISA method according to kit instructions with 3 re-
20 min, rinsed in distilled water 3–4 times, disinfected in 75% alcohol plicates.
for 10 min, and then rinsed in distilled water 3–4 times. After the outer
bracts, flower primordial residue, and membranous bracts were 3. Results
stripped, and the flower stems were cut, the remainder were introduced
in the media with two explants per bottle. The medium was basic 3.1. Induction of adventitious buds from inflorescence
Murashige and Skoog medium (Murashige and Skoog, 1962) with
30 g L−1 sucrose and 7.5 g L−1 agar added and the pH adjusted to 6.2. Twelve variants of media were designed and tested to determine the
At this step, the medium was supplemented with various types and effect of different plant growth regulators (KT, 6-BA, and NAA) and
concentrations of plant growth regulators, 6-Benzylaminopurine (6- their concentrations on the efficiency of adventitious bud regeneration.

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B. Fan et al. Scientia Horticulturae 225 (2017) 498–504

Table 2
Influence of plant growth regulators on adventitious buds proliferation and bulblets formation.

Media plant growth regulators Number of Number of adventitious Number of Number of adventitious Induction ratio of propagation
(mg L−1) explants buds bulblets buds per explant bulblets per bud coefficient

6-BA KT NAA

1 3 0 0 12 46 44 3.83aA 95.65bB 3.67aA

2 2 0 0 17 104 101 6.12bC 97.12dD 5.94bB
3 1 0 0 16 82 81 5.13bC 98.78eC 5.06bB
4 3 0 0.1 24 75 72 3.13aA 96.00cC 3.00aA
5 2 0 0.1 16 95 91 5.94bC 95.79bB 5.69bB
6 1 0 0.1 18 88 85 4.89aAB 96.59cC 4.72bB
7 0 3 0 18 93 90 5.17bBC 96.77cD 5.00bB
8 0 2 0 15 156 151 10.40dE 96.79cC 10.07dD
9 0 1 0 12 95 88 7.92cD 92.63aA 7.33cC
10 0 3 0.1 14 95 93 6.79bC 97.89dE 6.64cC
11 0 2 0.1 11 121 117 11.00eF 96.69cC 10.64dD
12 0 1 0.1 14 55 54 3.93aA 98.18eF 3.86aA

Note: the lower case letters indicates the significant difference at 5% level (p < 0.05) and the capital letters indicate the significant difference at 1% (p < 0.01).

The final rates of adventitious bud production were measured to de-
termine the optimized culture conditions (Table 2).
Table 2 shows that the induction rate of medium 2 was the highest
compared to medium 1 through 6 with the number of adventitious buds
induced by each inflorescence reaching 6.12. The medium without NAA
had a higher induction rate under the same 6-BA concentrations, in-
dicating that 6-BA had a much higher effect than NAA. Medium 11 had
the highest induction rate compared to medium conditions 7 through
12 with the number of adventitious buds induced by each inflorescence
reaching 11. The medium with NAA had a higher induction rate under
the same KT concentrations, showing that KT and NAA had a co-
operative effect on the induction of adventitious buds. The data in
Table 2 also shows that KT is more suitable than 6-BA for induction of
adventitious buds because the induction ratio of culture medium 7–12
was higher than culture medium 1–6. Therefore, the optimum medium
was medium 11 (MS + 2.0 mg L−1 KT + 0.1 mg L−1 NA-
A + 30.0 g L−1 sucrose + 7.5 g L−1 agar, pH = 6.2).
The different stages of adventitious buds induction in optimal media
are as follows (Fig. 1): The inflorescences following introduction to
culture media (Fig. 1a). The inflorescences had meristems on the sur-
face and expanded in a week or so. The cut surfaces became thicker and
formed multiple shoots with most densely growing at the middle and
lower parts (Fig. 1b). Translucent adventitious buds continued to grow
up to 2 cm and turned tender green 2 weeks later (Fig. 1c, d). After 4
weeks, multiple shoots had formed (Fig. 1e) and could be transplanted
into induction media for bulblet induction 6 weeks later (Fig. 1f).
3.2. Induction of bulblets from adventitious buds
Induction of bulblets was performed to improve reproductive effi-
ciency, because many buds at the base of the inflorescences were dif-
ficult to ensure real root induction from every plantlet and transplan-
tation. The inductive medium for bulblets was: MS + 120.0 g L−1 soft
white sugar + 6.8 g L−1 agar, pH = 7.5. The adventitious buds were
just transplanted into media (Fig. 2a), the basal part of the buds became
swollen 1 week later (Fig. 2b), and the plantlets became withered after
2 weeks (Fig. 2c). The bulb tunics gradually turned purple after 3 weeks
(Fig. 2d), the most bulb tunics became purple and green leaves had
withered 4 weeks later (Fig. 2e), and the bulblets matured after 6 weeks
(Fig. 2f). The bulblets were taken out, washed, and then dried in the
shade.
The bulblets induction frequency of adventitious buds was over
90%, with a maximum of 98.78%. Additionally, the number of bulblets
per inflorescence was as many as 10.64, and the propagation coefficient

Fig. 1. Induction of adventitious buds from garlic

inflorescences.
a. Introduction of plant explants into tissue culture in
June 2015. b. The inflorescences had meristems on
the surface and expanded, the cut surfaces became
thicker and formed multiple shoots. c. Translucent
adventitious buds continued to grow up to 2 cm and
turned tender green. d. The green leaves elongated to
3 cm. e. The length of green leaves was about 4 cm. f.
The multiple shoots could be transplanted into in-
duction media. (bar = 1 cm).

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B. Fan et al.
Fig. 3. The correlation analysis of adventitious buds number and bulblets number.
of bulblets induction was more than 2 times that of traditional planting
pattern (Table 2). There was a significant positive correlation between
the number of adventitious buds and bulblets induced by inflorescence
(p < 0.05) (Fig. 3)
3.3. Harvest and storage of bulblets
The total number of bulblets induced by garlic inflorescences was
1260. The mean large-diameter of bulblets was 6.65 mm, the mean
medium-diameter bulblets was 4.23 mm, and the mean small-diameter
bulblets was 1.68 mm. The bulblets harvested from culture bottle
showed clusters and had up to 25 bulbs per cluster (Fig. 4a, b). The bulb
tunics were purple like Baodi garlic bulbs (Fig. 4c). The bulblets all had
growing points observed under the dissection microscope (Fig. 4e–g),
which could grow normally after breaking dormancy and planting in
the field.
3.4. Statistical analysis of regenerated plant bulbs
The number of regenerated plants from bulblets was 539 after one
season of field cultivation. There were 530 single-clove garlics, 8 two-
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Scientia Horticulturae 225 (2017) 498–504
Fig. 2. Induction of bulblets from adventitious buds.
a. Transplantation of the plantlets with green leaves
into culture medium for bulb formation. b–f.
Progress in bulblets formation and development
during 6 weeks of cultivation (bar = 1 cm).
clove garlics, and 1 four-clove garlic in harvested bulbs with a max-
imum diameter of 22.14 mm and a minimum diameter of 3.16 mm.
There also was one secondary growth plant (Fig. 5).
3.5. Virus detection
Virus detection of regenerated plant leaves was performed by DAS-
ELISA technique using OYDV, LYSV, and GMV kits. The absorbance
value (wavelength = 405 nm) was read from leaves with an enzyme-
labeled instrument and compared with that from the negative and po-
sitive control. Virus infection was more serious in garlic cultvars such as
OYDV, LYSV, and GMV, but the degree of virus infection was different
depending on which viruses were present in the coinfection (Table 3).
The detection results of OYDV, LYSV, and GMV were 3.10, 6.11, and
3.11 times that of the negative control, respectively, which could ne-
gatively affect garlic yield and quality. However, the detection results
of OYDV, LYSV, and GMV from regenerated plants were respectively
2.9, 4.3, and 1.2 times that of the negative control (Table 3), showing a
partial virus elimination.
4. Discussion
Commercial garlic cultivars are propagated only vegetatively using
bulbs and bulbils, which often lead to virus accumulation in cloves,
which negatively influences garlic yield and quality. Because of this,
high quality and disease-resistant varieties of garlic are urgently
needed. Tissue culture is one of the most efficient methods for rapidly
propagating and obtaining virus-free. In this study, we present a com-
plete technical system for virus elimination and rapid propagation of
Baodi garlic “Liu Ban Hong” using inflorescences as explants. The core
of this system is optimization of high frequency induction of bulblets
and vernalization to achieve production of virus-free garlic in a short
period.
4.1. Induction of adventitious buds from inflorescences
Garlic inflorescences can form many bulbils, indicating their strong
regeneration potential. Induction of adventitious buds is affected by
various factors, the most important of which are plant growth reg-
ulators type and concentration. This research identified the optimal
system for the induction of adventitious buds by screening relative
B. Fan et al. Scientia Horticulturae 225 (2017) 498–504

Fig. 4. Morphology, size and anatomy of bulblets in

vitro.
a. Clustered bulblets harvested from the induction of
bulblets 8 weeks later. b. All bulblets each cluster. c.
1260 bulblets harvested from culture bottle. d. Garlic
clove (left) in comparison with bulblets obtained
from in vitro propagation in July 2016. The sizes
were 2 cm, 1.1 cm, 0.55 cm, and 0.35 cm. (a–d:
bar = 1 cm). e–g. The growing points (arrows) of
bulblets under a dissection microscope
(bar = 0.2 cm)

Table 3
Virus detection results of garlic leaves by ELISA technique.

Sample number OYDV LYSV GMV

Negative control 0.10 0.10 0.09

Positive control 3.80 3.80 3.70
Common garlic 0.31(+++) 0.61(++++) 0.28(+)
Regenerated plants 0.29(++) 0.43(+++) 0.12(−)

Note: The ratio between absorbance value of sample and negative control ≤2 indicates
negative result (−), the ratio ≥2 indicates positive result (+), the ratio ≥3 indicates
positive result (++), the ratio ≥4 indicates positive result (+++), the ratio ≥5 in-
dicates positive result (++++).

improved significantly by adding NAA, but NAA had no significant ef-

Fig. 5. Regenerated plants and bulbs from bulblets in vitro.
a. Regenerated plant in the ground. b. Harvested regeneration plant. c. Single-clove
garlic. d. Split garlic. e. Secondary growth garlic (Bar = 1.9 cm).
optimal medium components with concentrations of several kinds of
plant growth regulators. The results showed that 3 kinds of plant
growth regulators (6-BA, KT and NAA) could induce adventitious buds,
but with different inductive effects. KT had a greater effect on ad-
ventitious buds induction than 6-BA, and induction ratio of buds in-
creased initially before decreasing with the continued increase of plant
growth regulators concentration. The induction effect of KT was
fect on 6-BA. This demonstrates that KT and NAA have a synergistic
effect on adventitious buds induction.
In this work, young inflorescences of garlic were used for inducing
adventitious buds directly, so the genetic traits could be stably in-
herited. Importantly, the inflorescence at the apex of the plant, the
probability of being infected by virus is lower. Accordingly, using the
inflorescence as explants with tissue culture methods can result in virus-
free garlic, consistent with the results of previous studies (Ma et al.,
1994; Xiong et al., 2000). Induction of adventitious buds is affected by
various factors, especially cytokinin, but cytokinin and auxin cooperate
well to increase induction frequency. The inflorescence of garlic was
stored for 2 weeks at 4 °C, which is favorable to the induction of buds.
Using the inflorescence for producing adventitious buds has many ad-
vantages including faster growth, more adventitious buds, and con-
sistent yield.

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B. Fan et al.
4.2. Induction of bulblets from adventitious buds
The bulblets can be induced from the adventitious buds directly,
which have a higher survival rate, do not require hardening, and are
easy to manage. The number of adventitious buds has a positive re-
lationship to the number of bulblets, suggesting that the induction of
buds in the optimal media will produce more bulblets. The bulblets
were transplanted in the test field and the underground bulb of the filial
generation was harvested and compared with other inductive pathways
(Mohamed-Yasseen and Costanza, 1996; Wang and Sun, 1996). Planted
bulblets had higher survival rates and propagation coefficients, lower
maintenance and cost, and shorter growth periods than planted plant-
lets. A large number of laboratory-derived bulblets can be planted in the
field directly, avoiding the technical bottleneck of domestication of
plantlets, and thus has extreme practical value to the agricultural pro-
duction of garlic (Zhang and Li, 2006).
Most harvested garlics are single clove and have no bolting, a
phenomenon that is associated with temperature and the clove size at
planting. The development of traits such as the split, weight, and size of
the garlic clove depends on vegetative growth. Garlic growing during a
period of short days and low temperatures can accumulate nutrients,
which promotes differentiation (Liu et al., 2006). Thus, the bulblets
would need to be planted between the end of October and early No-
vember. The following factors can help prevent secondary growth:
suitable varieties, controlling the amount of nitrogen fertilizer, opti-
mizing the watering period, suitable early sowing, topdressing striking
root fertilizer, and the rational use of phosphorus potassium fertilizer
(Su and Gao, 2011). The split of garlic bulbs can increase the propa-
gation coefficient, and the phenomenon of secondary growth influences
garlic quality, so the cultivation system should be optimized to increase
the propagation coefficient and quality of garlic.
4.3. The detection of virus-free effect of regeneration plant
The bulbil of the garlic inflorescence can achieve virus-free effects
to a certain extent, because the inflorescence has properties that make it
more resistant to virus infection (Xiong et al., 2000). Our results show
that garlic was easily coinfected by 3 viruses (OYDV, LYSV, and GMV),
but that there was a significant difference in the sensitivity of Baodi
garlic to different viruses. Enzyme-linked immunoassay is based on an
antibody-antigen reaction and detects virus by chromogenic methods.
The results show that using inflorescence could result in garlic that was
effectively virus-free, providing a more effective method for producing
high quality, virus-free garlic.
5. Conclusions
In this research, we investigated the effect of different plant growth
regulators combinations on the induction of adventitious buds from
inflorescence in garlic. We identified the optimized culture medium as:
MS + 2.0 mg L−1 KT + 0.1 mg L−1 NAA + 30.0 g L−1 sucrose
+ 7.5 g L−1 agar, pH = 6.2. Under these conditions, there were up to
11 adventitious buds per inflorescence. The virus detection results from
ELISA showed that the regenerated plants had achieved definite virus-
free effect. In this study, we constructed a complete technical system of
garlic using inflorescence of Baodi garlic “Liu Ban Hong” as explants.
Through high frequency induction and vernalization of bulblets, and
optimization of cultivation conditions, we provide a system to improve
the reproduction efficiency, save culture time, and reduce the produc-
tion cost of high quality garlic.
Acknowledgments
This work was supported by the Tianjin agriculture science and
technology achievement transformation and promotion project
(201302030), and Tianjin Science and Technology Program
503
Scientia Horticulturae 225 (2017) 498–504
(14JCYBJC29900). We thank Editage company for critical reading of
English manuscript.
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